REVIEW www.rsc.org/npr | Natural Product Reports

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FView Article Online / Journal Homepage / Table of Contents for this issue

Total biosynthesis: in vitro reconstitution of polyketide and nonribosomalpeptide pathways

Elizabeth S. Sattely,a Michael A. Fischbachb and Christopher T. Walsh*a

Received 28th February 2008

First published as an Advance Article on the web 23rd May 2008

DOI: 10.1039/b801747f

Covering: 1990 to February 2008

This review surveys efforts to reconstitute key steps in polyketide and nonribosomal peptide

biosynthetic pathways with purified enzymes and substrates; 344 references are cited.

1 Introduction

2 Chemical logic and enzymatic machinery of NRPS and

PKS assembly lines

2.1 Chemical logic

2.2 Enzymatic machinery

2.3 Type I and type II enzymes

2.4 Four stages in NRP and PK biosynthesis

3 Phosphopantetheinyl transferases: the essential post-

translational priming catalysts

4 Just-in-time fashioning of dedicated monomers as

building blocks for assembly lines

4.1 Hydroxyphenylglycine and dihydroxyphenyglycine

building blocks for glycopeptide antibiotics

4.2 Halogenation of amino acid building blocks

4.3 b-Amino acid building blocks

4.4 Other nonproteinogenic building blocks

5 Assembly line action

5.1 Chain initiation: monomer selection, loading and

acylation

5.2 Chain elongation: Claisen, amide, and ester conden-

sations

5.2.1 C domains: condensation and heterocyclization

5.2.2 C domains that make ester linkages

5.3 NRP-PK hybrids: module switching and chain elon-

gation

6 Chain tailoring on the assembly line

6.1 Epimerization at Ca

6.2 N- and C-Methylations at Ca and Cb

6.3 Cb branches

6.4 Oxidative transformations on assembly lines

7 Chain release mechanisms and catalytic machinery

8 Post-assembly-line tailoring reactions

8.1 Post-assembly tailoring to produce glycopeptide and

lipoglycopeptide antibiotics

8.2 Tandem glycosylations of aromatic polyketide agly-

cones

8.3 C-Glycosylation: enterobactin to salmochelin

8.4 Post-assembly-line oxygenases

aDepartment of Biological Chemistry and Molecular Pharmacology,Harvard Medical School, 240 Longwood Ave., Boston, MA, 02115, USAbBroad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge,MA, 02142, USA

This journal is ª The Royal Society of Chemistry 2008

9 In vitro reconstitution of complete pathways

9.1 Enterobactin to salmochelin to microcin E492: three

layers of secondary metabolism

9.2 Yersiniabactin reconstitution: an NRP-PK hybrid

9.3 Aromatic polyketide scaffolds from malonyl-CoA

monomers

9.4 Enterocin: a type II PKS pathway that builds a tricyclic

scaffold

9.5 Aminocoumarins: four-component assembly of the

novobiocin scaffold and seven-component assembly of

coumermycin

9.6 Rebeccamycin, staurosporine, violacein, and terrequi-

none: reconstruction of pathways involving tryptophan

oxidative dimerization

9.6.1 Rebeccamycin and staurosporine

9.6.2 Violacein

9.6.3 Terrequinone

10 Lessons learned

11 Acknowledgements

12 References

1 Introduction

Bacterial and fungal genome sequences have radically altered the

framework for the study of natural product biosynthesis. This is

particularly true for molecular scaffolds of nonribosomal peptide

(NRP) and polyketide (PK) origin,1 where biosynthetic genes are

clustered together and can be readily detected by bioinformatic

algorithms. It has long been known that some bacterial phyla are

more prolific producers of NRP and PK products than others,

but a recent review by Donadio et al. in this journal provides

a clear and comprehensive view of NRP, PK and hybrid

NRP-PK biosynthesis from the sequenced genomes of 223

bacteria.2 Donadio et al. noted that in the current genomic

database, g-proteobacteria, actinobacteria, b-proteobacteria,

and firmicutes, in decreasing order, have the highest number of

nonribosomal peptide synthetase (NRPS) and polyketide

synthase (PKS) genes. They also made the correlation that PK

and NRP natural products are rare in bacteria with genomes

#3 Mb. For bacteria with genomes >5 Mb, there is a linear

correlation between genome size and number of PK and NRP

gene clusters, as though these classes of molecules are luxury

items added after a basic self-sustaining metabolic capacity has

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been achieved. NRP genes encoding siderophores3 are the most

common among all the NRPS and PKS encoding genes yet

detected, consistent with the observation that iron scarcity often

limits bacterial growth.4

The current bacterial genome database is biased towards the

genomes of pathogens, reflecting strong initial interest in using

genome information to identify new targets for antibiotics. As

bacteria with larger genomes are sequenced,5–9 it is likely that the

library of predicted novel PKS and NRPS biosynthetic gene

clusters will grow and serve as a beacon for selecting organisms

from which to isolate biologically active small molecules with

novel molecular scaffolds.

Microbial genomics and associated bioinformatic analyses

indicate the potential NRP and PK natural product biosynthetic

capacity of a producer organism, but they yield little information

about: (i) which pathways are active or cryptic under given

conditions, (ii) the structures of the encoded natural products,

and (iii) what chemical steps have been achieved by particular

protein catalysts. To close the gap between genome-guided

prediction and current chemical observation, two routes are

possible. One is to culture predicted secondary metabolite

producers, whose genomes have not yet been sequenced, under

a matrix of conditions to elicit the production of secondary

metabolites in the laboratory setting. This is the focus of much

current effort in PK and NRP discovery.10

A complementary approach is to overproduce some or even all

of the proteins in a predicted pathway, characterize their

catalytic activities, and look for intermediates and/or new

Elizabeth S: Sattely

Elizabeth S. Sattely received her

Ph.D. in 2006 from Boston

College with Amir Hoveyda,

where her research focused on

catalytic asymmetric olefin

metathesis and alkaloid total

synthesis through a collabora-

tion with Richard Schrock at

MIT. She is currently a Damon

Runyon Cancer Research Foun-

dation Fellow in the laboratory

of Christopher Walsh, where she

is studying siderophore biosyn-

thetic pathways.

Christopher T: Walsh

Christopher T. Walsh is the Hamilt

macology at HMS. He has served as

and the Department of BCMP at HM

Cancer Institute (1992–1995). His r

758 | Nat. Prod. Rep., 2008, 25, 757–793

products as a way to elucidate the metabolic logic of their

biosynthesis. This topic—the chemical transformations enacted

by NRPS and PKS catalysts—is the subject of this article and

should serve as a complement and companion piece to the recent

genomics review by Donadio et al. in this journal.2

In vitro studies on one or more enzymatic components of

a microbial natural product pathway allow the dissection of its

chemical logic. This approach also sheds light on the means by

which simple monomeric building blocks—typically acyl-CoAs

and amino acids—are forged into molecular scaffolds of

remarkable architectural diversity and functional group

complexity. Understanding the fundamental steps of acyl chain

initiation, elongation, termination, and tailoring is an essential

first step toward investigating how metabolic pathways evolve

and how they may be re-engineered to produce new molecules.

The catalytic rules for assembly line enzymes that have been

deciphered thus far allow the scaffolds of some natural products to

be predicted through bioinformatic analysis of gene sequences.11

However, the information in most gene clusters cannot yet be

read to predict end product structures, especially for gene clusters

with multiple tailoring enzymes, biosynthetic genes in

unusual orientations, or ‘missing’ genes, and for gene clusters that

encode novel small molecules or molecules produced by assembly

line enzymes that operate in an unconventional fashion.12

This review recounts some of the recent progress that has been

made to characterize enzymes in NRP, PK, and NRP-PK

pathways with emphasis on novel chemical transformations and

unusual functional groups that underlie the pharmacologic

Michael A: Fischbach

Michael A. Fischbach received

his Ph.D. in 2007 from Harvard

University, where he studied

siderophore-mediated iron

acquisition in the laboratories of

Christopher Walsh and David

Liu. He is currently an

Instructor of Medicine at

Harvard Medical School, where

he leads a collaborative effort

between HMS and the Broad

Institute focused on actinomy-

cete genome sequencing and

natural products research.

on Kuhn Professor of Biological Chemistry and Molecular Phar-

the Chair of the Department of Chemistry at MIT (1982–1987)

S (1987–1995), and the President and CEO of the Dana Farber

esearch has focused on enzymes, enzyme inhibitors, and antibiotics.

This journal is ª The Royal Society of Chemistry 2008

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activity and therapeutic interest in many of these molecular

classes. While there have been many studies on individual

enzymes, our primary focus will be on sets of enzymes that act in

tandem to carry out a series of chemical transformations. In

a few cases, reconstitution of a complete pathway has been

achieved, providing insights into how the constituent enzymes

work together and enabling the in vitro biosynthesis of unnatural

analogs by mixing components from different pathways.

Hertweck and colleagues13 have recently reviewed type II PKSs

that construct aromatic natural products; in complementary

fashion, we will focus primarily on type I PKS and NRPS

assembly lines. Our intention is not to provide an exhaustive list

of examples; rather, we describe a few representative cases that

can serve as a gateway to the literature for interested readers.

Fig. 1 Monomers for FA, PK, and NRP biosynthesis. Malonyl-

coenzyme A (malonyl-CoA) is the monomer for fatty acid biosynthesis,

while malonyl-CoA and methylmalonyl-CoA are both monomers for PK

biosynthesis. Amino acids and aryl acids are the most common mono-

mers for NRP biosynthesis.

Fig. 2 Nascent PK and NRP chains grow as a series of elongating, protein-bo

the first four modules of the nystatin PKS are shown, featuring acyl or peptid

groups of thiolation (T) domains.

This journal is ª The Royal Society of Chemistry 2008

2 Chemical logic and enzymatic machinery of NRPSand PKS assembly lines

2.1 Chemical logic

The assembly of fatty acids (FAs), polyketides (PKs), non-

ribosomal peptides (NRPs), and NRP-PK hybrids involves

equivalent logic for converting simple acid and amino acid

building blocks into linear condensed polymers that may

undergo further elaboration.1,14 FA and PK chains grow by the

iterated addition of malonyl units (or methylmalonyl units for

PKs) in which the C–C bonds are forged during chain elongation

by decarboxylative thio-Claisen condensations. NRPs are

formed by the condensation of amino acids, by C–N (amide)

rather than C–C bond formation.

Both the C–C bond-forming enzymes in FASs and PKSs and

the C–N bond-forming catalysts in NRPs use acyl or aminoacyl

thioesters as activated monomer units (Fig. 1). Hydrolysis of one

of the reacting acyl thioesters drives the equilibrium in favor of

C–C or C–N bond formation with concomitant chain elonga-

tion. The process is repeated several times in succession; for

example, seven times by a FAS to build C16 fatty acids,15,16 or six

times by a PKS to create the fourteen-membered macrolactone

scaffold of erythromycin.17

The FA, PK and NRP chains thus grow as a series of

elongating acyl/peptidylthioesters in which the sulfur atom of

the thiol group is the terminus of a phosphopantetheine

arm covalently attached to carrier protein domains

embedded in the assembly line enzyme (Fig. 2; see also

Fig. 8 and Section 3). This process is sometimes referred to

as thiotemplating.18–21

und acyl or peptidylthioesters. Schematics of the echinomycin NRPS and

yl thioester intermediates tethered to the phosphopantetheine prosthetic

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2.2 Enzymatic machinery

FAS and PKS enzymes have a core of catalytic domains along

with a carrier protein domain that constitutes the minimal

machinery for chain assembly, to which additional catalytic

domains can be added for tailoring purposes (Fig. 3).1 For PKSs,

the minimal domains are: (i) the C–C bond-forming ketosyn-

thase (KS); (ii) the acyltransferase (AT), which introduces

malonyl or methylmalonyl units during each cycle of chain

elongation; and (iii) the carrier protein domain, also known as

a thiolation (T) domain, on which the acyl chain is assembled and

elongated. Optional tailoring domains include ketoreductase

(KR), dehydratase (DH) and enoylreductase (ER).17,22

NRPS enzymes have a comparable minimal set of two cata-

lytic domains and one carrier protein domain.23 The peptide

bond-forming condensation (C) domain and the amino acid

Fig. 3 Core PKS and NRPS domains. Schematic abbreviations used

throughout the text are shown.

Fig. 4 Organization of assembly line enzyme domains. Type I PKSs and N

proteins. Type II PKSs and NRPSs have protein domains that are not covale

760 | Nat. Prod. Rep., 2008, 25, 757–793

adenylation (A) domain interact with a phosphopantetheine-

primed T domain to build the peptide chain as an elongating

thioester. Optional tailoring domains in NRPSs include epim-

erase (E) and methyltransferase (MT) domains.24 Since FASs,

PKSs, and NRPSs all build chains as elongating thioesters, they

must also have a catalytic domain to release full length acyl/

peptidyl chains from their covalent attachment to the assembly

line enzyme. This process is usually carried out by a thioesterase

(TE) domain.25,26

2.3 Type I and type II enzymes

Assembly line enzymes have been divided into two subclasses

known as type I and type II organizations.27 Type I enzymes have

their catalytic and carrier protein domains stitched together like

beads on a string, while the domains in type II enzymes are

separate proteins that interact only transiently during acyl/

peptidyl chain growth (Fig. 4). Yeast and humans use a type I

FAS,15,16 while bacteria use either a type I or a type II FAS.28,29

Assembly line enzymes are also divided into modular and

iterative systems (Fig. 5). In modular assembly lines, each

catalytic and carrier protein domain is used only once as the

growing chain passes from the N-terminal module to the

C-terminal module. The seven-module erythromycin PKS,

distributed over three enzymes, is a paradigm for modular

organization.30,31 Most NRPSs are also modular, type I assembly

lines.32 In contrast, many fungal type I enzymes33 and the

mammalian FAS34 comprise only a single module that is used

iteratively during cycles of chain elongation. For example, the

mammalian FAS catalyzes eight cycles of chain elongation—

with the growing chain anchored to a single carrier protein

domain—before releasing a C18 fatty acid. A more recently

characterized example is the bacterial protein family PKSE,

a group of type I modular PKSs that catalyze seven cycles of

elongation to synthesize a polyene fatty acid that is the precursor

to the highly reactive enediyne warhead of polyketides like

C-1027 and maduropeptin.35

Aromatic polyketides, such as the antibiotic tetracycline and

the anticancer drug daunomycin, are made by type II PKSs (see

Hertweck et al.13 for a recent review). The catalytic and carrier

protein domains are separate proteins that interact transiently.

The PK chain grows on the phosphopantetheine arm of the

carrier protein, which is visited iteratively by its catalytic

partners.36 One might conjecture that type II systems evolved

first, and subsequently became fused into type I enzymes

RPSs have covalently connected domains that form large, multi-domain

ntly connected; they are separate proteins that associate non-covalently.

This journal is ª The Royal Society of Chemistry 2008

Fig. 5 Mode of assembly line enzyme chain elongation. Modular PKSs and NRPSs use each catalytic and carrier protein domain once per molecule

synthesized, while iterative PKSs and NRPSs use catalytic and carrier protein domains for multiple cycles of chain elongation per molecule synthesized.

The lowercase ‘kr’ domain in DEBS 2 indicates an inactive ketoreductase domain.

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comprised of a single module that acts iteratively. Duplication of

these type I modules could then have led to the evolution of

modular type I assembly lines.37

A third category of PKSs, known as type III PKSs, differ from

type I and II PKSs primarily in that they use coenzyme A

thioesters directly as substrates—i.e., without the need for an

acyl carrier protein. While type III PKSs will not be covered in

detail in this review, readers are encouraged to read two recent

and interesting reviews on the topic.38,39

2.4 Four stages in NRP and PK biosynthesis

The logic of converting monomeric building blocks into

condensed natural product scaffolds by FAS, PKS, and NRPS

catalysts can be divided into four stages: (1) post-translational

priming of T domains to convert them from inactive apo to active

holo forms; (2) provision of unusual or dedicated monomeric

building block metabolites to feed into the assembly lines; (3) the

steps of chain initiation, chain elongation, and chain termination

during scaffold assembly; and (4) post-assembly-line tailoring of

nascent released products (Fig. 6). Enzymatic steps that have

been characterized from these four stages comprise the next four

sections of this review.

3 Phosphopantetheinyl transferases: the essentialpost-translational priming catalysts

The post-translational phosphopantetheinylation of all carrier

protein domains used in an FAS, PKS, or NRPS is the enabling

event that creates active protein machinery (Fig. 7).40–42 Coen-

zyme A (CoASH) is the phosphopantetheinyl donor and a serine

side chain in an elbow of the folded carrier protein domain is the

nucleophile, attacking CoASH at its pyrophosphate linkage.

This journal is ª The Royal Society of Chemistry 2008

This transfers the phosphopantetheinyl (Ppant) moiety of

CoASH onto the serine side chain, creating a stable phospho-

diester link to the 20 A long Ppant arm with a terminal thiol

group to which elongating FA, PK, and NRP chains are

covalently tethered (Fig. 8).

Given that phosphopantetheinyl transferase (PPTase)-

catalyzed priming is essential for assembly line function, it is not

surprising that PPTases are often encoded in natural product

gene clusters. Early on, it was discovered that the E. coli PPTase

that primed its FAS would not work on NRPS carrier protein

domains and only fitfully on PKS carrier protein domains.43 This

led to the discovery of different families of PPTases,40 with the B.

subtilis PPTase Sfp serving a particularly useful function since it

acts promiscuously on many types of carrier protein domains.44

Other characterized PPTases displaying relaxed specificity

include Svp from S. verticillus.45 Dozens of examples have now

been reported where either (i) coexpression of Sfp with PKS or

NRPS proteins in E. coli has led to primed, active assembly

lines;46,47 or (ii) incubation of purified carrier proteins with

purified Sfp has enabled in vitro assembly line priming.48–51

While Sfp will not accept dephospho-CoA as a Ppant donor

substrate, it will accept an almost limitless variety of acylated

CoA derivatives, therefore enabling the installation of a host of

acyl-S-pantetheinyl-phosphate moieties52–56 on almost any

carrier protein of interest. This has facilitated the investigation of

many alternate substrates in natural product assembly lines, both

for mechanistic study and for preparative uses.57,58 In a very real

sense, the discovery and characterization of Sfp and related

PPTases was a necessary precondition for in vitro studies of PKSs

and NRPSs. Prior to their availability, heterologous expression

of PKS and NRPS genes in E. coli was a poor substitute since it

yielded, at best, small fractions of an active assembly line enzyme

due to insufficient priming of the carrier proteins.

Nat. Prod. Rep., 2008, 25, 757–793 | 761

Fig. 6 Four stages in PK and NRP biosynthesis: (1) Post-translational priming of T domains; (2) Provision of dedicated monomers; (3) Chain

initiation, elongation, and termination; and (4) Post-assembly-line tailoring of nascent products. The example shown is the vancomycin NRPS.

Fig. 7 Post-translational phosphopantetheinylation of T domains. A serine side chain in the carrier protein attacks the pyrophosphate linkage of

coenzyme A, transferring the�20 A phosphopantetheine ‘arm’ to the carrier protein domain. During assembly line action, nascent PK and NRP chains

are tethered as thioesters to the terminal thiol group of the phosphopantetheine arm.

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4 Just-in-time fashioning of dedicated monomers asbuilding blocks for assembly lines

While some PKS assembly lines use the primary metabolite

malonyl-CoA as the chain extender unit, others use the

secondary metabolite methylmalonyl-CoA and more exotic

alkyl-, hydroxyl-, or aminomalonyl-CoAs.59,60 In cases where

PKS assembly lines use propionyl-, butyryl-, cyclohexyl- or

benzoyl-CoAs as starter units, monomer availability may

762 | Nat. Prod. Rep., 2008, 25, 757–793

constrain chain elongation machinery (see Moore and Hert-

weck61 for an excellent review).

NRPs feature a much wider range of monomeric building

blocks that yield peptidic scaffolds with unusual architecture and

functional groups. Over 350 nonproteinogenic amino acids are

known,62 and most of them are incorporated into one or more

NRP microbial metabolites. Since it would be energetically

wasteful and potentially deleterious formicrobial cells to generate

such nonproteinogenic amino acids except when they are needed

This journal is ª The Royal Society of Chemistry 2008

Fig. 8 Crystal structure of E. coli ACP, a T domain, showing the

phosphopantetheine arm in black (PDB entry 1L0I). This figure was

created using PyMOL (http://pymol.sourceforge.net/).

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as a monomer for natural product assembly, their synthesis is

coordinately regulated.63

While space limitations prevent a complete discussion of

dedicated building block synthesis, we consider below three

examples of the enzymatic construction of dedicated monomers

for (i) the glycopeptide antibiotics vancomcyin and teicoplanin,

(ii) halogenated natural products such as the antitumor agent

rebeccamycin and the phytotoxin syringomycin, and (iii)

b-amino acid-containing natural products such as the antibiotic

Fig. 9 Provision of nonproteinogenic amino acid monomers during chloroe

tyrosine (b-OH-Tyr), 3,5-dihydroxyphenylglycine (3,5-Dpg), and 4-hydroxyp

encoded by the chloroeremomycin gene cluster.

This journal is ª The Royal Society of Chemistry 2008

andrimid and the enediyne antitumor agent C-1027. Studies with

purified enzymes were required to decipher the mechanisms by

which these monomers are synthesized. These efforts have

revealed examples of novel biological catalysis.

4.1 Hydroxyphenylglycine and dihydroxyphenyglycine building

blocks for glycopeptide antibiotics

Most of the NRPS gene clusters that encode unusual NRP

scaffolds also encode genes for nonproteinogenic building

blocks. For example, five of the seven residues in the antibiotic

chloroeremomycin64,65 are nonproteinogenic: two b-hydroxy-3-

chlorotyrosines,66 two 4-hydroxyphenylglycines (Hpg),67 and one

3,5-dihydroxyphenylglycine (Dpg)68,69 (Fig. 9). When the

chloroeremomycin gene cluster is transcribed, these non-

proteinogenic amino acids are produced in a ‘just-in-time’

inventory control fashion because nine of its ORFs encode

enzymes to construct these monomers from primary metabolites.

To produce b-hydroxy-3-chloro-Tyr, tyrosine is first activated

and tethered as thioester on the Ppant arm of a carrier protein,

and then hydroxylated at the benzylic carbon by a dedicated

cytochrome P450 oxygenase.70 Chorismate is diverted from the

aromatic amino acid biosynthetic pathway toward Hpg by

oxygenation and rearrangement of its scaffold to yield

p-hydroxyphenylglyoxalate, with a final transamination to form

Hpg.67 The pathway to Dpg is particularly remarkable: DpgA,

a type III PKS,38 condenses four molecules of malonyl-CoA

using decarboxylative thio-Claisen condensations to yield the

eight-carbon product dihydroxyphenylacetyl-CoA.71 DpgC,

a rare metal-independent dioxygenase, then catalyzes a four-

electron oxidation of the a-methylene group to the ketone to

form dihydroxyphenylglyoxalate,72 which is transaminated to

Dpg. During DpgC catalysis, one atom from O2 ends up in the

ketone of dihydroxyphenylglyoxalate and the other in its

carboxylate, suggesting a novel mechanism of oxygenation that

may proceed through a dioxetane intermediate.73

remomycin biosynthesis. The nonproteinogenic amino acids b-hydroxy-

henylglycine (4-Hpg) are synthesized by dedicated enzymatic pathways

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4.2 Halogenation of amino acid building blocks

Around 4000 halogenated natural products are known.74 For

almost all of them, the halogen is installed by enzymes

conducting oxidative chemistry to transfer the halide as Xc or

X+.75 When the cosubstrate oxidant is hydrogen peroxide, the

enzymes are haloperoxidases, which use either heme coenzymes

or vanadyl cofactors to convert hydrogen peroxide to the halo-

genating species HOX. When the cosubstrate oxidant is molec-

ular oxygen, O2 gets reductively activated and ultimately

fragmented. Two kinds of cofactors can be utilized, flavin or

mononuclear nonheme iron. FADH2 reacts with O2 to yield an

FAD-OOH on the way to nascent HOCl.76 HOCl may be trap-

ped by a lysine in the halogenase active site to yield a chloramine

as the proximal donor of a Cl+ equivalent.77 A separate

O2-consuming halogenase family uses mononuclear nonheme

iron as a cofactor and generates a high-valent oxoiron interme-

diate that can create substrate radicals and transfer Clc to form

the C–Cl bond in the product.78,79

While haloperoxidases are often involved in the chlorination of

isoprenoid natural products,80,81 O2-dependent halogenase genes

predominate in NRPS and PKS gene clusters, suggesting that

these chemistries are used to halogenate NRP and PK scaffolds.75

Studies with purified enzymes have validated that chlorination of

tryptophan at the 5, 6, or 7 positions of the indole ring is enacted

Fig. 10 Flavin-dependent halogenases. PltA catalyzes the 4,5-dichlori-

nation of pyrrolyl-S-PltL during pyoluteorin biosynthesis.

Fig. 11 Fe/a-KG dependent halogenases. BarB2 and BarB1 catalyze the

trichlorination of Leu-S-BarA during barbamide biosynthesis.

Fig. 12 Cryptic chlorination as a strategy for cyclopropane formation. Follo

chloride-displacing cyclopropanation as the penultimate step of coronamic a

764 | Nat. Prod. Rep., 2008, 25, 757–793

by three distinct FADH2-dependent halogenases.82–85 Bischlori-

nation of a pyrrole ring at positions 4 and 5 during pyoluteorin

biosynthesis is likewise mediated by an FADH2-utilizing

halogenase,86 working on a pyrrolyl-2-carboxyl-S-carrier protein

species (Fig. 10). Additionally, chlorination of a b-Tyr-S-carrier

protein substrate occurs during C-1027 assembly.87

These studies, garnered from purified enzyme incubations,

allow the generalization that electron-rich aromatic rings such as

phenols, pyrroles, and indoles will be halogenated by FADH2-

utilizing enzymes that deliver Cl+ equivalents. Bioinformatic

analysis predicts that the gene clusters for halogenated natural

products such as the NRP antibiotic chloroeremomycin64 and the

PK antitumor agent ansamitocin harbor genes encoding flavin-

dependent halogenases.88,89 However, bioinformatic analysis

does not reveal the timing of halogenation. During rebeccamycin

biosynthesis, chlorination of a free amino acid is an initiating

step,82 while in pyoluteorin86 and C-102787 construction, the

substrate for chlorination is an aminoacyl/peptidyl-S-carrier

protein. Each system must be investigated biochemically to

understand the specificity of its tailoring halogenases and the

timing of their action in a pathway.

The biosynthetic gene clusters for some halogenated natural

products harbor no FADH2-dependent halogenases. Instead,

there are homologs of a-ketoglutarate-dependent mononuclear

nonheme iron oxygenases.78,90 These enzymes have turned out to

be halogenases for unactivated carbon centers, which are not

electron-rich and are therefore unreactive with Cl+ equivalents.

To generate a more potent halogenating species, FeII centers in

the iron halogenases have chloride (or bromide) ions in the first

coordination shell; when the FeIV]O intermediate is generated,

Clc rather than OHc is transferred to unactivated aliphatic

carbon sites (methyls or methylenes) in aminoacyl substrates,

presented as aminoacyl-S-carrier protein species. The remark-

able trichlorination of a Val-S-carrier protein substrate during

barbamide biosynthesis is mediated by the tandem action of two

such halogenases (Fig. 11).91

Studies on a purified nonheme iron halogenase in the

biogenesis of the aminocarboxycyclopropane coronamic acid

(a building block for the NRP-PK phytotoxin coronatine) has

established that cryptic chlorination is one of Nature’s chemical

strategies for cyclopropane construction.57 An allo-Ile-S-carrier

protein is chlorinated on the unactivated methyl side chain by the

nonheme iron halogenase CmaB (Fig. 12). The resultant Cl-allo-

Ile-S-carrier protein is then a substrate for CmaC, which

catalyzes the remarkable intramolecular cyclization of C2 onto

C4 with the displacement of chloride ion as the leaving group,

forming a cyclopropane ring.92 The substrate C2 carbanion

required for this intramolecular C–C bond to form is enabled by

wing CmaB-catalyzed chlorination of allo-Ile-S-CmaD, CmaC catalyzes

cid biosynthesis.

This journal is ª The Royal Society of Chemistry 2008

Fig. 14 The kutznerides, a family of NRPs entirely comprised of non-

proteinogenic amino acids. Kutzneride 2 is shown with the building

blocks b-hydroxyglutamate, O-methylserine, 3-chloropiperazate, tert-

butylglycoylate, 2-(1-methylcyclopropyl)glycine, and dichlorohydrox-

yhexahydropyrroloindole-2-carboxylate highlighted.

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its thioester linkage, since the carbanion is a stabilized thioester

enolate, and therefore kinetically accessible in the active site of

CmaC.

4.3 b-Amino acid building blocks

SomeNRP scaffolds contain isopeptide bonds in place of peptide

bonds, arising from the incorporation of b-amino acid mono-

mers rather than the usual a-amino acid building blocks. Notable

examples include the anticancer drug taxol,93 the enediyne

antibiotic C-1027,94 the protease inhibitor bestatin,95 and the

antibiotic andrimid,96 all of which contain either b-Phe or b-Tyr.

The gene clusters for C-102794 and for andrimid96 harbor ORFs

with homology to phenylalanine ammonia lyase, an enzyme

prevalent in plants that converts Phe to cinnamic acid and

ammonia as the initial step in phenylpropanoid biosynthetic

pathways (Fig. 13).97 Phe and His ammonia lyases were the first

examples of enzymes found to contain an autocatalytic modifi-

cation of the enzyme’s primary structure in which an Ala-Ser-Gly

sequence in the enzyme’s backbone is converted into the cyclic

4-methylideneimidazol-5-one (MIO) cofactor.98 MIO has an

electrophilic enone functional group that enables the fragmen-

tation of Phe or His to cinnamate or urocanate via the loss of Hb

and ammonia. With this precedent, it was anticipated that SgcC4

from the C-1027 pathway,99–102 AdmH from the andrimid gene

cluster,103 and the Taxus PAM enzyme in taxol biosynthesis93

would be aminomutases, generating cinnamate transiently and

then catalyzing re-addition of ammonia with opposite

regiochemistry. These expectations have been validated with

purified enzymes, and an X-ray structure of the tyrosine

aminomutase SgcC4 clearly shows the auto-modified MIO

cofactor in the active site.99 The three-step branch of the C-1027

pathway that builds the Cl-b-tyrosine building block has now

been reconstituted: aminomutase-catalyzed conversion of Tyr to

b-Tyr, its installation as a b-Tyr-S-carrier protein substrate, and

finally its FADH2-dependent chlorination.87,100–102,104

4.4 Other nonproteinogenic building blocks

Much of the useful functional group diversity and the attendant

biological activity of nonribosomal peptides arise from their

unusual nonproteinogenic amino acid building blocks. The three

Fig. 13 Aminomutases. AdmH converts phenylalanine to b-phenylala-

nine during andrimid biosynthesis, and SgcC4 converts tyrosine to b-

tyrosine as a monomer for C-1027 biosynthesis.

This journal is ª The Royal Society of Chemistry 2008

examples noted above are only a partial list of what could be

enumerated. They illustrate the diverse chemistry practised by

enzymes encoded in the biosynthetic gene clusters to carry out

just-in-time inventory control as natural product biosynthetic

pathways are activated in microbial producers. Understanding

the chemical mechanisms for building block construction will

enable components from different pathways to be mixed to

create architectural diversity in natural product libraries.

Attention should continue to be focused on the chemical

strategies and types of enzymes needed for nonproteinogenic

amino acid elaboration. In this context, the kutznerides (Fig. 14),

antifungal agents from a Scandanavian soil actinomycete, are

cyclic hexapeptidolactones, notable for the diversity of their

building blocks.105 One residue is tert-butylglycolate, a hydroxy

acid that engages in the ester linkage in the scaffold. The other

five building blocks are all non-proteinogenic amino acids,

ranging from the relatively prosaic O-methylserine and

b-hydroxyglutamate to the downright unusual 3-chloropiper-

azate, 2-(1-methylcyclopropyl)glycine, and dichlorohydroxy-

hexahydropyrroloindole-2-carboxylate. Characterization of the

enzymes that fashion these monomers for assembly line use will

surely turn up new chemistry and strategies for combinatorial

biosynthesis.

In passing, we note that knowledge of the scope of reactions

catalyzed by the two types of O2-consuming halogenases

discussed in the previous section suggested that both types

should be encoded in the kutzneride biosynthetic gene cluster.

Indeed, gene probes for FADH2-halogenases and mononuclear

iron halogenases facilitated cloning of the �50 kb kutzneride

gene cluster105 and points the way for cloning of other gene

clusters encoding halogenated and cyclopropane-containing

natural product scaffolds,106 e.g. from environmental DNA.

5 Assembly line action

The enzyme components of type I PKS and NRPS assembly lines

have been a more difficult set to study than the enzymes, just

discussed, that fashion the monomeric building blocks to be

incorporated by assembly line action. The difficulties arise from

Nat. Prod. Rep., 2008, 25, 757–793 | 765

Fig. 15 The size of assembly line enzymes. The daptomycin NRPS comprises 13 modules distributed over three proteins to form a�1.7 MDa assembly

line, while the avermectin PKS is composed of 13 modules distributed over four proteins to form a �2.2 MDa assembly line.

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the multimodular nature and imposing size of many type I

assembly line proteins (Fig. 15). The thirteen-module assembly

line for the PK avermectin is distributed over four proteins,

AVES 1–4, each in the 400–700 kDa size range.107 These proteins

contain catalytic and carrier protein domains in each module.

Analogously, the thirteen modules of the daptomycin synthetase

are distributed in a five-, six-, and two-module array across three

large enzymes.108 At one limit, all catalytic and carrier protein

domains in an assembly line could be stapled together in a single

megaenzyme. Such is the case for the cyclosporine synthetase,

which has 47 domains arrayed in tandem in a single polypeptide

of�15,000 amino acids.109 It is a challenging prospect to express,

purify and characterize such megasynthases. At the other end of

the spectrum are type II FASs and PKSs in which each domain,

catalytic and carrier, are separate proteins.13 Historically, these

type II systems were the first ones characterized, enabling the

elucidation of fundamental chemical steps, partial reactions, and

iterative catalytic cycles.110–113

In this section, we focus on type I PKS, NRPS, and NRPS-

PKS assembly lines to illustrate approaches to (i) deconvolute

the role of individual catalytic and carrier protein components,

(ii) analyze the process of chain elongation from one carrier

protein to the next, and (iii) to understand how full-length acyl/

peptidyl chains are disconnected from the assembly line when

Fig. 16 Loading/initiation modules in PKSs and NRPSs. The two-domain

shown; the two domains are adjacent in the bacitracin NRPS, while they are

766 | Nat. Prod. Rep., 2008, 25, 757–793

they have reached the final carrier protein domain in an assembly

line.

Type I assembly line enzymes are multidomain proteins. While

modular type I enzymes require a module-by-module examina-

tion for reconstitution, their architecture makes possible the

evaluation of intermediates tethered transiently to each carrier

protein domain, e.g. by mass spectroscopic analysis.114 Fungal

iterative type I enzymes such as LovF (lovastatin)33 and PKS4

(bikaverin)115 require purification of only a single module, but

since they act iteratively, it has been much more difficult to stop

catalysis at intermediate cycles to examine the tailoring reactions

occurring during chain growth. In either type I variant (modular

or iterative), full reconstitution from purified components can be

daunting. First, we examine studies to decipher parts of assembly

line action before turning in a subsequent section to look at

examples of full assembly line reconstitution. To that end, we

take up chain initiation, chain elongation, and chain termination

processes in that order.116

5.1 Chain initiation: monomer selection, loading and acylation

The first modules of PKS and NRPS assembly lines are the way-

stations where acyl/peptidyl chain growth is initiated. Sometimes

these are referred to as loading modules to emphasize their role

loading modules of the bacitracin NRPS and the stigmatellin PKS are

separated in the stigmatellin PKS.

This journal is ª The Royal Society of Chemistry 2008

Fig. 18 Noncanonical starter units for NRPSs. A fatty acid such as

decanoic acid is the starter unit for the daptomycin NRPS, while 2,3-

dihydroxybenzoate (2,3-DHB) is the starter unit for the vibriobactin

NRPS.

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as the starting point for synthesis. NRPS initiation modules

comprise, at a minimum, an adenylation (A) domain for amino

acid selection and activation, and a carrier protein domain that

bears the Ppant thiol group (Fig. 16). We will use the term T

(thiolation) for carrier protein domains in the following discus-

sions. By analogy, PKS loading modules would have—at

a minimum—an acyltransferase (AT) domain and a T domain to

be loaded by the AT domain, e.g. with malonyl-CoA as the

substrate. Many PKS assembly lines have a KS domain in the

loading module (KS-AT-T) that decarboxylates a malonyl unit

to an acetyl group to start the PK chain.117

More interesting are variations from the canonical acetyl

and aminoacyl starter units for PKS and NRPS assembly lines

(Fig. 17). Acyl-CoAs such as benzoyl-CoA118 or dihydroxy-

cyclohexenyl-CoA119 initiate enterocin and rapamycin biosyn-

thesis, while ansa-bridged scaffolds such as rifamycin and

geldanamycin use 3-amino-5-hydroxybenzoate (AHBA) as

a starter unit.120–123 At the end of the assembly line, the 3-amino

moiety of AHBA engages in intramolecular amide bond

formation to construct the ansa bridge.

A large family of NRPs are N-acylated peptide scaffolds

(Fig. 18). The A-T didomain of the chain initiation module

selects and loads the first aminoacyl group, which undergoes

acylation before chain elongation commences. A condensation

(C) domain in the initiation module (C1-A1-T1) may catalyze

N-acylation.124 Simple saturated or unsaturated fatty acyl

groups, diverted from the producer organism’s fatty acid

biosynthetic pathway, may serve as acyl group donors.125 During

the biosynthesis of the Bacillus subtilisNRPmycosubtilin, a fatty

acid is activated by the assembly line’s first A domain to the acyl-

AMP intermediate for capture by aminoacyl-S-T1.126 During the

fermentation of daptomycin, decanoic acid is added exogenously

and is preferentially incorporated into the lipopeptidolactone.125

A variety of N-acylpeptides start with b-hydroxy fatty acids,

which are also intermediates in fatty acid biosynthesis re-routed

towards the NRPS assembly line. The b-hydroxy group may

subsequently participate in chain-terminating macrocyclizations,

as will be noted below.127,128 A variant of functionalization of the

Fig. 17 Noncanonical starter units for PKSs. Benzoyl-CoA is the starter

unit for the enterocin PKS, while 3-amino-5-hydroxybenzoate (3,5-

AHBA) is the starter unit for the geldanamycin PKS.

This journal is ª The Royal Society of Chemistry 2008

long-chain fatty acid occurs duringmycosubtilin biosynthesis: the

chain initiation module uses a b-ketoacyl-CoA as an N-acylating

reagent, and the ketone undergoes reductive amination to the

b-amino group.129 This reductive amination is achieved by

a pyridoxamine-phosphate-utilizing transaminase fused into the

initiation module. This example demonstrates a rare occurrence

of the intersection of pyridoxal chemistry and PKS chemistry, but

it suggests that transaminase domains may be portable and

capable of being fused into other PKS modules to introduce

nitrogen into PK backbones. In the mycosubtilin scaffold,

subsequent chain termination occurs by intramolecular partici-

pation of this amino group,130 to form a macrolactam rather than

a macrolactone bond, with increased hydrolytic stability.

A large family of NRPs are iron-chelating molecules that

bacteria produce in response to iron starvation. These natural

products, known as siderophores, are virulence-conferring

factors for their bacterial producers.3 Siderophores contain

different varieties of ferric iron-coordinating groups, including

catechols, phenols, hydroxamates, and heterocycles (thiazolines

and oxazolines). The phenols and catechols are selected in chain

initiation steps in which salicylate or 2,3-dihydroxybenzoate are

aryl acids used toN-acylate aminoacyl1-S-T1 intermediates at the

start of the siderophore assembly lines.131,132

One additional example to note is the occurrence of pyrrole-2-

carboxy moieties at the N-termini of some nonribosomal

peptides. These do not arise as a result of N-pyrrolylation of an

aminoacyl1-S-T1, but instead by the selection and loading of

proline by A1-T1 to yield the prolyl1-S-T1 intermediate.133 This is

subjected to two cycles of dehydrogenation on the assembly line

to yield pyrrolyl1-S-T1.134 During pyoluteorin biosynthesis this

pyrrolyl-thioester is the substrate for dichlorination by an

FADH2-utilizing halogenase, as noted in Section 4.2.86

As a transition to the chain elongation section, we note that

the myxobacterial antitumor agent epothilone is a hybrid NRP-

PK that has a thiazole group fashioned from acetyl and cysteinyl

Nat. Prod. Rep., 2008, 25, 757–793 | 767

Fig. 20 b-Carbon tailoring domains in PKSs. The ketoreductase (KR)

domain catalyzes the two-electron reduction of the b-keto group to

a b-hydroxyl, the dehydratase (DH) domain dehydrates the b-hydroxyl to

an a,b-enoyl species, and the enoylreductase (ER) domain reduces the

a,b-enoyl to a fully saturated acyl group.

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moieties.135 This thiazole is cyclized during chain elongation

rather than chain initiation.

5.2 Chain elongation: Claisen, amide, and ester condensations

The transfer of growing chains from module to module is cata-

lyzed by C (condensation) domains in NRPS assembly lines and

KS (ketosynthase) domains in FAS and PKS assembly lines.1

C and KS domains make amide and carbon–carbon bonds

between upstream and downstream substrates tethered as Ppant

thioesters on T domains (Fig. 19). Condensation is directional,

from upstream where the growing acyl/peptidyl chain acts as the

electrophilic donor, to downstream where the thioester-tethered

monomer acts as the nucleophilic acceptor. In NRPS assembly

lines, the deprotonated amino group of an aminoacyl-S-Tn

attacks the peptidyl thioester on Tn�1, and amide bond forma-

tion is concomitant with chain translocation from Tn�1 to Tn.32

Correspondingly, in FAS and PKS assembly lines, the

downstream malonyl-S-Tn is decarboxylated to yield a two-

carbon thioester enolate nucleophile that attacks the KS-bound

thioester previously on Tn�1. Carbon–carbon bond formation

moves the growing chain from Tn�1 to Tn.17

Tailoring of the growing chain depends on which optional

catalytic domains are embedded within a module. In PKS

assembly lines, the minimal KS-AT-T module can also contain

a ketoreductase (KR), a dehydratase (DH), and an enoylre-

ductase (ER), which can respectively process the initial

b-ketoacyl-S-Tn moiety to the b-hydroxyl, then to the a,b-enoyl,

and finally to the fully saturated acyl chain elongated by two

carbons (Fig. 20).1 If one or more of the KR, DH, or ER domains

is absent or catalytically defective, intermediate oxidation states

persist and are subsequently carried forward as chain elongation

proceeds to the next module.136

Studies on KR domains and on purified PKS modules con-

taining KR domains have revealed that reduction to either the

S-alcohol or the R-alcohol is possible, and stereoselectivity is

correlated with the presence or absence of a key aspartyl residue

in the KR domain active site.137–139 There has been much interest

Fig. 19 Action of ketosynthase (KS) and condensation (C) domains in PK

cysteine with the upstream chain, decarboxylate the downstream (methyl)ma

enolate on their acyl-S-enzyme intermediate, forming a C–C bond and trans

attack of the downstream aminoacyl group on the upstream peptidyl group, tr

species.

768 | Nat. Prod. Rep., 2008, 25, 757–793

in evaluating the role of the b-KR domains in setting the adjacent

a-methyl stereochemistry when methylmalonyl-CoA is the

building block, as in the erythromycin PKS.140–144 Proposals for

epimerase and reductase activities in KR domains have been

made to explain syn and anti dispositions of the b-hydroxy-a-

methyl during chain elongation.144 Subsequent dehydration

would remove the b-substituent but leave the a-CH3 groups: in

erythromycin the methyls are 2R, 4R, 6S, 8R, 10R, and 12S,

respectively.145

5.2.1 C domains: condensation and heterocyclization. In

analogy to PKSs, the minimal domain set for NRPS elongation

modules is C-A-T, and only rarely are additional catalytic

S and NRPS chain elongation. KS domains autoacylate an active site

lonyl-S-T monomer, and then catalyze attack of the resulting thioester

locating the chain to the downstream T domain. C domains catalyze the

anslocating the chain without the known intermediacy of an acyl–enzyme

This journal is ª The Royal Society of Chemistry 2008

Fig. 21 Action of cyclization (Cy) domains in NRPS chain elongation and heterocycle formation. Cy domains are variants of C domains; both domains

catalyze peptide bond formation with concomitant chain translocation. The Cy domain then catalyzes the attack of the b-nucleophile (either a hydroxyl

as shown, or a thiol) on the upstream amide carbonyl, forming a five-membered adduct that dehydrates to an oxazoline or a thiazoline.

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domains found in elongation modules, although we shall note

methyltransferase domains below. Perhaps the most chemically

intriguing and consequential aspect of NRPS elongation

modules is that sometimes the C domain is substituted by

a variant known as a cyclization (Cy) domain (Fig. 21).146 This C

domain subclass operates on Cys, Ser, and Thr-S-Tn as down-

stream partner nucleophiles to yield acyl-Cys/Ser/Thr-S-Tn

adducts from the amide bond-forming condensation step. Then,

before the chain is translocated to the next module, cyclo-

dehydration is catalyzed in a second Cy-mediated step to yield

the thiazolinyl-S-Tn, oxazolinyl-S-Tn, or methyloxazolinyl-S-Tn

as product.147 This cyclization to the five-membered dihydro-

heterocyclic rings radically alters peptide backbone connectivity

and also creates chelating groups for FeIII, so one sees such

oxazolines and thiazolines in siderophores such as mycobactin

fromMycobacterium tuberculosis,148 yersiniabactin fromYersinia

pestis,149 and vibriobactin from Vibrio cholerae.150

5.2.2 C domains that make ester linkages. A large number of

PK and NRP natural products have macrolactone or macro-

lactam linkages. As will be noted in a subsequent section, mac-

rocyclization is catalyzed by the chain-terminating thioesterase

domain at the end of NRPS and PKS assembly lines. A subset of

nonribosomal peptide scaffolds have multiple ester linkages

replacing the normal amide backbone linkages: these are made

by condensation domains that accept a-hydroxyacyl-S-Tn as

a downstream acceptor, thereby acting as ester synthases rather

than amide synthases (Fig. 22).151

The a-hydroxyacyl units can be installed by two different

routes. The cyclodepsipeptides of the enniatin152–154 and beau-

vericin155 family are cyclic molecules in which N-methyl-L-amino

acids alternate with D-hydroxyisovalerate moieties (D-HIV). One

module of the two-module enniatin NRPS activates the L-amino

Fig. 22 Formation of an ester bond by a C domain during cereulide biosynth

is an a-hydroxyacyl-S-T rather than an a-aminoacyl-S-T, forming an ester b

This journal is ª The Royal Society of Chemistry 2008

acid and has an MT domain for N-methylation. The other

module activates D-HIV rather than an amino acid. One C

domain in the two-module enniatin synthetase is an amide bond-

forming catalyst, while the other C domain is an ester synthase.

A second route for a-hydroxyacyl unit installation occurs

during the biosynthesis of the potassium ionophores valinomy-

cin156 and cereulide.151,157 In their assembly lines, the modules

incorporating the hydroxy acid monomers have the four-domain

architecture C-A*-KR-T. The A* domain activates a-keto acids

(rather than a-hydroxy or a-amino acids) and installs them on

the downstream T domain as thioesters. The embedded KR

domain is an a-ketoreductase, converting the achiral a-ketoacyl-

S-Tn in situ to a chiral D- or L-a-hydroxyacyl-S-Tn intermediate.

These are the donor species for the downstream Cn+1 domains,

which use a-aminoacyl-S-Tn+1 species as nucleophiles. The C

domains in these modules are chiral ester synthases rather than

amide synthases, and in accordance with the nomenclature from

Section 6.1 below, they act as DCL catalysts.

5.3 NRP-PK hybrids: module switching and chain elongation

Natural products that are hybrid NRP-PKs have been known for

decades; these include the clinically-used immunosuppressant

FK506 and antitumor agent bleomycin. Given that NRP and PK

metabolites can be made by modular type I assembly lines using

similar chemical logic, it appeared that the two types of assembly

lines must be able to mix and match components to form hybrid

assembly lines.14 Sequences of the gene clusters for these hybrid

molecules confirmed module mixing,158,159 and raised questions

of catalyst specificity and domain portability to create new

hybrid assembly lines and variant hybrid products. A common

blueprint appears to involve switching between NRPS and PKS

assembly lines and back by interchanges of whole modules

esis. The terminal C domain of CesA uses a nucleophilic co-substrate that

ond rather than an amide bond.

Nat. Prod. Rep., 2008, 25, 757–793 | 769

Fig. 23 PKS-NRPS interfaces in hybrid assembly line enzymes. Bottom: a PKS-to-NRPS interface from the myxalamid synthase, in which the

upstream co-substrate of the MxaA C domain is a PK rather than a NRP. Top: an NRPS-to-PKS interface from the bleomycin synthetase, in which the

upstream co-substrate of the BlmVIII KS is a NRP rather than a PK.

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(Fig. 23). The bleomycin assembly line is mostly an NRPS

assembly line with one PKS module functioning in the middle.159

At the other end of the continuum, the myxalamid assembly line

is entirely composed of PKS modules until the end where MxaA,

a four-domain NRPS module with the domain structure C-A-

T-Re, is found.160 The A domain of this module selects and

installs alanine, and the C domain uses the alanyl amine as

a nucleophile and inserts it through an amide linkage into the

growing PK chain. This NRPS module is like a plug-in device for

inserting an amino acid into the middle of a PK scaffold and

represents one mode for introducing nitrogen into the backbone

of PK molecules.

Another example of note occurs in the epothilone PKS-

NRPS.135,161,162 The first three enzymes, EpoA, EpoB, and EpoC,

are a PKS module, an NRPS module, and a PKS module,

respectively. EpoA generates acetyl-S-T1 as starter unit using

typical PKS logic. EpoB (Cy-A-Ox-T2) activates cysteine, and in

a step that constitutes the PKS-NRPS interface, the Cy domain

transfers the acetyl group to make N-acetyl-Cys-S-T2.49 Since

this is a Cy domain rather than a C domain, the N-acetyl-Cys

moiety is subsequently cyclodehydrated to form thiazolinyl-

S-T2.163 EpoC (KS-AT-DH-KR-T3) is a typical PKS module,

transferring methylmalonyl onto T3. The KS domain acts as the

NRPS to PKS interface, forming a carbon–carbon bond and

transitioning the growing chain back onto a PKS track.164 Study

of the EpoA-EpoB and the EpoB-EpoC interactions with

purified proteins164 has revealed mechanisms and specificity for

prototypic PKS-NRPS and NRPS-PKS interfaces and the

characteristics of the key C and KS domains that facilitate bond

formation at the hybrid junctions. The collective action of

purified EpoABC yields the methylthiazolyl-methylacrylyl-S-T3

chain that persists in the final epothilone scaffold.164 Variant

heterocycles can be assembled by the use of different substrates

770 | Nat. Prod. Rep., 2008, 25, 757–793

for EpoA and EpoB,165 providing an instructive lesson for

engineering NRPS and PKS interfaces in future hybrid systems.

Many other examples of NRP-PK hybrids and their gene

clusters have been reported in recent years, including two

molecules in which valine is extended by a malonyl unit

(barbamide166 and andrimid).96 We will note the complete

reconstitution of the yersiniabactin NRPS-PKS in Section 9.2.

Some recently characterized PKSs andNRPSs deviate from the

conventional rules of domain organization or action described

above, for example by using AT domains that act in trans rather

than in cis, or by harboring modules that act iteratively or are

skipped entirely. While these unconventional but increasingly

prevalent enzymes are not described in detail here, readers are

encouraged to read two interesting reviews on the topic.12,27

6 Chain tailoring on the assembly line

In Section 5.2, we noted that most PKS modules contain one or

more of the optional KR, DH, and ER domains that tailor the

redox state of the b-ketoacyl-S-Tn chain in each PKS module

before the chain passes to the next module. It is the rule rather

than the exception that PKS modules contain one, two, or three

of these chain-tailoring domains.

In NRPSs, chain-tailoring domains are an exception rather

than a rule. Up to this point, we have noted one kind of tailoring

step during NRP chain elongation: the Cy variant of C domains

that catalyze cyclodehydration to form thiazolinyl, oxazolinyl,

and methyloxazolinyl-S-T species (see Section 5.2.1). In several

cases, the modules containing Cy domains also have embedded

FAD-dependent oxidase (Ox) domains to convert the

dihydroaromatic thiazolines and oxazolines to thiazoles and

oxazoles (Fig. 24). This is the case for EpoB (mentioned above

in Section 5.3), which creates a methylthiazole species,167 and

This journal is ª The Royal Society of Chemistry 2008

Fig. 24 Conversion of a thiazoline to a thiazole by a flavin-dependent oxidase domain during epothilone biosynthesis. After the EpoB Cy domain

catalyzes condensation, cyclization, and dehydration to form methylthiazolinyl-S-EpoB, the EpoB Ox domain catalyzes its FAD-dependent oxidation

to the fully aromatic methylthiazolyl-S-EpoB.

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more famously in the bleomycin NRPS-PKS,159 in which

a tandem thiazolinyl-thiazolinyl-S-T intermediate is doubly

oxidized to the planar bithiazole. The bithiazole moiety can

intercalate between base pairs in DNA and is the DNA-targeting

pharmacophore of this drug.168 In principle, the knowledge

gained from the enzymatic studies on Cy and Ox catalysts could

allow the insertion of a pair of Cys-specific modules (Cyn-An-

Oxn-Tn-Cyn+1-An+1-Oxn+1-Tn+1) into an NRP, PK, or hybrid

assembly line to produce a bithiazole moiety that should target

the new natural product to DNA.

6.1 Epimerization at Ca

In addition to the use of nonproteinogenic amino acid

monomers, one of the hallmarks of nonribosomal peptides is the

common occurrence of D-residues in the backbone scaffolds.

While in principle it could be the A domains in those modules

that select and activate D-amino acids rather than the available

L-amino acids,169 most of the D-residues in NRP scaffolds arise

by activation of L-amino acid monomers followed by the

epimerization of assembly line-tethered intermediates.51,170,171 In

some assembly lines, e.g. from Bacillus strains,172 there are

50 kDa epimerization (E) domains embedded in modules that

elongate D-residues (Fig. 25). The E domains epimerize either the

free L-aminoacyl-S-Tn to D-aminoacyl-S-Tn species before

condensation or act after condensation by Cn and before action

of Cn+1 on the peptidyl-S-Tn intermediate.173 Epimerization of C2

chirality is enabled by the kinetic acidity of the C2-H in

aminoacyl and peptidyl thioesters, since the resultant carbanion

is a stabilized thioester enolate.

Coupled to such on-line epimerizations must be chiral recog-

nition by the downstream C domain (Cn+1) to accept the

D-stereocenter in the upstream donor. That is, the Cn+1 must be

a DCL amide bond-forming catalyst. These predictions have been

borne out by studies with purified modules elongating D-residue

peptide chains.174 There are many other NRPS assembly lines

This journal is ª The Royal Society of Chemistry 2008

that also elongate D-residues but do not contain epimerization

domains. Studies on the purified modules have shown that the

C domains have both epimerization and condensation activity

(see Fig. 25).175 Again, the pairwise cooperation of tandem

C domains in assembly lines sets the chirality of the elongating

residues in the NRP chain. Any efforts in combinatorial

biosynthesis of NRPs to mix and match D- and L-amino acid

residues in backbone scaffolds must take into account the

existence of LCL and DCL chirality in C domains.176 To date noDCD condensation domains have been detected, suggesting that

D-D residues, e.g. as found at residues 4 and 5 in the vancomycin

scaffold (see Fig. 27), are generated by tandem action of DCL

domains with epimerization of a D-L- to a D-D-peptidyl-S-T

intermediate occurring before the second DCL domain acts.

6.2 N- and C-Methylations at Ca and Cb

Two other ‘‘on-assembly-line’’ tailoring events are of note: (1) N-

andC-methylations and (2) b-branching in polyketide backbones

(Fig. 26). A variety of NRP natural products are produced by

assembly lines with N-methyltransferase (N-MT) domains

embedded between the A and T domains (C-A-MT-T) in

modules that carry out N-methylation during amino acid

incorporation and elongation.32 The cosubstrate for methylation

is S-adenosylmethionine (SAM), which provides an electrophilic

‘‘CH3+’’ equivalent that is captured by the NH2 of the aminoacyl-

S-Tn domain to yield N-Me-aminoacyl-S-Tn before the next

elongation step.177

Less common are MT domains that effect C-methylation of

PK and hybrid PK-NRP products during chain elongation.1,178

The required nucleophile tethered to the growing substrate

chain is provided just after a KS domain has acted to produce

a b-ketoacyl-S-Tn intermediate. The methylene hydrogens on Ca

are acidic since the resultant carbanion is delocalized as the

thioester enolate. Studies with purified yersiniabactin synthetase

components established that mono- and di-C-methylation occur

Nat. Prod. Rep., 2008, 25, 757–793 | 771

Fig. 25 Generation of D-amino acid residues on NRP assembly lines. (a) Timing of epimerization and condensation in tyrocidine biosynthesis. (b)

Epimerase activity of C domain embedded in arthrofactin synthetase.

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to yield the a,a-dimethyl-b-ketoacyl-S-Tn species by C-MT

domain action, prior to reduction of the ketone to the alcohol by

the KR domain in that module (Fig. 37). A comparable

C-methylation occurs in the PK scaffold of epothilones

(Fig. 26).135 Anexample ofC-methylation of anNRP fragment by

an embedded C-MT domain occurs in the last module of yersi-

niabactin synthetases where C2 of the thiazolinyl-S-Tn, derived

from Cy domain action on an an acyl-Cys-S-Tn, is methylated

prior to release of the full length yersiniabactin chain.179

The SAM-dependent mechanism just noted leads to C-methyl-

ations at Ca of polyketide chains. A more common route to Ca

methyl substitution is the use of methylmalonyl-CoA monomers

by KS domains in PKS elongation. That is the strategy by which

DEBS 1, 2, and 3 introduce a-branching at carbons 2, 4, 6, 8, 10,

12 in the erythromycin scaffold.31

Although not discussed in detail here, post-assembly-line

methylation has also been noted as a point of structural

diversification, for exampleN-methylation in vancomycin,180 and

O-methylation of sugars181,182 and carboxylic acids.183

6.3 Cb branches

There are a small number of polyketides, including mupirocin,184

jamaicamide,185 bacillaene,186 and myxovirescin,187 that contain

b-methyl branches (Fig. 26). In vitro studies with five purified

enzymes, encoded as a cassette in the bacillaene and myxovir-

escin gene clusters, have revealed a SAM-independent pathway

772 | Nat. Prod. Rep., 2008, 25, 757–793

that represents an intersection of isoprene and polyketide

biochemistry.188,189

The first enzyme of the cassette is a homolog of b-hydroxy-

methylglutaryl (HMG)-CoA synthase (HCS) which is found in

isoprenoid biosynthetic pathways. HCS condenses the C2 anion

of acetyl-CoA onto the C3 ketone of acetoacetyl-CoA, making

the C–C bond in the branched chain HMG-CoA product. In

analogy, HCS acts on a b-ketoacyl intermediate on PKS

assembly lines, adding the C2 acetyl anion onto the 3-ketoacyl-S-

Tn intermediate to yield a product with a CH2COOH branch.

This intermediate then undergoes elimination to the conjugated

enoyl-S-Tn species. The resulting vinylogous carboxylate is lost

through decarboxylation by the next enzyme. Regiospecific

protonation of the thioester dienolate yields a D2-isoprenyl-S-Tn

tethered chain. The methyl group, derived ultimately from C2 of

the initial acetyl moiety, is in the b-position of the growing PK

scaffold. Transfer of this branched chain to the next module of

the PKS assembly line allows for continued chain elongation.

This machinery is likely responsible for subsequent elaboration

of b-branched species to the cyclopropane group in curacin190

and the vinyl chloride branch in jamaicamide.185

6.4 Oxidative transformations on assembly lines

In Sections 5.1 and 6, we have noted in passing two oxidative

dehydrogenations that occur while monomers or growing chains

are tethered on assembly lines. The first example is the double

This journal is ª The Royal Society of Chemistry 2008

Fig. 26 Strategies for methylation during NRP and PK biosynthesis. (a) SAM-dependent N-methylation. (b) Two sources of a-methyl substituents in

PKs: SAM-dependent methyl transfer in epothilone biosynthesis and incorporation of methylmalonyl CoAmonomers by the erythromycin synthase. (c)

SAM-independent b-carbon methylation by HMG-CoA synthase homologs.

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dehydrogenation of prolyl-S-T intermediates to pyrrolyl-S-T191

which occurs not only during pyoluteorin biosynthesis (Fig. 10)

but also in the biogenesis of the first pyrrole ring of the tripyrrolic

prodiginine pigments192 and in the pyrrolyl moieties of the

aminocoumarin antibiotics clorobiocin and coumermycin193

(Section 9.5), as established by studies with purified enzymes.

The second is the desaturation of thiazoline and oxazoline rings

in NRP assembly lines for making epothilone or oxazole analogs

of epothilone (Fig. 24).165 All of the above desaturations to

heteroaromatic systems are effected by flavoproteins, either

embedded in the samemodules as Cy domains (epothilones) or as

partner proteins to the proline activation adenylation domains.

Oxidations of elongating chains involving molecular oxygen

are at present best characterized by a set of three to four

This journal is ª The Royal Society of Chemistry 2008

hemeproteins of the cytochrome P450 superfamily which

perform the aryl–ether and aryl–aryl coupling chemistry in the

vancomycin (3 crosslinks) and teicoplanin (4 crosslinks) family of

antibiotics65 (Fig. 27). The biosynthetic gene clusters for this

antibiotic family encode three or four hemeproteins respectively,

OxyABC192 or StaFGHJ.194–196 Each of these O2-dependent

enzymes successively makes one of the 2–4 (Tyr2–Hpg4), 4–6

(Hpg4–Tyr6) or 1–3 (Hpg1–Hpg3) aryl ether linkages before

formation of the 5–7 direct C–C linkage between Hpg5 and Dpg7.

In the vancomycin series, the 4–6 ether crosslink is made first,

then the 2–4, and finally the 5–7.197 In the teicoplanin series, the

4–6 and then the 1–3 aryl ether crosslinks are formed in that

order.198 The net result is the conversion of the acyclic

heptapeptide to the highly rigidified, cup-shaped architecture of

Nat. Prod. Rep., 2008, 25, 757–793 | 773

Fig. 27 On assembly-line oxidative crosslinking of aglycone vancomycin and teicoplanin scaffolds by trans-acting cytochrome P450 oxidases.

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the vancomycin/teicoplanin aglycone. Enzymatic studies by

Robinson and colleagues199–201 have indicated that the cross-

linking steps occur while the heptapeptide is still tethered in

thioester linkage to T7. Chain release by the adjacent TE domain

may be controlled by the architectural changes introduced by the

crosslinks. The mechanism of these P450 crosslinking enzymes is

not yet deconvoluted, although phenoxy radical intermediates

are reasonable candidates as intermediates for the action of each

of the regioselective hemeprotein tailoring enzymes.202

7 Chain release mechanisms and catalytic machinery

When the growing ketidyl/peptidyl chains reach the most

downstream T domain and all the ‘‘on-assembly-line’’ tailoring

steps have been completed, the mature chain must be discon-

nected from its covalent thioester linkage to the Ppant prosthetic

group. The vast majority of PKSs and NRPSs have a thioesterase

(TE) domain at the C-terminus of their final module. The TE is

the catalyst for acyl/peptidyl thioester disconnection.

Two major routes of PK- and NRP-S-T disconnection are

known for subclasses of TE domains (Fig. 28). The first is

hydrolysis, where the linear natural product acid is released by

TE-catalyzed intermolecular attack of water on the ester linkage.

For example, this is the outcome for vancomycin,65 yersinia-

bactin,203 and the ACV tripeptide precursor of b-lactam anti-

biotics.204,205 A more intriguing subclass of TE domains

encompasses those that carry out intramolecular regiospecific

macrocyclization by a nucleophilic hydroxyl or amine in the PK/

NRP chain to create a cyclic lactone or lactam.25,26This is the fate

of the chain tethered to DEBS 3 in erythromycin biosynthesis206

and of the daptomycin chain207 tethered to DptD.

Among the most spectacular of the TE-mediated cyclizations

are the actions of the TE domain at the end of the four-domain

(C-A-T-TE) protein EntF which comprises the second (of two)

modules in enterobactin synthetase.208 The E. coli siderophore3

enterobactin is a cyclic trilactone of N-(2,3-dihydroxy-

benzoyl)serine, sent out to scavenge ferric iron, which it binds

using three pairs of catechol oxygens. EntF loads a 2,3-dihy-

droxybenzoyl-Ser (DHB-Ser) moiety covalently on the T domain

and then passes DHB-Ser to the active site serine side chain of the

TE domain where it is protected from adventitious hydrolysis

long enough for another DHB-Ser to accumulate on the T

774 | Nat. Prod. Rep., 2008, 25, 757–793

domain. Next, the two tethered DHB-Ser chains are condensed

using the side chain of the Ser moiety of one of the DHB-Ser

residues to build up a DHB-Ser-O-DHB-Ser dimer on the TE

domain. The T domain reloads a third time, builds the linear

(DHB-Ser)3-O-TE, and cyclizes it to release the trilactone

enterobactin. Studies with purified forms of EntF with mutant

TE domains have detected accumulation of the (DHB-Ser)2-O-

TE intermediate, and such results were central in formulation of

this mechanism.208

Since regiospecific macrocyclizations build in conformational

constraints in the released product and contribute dramatically

to medicinal activity, this class of TE cyclization catalysts has

generated much interest.209 Excision of the 30–35 kDa TE

domains from megasynthase assembly lines indicates they retain

their regioselective cyclization capacities and they can be

used for chemoenzymatic syntheses of macrocycles with soluble

acyl/peptidyl-thioester substrates to make new molecules

(Fig. 28). Use of a hybrid PK-NRP acyl donor with the excised

TE from the tyrocidine synthetase led to enzymatic production

of novel hybrid PK-NRP heterocycles,210,211 while studies with

the daptomycin synthetase TE have led to novel antibiotic

analogs.125,212

An intriguing variant of TE domain catalysis has been

deciphered by recent reconstitution of the fungal metabolite

terrequinone from purified enzymes,213 discussed in Section 9.6.3.

The TE domain of the lone NRPS module in the pathway

performs a remarkable feat: the double Claisen condensation

of two tethered indolepyruvyl–enzyme intermediates to construct

a benzoquinone ring during release of the dimerized indolyl-

benzoquinone.

A third and fundamentally distinct mode of chain release is

seen in PK and NRP assembly lines where the C-terminal TE

domain is replaced with a domain homologous to short chain

dehydrogenases214 and so has been annotated as a reductase

(Red) domain (Fig. 29). Reductive release requires NAD(P)H

as a cosubstrate. Hydride transfer from NADPH to the acyl/

peptidyl thioester linkage yields a hemithioacetal linkage which

unravels spontaneously to the released PK/NRP aldehyde and

the free thiolate of the pantetheinylated T domain. This is the

route for the reductive release of aminoadipoyl-S-T during lysine

biosynthesis in yeast.215 Intramolecular capture of the newly-

formed aldehyde by an amino group is proposed for the safracin

This journal is ª The Royal Society of Chemistry 2008

Fig. 28 Two modes of thioesterase (TE)-mediated chain termination: (a) TE-catalyzed hydrolysis by the ACV synthetase to yield the linear ACV-

tripeptide-carboxylic acid. (b) TE-catalyzed attack by an internal nucleophile: macrocylization yielding the daptomycin depsipeptide, and selective

trimerization by EntF to deliver the lactone enterobactin. (c) Excised TE domains can be used to generate novel hybrid macrocylic products of the

tyrocidine and daptomycin family.

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and saframycin antitumor antibiotics,216 and has been charac-

terized in vitro in the case of nostocylopeptide imines217 (Fig. 29).

There are some examples where reductive chain release is a net

four-electron rather than a two-electron process, taking the acid

oxidation state down to that of an alcohol. In lyngbyatoxin

biosynthesis, the reductase domain terminus of LtxA has been

shown to catalyze two iterations of hydride transfer from

NADPH.218 Reductive cleavage of the thioester of N-methyl-

Val-Trp-S-T to the aldehyde is followed by a second addition of

hydride to give the alcohol. For myxochelin A biosynthesis, net

four-electron reduction occurs through an analogous route.219

Reconstitution of this system has been accomplished through

incubation of DHB and L-Lys with the myxochelin synthetase as

shown in Fig. 29.

Not all modes of PK and NRP chain release have yet been

characterized (Fig. 30). In gliotoxin biosynthesis220 and ergota-

mine formation221 release of chains as cyclic diketopiperazines

occur.222 In the myxobacterial aurafuranone pathway223 the last

PKS module is lacking a TE domain so an oxidative Baeyer–

Villiger process followed by chain release has been proposed but

not yet validated.

This journal is ª The Royal Society of Chemistry 2008

8 Post-assembly-line tailoring reactions

In Section 6 we have noted sets of PK and NRP chain modifi-

cations that can occur by domains embedded within specific

modules of assembly lines. But this does not exhaust the catalytic

repertoire of the proteins encoded within the PK and NRP

biosynthetic gene clusters.224 In many cases, genes in these

clusters encode tailoring enzymes that act after release of the PK,

NRP, NRP-PK hybrid nascent product from assembly lines.24

The product that rolls off the assembly line is properly consid-

ered a late-stage intermediate rather than the final product. Some

of the subsequent tailoring enzyme steps can dramatically

resculpt the scaffold connectivity and decorate the periphery.225

We describe below examples of post-assembly tandem

glycosylations of both NRP and PK aglycones.226 One of the

characteristics of glycosylated PK and NRP natural products is

that the hexoses are often not the normal D-glucose, mannose, or

galactose monomers of primary metabolism.227,228 Instead,

nucleoside diphospho-esters of deoxy and aminodeoxyhexose

variants with balanced hydrophobic and charge properties

contribute important pharmacophores to natural products.

Nat. Prod. Rep., 2008, 25, 757–793 | 775

Fig. 30 Twomodes of presently uncharacterized chain release. (a) Spontaneous diketopiperazine formation exemplified by the gliotoxin synthetase. (b)

Baeyer–Villiger-type oxidation, followed by hydrolysis of the resulting hemiacetal, sets the stage for heterocycle formation in aurafuron A biosynthesis.

Fig. 29 Reductive chain release is catalyzed by a C-terminal reductase domain (Re) with NAD(P)H as the cosubstrate. Intramolecular capture of the

resulting aldehyde by an amine nucleophile delivers the nostocyclopeptide class of macrocyclic imines, while further reduction results in the alcohol of

lyngbyatoxin and myxochelin.

Fig. 31 Conversion of UDP-D-glucose to TDP-L-epivancosamine by

a dedicated, multi-step pathway.

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Thus, biosynthetic gene clusters for glycosylated PKs and NRPs

typically encode genes for making these dedicated NDP-hexoses

from the abundant metabolites UDP-glucose or TDP-glucose

(Fig. 31). For example, the gene cluster for chloroeremomycin,

discussed in Section 8.1, encodes six enzymes that convert TDP-

D-glucose to TDP-L-epivancosamine.229 While efforts over the

past 20 years have revealed the mechanisms and strategies for

many such NDP-deoxysugar-forming enzymes to make 2-deoxy,

3-deoxy, 4-deoxy, and 6-deoxy sugars,227,230 the in vitro

reconstitution of the five-step pathway from TDP-glucose

to TDP-L-epivancosamine is one of few examples where purified

776 | Nat. Prod. Rep., 2008, 25, 757–793 This journal is ª The Royal Society of Chemistry 2008

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enzymes have been shown to recapitulate the in vivo pathway to

the dedicated L-aminodeoxysugar building block for the post-

assembly-line tailoring of antibiotics. In vitro analysis of the

TDP-L-mycarose pathway has also been described.231

8.1 Post-assembly tailoring to produce glycopeptide and

lipoglycopeptide antibiotics

Among the best-studied late-stage tailoring enzymes are those

involved in the maturation of two clinically used glycopeptide

antibiotics, teicoplanin and vancomycin, and a close relative,

chloroeremomycin.232 We have noted the enzymatic construction

of the dedicated monomers incorporated at residues 4,5,7 of the

heptapeptide scaffold (Section 4.1) and their oxygenative cross

linking (Section 6.4) that account for 14 of the tailoring enzymes

encoded in these biosynthetic gene clusters.

The most notable remaining features of vancomycin and its

close relative chloroeremomycin are the disaccharide chain

attached to the phenolic hydroxyl of Hpg4 of each crosslinked

heptapeptide scaffold and the monosaccharide attached to the

b-hydroxyl of Tyr6 of chloroeremomycin. The three glycosyl-

transferases (Gtfs) in the chloroeremomycin cluster64 and the two

Gtfs in the vancomycin cluster233 have been purified and char-

acterized both kinetically234,235 and structurally236–238 to establish

order of action, mechanism, and selectivity (Fig. 32A). The

glucosyl moiety is added by the first Gtf (GtfB or GtfE) from

UDP-glucose to the Hpg4-hydroxyl of the crosslinked hepta-

peptide aglycone. The 2-hydroxyl of the glucosyl group is then

the nucleophile for GtfD-mediated transfer of L-vancosaminyl

moiety from TDP-L-vancosamine (vancomycin) or GtfC-

promoted addition of the epimeric L-epivancosaminyl sugar

in chloroeremomycin maturation. GtfA also uses TDP-L-epi-

vancosamine as a sugar donor, but is regiospecific for transfer

to the benzylic alcohol of b-hydroxy-Tyr6 of the crosslinked

Fig. 32 Action of post-assembly-line tailoring glycosyltransferases. (a) Site-s

and teicoplanin aglycones to the mature glycopeptide antibiotics. (b) Glycosy

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scaffold of chloroeremomycin. X-Ray structures reveal the

location of substrates and the basis for altered regiochemistry

between GtfA and GtfC.237 The monoglucosylated vancomycin

intermediate has also been a testing ground for chemoenzymatic

glycorandomization and neoglycosylation strategies to build

semisynthetic glycopeptide antibiotic variants.239–241

Teicoplanin, the second glycopeptide family member used as

a human antibiotic, has three differences from vancomycin.195,196

First is the replacement of Val1 and Asn3 of vancomcyin with

Tyr1 and Hpg3 in teicoplanin, allowing a fourth crosslink and so

requiring a fourth tailoring oxygenase (see discussion in Section

6.4). Second, there is only a monosaccharide, D-2-aminoglucose,

in glycosidic linkage to HPG4. There is an N-acetylglucosamine

attached to residue six, and finally there is a long-chain acyl

group linked to the 2-amino group of the D-glucosamine residue.

Studies with the two purified teicoplanin glycosyltransferases

and the acyltransferase242 have established the order of those

three tailoring steps: glucosamine addition to HPG4 first, then

N-acylation and subsequent installation of the N-acetylglucos-

amine moiety at residue six of the crosslinked peptide scaffold.

Knowledge of the order of action of the tailoring Gtfs and

acyltransferase enable strategies for design and enzymatic

synthesis of lipoglycopeptide variants of teicoplanin.

There are several NRP and PK lipid-containing natural

products that have undergone prenylation reactions at some

stage in their biosynthesis rather than acylation. Two examples

(Fig. 33) reflect the capture of electrophilic prenyl units by

cosubstrate nucleophiles. The aminocoumarin antibiotics, noted

in Section 9.5, all contain a prenylated hydroxybenzoate unit

which arises by attack of the C3 carbanion, generated through

the adjacent C4-hydroxyl of 4-hydroxybenzoate, on C1 of D2-

isopentenyl diphosphate.243,244 Lyngbyatoxin, a cyanobacterial

metabolite, is assembled from an N-methyl-Val-Trp core by

a two-module NRPS with reductive cleavage of the Trp moiety

elective glycosylation converts the NRP chloroeremomycin, vancomycin

l-transfer to aromatic PKs.

Nat. Prod. Rep., 2008, 25, 757–793 | 777

Fig. 33 Action of prenyltransferases during the maturation of novobi-

ocin and lyngbyatoxin.

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(see Fig. 29) to the alcohol mentioned in Section 7. This is

followed by cytochrome P450-mediated addition of theN-methyl

group to C4 of the indole side chain of the tryptophanol

(presumably via an arene oxide intermediate) and finally

geranylation at C7 of the indole ring (Fig. 33).245

8.2 Tandem glycosylations of aromatic polyketide aglycones

The daunomycin and aclacinomycin family of aromatic poly-

ketides are clinically used antitumor agents246 whose tetracyclic

aromatic ring scaffold is assembled by type II PKSs.13 Subsequent

regiospecific glycosylation of the 7-hydroxy substituent is cata-

lyzed by tailoring PKGtfs (Fig. 32B).24 Daunomycin has a single

L-aminodeoxysugar, L-daunosamine, while aclacinomycin A has

tethered L-rhodosamine, 2-deoxy-L-fucose, and L-cinerulose. This

trisaccharide chain is involved in binding to the minor groove of

DNA, while the anthracycline intercalates between base

pairs.247,248 There is one Gtf in the daunomycin-tailoring

Fig. 34 C-Glycosylation of enterob

778 | Nat. Prod. Rep., 2008, 25, 757–793

pathway36 and two, AknS and AknK, in the aclacinomycin-

tailoring pathway. With purified enzymes it has been possible

to show that AknS acts first but only in the presence of a second

protein AknT,249,250 to add L-rhodosamine from TDP-L-rhodos-

amine to the polyketide aglycone. Then AknK251 can add the

second and thirddeoxysugar residues. There are other examples in

polyketide maturation where a single Gtf will add tandem

deoxyhexose residues to build an oligosaccharide chain.252 AknS

joins a small family of other polyketide glycosyltransferases,

among them theGtfs that glycosylate the erythromycin scaffold at

the C3 hydroxyl,253,254 where a second (regulatory?) subunit (here

AknT) is required for active catalysis, a feature that could only be

gleaned from in vitro reconstitutions. It will be possible to use

variant TDP-L-aminodeoxysugars to alter the sugar residues in

the tailoring steps as a tool to evaluate structure activity

relationships for antitumor efficacy.253

8.3 C-Glycosylation: enterobactin to salmochelin

Although the vast majority of glycosides in primary metabolites

and secondary natural products are O-glycosides, there are

a small number of C-glycoside natural products in which C1 of

the glycosyl unit is attached by a direct C–C bond to a carbon of

the aglycone that is presumed to have acted as a nucleophile

during C–C bond formation.255 C-Glycosides would be resistant

to hydrolysis by the suite of glycosidase enzymes and so represent

a metabolically stable linkage. AC-glycosyltransferase, IroB, has

been purified from a uropathogenic E. coli strain that converts

the catecholic NRP siderophore enterobactin to diglucosyl-

enterobactin (DGE), known as salmochelin S4 because of its

initial detection as a Salmonella-derived iron chelator (Fig. 34).256

Both glucosyl units are connected by C-glycosidic bonds, from

C1 of glucose to C5 of two of the three DHB moieties. The

regioselectivity of C-glucosylation, meta or para to the existing

actin to form the salmochelins.

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catechol hydroxyls of the DHB units, make direct C-glucosyla-

tion more likely than O-glucosylation followed by a sterically

demanding O-to-C glucosyl transfer across the face of the DHB

ring. UDP-glucose is the glucose donor and enterobactin the

acceptor, suggesting that ionization of the C2-hydroxyl of a DHB

ring to the phenolate would initiate catalysis. The C5 anion,

a resonance contributor to the phenolate anion, is the proposed

nucleophile that attacks C1 of the glucosyl donor UDP-glucose.

The C-glucosyl units in DGE do not compromise the ferric

iron-scavenging function but the modified siderophore no longer

binds to siderocalin, an innate immune protein from the

vertebrate host. Siderocalin binds ferric enterobactin, preventing

its uptake by bacteria, and thus reducing virulence as part of the

innate immune response. The C-glucosyl units in DGE sterically

block sequestration by siderocalin and allow ferric-DGE uptake

by its Gram-negative enteric producers.1,257

8.4 Post-assembly-line oxygenases

Most enzymes that have evolved to catalyze the reductive

activation and fragmentation of O2 with insertion of one or both

oxygen atoms into a cosubstrate utilize either the conjugated

organic cofactor FAD258 or the redox active transition metal

iron, in both heme and nonheme active site environments.259–261

Both categories are found as tailoring enzymes for PK and NRP

natural products as illustrated by some type I examples

(Fig. 35).24

In passing we note that metal-independent oxygenases,

exemplified by DpgC72,73,262 (Fig. 9), are also found in quinone-

forming steps of aromatic polyketides where phenolic ring-

containing polycyclics are prone to one electron oxidation,

generating phenoxy radicals reactive with triplet O2.

FAD-dependent enzymes that carry out Baeyer–Villiger ring

expansion reactions of cyclic ketones in type II PKS products

Fig. 35 Post-assembly-line tailoring by oxygenases of the (a) flavin-dep

This journal is ª The Royal Society of Chemistry 2008

appear to be prevalent in maturation steps of aromatic polyke-

tide families.13 In the biosynthesis of the anticancer agent

mithramycin, a late-stage tetracyclic intermediate, premi-

thramycin B, is substrate for purified MtmOIV.263,264 This FAD

enzyme performs a Baeyer–Villiger ring expansion of a six-

membered ring enone to a seven-membered ring lactone.

Subsequent hydrolytic ring opening of the lactone and decar-

boxylation may be spontaneous or enzyme-assisted. Post-

assembly-line oxidation of a secondary alcohol catalyzed by

flavin-dependent dehydrogenase SpnJ has also been recently

described in the context of spinosyn biosynthesis.265

In vitro studies with purified oxygenases have allowed evalu-

ation of catalytic parameters and specificity determinants for

maturation of both polyketide and nonribosomal peptide

scaffolds. In the erythromycin pathway, 6-deoxyerythronolide B

released by the PKS assembly line undergoes stereo- and regio-

specific hydroxylation at C6 by the cytochrome P450 EryF.266

This product is then a substrate for the two glycosyltransferases

that attach deoxysugars to the 3-hydroxyl and 5-hydroxyl of the

macrolactone scaffold.254 At this juncture a second cytochrome

P450 monooxygenase, EryK,267 acts to hydroxylate C12,

completing a four-step tailoring sequence of two oxygenations

and two glycosylations of the macrolide. In the maturation of the

polyene macrolide pimaricin, a late-step regioselective epoxida-

tion of the C4–C5 double bond is catalyzed by the hemeprotein

epoxidase PimD.268 In analogy, the epoxidase EpoK acts in the

last step of epothilone tailoring to introduce the C12,C13 epoxide

in epothilones A and B.269

In post-assembly-line oxidative tailoring of nonribosomal

peptide scaffolds requiring dioxygen, the most celebrated trans-

formations are the generation of the 5,4- (penicillin) and 6,4-

(cephalosporin) fused ring systems of the b-lactam antibiotics

from the acyclic precursor aminoadipoyl-cysteinyl-valine (ACV).

ACV synthetase204 is a ten-domain, three-moduleNRPSassembly

endent, (b) cytochrome P450, and (c) Fe/a-KG-dependent classes.

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line protein that releases theACVwhere the L-valine unit has been

epimerized to D-Val on the assembly line by an epimerization

domain (see Fig. 28). The next enzyme is isopenicillin N synthase

(IPNS), an a-ketoglutarate- and O2-requiring nonheme

mononuclear FeII enzyme that makes both the four-ring b-lactam

and the five-ring thiolane while O2 is reduced by four electrons to

two molecules of H2O.270 The subsequent conversion of the

penicillin scaffold to the ring-expanded cephem nucleus is then

mediated by another member of the FeII monooxygenase

superfamily, deacetoxycephalosporinC synthase (DAOCS).271,272

The structural and mechanistic scrutiny these two enzymes have

received is due, in large part, to the central importance of b-lactam

antibiotics in anti-infective therapy.

9 In vitro reconstitution of complete pathways

In this closing section we note six case studies in which full

reconstitution of NRP, PK, or hybrid pathways have been

achieved in vitro with purified enzymes. We conclude with

a discussion of lessons learned from the characterization of

catalysts that function in tandem.

9.1 Enterobactin to salmochelin to microcin E492: three layers

of secondary metabolism

Siderophores are secreted by many genera of bacteria as

a response to a variety of iron-limited microenvironments,

ranging from the ocean to mammalian serum.3 NRP genes

encoding siderophore biosynthesis are among the most

widespread of the thiotemplated biosynthetic pathways found in

the 223 sequenced bacterial genomes,2 emphasizing the vital role

that the iron-chelating molecules play in bacterial survival. The

Fig. 36 Reconstitution of the microcin E492 biosynthetic pa

780 | Nat. Prod. Rep., 2008, 25, 757–793

reconstituted biosynthesis of the DHB-derived siderophore

myxochelin219 has outlined one such pathway (Section 7).

In our discussion of assembly-line chain release mechanisms

(Section 7) we have also noted that the Gram-negative bacterial

siderophore enterobactin is made on a two-module NRPS

assembly line and that, in some virulent bacteria, it is C-gluco-

sylated at C5 of two of the three DHB residues to yield

salmochelin (DGE) as a second layer of maturation256 (Section

8.3). A third layer of secondary metabolism occurs in enteric

bacteria elaborating the 84-residue polypeptide microcin E492

(MccE492) (Fig. 36). The C-terminal Ser of this peptide is

enzymatically modified by a linearized monoglucosylenter-

obactin via an oxoester bond between the Ser84 carboxyl and the

C6-hydroxyl of glucose. The resultant MccE492 can now be

taken up by neighboring cells that express siderophore-specific

outer membrane permeases. Once internalized into the

periplasmic space, MccE492 molecules self-assemble into inner

membrane pores that depolarize target cells.273

Eight enzymes are required to reconstitute the three layers

of secondary metabolism. Four purified proteins, the two-

module NRPS assembly line composed of EntEBF and the

phosphopantetheinyltransferase EntD, are required to make

enterobactin.131 Two enzymes, MceC (an IroB homolog) and

MceD, are a C-glucosyltransferase and a regiospecific trilactone

hydrolase, respectively. MceI and MceJ form a heterodimer that

catalyzes the attachment of the linearized MGE to the MccE492

C-terminus.274 Among the unusual chemistry revealed in these

three enzymatic layers of natural product biosynthesis are: (1)

identification of the EntF TE domain as a cyclotrimerizing

macrolactone synthase; (2) demonstration of C–C bond

formation by C-glucosyltransfer (IroB/MceC); and (3)

construction of a glucose bridge between a ribosomal peptide

thway illustrating three layers of secondary metabolism.

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(pre-MccE492) and a nonribosomal peptide scaffold (enter-

obactin). Close inspection of MceIJ biochemistry revealed that

the initial product is the MccE492 ester attached to the

4-hydroxyl of the glucosyl bridge; nonenzymatic rearrangement

at physiological pH moves the 84-residue peptide from the

4-hydroxyl to the 6-hydroxyl of the bridging glucose.

9.2 Yersiniabactin reconstitution: an NRP-PK hybrid

Full reconstitution of the NRP-PK hybrid siderophore yersi-

niabactin,149 a virulence-conferring metabolite made by the

plague bacterium Yersinia pestis, has been achieved from four

purified proteins, High Molecular Weight Proteins 1 and 2

(HMWP1 and HMWP2), YbtE (a salicylate-adenylating

domain) and YbtU, a thiazoline reductase203 (Fig. 37). YbtE

activates salicylate as salicyl-AMP then transfers it to T1 of the

next protein, the 220 kDa HMWP2. HMWP2 is a two-module

NRPS with three carrier protein (T) domains and two cyclization

domains, building a hydroxyphenyl-thiazolinyl-thiazolinyl-S-T3

intermediate.275,276 HMWP1 is a 330 kDa two module hybrid,

with a PKS module followed by an NRPS module.48 Transfer of

the bisthiazolinyl chain from T3 on HMWP2 to malonyl-S-T4 in

the PKS module constitutes an NRPS-PKS interface where the

KS is the catalytic domain. Double Ca-methylation by an MT

domain in the PKS module occurs on the b-ketoacyl-S-T4

condensation product before reduction to the b-hydroxy,

a,a-dimethyl intermediate. The chain is then transferred to the

downstream NRPS module of HMWP1 where the Cy domain

constitutes the PKS to NRPS interface. This third NRPS

module, like the first two on HMWP2, activates cysteine and

cyclizes the chain after amide bond formation to a thiazolinyl-S-

T5. Another C-methylation occurs by a second embedded MT

domain, this time at Ca of the third thiazolinyl ring before

hydrolytic chain release.179 The fourth protein, YbtU, acts in

trans as a tailoring catalyst to reduce the middle thiazoline ring to

the tetrahydrothiazolidine oxidation state. All told, 22 chemical

operations happen on the five T domains of HMWP2/1 with

a net throughput of mature yersiniabactin of 1.4 product

molecules per minute.203

Reconstitution of an NRP-PK-NRP hybrid assembly line

from purified proteins and building blocks revealed the chemical

logic and plasticity of KS and Cy domains for switching growing

chains from one type of building block (acyl-CoAs) to the other

Fig. 37 Reconstitution of the yersiniabactin biosynthetic pathw

This journal is ª The Royal Society of Chemistry 2008

(amino acids). Characterization of this assembly line also

illuminated the tandem construction of bisthiazolines and their

redox adjustment as well as two modes of C-methylation.

9.3 Aromatic polyketide scaffolds from malonyl-CoA

monomers

Polycyclic aromatic scaffolds are assembled both by type I

(iterative) fungal PKS17 and by bacterial type II PKS

enzymes.13,277 The separate subunits of minimal PKS catalysts

have been purified and reconstituted from several streptomycete

systems (Fig. 38). The type II minimal set consists of the KS

catalytic subunit, KSa and its regulatory partner KSb (also

termed CLF (chain length factor) because it controls polyketide

length), that form a KS-CLF (KSa-KSb) heterodimer. A malonyl

acyl transferase (AT) delivers malonyl units from malonyl-CoA

to a fourth protein, the holo form of the acyl carrier protein

(T domain).278 When these four proteins from the actinorhodin

system279 are combined with the aromatase/cyclase and a sixth

protein, the free-standing ketoreductase (KR),280 chain elonga-

tion to an octaketide is observed.281 The first ring is regiospe-

cifically cyclized with the anticipated C7–C12 connectivity, but

the remaining polyketones undergo off-pathway intramolecular

cyclization and dehydration.

Mechanistic282 and structural studies283 of the CLF subunits

have revealed that volume control in the active site pocket is

a major factor in determining how many cycles of chain elon-

gation occur before chain release (typically 8–16 for aromatic

polyketides from bacterial type II PKSs). For control of

subsequent cyclization chemistry, it has been suggested that the

regioselective reduction of the C9 keto group in growing

polyketone chains may be a crucial step in biasing subsequent

ring closure aldol chemistry.280,284 Conversion of the sp2 ketone

to sp3 alcohol is likely to introduce a kink in the polyketone

(polyenolate) chain and constrain the acyclic product in

a conformer poised for intramolecular ring formation. Cyclases

may also have a chaperone like function in addition to aldol

condensation catalysis by binding the polyketone-S-T

intermediates, before and after C9 keto reduction, to prevent off

pathway intramolecular cyclizations.13 The role of cyclases for

control of topologies of the second, third, and fourth ring

cyclizations in tetracyclic aromatic polyketides was originally

determined by genetic studies but has since been examined by

ay demonstrates catalysis across an NRPS/PKS interface.

Nat. Prod. Rep., 2008, 25, 757–793 | 781

Fig. 38 Reconstitution of the biosynthetic pathways of aromatic polyketides. (a) Action of minimal Type II PKS in the early steps of actinorhodin

biosynthesis. (b) Chain elongation, aromatization, and tailoring to produce tetracenomycin C. (c) Mechanism of chain initiation and elongation in the

biosynthesis of the R1128 polyketides. (d) Bikaverin synthase and the use of alternative starter units.

782 | Nat. Prod. Rep., 2008, 25, 757–793 This journal is ª The Royal Society of Chemistry 2008

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purified cyclases from the tetracenomycin,285 nogalamycin,286

aclacinomycin,287 and doxorubicin288 type II PKS pathways.

Full reconstitution of the tetracyclic backbones of the scaf-

folds in doxorubicin36 and oxytetracycline289 purified systems

remain to be established, although studies with specific genes on

plasmids in engineered strains of streptomyces have gone a long

way to facilitate identification of the roles of particular protein

components, accumulation of intermediates in blocked strains,

and re-engineering to produce novel products.13 In the case of

tetracenomycin, biochemical characterization of the Tcm poly-

ketide synthase in vivo290 has been supplemented with detailed

mechanistic studies of isolated proteins.291 TcmN has been

shown to initiate aromatization of the decaketide interme-

diate,292 while TcmI functions to close the last ring of the tetra-

cycle.285,293 TcmH,294 N,292 OP and G295 then act to tailor the

tetracenomycin core. In the R1128 Type II PKS system, studies

with purified proteins that carry out chain initiation296,297 and

then chain elongation steps298 show orthogonal specificity of the

KS components for the initiation or elongation T domain,

indicating approaches to use an alternate starter acyl-CoA to

reprogram those pathways.299,300

The fungus Gibberella fujikuroi has an iterative type I PKS

known as PKS4 that elaborates the tetracyclic metabolite bika-

verin, which has anticancer activity.115 The single PKS4 module

is used nine times to build a tetracyclic nonaketide scaffold from

malonyl-CoA monomers. PKS4 can be expressed in and purified

from E. coli in the active, holo form. Addition of malonyl-CoA

monomers leads to the tetracyclic product SMA76a, a non-

aketide that has undergone two dehydrations. In addition to full

chain length control, purified PKS4 catalyzes the four regiospe-

cific cyclizations found in the bikaverin scaffold: an aldol

condensation to connect C2 and C7, a Claisen condensation to

connect C10 to C1, and attack of the C9 phenolate on the C13

carbonyl to yield the tricyclic naphthopyrone. This three-ring

connectivity is also seen in action of the Aspergillus nidulans wA

synthase.301 The last ring then presumably arises by C12–C17

aldol-type condensation. Use of different starter acyl-CoAs such

as octanoyl-CoA reroutes flux to a benzopyrone hexaketide,

reminiscent of volume control of chain length by CLFs in the

type II bacterial PKSs noted above. The PKS4 module can be

mutated in its terminal cyclase domain and then reconstituted in

trans with purified cyclases to establish alternate ring-closing

topologies to generate other aromatic scaffolds.302

Fig. 39 Complete reconstitution of a type II PKS biosynthetic pathway. The c

with the enterocin synthase results in formation of the complex tricyclic scaf

This journal is ª The Royal Society of Chemistry 2008

While we do not cover them in detail here, we note that much

progress has been made in reconstituting the biosynthesis of type

III PKs in vitro, including structural studies, the identification of

residues that control chain length, and successful efforts to

engineer these enzymes to produce novel products.38,39,303–307

9.4 Enterocin: a type II PKS pathway that builds a tricyclic

scaffold

The antibiotic enterocin, produced by Streptomyces maritimus, is

derived from an octaketide scaffold that is enzymatically

processed after chain elongation to a tricyclic core308 (Fig. 39). It

is assembled by a type II PKS system that uses benzoate as an

unusual starter unit309 followed by seven malonyl CoAs as

extender units. The minimal PKS requires the KS and a regula-

tory CLF type subunit (EncAB), the holo form of the T domain

(EncC), and an acyltransferase (FabD) borrowed from the

organism’s type II FAS. The ATP-dependent benzoate ligase

(EncN) generates benzoyl-AMP as the thermodynamically

activated starter unit. In the absence of tailoring enzymes, the

octaketone intermediate is rerouted to cyclic off-pathway struc-

tures,310 as is typical of type II PKS systems in the absence of

their cognate-tailoring enzymes. When EncM, EncK, and EncR

are provided, full reconstitution of enterocin is enabled with the

eight purified enzymes.310 EncM is the key rearrangement

catalyst, a proposed ‘‘Favorskiiase’’ that converts the C13 ketone

into an ester and yields the tricyclic scaffold.311 Further tailoring

involvesO-methylation (EncK) and installation of the 5-hydroxy

group (provided by the CytP450-type monoxygenase EncR).

EncM presumably uses a triketonic intermediate and makes the

C6–C11 and C7–C14 bonds by aldol chemistry. This test tube

recapitulation of the enterocin pathway installs the ten C–C

bonds, the five C–O bonds, and creates the seven chiral centers in

the natural product; most remarkable is the Favorskii-style

rearrangement. Only in this context can studies with purified

EncM shed light on the precise mechanism of tricyclic scaffold

generation.

9.5 Aminocoumarins: four-component assembly of the

novobiocin scaffold and seven-component assembly of

coumermycin

The aminocoumarin antibiotics (novobiocin, clorobiocin, and

coumermycin) act by targeting bacterial type II topoisomerases,

ombination of a benzoate starter unit and seven malonyl CoAmonomers

fold.

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thereby blocking DNA replication.312–314 Novobiocin and

clorobiocin differ only in their aminocoumarin C8 substituents

(chlorine vs. methyl) and their L-noviosyl 30-substituents

(carbamoyl vs. 5-methylpyrrole-2-carboxyl) (Fig. 40 and Fig. 41).

Coumermycin is a pseudosymmetric dimer built around

a pyrrole scaffold with the CH3 substituent on the amino-

coumarins and the methylpyrrolyl-ester on the noviose rings.

Cloning of the gene clusters315–317 has enabled subsequent

reconstitution studies with purified Nov, Clo, and Cou enzymes

such that all but one step in the four-part assembly of the three

aminocoumarin antibiotics have been described.

The eponymous bicyclic aminocoumarin ring is formed on

a free-standing A-T didomain enzyme (NovH) which activates

L-Tyr and installs it as the Tyr-S-T thioester in preparation for

b-hydroxylation by a partner cytochrome P450 type mono-

oxygenase (NovI).318 The newly introduced benzylic hydroxyl is

then oxidized by a heterodimeric NAD enzyme (NovJK) to yield

the b-keto-Tyr-S-T intermediate.319 A presumed hydroxylation,

by an as yet uncharacterized enzyme, sets up intramolecular

lactonization as the bicyclic aminocoumarin gets released from

the pantetheinyl thiol tether. The second building block is the

Fig. 40 (a) Biosynthesis of the aminocoumarin ring. (b) Production of th

novobiocin and clorobiocin biosynthetic pathways is centered on elaboration

784 | Nat. Prod. Rep., 2008, 25, 757–793

3-prenyl-4-hydroxybenzoate, derived from tyrosine by oxidative

enzymology (CloR)320 and prenyltransferase (CloQ) catalysis.244

The aminocoumarin and the prenylated hydroxybenzoate frag-

ments are ligated (NovL)321 by an ATP-dependent ligase that

generates the acyl-AMP intermediate that is captured by the

amine of the aminocoumarin to yield novobiocic acid. This

product must be enzymatically tailored at C8 of the

aminocoumarin before the hexosyl unit (L-noviose) is added to

the phenolic hydroxyl. C-methylation requires the carbanion at

C8, which is generated on deprotonation of the phenol hydroxyl

in the active site of NovO.322 Subsequent attack of the activated

methyl group of SAM by the resonance-stabilized carbanion

results in the new C–C bond. Glycosylation is then effected by

NovM, using TDP-L-noviose as glycosyl donor.323 The enzymes

converting TDP-glucose to the dedicated building block TDP-

L-noviose are also encoded in the biosynthetic gene cluster, and

several of the steps have been characterized.324,325 The product

from NovM action requires two final tailoring steps to yield the

active antibiotic. NovP326 is a 40-O-methyltransferase, again

using SAM as cosubstrate methyl donor. The last step is cata-

lyzed by the carbamoyl transferase NovN, using carbamoyl

e 3-prenyl-4-hydroxybenzoate building block. (c) Reconstitution of the

of the eponymous aminocoumarin ring.

This journal is ª The Royal Society of Chemistry 2008

Fig. 41 Ligation of two aminocoumarin fragments to a methyl pyrrole is demonstrated in the reconstitution of the coumermycin biosynthetic pathway.

Characterization of the aminocoumarin enzyme set has enabled the synthesis of several structural analogs through combinatorial biosynthesis.

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phosphate as the carbamoyl donor.327 Carbamoylation results in

a 200-fold enhancement of antibiotic activity, indicating the

30-O-acyl substituent on the novose sugar moiety is a key

pharmacophore.328 Ten purified enzymes have been studied to

reconstruct the conversion of two molecules of tyrosine,

carbamoyl phosphate, D2-isopentenyl diphosphate, and TDP-L-

noviose to the antibiotic novobiocin, with one enzymatic step as

yet uncharacterized.

The clorobiocin pathway differs in that the C-methyl-

transferase NovO (and CouO) is replaced by the FADH2-

dependent halogenase, Clo halogenase, which is presumed to add

a Cl+ equivalent analogously at the clorobiocic acid stage.329 The

other main difference is that the 30-O-carbamoyl transferase

gene, novN, is replaced by seven contiguous ORFs in both the

clorobiocin and coumermycin clusters: cloN1–7 and couN1–7.193

Studies with purified CouN3, Clo/CouN4, and Clo/CouN5 have

confirmed that Clo/CouN4 is the proline-specific adenylyl-

transferase, Clo/CouN5 is the T domain, and Clo/CouN3 is the

flavin-dependent dehydrogenase that catalyzes the double

dehydrogenation of prolyl-S-T (where T is Clo/CouN5) to

pyrrolyl-S-T.134 The proteins Clo/CouN2 and Clo/CouN7 have

been predicted to be acyl transferases and Clo/CouN1 a variant

of a T domain. These three proteins presumably act in a sequence

where Clo/CouN2 transfers the pyrrolyl moiety fromClo/CouN5

to Clo/CouN1. Clo/CouN7 would then act as the final pyrrolyl

transferase to move the pyrrole-2-carboxyl moiety to the

clorobiocin/coumermycin penultimate intermediate where the

30-hydroxyl of the 40-O-Me-noviosyl ring is the nucleophile.

Indeed, the apo form of CouN1 can be primed with a Ppant

prosthetic group and the pyrrolyl moiety installed via Sfp action

with pyrrolyl-CoA as its substrate. Pyrrolyl-S-CouN1 is a good

This journal is ª The Royal Society of Chemistry 2008

substrate for coupling with the clorobiocin, coumermycin and

novobiocin scaffolds in a regiospecific acyl transfer catalyzed by

purified CouN7.58,330 With purified apo CouN1, CouN7 and

a variety of synthetic pyrrolyl-CoA analogs, the Sfp-mediated

loading of CouN1 has demonstrated that CouN7 will transfer

two dozen heterocyclic variants of the pyrrole-2-carbonyl moiety

to make variant antibiotics.58 Thus, in the Clo/CouN1–7 arm of

the pathway, five of the seven proteins have been purified and

characterized. To date, Clo/CouN2 has been insoluble on

heterologous expression but can be bypassed by using Sfp to load

CouN1. Clo/CouN6 is predicted to be a novel C-methylation

catalyst making the CH3–C5 bond in the 5-methylpyrrole-

2-carboxyl pharmacophore; Clo/CouN6 have yet to be

characterized but now can be approached.

Tracing the reconstitution of clorobiocin, 15 purified proteins

have so far been characterized and only the Clo halogenase,329

and the missing lactone-forming enzyme in aminocoumarin

construction are not yet evaluated. Finally, it is worth noting that

the more potent family member coumermycin is built from both

carboxyl groups of 2,4-dicarboxy-3-methylpyrrole as the central

scaffold. The ligase CouL adds 8-methylnovobiocic acid to both

arms of the dicarboxypyrrole to make two amide linkages.331

Both phenols in the two aminocoumarins are then noviosylated

by CouM and the noviosyl sugars are likewise eachO-methylated

by CouP.332 Use of the CouN1–CouN7 pair with Sfp and

pyrrolyl-CoAs allows enzymatic construction of coumermycin

variants.58,333 In one representative study, five different sites were

varied in the Cou scaffold by use of alternate substrate fragments

with the tandem action of the four enzymes CouLMP and

NovN.332 Clearly, one can tinker with very complex antibiotic

natural product scaffolds with purified biosynthetic enzymes.

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9.6 Rebeccamycin, staurosporine, violacein, and terrequinone:

reconstruction of pathways involving tryptophan oxidative

dimerization

Microbial oxidation of tryptophan at the C2 amino group is the

first step in a number of pathways which divert part of the pool of

this proteinogenic amino acid to bisindole-containing structures

with diverse biological functions.334 For example, the six-ring

indolocarbazole scaffold is found in the antitumor agent

rebeccamycin and the protein kinase inhibitor staurosporine. A

different connectivity is found in the purple bisindole pigment

violacein that gives Chromobacterium violaceum its name

(Fig. 42). These three metabolites have a pyrrole ring connector

between the two indole moieties. In contrast, the cytostatic

fungal metabolite terrequinone consists of a benzoquinone

scaffold between the indole pair. Recent studies with the purified

biosynthetic enzymes for these four natural products have

allowed deconvolution of some remarkable enzyme-mediated

C–C bond-forming redox chemistry.

Fig. 42 Reconstitution of biosynthetic pathways based on oxidative coupling

staurosporine and rebeccamycin, while violacein displays a rearranged scaffol

Claisen condensation.

786 | Nat. Prod. Rep., 2008, 25, 757–793

9.6.1 Rebeccamycin and staurosporine. A combination of

cloning, gene cluster sequencing, and genetic knockout studies

yielded hypotheses about the roles of several enzymes encoded in

the reb and sta gene clusters,334,335with further illumination of the

remarkable chemistry provided by studies with purified

RebHODPC or their Sta counterparts.336,337 The first reaction in

the Reb pathway, but not the Sta pathway, is mediated by the

FADH2-dependent halogenase RebH76,77,82 (noted in Section

4.2), to yield 7-chloro-Trp as the first committed substrate.

Oxidation of 7-Cl-Trp by the flavoenzyme amino acid oxidase

RebO yields the imino acid form of the 7-chloroindolopyruvate.

While the imine could hydrolyze after release from the active site

of RebO, two such molecules can be intercepted by the next

enzyme, the hemeprotein RebD, which appears to mediate C–C

bond formation by benzylic coupling. Loss of one NH3 and

intramolecular cyclization of the initial adduct yields the pyrrole

ring that is the key connecting element in the observed RebD

product (dichloro)-chromopyrrolic acid. This oxidative

chemistry,337 initiated and controlled by RebD, has built

of tryptophan. Direct coupling results in the chromopyrrolic acid core of

d. Dimerization to produce the terrequinones proceeds through a double

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a pyrrole connector between the two indoles in a five-ring

pathway intermediate. The sixth ring is then formed by RebP,

a second hemeprotein, which acts to make the bond between the

two C2 positions in the indole moieties, probably via one-electron

chemistry.336,338 Remarkably, three products differing in the

oxidation state of the pyrrole ring are found in incubations of

chromopyrrolic acid with purified RebP/StaP. Addition of the

flavoenzyme RebC reroutes the flux to the aglycone of rebecca-

mycin. On the other hand, StaC sends the flux towards the

stauosporine amide, which lacks the second carbonyl of rebec-

camycin, installed through four-electron oxidation.

From the studies with the purified enzymes, we now know that

the four enzymes RebODPC catalyze a net fourteen-electron

oxidation as the two Trp substrate molecules are converted to the

six-ring indolocarbazole scaffold. Each enzyme has a redox

cofactor: FAD, heme, heme, and FAD, respectively, and O2 is

a cosubstrate for each of the four enzymes. This is a short but

impressive lesson in Nature’s redox catalyst capacity. The

aglycones in the rebeccamycin and staurosporine pathway then

get N-glycosylated (and O-methylated) by Gtfs that have not yet

been characterized biochemically. The staurosporine pathway

has one final twist in which C5 of the N-glycosyl deoxysugar

moiety is subjected to one more hemeprotein oxidative reaction,

setting up a second N–C glycosidic linkage. The resulting bridged

system of the staurosporine protein kinase inhibitor features

attachment of the deoxysugar to both indole nitrogens.

9.6.2 Violacein. Chromobacterium violaceum is found glob-

ally distributed in tropical waters and it is likely that the deep

violet color of violacein protects the producer organism’s

genome against UV damage.339 Violacein is most noteworthy

for a 1,2-indole shift that has occurred on the pyrrole ring

connecting scaffold340 as compared to the rebeccamycin and

staurosporine pathways.

The Vio pathway starts the same way as the Reb and Sta

pathways: with enzymatic oxidation of Trp to the iminopyruvate

by a flavoenzyme oxidase VioA. VioB is a hemeprotein analog of

RebD/StaD, and indeed, tandem action of purified VioA and

VioB yields chromopyrrolic acid (CPA). In violacein biosyn-

thesis, however, CPA is an off-pathway side product.

On-pathway, flux requires a third purified protein, VioE,341 that

has been found to mediate the 1,2-indole shift to yield

prodeoxyviolacein. Finally, action of purified VioD and VioC

lead to the final indole ring hydroxylations/oxidations to produce

violacein. Even more impressively, VioE will reroute the inter-

mediate made by RebOD away from chromopyrrolic acid to the

rearranged violacein scaffold. As yet, the mechanism of the

remarkable 1,2-indole shift is not understood, but the purified

VioBE combination offers the chance for mechanistic dissection

in both the Reb/Sta and Vio systems.

9.6.3 Terrequinone. The five-enzyme pathway to the Asper-

gillus metabolite terrequinone (a member of a family of asterri-

quinones) has recently been identified by a combination of fungal

genomics342,343 and purified enzyme analysis.213,344 Three

unanticipated features of enzyme chemistry have emerged. First,

the usual oxidation at the C2-amino group of Trp occurs;

however, in the terrequinone pathway, a PLP enzyme (TdiD),

rather than a flavoenzyme, mediates this transformation. The

This journal is ª The Royal Society of Chemistry 2008

PLP enzyme is a transaminase, and therefore the direct product is

the phenylpyruvate rather than the iminophenylpyruvate

generated by the flavoenzymes RebO and VioA. This detail

becomes relevant as the nitrogen of the rebeccamycin pyrrole is

lost before enzymatic dimerization occurs.

The second enzyme TdiA is a single module NRPS with an

A-T-TE tridomain organization. However, the A domain is

specific for selection and activation of the a-keto acid

phenylpyruvate, rather than the a-amino acid Trp. Such keto

acid activating domains are unusual but have now been detected

in the NRPS modules of valinomycin, cereluide, and barbamide

biosynthesis (vide supra). TdiA has some analogies to the enter-

obactin synthetase EntF in its TE domain: they both act as

cyclization catalysts, holding one acyl group as a thioester on the

Ppant arm of the T domain and the other as an oxoester at the

active site Ser residue of the TE domain. Because the two

tethered acyl chains here are phenylpyruvyl moieties, they can

react as enolates in C–C bond formation. Double Claisen-type

condensation can happen in head-to-tail fashion to release the

bisindolylbenzoquinone. This is novel cyclization chemistry for

an NRPS TE domain, set up by the A domain that activates

a keto acid rather than an amino acid. Note that the scaffold

connecting the two indole moieties is a six-membered benzo-

quinone rather than the five-membered pyrrole found in chro-

mopyrrolic acid in the Reb and Sta pathways. This harks back to

the use of a PLP transaminase rather than an FAD-oxidase in the

initial oxidation of the two Trp substrate molecules.

The remaining three proteins have the following roles. TdiC is

an NADH-dependent quinone reductase creating the hydroqui-

none oxidation state. The hydroquinone is required for C3 of the

ring to act as nucleophile in the active site of the next enzyme

TdiB, a prenyltransferase. TdiB catalyzes C–C bond formation

between C3 of the hydroquinone and C1 of the D2-isopentenyl

diphosphate partner subsbtrate. The C-monoprenyl product is

the substrate for a second prenyl transfer by TdiB, this time to C2

of one of the indole rings. The double prenylation by TdiB has

one additional novel feature: the second prenylation proceeds

with reverse regiochemistry (SN20), that results in capture of the

of the second D2-isopentenyl-PP moiety at C3, rather than at C1,

for C–C bond formation. Finally, TdiE appears to be a

chaperone which directs flux in the first prenylation step to

on-pathway C–C bond formation. In the absence of TdiE,

incubations of TdiC/TdiB result in off-pathwayO-prenylation of

the quinone. In loose analogy to VioE, noted in the violacein

pathway above, TdiE is a protein factor that keeps reactant flux

on-pathway by an as-yet unknown mechanism.

In summary, the studies of rebeccamycin, staurosporine,

violacein, and terrequinone biosynthesis with purified enzymes

have revealed some remarkable chemical strategies and mecha-

nisms for dimerizing tryptophan to a variety of scaffolds. They

also show the versatility of enzymes with FAD, PLP, and heme

iron redox cofactors to control and direct the flux of highly

reactive intermediates for regiospecific construction of the diverse

molecular frameworks of these biologically active metabolites.

10 Lessons learned

In vitro studies with purified enzymes bridge the gap between

bioinformatic predictions, resulting from sequence annotation

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and gene knockouts, and the direct observation of gene product

function in NRPS, PKS, or hybrid NRPS-PKS biosynthetic

pathways. Experiments with encoded enzymes can uncover novel

chemistry and set the stage for rational manipulation of partic-

ular steps in natural product biosyntheses. Characterization of

enzymes in the four stages of type I PK and NRP synthesis have

revealed mechanisms and selectivity in: (1) the T domain priming

steps, (2) the pre-assembly line provision of dedicated monomer

units, (3) the multiple steps of assembly line operations, including

chain initiation, elongation, and termination, and (4) post-

assembly-line tailoring reactions to create mature natural

product scaffolds.

Investigation of the phosphopantetheinyl transferase

superfamily has greatly facilitated in vitro PKS and NRPS

reconstitutions by enabling the reliable priming of apo T domains

with the essential phosphopantetheinyl arm. PPTase studies have

revealed the molecular basis for discrimination of subclasses of T

domains—FAS and PKS acyl carrier proteins vs NRPS peptidyl

carrier proteins—and have shown that these enzymes can load

many forms of acyl-S-pantetheinyl chains onto a range of apo T

domains for mechanistic analysis, biosynthetic reconstitutions,

and the generation of novel natural product derivatives.

Work with purified enzymes that provide dedicated monomer

units has indicated which ORFs need to be transferred to cells for

effective reconstitution of functional assembly lines in new

producer organisms. In addition, the study of monomer-unit

provision has illuminated novel chemical strategies for the

construction of nonproteinogenic amino acid building blocks;

for example, the action of the metal-free dioyxgenase DpgC in

the vancomycin/teicoplanin pathways.

Efforts to reconstitute the action of assembly line modules

have also decifered mechanisms for chain initiation, including

the intersection of PLP-enzymes and PKS modules for the

introduction of nitrogen into polyketide scaffolds. Studies of

subsets of condensation domains have demonstrated variations

of C domain function both as chiral (DCL) amide and ester

synthases during chain elongations and epimerization catalysts.

The Cy subset of C domains are extraordinary catalysts,

converting acyl-Cys-S-Tn and acyl-Ser-S-Tn to tethered hetero-

cyclic thiazolinyl and oxazolinyl-S-Tn.

Insight into how isoprenoid and polyketide metabolism can

intersect on assembly lines has come from examination of a six-

protein cassette that includes an HMG CoA synthase homolog.

Experiments with the HMG synthase and a pair of tandem-

acting enoyl CoA hydratase family members that catalyze

dehydration, then decarboxylation, have revealed the strategy

and mechanism for in trans tailoring of elongating polyketides

that results in the introduction of b-branches in the chain

backbone.

Characterization of hemeproteins OxyA and OxyB has shown

how the peptide scaffold of the vancomycin family of glyco-

peptide antibiotics is rigidified through crosslinking. This mode

of peptide tailoring exemplifies enzymatic installation of archi-

tecturally constraining ether and C–C crosslinks. Examination of

the SyrB1,B2, and SyrC enzymes in the syringomycin pathway

has revealed Nature’s strategy for chlorination of threonine on

a side module of the assembly line followed by transfer to the

ninth module of the megasynthetase SyrE. Study of the RapP

NRPSmodule has shown how a pipecolyl unit is inserted into the

788 | Nat. Prod. Rep., 2008, 25, 757–793

middle of the polyketide backbone in rapamycin and FK506

immunosuppressants. Characterization of the chain-terminating

TE domains of NRPS and PKS assembly lines have illustrated

the regio- and stereospecific incorporation of macrocyclic

constraints into nascent scaffolds as they are released.

Investigation of the post-assembly-line tailoring reactions of

acylation, glycosylation, and oxidation has revealed the timing

and mechanism of natural product maturation and provided

information required for the manipulation of these late-stage

transformations for combinatorial engineering of new structures.

Finally, full reconstitution of natural product pathways in

both type I and type II contexts with pure enzymes in test tubes

has allowed the elaboration of remarkable complexity

from simple building blocks, as in enterocin, coumermycin,

rebeccamycin, and terrequinone scaffold construction. These

efforts enable future investigation of some remarkable, unsolved

catalytic transformations such as the Favorskii reaction in

enterocin assembly and the 1,2-indole shift reaction during

violacein biosynthesis. The studies on syringomycin and on

coronamic acid formation have revealed a striking class of

biological halogenases that utilize nonheme mononuclear high-

valent oxoiron intermediates for selective C–H activation and

halogen transfer to unactivated substrates. Undoubtedly, more

novel enzyme chemistry remains to be deciphered through the

deconvolution of NRP and PK biosynthetic pathways.

11 Acknowledgements

We acknowledge all of our colleagues whose names are listed in

the specific references for their efforts in natural product enzyme

characterization and pathway reconstitution.

12 References

1 M. A. Fischbach and C. T. Walsh, Chem. Rev., 2006, 106, 3468–3496.

2 S. Donadio, P. Monciardini andM. Sosio,Nat. Prod. Rep., 2007, 24,1073–1109.

3 J. H. Crosa and C. T. Walsh, Microbiol. Mol. Biol. Rev., 2002, 66,223–249.

4 S. C. Andrews, A. K. Robinson and F. Rodriguez-Quinones, FEMSMicrobiol. Rev., 2003, 27, 215–237.

5 S. Schneiker, O. Perlova, O. Kaiser, K. Gerth, A. Alici,M. O. Altmeyer, D. Bartels, T. Bekel, S. Beyer, E. Bode,H. B. Bode, C. J. Bolten, J. V. Choudhuri, S. Doss,Y. A. Elnakady, B. Frank, L. Gaigalat, A. Goesmann,C. Groeger, F. Gross, L. Jelsbak, L. Jelsbak, J. Kalinowski,C. Kegler, T. Knauber, S. Konietzny, M. Kopp, L. Krause,D. Krug, B. Linke, T. Mahmud, R. Martinez-Arias,A. C. McHardy, M. Merai, F. Meyer, S. Mormann, J. Munoz-Dorado, J. Perez, S. Pradella, S. Rachid, G. Raddatz, F. Rosenau,C. Ruckert, F. Sasse, M. Scharfe, S. C. Schuster, G. Suen,A. Treuner-Lange, G. J. Velicer, F. J. Vorholter, K. J. Weissman,R. D. Welch, S. C. Wenzel, D. E. Whitworth, S. Wilhelm,C. Wittmann, H. Blocker, A. Puhler and R. Muller, Nat.Biotechnol., 2007, 25, 1281–1289.

6 M. Oliynyk, M. Samborskyy, J. B. Lester, T. Mironenko, N. Scott,S. Dickens, S. F. Haydock and P. F. Leadlay,Nat. Biotechnol., 2007,25, 447–453.

7 D. W. Udwary, L. Zeigler, R. N. Asolkar, V. Singan, A. Lapidus,W. Fenical, P. R. Jensen and B. S. Moore, Proc. Natl. Acad. Sci.U. S. A., 2007, 104, 10376–10381.

8 H. Ikeda, J. Ishikawa, A. Hanamoto, M. Shinose, H. Kikuchi,T. Shiba, Y. Sakaki, M. Hattori and S. Omura, Nat. Biotechnol.,2003, 21, 526–531.

This journal is ª The Royal Society of Chemistry 2008

Dow

nloa

ded

by U

nive

rsity

of

Chi

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on

03/0

5/20

13 1

6:08

:31.

Pu

blis

hed

on 2

3 M

ay 2

008

on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/B80

1747

F

View Article Online

9 S. Omura, H. Ikeda, J. Ishikawa, A. Hanamoto, C. Takahashi,M. Shinose, Y. Takahashi, H. Horikawa, H. Nakazawa,T. Osonoe, H. Kikuchi, T. Shiba, Y. Sakaki and M. Hattori, Proc.Natl. Acad. Sci. U. S. A., 2001, 98, 12215–12220.

10 H. B. Bode, B. Bethe, R. Hofs and A. Zeeck,ChemBioChem, 2002, 3,619–627.

11 I. de Bruijn, M. J. de Kock, M. Yang, P. de Waard, T. A. van Beekand J. M. Raaijmakers, Mol. Microbiol., 2007, 63, 417–428.

12 S. C. Wenzel and R. Muller, Curr. Opin. Chem. Biol., 2005, 9, 447–458.

13 C. Hertweck, A. Luzhetskyy, Y. Rebets and A. Bechthold, Nat.Prod. Rep., 2007, 24, 162–190.

14 D. E. Cane and C. T. Walsh, Chem. Biol., 1999, 6, R319–325.15 I. B. Lomakin, Y. Xiong and T. A. Steitz, Cell, 2007, 129, 319–332.16 S. Jenni, M. Leibundgut, D. Boehringer, C. Frick, B. Mikolasek and

N. Ban, Science, 2007, 316, 254–261.17 J. Staunton and K. J. Weissman, Nat. Prod. Rep., 2001, 18, 380–416.18 O. Froyshov, Acta Microbiol. Acad. Sci. Hung., 1975, 22, 427–432.19 S. G. Laland and T. L. Zimmer, Essays Biochem., 1973, 9, 31–57.20 P. Zuber, Curr. Opin. Cell Biol., 1991, 3, 1046–1050.21 H. Kleinkauf and H. von Dohren, Eur. J. Biochem., 1990, 192, 1–15.22 D. A. Hopwood, Chem. Rev., 1997, 97, 2465–2498.23 D. Schwarzer, R. Finking and M. A. Marahiel, Nat. Prod. Rep.,

2003, 20, 275–287.24 C. T. Walsh, H. Chen, T. A. Keating, B. K. Hubbard, H. C. Losey,

L. Luo, C. G. Marshall, D. A. Miller and H. M. Patel, Curr. Opin.Chem. Biol., 2001, 5, 525–534.

25 R. M. Kohli and C. T. Walsh, Chem. Commun., 2003, 297–307.26 F. Kopp and M. A. Marahiel, Nat. Prod. Rep., 2007, 24, 735–749.27 B. Shen, Curr. Opin. Chem. Biol., 2003, 7, 285–295.28 Y. J. Lu, Y. M. Zhang and C. O. Rock, Biochem. Cell Biol., 2004, 82,

145–155.29 E. Schweizer and J. Hofmann, Microbiol. Mol. Biol. Rev., 2004, 68,

501–517, table of contents.30 C. Khosla, Chem. Rev., 1997, 97, 2577–2590.31 C. M. Kao, L. Katz and C. Khosla, Science, 1994, 265, 509–512.32 S. A. Sieber and M. A. Marahiel, Chem. Rev., 2005, 105, 715–738.33 C. R. Hutchinson, J. Kennedy, C. Park, S. Kendrew, K. Auclair and

J. Vederas, Antonie Van Leeuwenhoek, 2000, 78, 287–295.34 T. Maier, S. Jenni and N. Ban, Science, 2006, 311, 1258–1262.35 J. Zhang, S. G. Van Lanen, J. Ju, W. Liu, P. C. Dorrestein, W. Li,

N. L. Kelleher and B. Shen, Proc. Natl. Acad. Sci. U. S. A., 2008,105, 1460–1465.

36 C. R. Hutchinson, Chem. Rev., 1997, 97, 2525–2536.37 H. Jenke-Kodama, A. Sandmann, R. Muller and E. Dittmann,Mol.

Biol. Evol., 2005, 22, 2027–2039.38 K. Watanabe, A. P. Praseuth and C. C. Wang, Curr. Opin. Chem.

Biol., 2007, 11, 279–286.39 M. B. Austin and J. P. Noel, Nat. Prod. Rep., 2003, 20, 79–110.40 R. H. Lambalot, A. M. Gehring, R. S. Flugel, P. Zuber, M. LaCelle,

M. A. Marahiel, R. Reid, C. Khosla and C. T. Walsh, Chem. Biol.,1996, 3, 923–936.

41 C. T. Walsh, A. M. Gehring, P. H. Weinreb, L. E. Quadri andR. S. Flugel, Curr. Opin. Chem. Biol., 1997, 1, 309–315.

42 A. C.Mercer andM. D. Burkart,Nat. Prod. Rep., 2007, 24, 750–773.43 J. Crosby, D. H. Sherman, M. J. Bibb, W. P. Revill, D. A. Hopwood

and T. J. Simpson, Biochim. Biophys. Acta, 1995, 1251, 32–42.44 L. E. Quadri, P. H. Weinreb, M. Lei, M. M. Nakano, P. Zuber and

C. T. Walsh, Biochemistry, 1998, 37, 1585–1595.45 C. Sanchez, L. Du, D. J. Edwards, M. D. Toney and B. Shen, Chem.

Biol., 2001, 8, 725–738.46 K. Watanabe, M. A. Rude, C. T. Walsh and C. Khosla, Proc. Natl.

Acad. Sci. U. S. A., 2003, 100, 9774–9778.47 S. Gruenewald, H. D.Mootz, P. Stehmeier and T. Stachelhaus,Appl.

Environ. Microbiol., 2004, 70, 3282–3291.48 Z. Suo, C. C. Tseng and C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A.,

2001, 98, 99–104.49 H. Chen, S. O’Connor, D. E. Cane and C. T. Walsh, Chem. Biol.,

2001, 8, 899–912.50 M. R. Mofid, R. Finking, L. O. Essen and M. A. Marahiel,

Biochemistry, 2004, 43, 4128–4136.51 D. B. Stein, U. Linne andM. A.Marahiel, FEBS J., 2005, 272, 4506–

4520.52 J. Yin, A. J. Lin, D. E. Golan and C. T. Walsh,Nat. Protocols, 2006,

1, 280–285.

This journal is ª The Royal Society of Chemistry 2008

53 J. Yin, F. Liu, X. Li and C. T. Walsh, J. Am. Chem. Soc., 2004, 126,7754–7755.

54 J. Yin, F. Liu, M. Schinke, C. Daly and C. T. Walsh, J. Am. Chem.Soc., 2004, 126, 13570–13571.

55 J. Yin, P. D. Straight, S. Hrvatin, P. C. Dorrestein, S. B. Bumpus,C. Jao, N. L. Kelleher, R. Kolter and C. T. Walsh, Chem. Biol.,2007, 14, 303–312.

56 J. Yin, P. D. Straight, S. M. McLoughlin, Z. Zhou, A. J. Lin,D. E. Golan, N. L. Kelleher, R. Kolter and C. T. Walsh, Proc.Natl. Acad. Sci. U. S. A., 2005, 102, 15815–15820.

57 F. H. Vaillancourt, E. Yeh, D. A. Vosburg, S. E. O’Connor andC. T. Walsh, Nature, 2005, 436, 1191–1194.

58 M. Fridman, C. J. Balibar, T. Lupoli, D. Kahne, C. T. Walsh andS. Garneau-Tsodikova, Biochemistry, 2007, 46, 8462–8471.

59 J. Ligon, S. Hill, J. Beck, R. Zirkle, I. Molnar, J. Zawodny, S.Moneyand T. Schupp, Gene, 2002, 285, 257–267.

60 Y. A. Chan, M. T. Boyne, 2nd, A. M. Podevels, A. K. Klimowicz,J. Handelsman, N. L. Kelleher and M. G. Thomas, Proc. Natl.Acad. Sci. U. S. A., 2006, 103, 14349–14354.

61 B. S. Moore and C. Hertweck, Nat. Prod. Rep., 2002, 19, 70–99.62 S. Caboche, M. Pupin, V. Leclere, A. Fontaine, P. Jacques and

G. Kucherov, Nucleic Acids Res., 2008, 36, D326–331.63 N. J. Ryding, T. B. Anderson and W. C. Champness, J. Bacteriol.,

2002, 184, 794–805.64 A. M. van Wageningen, P. N. Kirkpatrick, D. H. Williams,

B. R. Harris, J. K. Kershaw, N. J. Lennard, M. Jones, S. J. Jonesand P. J. Solenberg, Chem. Biol., 1998, 5, 155–162.

65 B. K. Hubbard and C. T. Walsh, Angew. Chem., Int. Ed., 2003, 42,730–765.

66 O. Puk, P. Huber, D. Bischoff, J. Recktenwald, G. Jung,R. D. Sussmuth, K. H. van Pee, W. Wohlleben and S. Pelzer,Chem. Biol., 2002, 9, 225–235.

67 B. K. Hubbard, M. G. Thomas and C. T. Walsh, Chem. Biol., 2000,7, 931–942.

68 V. Pfeifer, G. J. Nicholson, J. Ries, J. Recktenwald, A. B. Schefer,R. M. Shawky, J. Schroder, W. Wohlleben and S. Pelzer, J. Biol.Chem., 2001, 276, 38370–38377.

69 H. Chen, C. C. Tseng, B. K. Hubbard and C. T. Walsh, Proc. Natl.Acad. Sci. U. S. A., 2001, 98, 14901–14906.

70 O. Puk, D. Bischoff, C. Kittel, S. Pelzer, S. Weist, E. Stegmann,R. D. Sussmuth and W. Wohlleben, J. Bacteriol., 2004, 186, 6093–6100.

71 C. C. Tseng, S. M. McLoughlin, N. L. Kelleher and C. T. Walsh,Biochemistry, 2004, 43, 970–980.

72 C. C. Tseng, F. H. Vaillancourt, S. D. Bruner and C. T. Walsh,Chem. Biol., 2004, 11, 1195–1203.

73 P. F. Widboom, E. N. Fielding, Y. Liu and S. D. Bruner, Nature,2007, 447, 342–345.

74 G. W. Gribble, Chemosphere, 2003, 52, 289–297.75 F. H. Vaillancourt, E. Yeh, D. A. Vosburg, S. Garneau-Tsodikova

and C. T. Walsh, Chem. Rev., 2006, 106, 3364–3378.76 E. Yeh, L. J. Cole, E. W. Barr, J. M. Bollinger, Jr., D. P. Ballou and

C. T. Walsh, Biochemistry, 2006, 45, 7904–7912.77 E. Yeh, L. C. Blasiak, A. Koglin, C. L. Drennan and C. T. Walsh,

Biochemistry, 2007, 46, 1284–1292.78 F. H. Vaillancourt, J. Yin and C. T. Walsh, Proc. Natl. Acad. Sci.

U. S. A., 2005, 102, 10111–10116.79 D. P. Galonic, E. W. Barr, C. T. Walsh, J. M. Bollinger, Jr. and

C. Krebs, Nat. Chem. Biol., 2007, 3, 113–116.80 A. Butler and J. N. Carter-Franklin, Nat. Prod. Rep., 2004, 21, 180–

188.81 J. N. Carter-Franklin, J. D. Parrish, R. A. Tschirret-Guth,

R. D. Little and A. Butler, J. Am. Chem. Soc., 2003, 125, 3688–3689.82 E. Yeh, S. Garneau and C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A.,

2005, 102, 3960–3965.83 C. Dong, S. Flecks, S. Unversucht, C. Haupt, K. H. van Pee and

J. H. Naismith, Science, 2005, 309, 2216–2219.84 S. Zehner, A. Kotzsch, B. Bister, R. D. Sussmuth, C. Mendez,

J. A. Salas and K. H. van Pee, Chem. Biol., 2005, 12, 445–452.85 C. Seibold, H. Schnerr, J. Rumpf, A. H. Kunzendorf, C. T. Wage,

A. J. Ernyei, D. Changjiang, J. H. Naismith and K. H. Van Pee,Biocatal. Biotransform., 2006, 24, 401–408.

86 P. C. Dorrestein, E. Yeh, S. Garneau-Tsodikova, N. L. Kelleherand C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A., 2005, 102,13843–13848.

Nat. Prod. Rep., 2008, 25, 757–793 | 789

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87 S. Lin, S. G. Van Lanen and B. Shen, J. Am. Chem. Soc., 2007, 129,12432–12438.

88 P. Spiteller, L. Bai, G. Shang, B. J. Carroll, T. W. Yu andH. G. Floss, J. Am. Chem. Soc., 2003, 125, 14236–14237.

89 T. W. Yu, L. Bai, D. Clade, D. Hoffmann, S. Toelzer, K. Q. Trinh,J. Xu, S. J. Moss, E. Leistner and H. G. Floss, Proc. Natl. Acad. Sci.U. S. A., 2002, 99, 7968–7973.

90 L. C. Blasiak, F. H. Vaillancourt, C. T. Walsh and C. L. Drennan,Nature, 2006, 440, 368–371.

91 D. P. Galonic, F. H. Vaillancourt and C. T. Walsh, J. Am. Chem.Soc., 2006, 128, 3900–3901.

92 W. L. Kelly, M. T. Boyne, 2nd, E. Yeh, D. A. Vosburg,D. P. Galonic, N. L. Kelleher and C. T. Walsh, Biochemistry,2007, 46, 359–368.

93 K. D. Walker, K. Klettke, T. Akiyama and R. Croteau, J. Biol.Chem., 2004, 279, 53947–53954.

94 W. Liu, S. D. Christenson, S. Standage and B. Shen, Science, 2002,297, 1170–1173.

95 H. Umezawa, T. Aoyagi, H. Suda, M. Hamada and T. Takeuchi,J. Antibiot. (Tokyo), 1976, 29, 97–99.

96 M. Jin, M. A. Fischbach and J. Clardy, J. Am. Chem. Soc., 2006,128, 10660–10661.

97 N. J. Bate, J. Orr, W. Ni, A. Meromi, T. Nadler-Hassar,P. W. Doerner, R. A. Dixon, C. J. Lamb and Y. Elkind, Proc.Natl. Acad. Sci. U. S. A., 1994, 91, 7608–7612.

98 L. Poppe, Curr. Opin. Chem. Biol., 2001, 5, 512–524.99 C. V. Christianson, T. J. Montavon, S. G. Van Lanen, B. Shen and

S. D. Bruner, Biochemistry, 2007, 46, 7205–7214.100 S. G. Van Lanen, P. C. Dorrestein, S. D. Christenson, W. Liu, J. Ju,

N. L. Kelleher and B. Shen, J. Am. Chem. Soc., 2005, 127, 11594–11595.

101 S. D. Christenson, W. Wu, M. A. Spies, B. Shen and M. D. Toney,Biochemistry, 2003, 42, 12708–12718.

102 S. D. Christenson, W. Liu, M. D. Toney and B. Shen, J. Am. Chem.Soc., 2003, 125, 6062–6063.

103 P. D. Fortin, C. T. Walsh and N. A. Magarvey, Nature, 2007, 448,824–827.

104 S. G. Van Lanen, S. Lin, P. C. Dorrestein, N. L. Kelleher andB. Shen, J. Biol. Chem., 2006, 281, 29633–29640.

105 D. G. Fujimori, S. Hrvatin, C. S. Neumann, M. Strieker,M. A. Marahiel and C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A.,2007, 104, 16498–16503.

106 M. Piraee and L. C. Vining, J. Ind. Microbiol. Biotechnol., 2002, 29,1–5.

107 H. Ikeda, T. Nonomiya, M. Usami, T. Ohta and S. Omura, Proc.Natl. Acad. Sci. U. S. A., 1999, 96, 9509–9514.

108 V. Miao, M. F. Coeffet-Legal, P. Brian, R. Brost, J. Penn,A. Whiting, S. Martin, R. Ford, I. Parr, M. Bouchard, C. J. Silva,S. K. Wrigley and R. H. Baltz, Microbiology, 2005, 151, 1507–1523.

109 H. Kleinkauf, J. Dittmann and A. Lawen, Biomed. Biochim. Acta,1991, 50, S219–224.

110 F. Malpartida and D. A. Hopwood, Mol. Gen. Genet., 1986, 205,66–73.

111 G. D’Agnolo, I. S. Rosenfeld and P. R. Vagelos, J. Biol. Chem.,1975, 250, 5283–5288.

112 J. Elovson and P. R. Vagelos, J. Biol. Chem., 1968, 243, 3603–3611.113 C. R. Hutchinson, C. W. Borell, M. J. Donovan, F. Kato,

H. Motamedi, H. Nakayama, R. L. Rubin, S. L. Streicher,R. G. Summers, E. Wendt-Pienkowski and W. L. Wessel, PlantaMed., 1991, 57, S36–43.

114 P. C. Dorrestein and N. L. Kelleher, Nat. Prod. Rep., 2006, 23,893–918.

115 S.M.Ma, J. Zhan, K.Watanabe, X. Xie, W. Zhang, C. C.Wang andY. Tang, J. Am. Chem. Soc., 2007, 129, 10642–10643.

116 T. A. Keating and C. T. Walsh, Curr. Opin. Chem. Biol., 1999, 3,598–606.

117 C. Bisang, P. F. Long, J. Cortes, J. Westcott, J. Crosby,A. L. Matharu, R. J. Cox, T. J. Simpson, J. Staunton andP. F. Leadlay, Nature, 1999, 401, 502–505.

118 L. Xiang and B. S. Moore, J. Bacteriol., 2003, 185, 399–404.119 P. A. Lowden, B. Wilkinson, G. A. Bohm, S. Handa, H. G. Floss,

P. F. Leadlay and J. Staunton, Angew. Chem., Int. Ed., 2001, 40,777–779.

120 C. G. Kim, T. W. Yu, C. B. Fryhle, S. Handa and H. G. Floss,J. Biol. Chem., 1998, 273, 6030–6040.

790 | Nat. Prod. Rep., 2008, 25, 757–793

121 W. He, L. Wu, Q. Gao, Y. Du and Y. Wang, Curr. Microbiol., 2006,52, 197–203.

122 P. R. August, L. Tang, Y. J. Yoon, S. Ning, R. Muller, T. W. Yu,M. Taylor, D. Hoffmann, C. G. Kim, X. Zhang,C. R. Hutchinson and H. G. Floss, Chem. Biol., 1998, 5, 69–79.

123 A. Rascher, Z. Hu, N. Viswanathan, A. Schirmer, R. Reid,W. C. Nierman, M. Lewis and C. R. Hutchinson, FEMSMicrobiol. Lett., 2003, 218, 223–230.

124 V. Miao, M. F. Coeffet-Le Gal, K. Nguyen, P. Brian, J. Penn,A. Whiting, J. Steele, D. Kau, S. Martin, R. Ford, T. Gibson,M. Bouchard, S. K. Wrigley and R. H. Baltz, Chem. Biol., 2006,13, 269–276.

125 R. H. Baltz, V. Miao and S. K. Wrigley, Nat. Prod. Rep., 2005, 22,717–741.

126 D. B. Hansen, S. B. Bumpus, Z. D. Aron, N. L. Kelleher andC. T. Walsh, J. Am. Chem. Soc., 2007, 129, 6366–6367.

127 S. D. Bruner, T.Weber, R.M. Kohli, D. Schwarzer,M. A.Marahiel,C. T. Walsh and M. T. Stubbs, Structure, 2002, 10, 301–310.

128 C. C. Tseng, S. D. Bruner, R. M. Kohli, M. A. Marahiel,C. T. Walsh and S. A. Sieber, Biochemistry, 2002, 41, 13350–13359.

129 Z. D. Aron, P. C. Dorrestein, J. R. Blackhall, N. L. Kelleher andC. T. Walsh, J. Am. Chem. Soc., 2005, 127, 14986–14987.

130 E. H. Duitman, L. W. Hamoen, M. Rembold, G. Venema, H. Seitz,W. Saenger, F. Bernhard, R. Reinhardt, M. Schmidt, C. Ullrich,T. Stein, F. Leenders and J. Vater, Proc. Natl. Acad. Sci. U. S. A.,1999, 96, 13294–13299.

131 A. M. Gehring, I. Mori and C. T. Walsh, Biochemistry, 1998, 37,2648–2659.

132 T. A. Keating, C. G. Marshall and C. T. Walsh, Biochemistry, 2000,39, 15522–15530.

133 M. G. Thomas, M. D. Burkart and C. T. Walsh, Chem. Biol., 2002,9, 171–184.

134 S. Garneau, P. C. Dorrestein, N. L. Kelleher and C. T. Walsh,Biochemistry, 2005, 44, 2770–2780.

135 L. Tang, S. Shah, L. Chung, J. Carney, L. Katz, C. Khosla andB. Julien, Science, 2000, 287, 640–642.

136 C. Y. Kim, V. Y. Alekseyev, A. Y. Chen, Y. Tang, D. E. Cane andC. Khosla, Biochemistry, 2004, 43, 13892–13898.

137 R. Carvalho, R. Reid, N. Viswanathan, H. Gramajo and B. Julien,Gene, 2005, 359, 91–98.

138 R. Reid, M. Piagentini, E. Rodriguez, G. Ashley, N. Viswanathan,J. Carney, D. V. Santi, C. R. Hutchinson and R. McDaniel,Biochemistry, 2003, 42, 72–79.

139 P. Caffrey, ChemBioChem, 2003, 4, 654–657.140 R. Castonguay, W. He, A. Y. Chen, C. Khosla and D. E. Cane,

J. Am. Chem. Soc., 2007, 129, 13758–13769.141 A. Baerga-Ortiz, B. Popovic, A. P. Siskos, H. M. O’Hare,

D. Spiteller, M. G. Williams, N. Campillo, J. B. Spencer andP. F. Leadlay, Chem. Biol., 2006, 13, 277–285.

142 A. T. Keatinge-Clay and R.M. Stroud, Structure, 2006, 14, 737–748.143 I. E. Holzbaur, A. Ranganathan, I. P. Thomas, D. J. Kearney,

J. A. Reather, B. A. Rudd, J. Staunton and P. F. Leadlay, Chem.Biol., 2001, 8, 329–340.

144 I. E. Holzbaur, R. C. Harris, M. Bycroft, J. Cortes, C. Bisang,J. Staunton, B. A. Rudd and P. F. Leadlay, Chem. Biol., 1999, 6,189–195.

145 J. Staunton and B. Wilkinson, Chem. Rev., 1997, 97, 2611–2630.146 T. A. Keating, C. G. Marshall, C. T. Walsh and A. E. Keating, Nat.

Struct. Biol., 2002, 9, 522–526.147 C. G. Marshall, M. D. Burkart, T. A. Keating and C. T. Walsh,

Biochemistry, 2001, 40, 10655–10663.148 L. E. Quadri, J. Sello, T. A. Keating, P. H.Weinreb and C. T.Walsh,

Chem. Biol., 1998, 5, 631–645.149 C. Pelludat, A. Rakin, C. A. Jacobi, S. Schubert and J. Heesemann,

J. Bacteriol., 1998, 180, 538–546.150 E. E. Wyckoff, J. A. Stoebner, K. E. Reed and S. M. Payne,

J. Bacteriol., 1997, 179, 7055–7062.151 N. A. Magarvey, M. Ehling-Schulz and C. T. Walsh, J. Am. Chem.

Soc., 2006, 128, 10698–10699.152 R. Pieper, A. Haese, W. Schroder and R. Zocher, Eur. J. Biochem.,

1995, 230, 119–126.153 S. C. Feifel, T. Schmiederer, T. Hornbogen, H. Berg,

R. D. Sussmuth and R. Zocher, ChemBioChem, 2007, 8, 1767–1770.

This journal is ª The Royal Society of Chemistry 2008

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.rsc

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i:10.

1039

/B80

1747

F

View Article Online

154 M. Krause, A. Lindemann, M. Glinski, T. Hornbogen, G. Bonse,P. Jeschke, G. Thielking, W. Gau, H. Kleinkauf and R. Zocher,J. Antibiot. (Tokyo), 2001, 54, 797–804.

155 H. Peeters, R. Zocher and H. Kleinkauf, J. Antibiot. (Tokyo), 1988,41, 352–359.

156 Y. Q. Cheng, ChemBioChem, 2006, 7, 471–477.157 M. Ehling-Schulz, M. Fricker, H. Grallert, P. Rieck, M.Wagner and

S. Scherer, BMC Microbiol., 2006, 6, 20.158 K. Wu, L. Chung, W. P. Revill, L. Katz and C. D. Reeves, Gene,

2000, 251, 81–90.159 L. Du, C. Sanchez, M. Chen, D. J. Edwards and B. Shen, Chem.

Biol., 2000, 7, 623–642.160 B. Silakowski, G. Nordsiek, B. Kunze, H. Blocker and R. Muller,

Chem. Biol., 2001, 8, 59–69.161 B. Julien, S. Shah, R. Ziermann, R. Goldman, L. Katz and

C. Khosla, Gene, 2000, 249, 153–160.162 C. T. Walsh, S. E. O’Connor and T. L. Schneider, J. Ind. Microbiol.

Biotechnol., 2003, 30, 448–455.163 W. L. Kelly, N. J. Hillson and C. T. Walsh, Biochemistry, 2005, 44,

13385–13393.164 S. E. O’Connor, H. Chen and C. T. Walsh, Biochemistry, 2002, 41,

5685–5694.165 T. L. Schneider, C. T.Walsh and S. E. O’Connor, J. Am. Chem. Soc.,

2002, 124, 11272–11273.166 Z. Chang, P. Flatt, W. H. Gerwick, V. A. Nguyen, C. L. Willis and

D. H. Sherman, Gene, 2002, 296, 235–247.167 T. L. Schneider, B. Shen and C. T. Walsh, Biochemistry, 2003, 42,

9722–9730.168 T. T. Sakai, J. M. Riordan and J. D. Glickson, Biochemistry, 1982,

21, 805–816.169 K. Hoffmann, E. Schneider-Scherzer, H. Kleinkauf and R. Zocher,

J. Biol. Chem., 1994, 269, 12710–12714.170 T. Stachelhaus and C. T. Walsh, Biochemistry, 2000, 39, 5775–5787.171 H. M. Patel, J. Tao and C. T. Walsh, Biochemistry, 2003, 42, 10514–

10527.172 H. D. Mootz and M. A. Marahiel, J. Bacteriol., 1997, 179, 6843–

6850.173 L. Luo, R. M. Kohli, M. Onishi, U. Linne, M. A. Marahiel and

C. T. Walsh, Biochemistry, 2002, 41, 9184–9196.174 S. L. Clugston, S. A. Sieber, M. A. Marahiel and C. T. Walsh,

Biochemistry, 2003, 42, 12095–12104.175 C. J. Balibar, F. H. Vaillancourt and C. T. Walsh, Chem. Biol., 2005,

12, 1189–1200.176 U. Linne, S. Doekel and M. A. Marahiel, Biochemistry, 2001, 40,

15824–15834.177 T. Hornbogen, S. P. Riechers, B. Prinz, J. Schultchen, C. Lang,

S. Schmidt, C. Mugge, S. Turkanovic, R. D. Sussmuth,E. Tauberger and R. Zocher, ChemBioChem, 2007, 8, 1048–1054.

178 A. M. Hill, Nat. Prod. Rep., 2006, 23, 256–320.179 D. A. Miller, C. T. Walsh and L. Luo, J. Am. Chem. Soc., 2001, 123,

8434–8435.180 D. P. K. O’Brien, P.N., S. W. O’Brien, T. Staroske, J. B. Spencer,

D. H. Williams, T. I. Richardson, D. A. Evans and A. Hopkinson,Chem. Commun., 2000, 103–104.

181 J. A. Salas and C. Mendez, J. Mol. Microbiol. Biotechnol., 2005, 9,77–85.

182 E. P. Patallo, G. Blanco, C. Fischer, A. F. Brana, J. Rohr,C. Mendez and J. A. Salas, J. Biol. Chem., 2001, 276, 18765–18774.

183 I. Muller and R. Muller, FEBS J., 2006, 273, 3768–3778.184 A. K. El-Sayed, J. Hothersall, S. M. Cooper, E. Stephens,

T. J. Simpson and C. M. Thomas, Chem. Biol., 2003, 10, 419–430.

185 D. J. Edwards, B. L. Marquez, L. M. Nogle, K. McPhail,D. E. Goeger, M. A. Roberts and W. H. Gerwick, Chem. Biol.,2004, 11, 817–833.

186 J. Moldenhauer, X. H. Chen, R. Borriss and J. Piel, Angew. Chem.,Int. Ed., 2007, 46, 8195–8197.

187 Y. Paitan, E. Orr, E. Z. Ron and E. Rosenberg,Microbiology, 1999,145, 3059–3067.

188 C. T. Calderone, D. F. Iwig, P. C. Dorrestein, N. L. Kelleher andC. T. Walsh, Chem. Biol., 2007, 14, 835–846.

189 C. T. Calderone, W. E. Kowtoniuk, N. L. Kelleher, C. T. Walsh andP. C. Dorrestein, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 8977–8982.

This journal is ª The Royal Society of Chemistry 2008

190 Z. Chang, N. Sitachitta, J. V. Rossi, M. A. Roberts, P. M. Flatt,J. Jia, D. H. Sherman and W. H. Gerwick, J. Nat. Prod., 2004, 67,1356–1367.

191 C. T. Walsh, S. Garneau-Tsodikova and A. R. Howard-Jones, Nat.Prod. Rep., 2006, 23, 517–531.

192 S. Pelzer, R. Sussmuth, D. Heckmann, J. Recktenwald, P. Huber,G. Jung and W. Wohlleben, Antimicrob. Agents Chemother., 1999,43, 1565–1573.

193 S. M. Li and L. Heide, Planta Med., 2006, 72, 1093–1099.194 J. Pootoolal, M. G. Thomas, C. G. Marshall, J. M. Neu,

B. K. Hubbard, C. T. Walsh and G. D. Wright, Proc. Natl. Acad.Sci. U. S. A., 2002, 99, 8962–8967.

195 M. Sosio, H. Kloosterman, A. Bianchi, P. de Vreugd, L. Dijkhuizenand S. Donadio, Microbiology, 2004, 150, 95–102.

196 T. L. Li, F. Huang, S. F. Haydock, T. Mironenko, P. F. Leadlay andJ. B. Spencer, Chem. Biol., 2004, 11, 107–119.

197 D. Bischoff, S. Pelzer, B. Bister, G. J. Nicholson, S. Stockert,M. Schirle, W. Wohlleben, G. Jung and R. D. Sussmuth, Angew.Chem., Int. Ed., 2001, 40, 4688–4691.

198 B. Hadatsch, D. Butz, T. Schmiederer, J. Steudle, W. Wohlleben,R. Sussmuth and E. Stegmann, Chem. Biol., 2007, 14, 1078–1089.

199 K. Zerbe, K. Woithe, D. B. Li, F. Vitali, L. Bigler andJ. A. Robinson, Angew. Chem., Int. Ed., 2004, 43, 6709–6713.

200 D. Bischoff, B. Bister, M. Bertazzo, V. Pfeifer, E. Stegmann,G. J. Nicholson, S. Keller, S. Pelzer, W. Wohlleben andR. D. Sussmuth, ChemBioChem, 2005, 6, 267–272.

201 K. Woithe, N. Geib, K. Zerbe, D. B. Li, M. Heck, S. Fournier-Rousset, O. Meyer, F. Vitali, N. Matoba, K. Abou-Hadeed andJ. A. Robinson, J. Am. Chem. Soc., 2007, 129, 6887–6895.

202 N. Geib, K. Woithe, K. Zerbe, D. B. Li and J. A. Robinson, Bioorg.Med. Chem. Lett., 2007.

203 D. A. Miller, L. Luo, N. Hillson, T. A. Keating and C. T. Walsh,Chem. Biol., 2002, 9, 333–344.

204 M. F. Byford, J. E. Baldwin, C. Y. Shiau and C. J. Schofield, Chem.Rev., 1997, 97, 2631–2650.

205 J. F. Martin, J. Antibiot. (Tokyo), 2000, 53, 1008–1021.206 C. Khosla, Y. Tang, A. Y. Chen, N. A. Schnarr and D. E. Cane,

Annu. Rev. Biochem., 2007, 76, 195–221.207 J. Grunewald, S. A. Sieber and M. A. Marahiel, Biochemistry, 2004,

43, 2915–2925.208 C. A. Shaw-Reid, N. L. Kelleher, H. C. Losey, A. M. Gehring,

C. Berg and C. T. Walsh, Chem. Biol., 1999, 6, 385–400.209 F. Kopp andM. A.Marahiel,Curr. Opin. Biotechnol., 2007, 18, 513–

520.210 R. M. Kohli, M. D. Burke, J. Tao and C. T. Walsh, J. Am. Chem.

Soc., 2003, 125, 7160–7161.211 R. M. Kohli, C. T. Walsh and M. D. Burkart, Nature, 2002, 418,

658–661.212 F. Kopp, J. Grunewald, C. Mahlert and M. A. Marahiel,

Biochemistry, 2006, 45, 10474–10481.213 C. J. Balibar, A. R. Howard-Jones and C. T. Walsh, Nat. Chem.

Biol., 2007, 3, 584–592.214 Y. Kallberg, U. Oppermann, H. Jornvall and B. Persson, Eur.

J. Biochem., 2002, 269, 4409–4417.215 D. E. Ehmann, A. M. Gehring and C. T. Walsh, Biochemistry, 1999,

38, 6171–6177.216 L. Li, W. Deng, J. Song, W. Ding, Q. F. Zhao, C. Peng, W. W. Song,

G. L. Tang and W. Liu, J. Bacteriol., 2008, 190, 251–263.217 F. Kopp, C. Mahlert, J. Grunewald and M. A. Marahiel, J. Am.

Chem. Soc., 2006, 128, 16478–16479.218 J. A. Read and C. T. Walsh, J. Am. Chem. Soc., 2007, 129, 15762–

15763.219 N. Gaitatzis, B. Kunze and R. Muller, Proc. Natl. Acad. Sci.

U. S. A., 2001, 98, 11136–11141.220 C. J. Balibar and C. T. Walsh, Biochemistry, 2006, 45, 15029–15038.221 T. Haarmann, I. Ortel, P. Tudzynski and U. Keller, ChemBioChem,

2006, 7, 645–652.222 D. M. Gardiner, P. Waring and B. J. Howlett, Microbiology, 2005,

151, 1021–1032.223 B. Frank, S. C. Wenzel, H. B. Bode, M. Scharfe, H. Blocker and

R. Muller, J. Mol. Biol., 2007, 374, 24–38.224 C. T. Walsh, Science, 2004, 303, 1805–1810.225 C. T. Walsh, Acc. Chem. Res., 2008, 41, 4–10.226 C. Walsh, C. L. Freel Meyers and H. C. Losey, J. Med. Chem., 2003,

46, 3425–3436.

Nat. Prod. Rep., 2008, 25, 757–793 | 791

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.rsc

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i:10.

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View Article Online

227 X. M. He and H. W. Liu, Annu. Rev. Biochem., 2002, 71, 701–754.228 S. Blanchard and J. S. Thorson, Curr. Opin. Chem. Biol., 2006, 10,

263–271.229 H. Chen, M. G. Thomas, B. K. Hubbard, H. C. Losey, C. T. Walsh

and M. D. Burkart, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 11942–11947.

230 C. Mendez and J. A. Salas, Ernst Schering Research Foundationworkshop, 2005, pp. 127–146.

231 H. Takahashi, Y. N. Liu, H. Chen and H. W. Liu, J. Am. Chem.Soc., 2005, 127, 9340–9341.

232 D. Kahne, C. Leimkuhler, W. Lu and C. Walsh, Chem. Rev., 2005,105, 425–448.

233 P. J. Solenberg, P. Matsushima, D. R. Stack, S. C. Wilkie,R. C. Thompson and R. H. Baltz, Chem. Biol., 1997, 4, 195–202.

234 H. C. Losey, M. W. Peczuh, Z. Chen, U. S. Eggert, S. D. Dong,I. Pelczer, D. Kahne and C. T. Walsh, Biochemistry, 2001, 40,4745–4755.

235 W. Lu, M. Oberthur, C. Leimkuhler, J. Tao, D. Kahne andC. T. Walsh, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 4390–4395.

236 A. M. Mulichak, W. Lu, H. C. Losey, C. T. Walsh andR. M. Garavito, Biochemistry, 2004, 43, 5170–5180.

237 A. M. Mulichak, H. C. Losey, W. Lu, Z. Wawrzak, C. T. Walsh andR. M. Garavito, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 9238–9243.

238 A. M. Mulichak, H. C. Losey, C. T. Walsh and R. M. Garavito,Structure, 2001, 9, 547–557.

239 M. Oberthur, C. Leimkuhler, R. G. Kruger, W. Lu, C. T. Walsh andD. Kahne, J. Am. Chem. Soc., 2005, 127, 10747–10752.

240 H. C. Losey, J. Jiang, J. B. Biggins, M. Oberthur, X. Y. Ye,S. D. Dong, D. Kahne, J. S. Thorson and C. T. Walsh, Chem.Biol., 2002, 9, 1305–1314.

241 R. H. Baltz, Nat. Biotechnol., 2006, 24, 1533–1540.242 A. R. Howard-Jones, R. G. Kruger, W. Lu, J. Tao, C. Leimkuhler,

D. Kahne and C. T. Walsh, J. Am. Chem. Soc., 2007, 129, 10082–10083.

243 S. Keller, F. Pojer, L. Heide and D. M. Lawson, Acta Crystallogr.,Sect. F: Struct. Biol. Cryst. Commun., 2006, 62, 1153–1155.

244 F. Pojer, E.Wemakor, B. Kammerer, H. Chen, C. T.Walsh, S.M. Liand L. Heide, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 2316–2321.

245 D. J. Edwards and W. H. Gerwick, J. Am. Chem. Soc., 2004, 126,11432–11433.

246 G. Minotti, P. Menna, E. Salvatorelli, G. Cairo and L. Gianni,Pharmacol. Rev., 2004, 56, 185–229.

247 D. Yang and A. H. Wang, Biochemistry, 1994, 33, 6595–6604.248 G. A. Leonard, T. W. Hambley, K. McAuley-Hecht, T. Brown and

W. N. Hunter, Acta Crystallogr., Sect. D, 1993, 49, 458–467.249 C. Leimkuhler, M. Fridman, T. Lupoli, S. Walker, C. T. Walsh and

D. Kahne, J. Am. Chem. Soc., 2007, 129, 10546–10550.250 W. Lu, C. Leimkuhler, G. J. Gatto, Jr., R. G. Kruger, M. Oberthur,

D. Kahne and C. T. Walsh, Chem. Biol., 2005, 12, 527–534.251 W. Lu, C. Leimkuhler, M. Oberthur, D. Kahne and C. T. Walsh,

Biochemistry, 2004, 43, 4548–4558.252 A. Luzhetskyy, M. Fedoryshyn, C. Durr, T. Taguchi, V. Novikov

and A. Bechthold, Chem. Biol., 2005, 12, 725–729.253 Y. Yuan, H. S. Chung, C. Leimkuhler, C. T. Walsh, D. Kahne and

S. Walker, J. Am. Chem. Soc., 2005, 127, 14128–14129.254 H. Y. Lee, H. S. Chung, C. Hang, C. Khosla, C. T. Walsh, D. Kahne

and S. Walker, J. Am. Chem. Soc., 2004, 126, 9924–9925.255 M. Mittler, A. Bechthold and G. E. Schulz, J. Mol. Biol., 2007, 372,

67–76.256 M. A. Fischbach, H. Lin, D. R. Liu and C. T. Walsh, Proc. Natl.

Acad. Sci. U. S. A., 2005, 102, 571–576.257 M. A. Fischbach, H. Lin, D. R. Liu and C. T. Walsh, Nat. Chem.

Biol., 2006, 2, 132–138.258 W. J. van Berkel, N. M. Kamerbeek and M. W. Fraaije,

J. Biotechnol., 2006, 124, 670–689.259 T. D. Bugg and S. Ramaswamy, Curr. Opin. Chem. Biol., 2008.260 V. Joosten and W. J. van Berkel, Curr. Opin. Chem. Biol., 2007, 11,

195–202.261 M. J. Cryle, J. E. Stok and J. J. De Voss, Aust. J. Chem., 2003, 56,

749–762.262 E. N. Fielding, P. F. Widboom and S. D. Bruner, Biochemistry,

2007, 46, 13994–14000.263 M. Gibson, M. Nur-e-alam, F. Lipata, M. A. Oliveira and J. Rohr,

J. Am. Chem. Soc., 2005, 127, 17594–17595.

792 | Nat. Prod. Rep., 2008, 25, 757–793

264 D. Rodriguez, L. M. Quiros, A. F. Brana and J. A. Salas,J. Bacteriol., 2003, 185, 3962–3965.

265 H. J. Kim, R. Pongdee, Q. Wu, L. Hong and H. W. Liu, J. Am.Chem. Soc., 2007, 129, 14582–14584.

266 J. F. Andersen and C. R. Hutchinson, J. Bacteriol., 1992, 174, 725–735.

267 R. H. Lambalot, D. E. Cane, J. J. Aparicio and L. Katz,Biochemistry, 1995, 34, 1858–1866.

268 M. V. Mendes, N. Anton, J. F. Martin and J. F. Aparicio, Biochem.J., 2005, 386, 57–62.

269 H. Ogura, C. R. Nishida, U. R. Hoch, R. Perera, J. H. Dawson andP. R. Ortiz de Montellano, Biochemistry, 2004, 43, 14712–14721.

270 P. L. Roach, I. J. Clifton, C. M. Hensgens, N. Shibata,C. J. Schofield, J. Hajdu and J. E. Baldwin, Nature, 1997, 387,827–830.

271 K. Valegard, A. C. van Scheltinga, M. D. Lloyd, T. Hara,S. Ramaswamy, A. Perrakis, A. Thompson, H. J. Lee,J. E. Baldwin, C. J. Schofield, J. Hajdu and I. Andersson, Nature,1998, 394, 805–809.

272 N. I. Burzlaff, P. J. Rutledge, I. J. Clifton, C. M. Hensgens,M. Pickford, R. M. Adlington, P. L. Roach and J. E. Baldwin,Nature, 1999, 401, 721–724.

273 D. Destoumieux-Garzon, X. Thomas, M. Santamaria, C. Goulard,M. Barthelemy, B. Boscher, Y. Bessin, G. Molle, A. M. Pons,L. Letellier, J. Peduzzi and S. Rebuffat, Mol. Microbiol., 2003, 49,1031–1041.

274 E. M. Nolan, M. A. Fischbach, A. Koglin and C. T. Walsh, J. Am.Chem. Soc., 2007, 129, 14336–14347.

275 T. A. Keating, Z. Suo, D. E. Ehmann and C. T.Walsh, Biochemistry,2000, 39, 2297–2306.

276 A. M. Gehring, I. Mori, R. D. Perry and C. T. Walsh, Biochemistry,1998, 37, 11637–11650.

277 B. Shen, Top. Curr. Chem., 2000, 209, 1–51.278 C. W. Carreras and C. Khosla, Biochemistry, 1998, 37, 2084–2088.279 C. W. P. Carreras, R. and C. Khosla, J. Am. Chem. Soc., 1996, 118,

5158–5159.280 A. T. Hadfield, C. Limpkin, W. Teartasin, T. J. Simpson, J. Crosby

and M. P. Crump, Structure, 2004, 12, 1865–1875.281 R. J. Zawada and C. Khosla, Chem. Biol., 1999, 6, 607–615.282 Y. Tang, S. C. Tsai and C. Khosla, J. Am. Chem. Soc., 2003, 125,

12708–12709.283 A. T. Keatinge-Clay, D. A. Maltby, K. F. Medzihradszky,

C. Khosla and R. M. Stroud, Nat. Struct. Mol. Biol., 2004, 11,888–893.

284 B. J. Rawlings, Nat. Prod. Rep., 2001, 18, 190–227.285 B. Shen and C. R. Hutchinson, Biochemistry, 1993, 32, 11149–11154.286 A. Sultana, P. Kallio, A. Jansson, J. S. Wang, J. Niemi, P. Mantsala

and G. Schneider, EMBO J., 2004, 23, 1911–1921.287 P. Kallio, A. Sultana, J. Niemi, P. Mantsala and G. Schneider,

J. Mol. Biol., 2006, 357, 210–220.288 S. G. Kendrew, K. Katayama, E. Deutsch, K. Madduri and

C. R. Hutchinson, Biochemistry, 1999, 38, 4794–4799.289 W. Zhang, K. Watanabe, C. C. Wang and Y. Tang, J. Biol. Chem.,

2007, 282, 25717–25725.290 B. Shen and C. R. Hutchinson, Science, 1993, 262, 1535–1540.291 W. Bao, E. Wendt-Pienkowski and C. R. Hutchinson, Biochemistry,

1998, 37, 8132–8138.292 B. Shen and C. R. Hutchinson, Proc. Natl. Acad. Sci. U. S. A., 1996,

93, 6600–6604.293 T. B. Thompson, K. Katayama, K. Watanabe, C. R. Hutchinson

and I. Rayment, J. Biol. Chem., 2004, 279, 37956–37963.294 B. Shen and C. R. Hutchinson, Biochemistry, 1993, 32, 6656–6663.295 B. Shen and C. R. Hutchinson, J. Biol. Chem., 1994, 269, 30726–

30733.296 Y. Tang, T. S. Lee, S. Kobayashi and C. Khosla, Biochemistry, 2003,

42, 6588–6595.297 Y. Tang, A. T. Koppisch and C. Khosla, Biochemistry, 2004, 43,

9546–9555.298 Y. Tang, H. Y. Lee, Y. Tang, C. Y. Kim, I. Mathews and C. Khosla,

Biochemistry, 2006, 45, 14085–14093.299 T. S. Lee, C. Khosla and Y. Tang, J. Am. Chem. Soc., 2005, 127,

12254–12262.300 Y. Tang, T. S. Lee and C. Khosla, PLoS Biol., 2004, 2, E31.301 I. Fujii, A. Watanabe, U. Sankawa and Y. Ebizuka, Chem. Biol.,

2001, 8, 189–197.

This journal is ª The Royal Society of Chemistry 2008

Dow

nloa

ded

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13 1

6:08

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Pu

blis

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on 2

3 M

ay 2

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on h

ttp://

pubs

.rsc

.org

| do

i:10.

1039

/B80

1747

F

View Article Online

302 S. M. Ma, J. Zhan, X. Xie, K. Watanabe, Y. Tang and W. Zhang,J. Am. Chem. Soc., 2008, 130, 38–39.

303 Y. Katsuyama, M. Matsuzawa, N. Funa and S. Horinouchi, J. Biol.Chem., 2007, 282, 37702–37709.

304 I. Abe, S. Oguro, Y. Utsumi, Y. Sano and H. Noguchi, J. Am. Chem.Soc., 2005, 127, 12709–12716.

305 I. Abe, Y. Utsumi, S. Oguro, H. Morita, Y. Sano and H. Noguchi,J. Am. Chem. Soc., 2005, 127, 1362–1363.

306 J. L. Ferrer, J. M. Jez, M. E. Bowman, R. A. Dixon and J. P. Noel,Nat. Struct. Biol., 1999, 6, 775–784.

307 M. B. Austin, M. Izumikawa, M. E. Bowman, D. W. Udwary,J. L. Ferrer, B. S. Moore and J. P. Noel, J. Biol. Chem., 2004, 279,45162–45174.

308 J. Piel, C. Hertweck, P. R. Shipley, D. M. Hunt, M. S. Newman andB. S. Moore, Chem. Biol., 2000, 7, 943–955.

309 M. Izumikawa, Q. Cheng and B. S. Moore, J. Am. Chem. Soc., 2006,128, 1428–1429.

310 Q. Cheng, L. Xiang, M. Izumikawa, D. Meluzzi and B. S. Moore,Nat. Chem. Biol., 2007, 3, 557–558.

311 L. Xiang, J. A. Kalaitzis and B. S. Moore, Proc. Natl. Acad. Sci.U. S. A., 2004, 101, 15609–15614.

312 A. Maxwell and D. M. Lawson, Curr. Top. Med. Chem., 2003, 3,283–303.

313 S. C. Kampranis, N. A. Gormley, R. Tranter, G. Orphanides andA. Maxwell, Biochemistry, 1999, 38, 1967–1976.

314 K. Drlica and X. Zhao, Microbiol. Mol. Biol. Rev., 1997, 61, 377–392.

315 F. Pojer, S. M. Li and L. Heide,Microbiology, 2002, 148, 3901–3911.316 M. Steffensky, A. Muhlenweg, Z. X. Wang, S. M. Li and L. Heide,

Antimicrob. Agents Chemother., 2000, 44, 1214–1222.317 Z. X. Wang, S. M. Li and L. Heide, Antimicrob. Agents Chemother.,

2000, 44, 3040–3048.318 H. Chen and C. T. Walsh, Chem. Biol., 2001, 8, 301–312.319 M. Pacholec, N. J. Hillson and C. T. Walsh, Biochemistry, 2005, 44,

12819–12826.320 F. Pojer, R. Kahlich, B. Kammerer, S. M. Li and L. Heide, J. Biol.

Chem., 2003, 278, 30661–30668.321 M. Steffensky, S. M. Li and L. Heide, J. Biol. Chem., 2000, 275,

21754–21760.322 M. Pacholec, J. Tao and C. T.Walsh, Biochemistry, 2005, 44, 14969–

14976.323 C. L. Freel Meyers, M. Oberthur, J. W. Anderson, D. Kahne and

C. T. Walsh, Biochemistry, 2003, 42, 4179–4189.

This journal is ª The Royal Society of Chemistry 2008

324 A. Freitag, S. M. Li and L. Heide, Microbiology (Reading, U. K.),2006, 152, 2433–2442.

325 T. T. Thuy, H. C. Lee, C. G. Kim, L. Heide and J. K. Sohng,Arch. Biochem. Biophys., 2005, 436, 161–167.

326 C. E. Stevenson, C. L. Freel Meyers, C. T.Walsh andD.M. Lawson,Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun., 2007, 63,236–238.

327 C. L. Freel Meyers, M. Oberthur, H. Xu, L. Heide, D. Kahne andC. T. Walsh, Angew. Chem., Int. Ed., 2004, 43, 67–70.

328 M. Gellert, M. H. O’Dea, T. Itoh and J. Tomizawa, Proc. Natl.Acad. Sci. U. S. A., 1976, 73, 4474–4478.

329 A. S. Eustaquio, B. Gust, T. Luft, S. M. Li, K. F. Chater andL. Heide, Chem. Biol., 2003, 10, 279–288.

330 C. J. Balibar, S. Garneau-Tsodikova and C. T. Walsh, Chem. Biol.,2007, 14, 679–690.

331 E. Schmutz, M. Steffensky, J. Schmidt, A. Porzel, S. M. Li andL. Heide, Eur. J. Biochem., 2003, 270, 4413–4419.

332 C. L. Freel Meyers, M. Oberthur, L. Heide, D. Kahne andC. T. Walsh, Biochemistry, 2004, 43, 15022–15036.

333 C. Anderle, S. Hennig, B. Kammerer, S. M. Li, L. Wessjohann,B. Gust and L. Heide, Chem. Biol., 2007, 14, 955–967.

334 C. Sanchez, C. Mendez and J. A. Salas, Nat. Prod. Rep., 2006, 23,1007–1045.

335 C. Sanchez, I. A. Butovich, A. F. Brana, J. Rohr, C. Mendez andJ. A. Salas, Chem. Biol., 2002, 9, 519–531.

336 A. R. Howard-Jones and C. T. Walsh, J. Am. Chem. Soc., 2006, 128,12289–12298.

337 A. R. Howard-Jones and C. T. Walsh, Biochemistry, 2005, 44,15652–15663.

338 A. R. Howard-Jones and C. T. Walsh, J. Am. Chem. Soc., 2007, 129,11016–11017.

339 R. V. Antonio and T. B. Creczynski-Pasa, Genet. Mol. Res., 2004, 3,85–91.

340 C. J. Balibar and C. T. Walsh, Biochemistry, 2006, 45, 15444–15457.

341 K. S. Ryan, C. J. Balibar, K. E. Turo, C. T. Walsh andC. L. Drennan, J. Biol. Chem., 2008, 283, 6467–6475.

342 S. Bouhired, M. Weber, A. Kempf-Sontag, N. P. Keller andD. Hoffmeister, Fungal Genet. Biol., 2007, 44, 1134–1145.

343 J. W. Bok, D. Hoffmeister, L. A. Maggio-Hall, R. Murillo,J. D. Glasner and N. P. Keller, Chem. Biol., 2006, 13, 31–37.

344 P. Schneider, M. Weber and D. Hoffmeister, Fungal Genet. Biol.,2008, 45, 302–309.

Nat. Prod. Rep., 2008, 25, 757–793 | 793