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PART II
BIOSYNTHESIS OF NATURALPRODUCTS
4NONRIBOSOMAL PEPTIDES
Georg Schoenafinger and Mohamed A. MarahielPhilipps-University of Marburg, FB Chemie/Biochemie, Marburg, Germany
Many microorganisms have evolved an unusual way of producing secondary peptidemetabolites. Large multidomain enzymatic machineries, the so-called nonribosomalpeptide synthetases (NRPSs), are responsible for the production of this structurallydiverse class of peptides with various functions, such as cytostatic, immunosuppres-sive, antibacterial, or antitumor properties. These secondary metabolites differ frompeptides of ribosomal origin in several ways. Their length is limited to a mere 20 build-ing blocks, roughly, and mostly a circular or branched cyclic connectivity is found.Furthermore, aside from the proteinogenic amino acids, a larger variety of chemicalgroups is found in these bioactive compounds: d-configurated amino acids, fatty acids,methylated, oxidized, halogenated, and glycosylated building blocks. These functionaland structural features are known to be important for bioactivity, and often naturaldefense mechanisms are thus evaded. In this chapter, we describe the enzymaticmachineries of NRPSs, the chemical reactions catalyzed by their subunits, and thepotential of redesigning or using these machineries to give rise to new nonribosomalpeptide antibiotics.
Nonribosomal peptide synthetases (NRPSs) compose a unique class of multido-main enzymes capable of producing peptides (1–4). In contrast to the ribosomalmachinery where the mRNA template is translated, the order of catalyticallyactive entities within these synthetases intrinsically determines the sequence ofbuilding blocks in the peptide product (Fig. 4.1). As a consequence, generallyspeaking, each NRPS can only produce one defined peptide product. This ischemically implemented by the fact that all substrates and reaction intermediatesare spatially fixed to the synthetase by covalent linkage—thereby eliminating
Natural Products in Chemical Biology, First Edition. Edited by Natanya Civjan.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.
111
112 NONRIBOSOMAL PEPTIDES
(a)
(b) Gene NRPS: ModulesNonribosomal
BiosynthesisPeptide
Gene mRNA: CodonsRibosomal
BiosynthesisProtein
Figure 4.1 Comparison of ribosomal and nonribosomal peptide synthesis. (a) In theribosomal information pathway, the sequence of codons in the mRNA determines thesequence of amino acids in the protein, whereas (b) the sequence of modules in the nonri-bosomal peptide synthetases intrinsically determines the primary sequence of the peptideproduct.
side product formation caused by diffusion. The catalytic entities responsiblefor the incorporation of a distinct building block into the product are calledmodules. Each module carries out several chemical steps required for the syn-thesis of nonribosomal peptides: Recognition of the building block, activation,covalent attachment, translocation, and condensation. In several cases, additionalmodifications are found, such as epimerization [Tyrocidine, (5)], cyclization[Gramicidin S, (6)], oxidation [Epothilone, (7)], reduction [linear gramicidin(8)], methylation [Cyclosporin, (9)], and formylation [linear gramicidin, (10)].In vitro studies have shown that each module can be subdivided into catalyticallyactive domains to which the different reactions mentioned above can be assigned(1–4). Thus, the so-called adenylation (A), peptidyl carrier protein (PCP), andthe condensation (C) domains were identified as being essential to all NRPSs. Inaddition, a second group of so-called optional domains exists: the epimerization(E), cyclization (TE or Cy), oxidation (Ox), reduction (R), N-methylation (Mt),and formylation (F) domains. Aside from NRPSs themselves, several enzymes areknown to act on some peptides while they are still bound to the synthetase or evenafter their release. These modifying enzymes can glycosylate [Vancomycin, (11)],halogenate [Vancomycin, (11)], or reduce [linear gramicidin, (8)] the peptidesin trans . With several hundred different building blocks found in nonribosomalpeptide products, it becomes evident that their diversity is vast (Fig. 4.2). Thischapter addresses the biologic background of these secondary metabolites, theenzymatic machineries of NRPSs, and the chemical reactions catalyzed by theirdomains. Furthermore, the possibility of manipulating NRPSs and using certaindomains to produce novel compounds is discussed.
4.1 BIOLOGIC BACKGROUND
Nonribosomal peptides are produced by a large number of bacteria, fungi, andlower eucaryotes. For most of these compounds, their biologic role is unknown.One might suspect that these secreted molecules are used for unknown formsof communication or simply to critically increase the chance of survival forthe producing cell in its habitat, because the metabolic cost of their produc-tion is enormous. However, the function of some nonribosomal compounds has
BIOLOGIC BACKGROUND 113
H2N
H2N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2NH2
ONH
HN
NH
HN
NH
O
O
O
OHO
O
OH
OCl
HN
O
O
OCl
HN
O
HO
HOOH
HO
O OHOH
OHO
O
OH
OH
NOH
O
NH
O
NOH
NH
O
NHO
O H
OH
H
O OH
HN
NH
HN
NH
O
O
O
O
NH
O
ONH
O
ONH
O O
NH
OHO
NH
O
HNO
HN
OHN
O
OH
O
HN
O
OHO
NH
O
OHO
HO
OH O
HN
O
HN
O O O
O
O
O
NH
NHO
NH
O
HO
OH
O
HN O
OH
OH
HN
O
N
ONH
O
HNO
HN
OOHN
O
O
NH
O
HN
O
NH
O
NH
O
HNNH
HN
NH
HN
NH
HN
NH
HN
NH
O HO
O
O
O
O
O
O
O
O
HN
HN
NH
HN
NH
O
O
O
O
HN HN
HN
NH
O
O
HN
OH
HN
O
N
ONH
O
HNO
HN
OHNO
N
O
HN
O
NH
O
NH
O
O O
NH
O
HN
O
HN
O NHO
NH
ONH
O
HNO
O
HO
O
OH
H2NS
NO
NH
O
HN
OHO
O
NH
O
HN
HN
O
NH
O
O
NH
OH
O
ONH
NH
NO
HNO
HN
OHN
O
NH3
NH2
Bacitracin Surfactin
TyrocidineVancomycin
Coelichelin
Daptomycin
Gramicidin SBacillibactin
linear Gramicidin
Figure 4.2 A selection of nonribosomal peptides. Chemical and structural features thatcontribute to the vast diversity of this class of metabolites are highlighted: Heterocycle(bacitracin), lactone (surfactin, daptomycin), ornithine and lactam (Tyrocidine), sugar,chlorinated aromats, C–C crosslink (Vancomycin), N-formyl groups (Coelichelin andlinear gramicidin), fatty acid (daptomycin), dihydroxybenzoate and trimeric organization(bacillibactin), dimeric organization (gramicidin S), and ethanolamine (linear gramicidin).
114 NONRIBOSOMAL PEPTIDES
been identified: The well-studied penicillin produced by Penicillium notatum ,for instance, is a weapon against nutrient competitors, and the siderophore bacil-libactin helps its producer, Bacillus subtilis , acquire iron and thereby preventiron starvation. For us, natural products produced by microorganisms attract con-siderable attention because their observed bioactivities range from antibiotic toimmunosuppressive and from cytostatic to antitumor. Not only have these sec-ondary metabolites been optimized for their dedicated function over millions ofyears of evolution, but they also represent promising scaffolds for the develop-ment of novel drugs with improved or altered activities.
4.2 CATALYTIC DOMAINS OF NONRIBOSOMAL PEPTIDESYNTHETASES
The catalytically active entities that NRPSs are composed of can be classified asbeing essential to all NRPSs or being responsible for special modifications. Onlywhen a set of domains correctly acts in appropriate order, the designated productcan be synthesized (3) (Fig. 4.3). The function, chemistry, and interactions ofthese domains are discussed in the following section (Fig. 4.4).
4.2.1 Essential Domains of NRPSs
Before any peptide formation can occur, the amino acids or, generally speaking,the building blocks to be condensed need to be recognized and activated (12). Theadenylation (A) domains are capable of specifically binding one such buildingblock. Once bound, the same enzyme catalyses the formation of the correspondingacyl-adenylate-monophosphate by consumption of ATP (Fig. 4.4). The resultingmixed anhydride is the reactive species that can be processed additionally bythe NRPS machinery. Sequence alignments, mutational studies, and structural
H2N
NH2
H3N
S
NO
NH O
HN
OHO
O
NH O
HN
HN
ONH
O
O
NH
OH
O
ONH
NH
NO
HNO
HN
OHN
O
Bacitracin
(a) (b)
A PCP Cy A PCP C A PCP C A PCP E C A PCP
BacA (598 kDa)module 1 module 2 module 3 module 4 module 5
C A PCP C A PCP E
BacB (297 kDa)module 6 module 7
C A PCP C A PCP E C A PCP C A PCP E C A PCP TE
BacC (723 kDa)module 8
module 9 module 10module 11
module 12
BacA
BacB
BacC
12
34
56
7 8
9
10
1112
Figure 4.3 The nonribosomal machinery (a) needed to produce bacitracin (b) consists ofthree separate synthetases: BacA, BacB, and BacC, composing a total of 12 modules and40 catalytic domains. These synthetases specifically interact with each other to producethe nonribosomal peptide bacitracin.
CATALYTIC DOMAINS OF NONRIBOSOMAL PEPTIDE SYNTHETASES 115
A PCP C A PCP C A PCP
OH OH OH
C
A PCP C A PCP C A PCP
SH SH SH
C
SO
SO
SO
O
A PCP C A PCP C A PCP C
SO
O
A PCP C A PCP C A PCP
SH SO
NHO
C
SO
NH
OH2N
H2N
H2N
H2N
O
HNO
NH2
NH2NH2NH2NH2
SHSH
A PCP C A PCP C A PCP C
(a)
CoA[Sfp]
3',5'-ADP
ATP,cognate substrates
AMP, PPi
[A domain]
(b)
[first C domain]
[second C domain]
(c) (d)
Figure 4.4 Single reactions in NRPSs and their timing. (a) After ribosomal syn-thesis of the apo-enzymes, the PCP domains are postsynthetically modified with 4′-Phosphopantetheine cofactors by a 4′Ppan transferase, e.g., Sfp. (b) In a second step,the A domains bind their cognate substrates as well as ATP and form the correspondingacyl-adenylate intermediates. These are transferred onto the cofactor of the neighboringPCP domains. (c) The C domains catalyze the condensation of two building blocks. Thespecificities of C domains and the affinities of aminoacyl-/peptidyl-holo-PCP domainsensure that no internal start reactions occur: (d) Only after the first condensation domainhas acted does the second C domain seem to process the intermediate. During synthesis,the growing product chain is continuously translocated toward the C-terminal end of theenzyme.
data have revealed that amino acid residues at certain positions in the enzymedetermine the specificity of an A domain (13). This result can be explained bythe thereby generated chemical and physical environment of the substrate bindingsite. Some A domains, however, are known to have a relaxed substrate specificity.In these cases, chemically or sterically similar amino acids also are recognized,processed analogously, and thus found at that very position in the product. Forexample, the A domain of the first module of the gramicidin synthetase LgrA(10) not only activates valine but also activates isoleucine, which is found in 5%of linear gramicidins extracted from producing strains.
When the first building block has been recognized and activated by the Adomain, the next essential domain comes into play: The peptidyl carrier protein(PCP) domain. Like the acyl carrier protein (ACP) in fatty acid biosynthesis,this PCP domain is responsible for keeping the reaction intermediates bound to
116 NONRIBOSOMAL PEPTIDES
the enzymatic machinery. Thus, a directed order of additional reaction steps canbe implemented by controlled translocation, and NRPSs are thus often describedas assembly line-like machineries. The PCP domain consists of 90 amino acidresidues, roughly, and is known to rearrange itself to at least three differenttertiary structures in aqueous solution, as is necessary for interaction with thesurrounding domains at certain stages of synthesis (14). Just like ACPs, the PCPdomains are also dependent on a posttranslational modification to function. Thismodification is the attachment of a 4′-phosphopantetheine cofactor to a con-served serine residue. The terminal thiol group of this cofactor is the nucleophilethat attacks the mixed anhydride (acyl-AMP) and therefore covalently binds theNRPS substrates via a thioester bond. After such an acylation, the PCP domaindirects the substrate toward the next processing domain. If we leave out anyoptional modifying domains at this point, this next domain would generally be acondensation (C) domain.
The C domain is needed for the condensation of two biosynthetic interme-diates during nonribosomal peptide assembly (15). The PCP-bound electrophilicdonor substrate is presented from the N-terminal side of the synthetase. On theother side, the nucleophilic acceptor substrate—bound analogously to the PCPdomain of the next module—reaches back to the active site of the C domainfrom the other direction (downstream). In the first condensation reaction of anNRPS, both of these substrates would typically be aminoacyl groups connectedto their PCP domains. Condensation is initiated by the nucleophilic attack ofthe α-amino group of the acceptor substrate onto the thioester group of thedonor substrate. The cofactor of the upstream PCP domain is released, and theresulting amide bond now belongs to the dipeptide, which remains bound tothe downstream PCP domain. Thus, a translocation of the condensation prod-uct toward the next module has occurred. All condensation reactions are strictlyunidirectional—always transporting the growing product chain toward the mod-ule closer to the C-terminus of the machinery. The elongated peptide then servesas the donor in a subsequent condensation step on the next module. Usually,there are as many condensation domains in an NRPS as there are peptide bondsin the linear peptide product. This general translocation model implies that thebiosynthesis is linear—altogether dependent on delicate, situationally changingaffinities that guarantee correct timing for each reaction and that prevents sidereactions (Fig. 4.4). Even though this model successfully puts the biosyntheticenzymes in relation with their products for most known NRPS systems, someexceptions are known: The structures of syringomycin (16) or coelichelin (17)cannot be sufficiently explained by merely deciphering the buildup of their NRPSswhen using this model. Obviously, other regulatory mechanisms and forms ofinter-domain communication are not yet fully understood.
When the last condensation reaction has occurred, the linear precursor needsto be released from the enzyme. For this important last step, several mecha-nisms are known: simple hydrolysis of the thioester (balhimycin, vancomycin),intramolecular cyclization leading to a lactam (tyrocidine, bacitracin) or a lac-tone (surfactin), or even reductive thioester cleavage (linear gramicidin). In some
CATALYTIC DOMAINS OF NONRIBOSOMAL PEPTIDE SYNTHETASES 117
cases, the linear precursor is dimerized (gramicidin S) or even trimerized (bacil-libactin, enterobactin) before cyclization (Fig. 4.2). Even though these reactionsare critical for the compound’s bioactivity, the catalytic domains responsiblefor the release are not found in all NRPS systems and will therefore be called“modifying” domains.
4.2.2 Modifying Domains of NRPSs
Apart from the essential domains in NRPSs, several so-called modifying domainsare not found in every NRPS system. Nevertheless, they are required for properprocessing of their designated substrate within their synthetase. Deletion or inacti-vation of these modifying domains usually results in the production of compoundswith bioactivities severely reduced or altogether abolished.
Most nonribosomal peptides have a cyclic connectivity. In these cases, aC-terminal, so-called thioesterase (TE) domain, is often found in the synthetase.These TE domains all share an invariant serine residue belonging to a catalytictriad (Asp-His-Ser), which is known to be acylated with the linear peptide beforecyclization (18). Once the substrate is translocated from the PCP domain ontothe TE domain, the regiospecific and stereospecific intramolecular attack of anucleophile onto the C-terminal carbonyl group of the substrate is directed bythe enzyme. This nucleophile can be the N-terminal α-amino group of the linearpeptide (tyrocidine, gramicidin S), a side-chain amino (bacitracin) or hydroxylgroup (surfactin). Since the ester bond between the substrate and the TE domainis cleaved by these cyclization reactions, the resulting lactams or lactones arereleased from the synthetic machinery by this step. In a few cases, the mod-ular arrangement of NRPSs suggests that only one half (gramicidin S) or onethird (bacillibactin, enterobactin) of the extracted peptide product can be pro-duced by one assembly line-like synthesis (Fig. 4.2). These synthetases areconsidered iterative (19) because they have to complete more than one linear pep-tide synthesis before one molecule of the secondary metabolite can be released.According to a proposed model, the first precursor is translocated onto the TEdomain, the second monomer is then produced and transferred to the TE domain-bound first monomer leading to a dimer. An analogous trimerization occurs—ifapplicable—and finally the product is released by cyclization.
Another modifying reaction that is commonly found in NRPSs is the epimer-ization (E) of an amino acid (5). E domains that are always situated directlydownstream of a PCP domain catalyze these reactions. The most C-terminalamino acid of the reaction intermediate is racemized by an E domain, no matterwhether the substrate is an aminoacyl group alone or a peptidyl group. The mech-anism of these E domains is so far unclear, even though a catalysis that involvesone or more catalytic bases to deprotonate the α-carbon atom as a first step seemslikely. The resulting planar double-bond species then needs to be reprotonatedfrom the other side to invert the absolute configuration of the building block. Thisresult can be accomplished by a nearby protonated catalytic base in the enzymeor water, which is positioned opposite of the first catalytic base. Nevertheless, a
118 NONRIBOSOMAL PEPTIDES
mixture of both stereoisomers always can be detected when the substrate boundto the enzyme is analyzed, which is indicative for either a nonstereoselective ora reversible reaction. Once the epimerized substrate undergoes the subsequentcondensation reaction, only the species with an inverted stereocenter is found inthe elongated product. Thus, the C domain only processes the inverted species.In some rare cases, the C domain also exhibits epimerization activity besides itsnormal function, and it is then called the “dual C/E” domain [Arthrofactin, (20)].
Another structural feature often found in NRPS products is N-methylatedamide bonds. The domain that introduces this C1 unit, the so-called methyl-transferase (Mt) domain, is situated between the A and the PCP domain (21).By consumption of S -Adenosyl-methionine, the α-amino group of the acceptorsubstrate is methylated before condensation with the donor.
In the case of linear gramicidin, the N-terminus of the nonribosomal peptidecarries a formyl group (10). Just like in the bacterial ribosomal synthesis, onlya formylated first building block is processed additionally by the correspondingenzymatic machinery. Thus, one can find a distinct formylation (F) domain atthe very N-terminus of the synthetase. Another formylated NRPS product iscoelichelin whose N-terminal ornithine residue is believed to be Nδ-formylated intrans by a formyltransferase genetically associated with the NRPS (17). Formyl-tetrahydrofolate is used as source of the formyl group by these enzymes.
The essential condensation domain mentioned above can, in some cases, notonly condensate but also catalyze a side-chain cyclization. It is then calledcyclization (Cy) domain. The cyclization is initiated by a nucleophilic attackof the side-chain heteroatom on the carbonyl group of the amide bond formedby the same domain. When water is eliminated, a stable pentacycle is inte-grated into the peptide chain without altering the rest of the backbone. Thenucleophiles known to be reactants in these Cy domain reactions are either thre-onine/serine [mycobactin A, (22)] or cysteine [bacitracin, (23)]. The former leadsto (methyl-)oxazoline heterocycles, whereas the latter gives rise to thiazoline-like units. Another domain sometimes associated with this heterocyclization isthe oxidation (Ox) domain [Epothilone, (7)]. It is located between A and PCPdomains, and it catalyzes the oxidation of oxa/thiazoline intermediates, whichleads to oxazoles or thiazoles, respectively.
4.3 TECHNIQUES FOR THE PRODUCTION OF NOVELNONRIBOSOMAL PEPTIDES
With a constantly growing number of pathogenic bacterial strains resistant to theknown antibiotics, the demand for novel antibiotics or, more generally speaking,therapeutic agents is evident. Because many NRPS products already have suchactivities and their chemical and structural diversity is so huge, efforts have beenmade to use NRPSs to broaden the known spectrum of therapeutics. In thissection, the possibilities of using NRPS machineries or parts of them to producenew bioactive compounds are addressed.
TECHNIQUES FOR THE PRODUCTION OF NOVEL NONRIBOSOMAL PEPTIDES 119
4.3.1 Module or Domain Exchange
When considering the modular buildup of NRPSs, the possibility of altering thepeptide product by insertion, deletion, or exchange of modules seems to be anobvious approach for the production of new compounds. Because the A domainsdetermine the specificity of each module, even an exchange of fractions smallerthan whole modules in a synthetase could lead to an altered product. In the past,various attempts have succeeded using these strategies (24, 25). For instance,the exchange of an A domain in the surfactin NRPS with other A domains ofboth bacterial and fungal origin lead to the formation of the expected variantsof surfactin (26). However, in all of these early studies, the apparent turnoverrates were significantly lower than in the wild-type systems. According to com-mon understanding of NRPSs, two explanations for the drastically slowed downsynthetic process can be given. First, the borders chosen to dissect and to fusethe catalytic domains might have been unsuitable. Even though the reoccurring,highly variable so-called linker regions between each pair of catalytic domainsseem not to exhibit secondary structures, their sequence and length might be crit-ical for proper inter-domain communication. So far, no structure of any enzymeconsisting of two or more NRPS domains has been published, which makes itdifficult to define the right domain border when preparing a cloning strategy forfusion or for dissection. Second, the specificity of the C domains might resultin a reduced product turnover. Even though a relaxed specificity for the donorsubstrate has been reported, the acceptor site seems to be highly specific, whichdiscriminates against artificial substrates (27). Both the mode of catalytic actionand the molecular and structural basis for the selectivity are not fully under-stood for C domains so that a straightforward approach for overcoming theselow turnover rates currently cannot be given.
4.3.2 Changing the Specificity Code for A Domains
Sequence alignments of A domains have revealed that domains activating thesame type of building block share a set of conserved residues in the primaryprotein sequence (13). With the A domain’s crystal structure, one can find thatthese residues form the substrate binding pocket (28). These residues are thereforereferred to as the “selectivity-conferring code” of NRPSs (13). One can now ratio-nally exchange these sets of residues and can obtain fully functional A domainswith altered substrate recognition. For example, this process has been done forthe first module of the surfactin synthetase srfAA, which activated glutamate(29). In this case, the only difference in the selectivity-conferring code comparedwith a glutamine activating A domain lies in one residue. Thus, the single muta-tion of Lys239 to Gln239 in the enzyme leads to the desired and predicted shiftof specificity. In another experiment, three residues were altered to change thesubstrate recognition of the aspartate activating A domain in srfB2 to asparagine.The corresponding bacterial strain was shown to produce the expected variantof surfactin containing asparagine at position 5. Even though this elegant way
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of manipulating NRPSs works, a few drawbacks are worth mentioning. On theone hand, product turnover rates are predicted to be very low. As discussed, theC domains that have to process the artificial substrates are predicted to discrim-inate against nonnatural substrates, which kinetically impede product formationdrastically. On the other hand, this method is limited to the building blocks thatother known A domains activate. Yet, the vast diversity and bioactivity of non-ribosomal peptides mainly arises from their unusual connectivities and a largenumber of postsynthetic modifications, which one cannot address when merelychanging the A domains’ specificities.
4.3.3 Chemoenzymatic Approaches
A very powerful method for producing novel antibiotics is the chemoenzymaticapproach (30). The idea behind this strategy is to leave out the enzymatic buildupof the linear peptide scaffolds and replace it by solid-phase peptide synthesis(Fig. 4.5). Once the desired peptide is produced, its C terminus needs to be syn-thetically activated (usually as a thioester) before the substrate can be subjected toenzymatic cyclization using a TE domain. The advantages of solid-phase synthe-sis (SPPS) are obvious: Virtually any oligo-peptide can be made in a short timeand in large quantities. Even though this is true for most oligo-peptides, someamino acid sequences seem very difficult to synthesize, and the popular Fmocprotective group strategy always imposes the risk of racemization. Many differ-ent building blocks (already modified with protective groups necessary for SPPS)can be purchased, and by automated parallel peptide synthesis whole libraries canbe produced very quickly. The reason why one can use such peptides as sub-strates lies in the relaxed substrate specificity of many TE domains. The TEdomain of the Tyrocidine synthetase, which carries out a head-to-tail cyclizationof the decapeptide DPhe-Pro-Phe-DPhe-Asn-Gln-Tyr-Val-Orn-Leu, for instance,only recognizes the two N-terminal and C-terminal amino acids of the naturalsubstrate. The side chains of the amino acids at other positions are not recog-nized by the enzyme, and experiments with substrates that carry substitutions toalanine in these positions still lead to analogous cyclodecamers (31). The majoradvantage of using TE domains for cyclization reactions is their regiospecificity,stereospecificity, and chemospecificity. Thus, no protective groups are neededduring these enzymatic cyclization reactions, and undesired side product for-mation is minimized. Additionally, these reactions are carried out under mildaqueous conditions, usually pH 7–8.
To follow this chemoenzymatic approach, the synthetic substrates must betransferred onto the catalytically active serine residue of the TE domain. Thistransfer can either be done directly or with the help of a PCP domain. In thenatural system, translocation is realized by the interaction between the PCP andthe TE domain. The substrate, which is bound to the 4′Ppan cofactor of the PCPdomain as a thioester, acylates the hydroxyl group of the serine. Chemicallyspeaking, the acylation of the TE is a result of a trans-esterification. When using
TECHNIQUES FOR THE PRODUCTION OF NOVEL NONRIBOSOMAL PEPTIDES 121
OH
C A PCP C A PCP C A PCP E C A PCP C A PCP C A PCP E
module 6 module 7 module 8 module 9 module 10 module 11
C A PCP TEC A PCP
module 12 module 13
module 1 module 2 module 3 module 4
C A PCP C A PCP E C A PCP C A PCP C A PCP
module 5
replaced by solid phase synthesis
FA
Trp
DAsn
Asp
Thr
Gly
Orn
Asp
DAla
Asp
Gly
DSer
mGlu
Kyn
O S-CoA
OH OH
PCP TE
O SOH
PCP TE
FA
Trp
DAsn
Asp
Thr
Gly
Orn
Asp
DAla
Asp
Gly
DSer
mGlu
Kyn
SH
PCP TE
OO
FA
Trp
DAsn
Asp
Thr
Gly
Orn
Asp
DAla
Asp
Gly
DSer
mGlu
Kyn
[Sfp]
-3',5'ADP Daptomycin
SH
PCP TE
(a)
O S
FA
Trp
DAsn
Asp
Thr
Gly
Orn
Asp
DAla
Asp
Gly
DSer
mGlu
Kyn OH
TE
TE
OO
FA
Trp
DAsn
Asp
Thr
Gly
Orn
Asp
DAla
Asp
Gly
DSer
mGlu
Kyn
OH
TE
single turnover
multiple turnover
(b)
(c)
Figure 4.5 Chemoenzymatic approaches for the production of novel bioactive com-pounds. In this example, the enzymatic buildup of the linear precursor of daptomycin byits NRPSs (DptA, DptBC, and DptD) is substituted by solid-phase synthesis (a). By usingthe 4′Ppan transferase Sfp and the CoA-thioester of the linear peptide, the apo-enzymePCP-TE and be modified, and after trans-esterification cyclized by the TE domain (b).Because the resulting holo-enzyme cannot be modified again, this is a single turnoverreaction. Another strategy uses thiophenole-esters of the linear peptides to be cyclized(c). When these compounds are used, no PCP domain is necessary. The TE domain isreadily acylated, and regiospecific and stereospecific cyclization toward daptomycin or,depending on the linear peptide provided, toward variants thereof occurs. Because theenzyme is not altered in any way after product release, this setup results in a multipleturnover.
TE domains, the substrate provided in trans must also have an appropriate acy-lation potential. Several key techniques have been developed to covalently attachsynthetic substrates to PCP and TE domains. In the first method, the relaxedsubstrate specificity of the 4′Ppan transferase Sfp is used to load acyl moietiesonto PCP domains enzymatically. Just like in the natural priming reaction inwhich the 4′Ppan part of CoA is transferred onto the conserved serine residueof the apo-PCP domain, Sfp does analogously attach S-acylated 4′Ppans, whichoriginates from S-acylated CoA substrates (32). These CoA substrates can readilybe obtained by one coupling reaction directly after solid phase synthesis. Withthis technique, virtually any substrate can be brought to a desired position inrecombinant NRPS enzymes containing an apo-PCP domain. This result is ofgreat value when elucidating the catalytic properties and substrate specificities of
122 NONRIBOSOMAL PEPTIDES
other domains. When investigating TE domains, for example, the correspondingapo-PCP-TE would be the starting point for screening the cyclization abilitiesof the TE domain with a synthetic substrate library. However, the major dis-advantage of this method becomes evident when looking closely at the enzymeafter the release of the product: The PCP domain is now in its holo-state, andtherefore, subsequent enzymatic loading of substrates with Sfp is impossible.Because product formation is limited to a single turnover, other methods havebeen developed to allow for multiple turnover.
For multiple turnover reactions, the TE domains must be supplied with sub-strates in trans; yet the acylation potential must be sufficient and the compoundmust be recognized by the enzyme. The first approach made was inspired bythe natural system where the substrate is activated as a thioester bound to the4′Ppan cofactor. The idea was to minimize the 4′Ppan moiety by replacing itwith N-acetyl-cysteamine (SNAc) (33). This method works fairly well; however,it seems in later studies that the acylation potential is of greater importance thanthe similarity to the natural situation and a variety of peptidyl-thioesters with bet-ter leaving groups than SNAc was tested. The fastest turnover rates were foundwhen thiophenol-esters were used (30, 34). Thiophenol has several advantages: Itdoes not have any functional groups other than the thiol group, it is inexpensive,and it can easily be separated from the product. This method has been success-fully used to shed light on the promiscuity of the TE domain of the daptomycinNRPS (35).
Even though these techniques allow for the production of new potentiallybioactive compounds, they are usually closely related to known substances, whichbasically implies an analogous mode of action, and the deviations normally alterquantitative parameters such as solubility and affinity. Nevertheless, hundreds ofnonribosomal systems still need to be explored, and the discoveries of new onesare frequently reported. The basic understanding of the catalytic functions ofnonribosomal domains and modules that we have today is a good starting pointfor additional exploration and use of systems that are not yet understood fully.
REFERENCES
1. Walsh CT. Polyketide and nonribosomal peptide antibiotics: modularity and versatil-ity. Science 2004;303:1805–1810.
2. Finking R, Marahiel MA. Biosynthesis of nonribosomal peptides1. Annu. Rev. Micro-biol. 2004;58:453–488.
3. Fischbach MA, Walsh CT. Assembly-line enzymology for polyketide and nonri-bosomal Peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 2006;106:3468–3496.
4. Keller U, Schauwecker F. Combinatorial biosynthesis of non-ribosomal peptides.Comb. Chem. High Throughput Screen. 2003;6:527–540.
5. Stein DB, Linne U, Hahn M, Marahiel MA. Impact of epimerization domains onthe intermodular transfer of enzyme-bound intermediates in nonribosomal peptidesynthesis. ChemBioChem. 2006;7:1807–1814.
REFERENCES 123
6. Kohli RM, Trauger JW, Schwarzer D, Marahiel MA, Walsh CT. Generality of pep-tide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptidesynthetases. Biochemistry 2001;40:7099–7108.
7. Chen H, O’Connor S, Cane DE, Walsh CT. Epothilone biosynthesis: assembly ofthe methylthiazolylcarboxy starter unit on the EpoB subunit. Chem. Biol. 2001;8:899–912.
8. Schracke N, Linne U, Mahlert C, Marahiel MA. Synthesis of linear gramicidinrequires the cooperation of two independent reductases. Biochemistry. 2005;41(44):8507–8513.
9. Velkov T, Lawen A. Mapping and molecular modeling of S-adenosyl-l-methioninebinding sites in N-methyltransferase domains of the multifunctional polypeptidecyclosporin synthetase. J. Biol. Chem. 2003;278:1137–1148.
10. Schoenafinger G, Schracke N, Linne U, Marahiel MA. Formylation domain: an essen-tial modifying enzyme for the nonribosomal biosynthesis of linear gramicidin. J. Am.Chem. Soc. 2006;128:7406–7407.
11. Hubbard BK, Walsh CT. Vancomycin assembly: nature’s way. Angew. Chem. Int.Ed. Engl. 2003;42:730–765.
12. Luo L, Burkart MD, Stachelhaus T, Walsh CT. Substrate recognition and selectionby the initiation module PheATE of gramicidin S synthetase. J. Am. Chem. Soc.2001;123:11208–11218.
13. Stachelhaus T, Mootz HD, Marahiel MA. The specificity-conferring code of adeny-lation domains in nonribosomal peptide synthetases. Chem. Biol. 1999;6:493–505.
14. Koglin A, Mofid MR, Lohr F, Schafer B, Rogov VV, Blum MM, et al. Confor-mational switches modulate protein interactions in peptide antibiotic synthetases.Science. 2006;14(312):273–276.
15. Keating TA, Marshall CG, Walsh CT, Keating AE. The structure of VibH repre-sents nonribosomal peptide synthetase condensation, cyclization and epimerizationdomains. Nat. Struct. Biol. 2002;9:522–526.
16. Bender CL, Alarcon-Chaidez F, Gross DC. Pseudomonas syringae phytotoxins: modeof action, regulation, and biosynthesis by peptide and polyketide synthetases. Micro-biol. Mol. Biol. Rev. 1999;63:266–292.
17. Lautru S, Deeth RJ, Bailey LM, Challis GL. Discovery of a new peptide natural prod-uct by Streptomyces coelicolor genome mining. Nat. Chem. Biol. 2005;1:265–269.
18. Kohli RM, Trauger JW, Schwarzer D, Marahiel MA, Walsh CT. Generality of pep-tide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptidesynthetases. Biochemistry 2001;40:7099–7108.
19. Gehring AM, Mori I, Walsh CT. Reconstitution and characterization of theEscherichia coli enterobactin synthetase from EntB, EntE, and EntF. Biochemistry1998;37:2648–2659.
20. Balibar CJ, Vaillancourt FH, Walsh CT. Generation of D amino acid residues in assem-bly of arthrofactin by dual condensation/epimerization domains. Chem. Biol. 2005;12:1189–1200.
21. Billich A, Zocher R. N-Methyltransferase function of the multifunctional enzymeenniatin synthetases. Biochemistry 1987;26:8417–8423.
22. Quadri LE, Sello J, Keating TA, Weinreb PH, Walsh CT. Identification of a Mycobac-terium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly ofthe virulence-conferring siderophore mycobactin. Chem. Biol. 1998;5:631–645.
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23. Eppelmann K, Doekel S, Marahiel MA. Engineered biosynthesis of the peptideantibiotic bacitracin in the surrogate host Bacillus subtilis. J. Biol. Chem. 2001;276:34824–34831.
24. Mootz HD, Kessler N, Linne U, Eppelmann K, Schwarzer D, Marahiel MA. Decreas-ing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame moduledeletion in the biosynthetic genes. J. Am. Chem. Soc. 2002;124:10980–10981.
25. Schauwecker F, Pfennig F, Grammel N, Keller U. Construction and in vitro analysis ofa new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides.Chem. Biol. 2000;7:287–297.
26. Stachelhaus T, Schneider A, Marahiel MA. Rational design of peptide antibiotics bytargeted replacement of bacterial and fungal domains. Science 1995;269:69–72.
27. Belshaw PJ, Walsh CT, Stachelhaus T. Aminoacyl-CoAs as probes of condensationdomain selectivity in nonribosomal peptide synthesis. Science 1999;284:486–489.
28. May JJ, Kessler N, Marahiel MA, Stubbs MT. Crystal structure of DhbE, an archetypefor aryl acid activating domains of modular nonribosomal peptide synthetases. Proc.Natl. Acad. Sci. U.S.A. 2002;99:12120–12125.
29. Eppelmann K, Stachelhaus T, Marahiel MA. Exploitation of the selectivity-conferringcode of nonribosomal peptide synthetases for the rational design of novel peptideantibiotics. Biochemistry. 2002;41:9718–9726.
30. Grunewald J, Marahiel MA. Chemoenzymatic and template-directed synthesis ofbioactive macrocyclic peptides. Microbiol. Mol. Biol. Rev. 2006;70:121–146.
31. Trauger JW, Kohli RM, Mootz HD, Marahiel MA, Walsh CT. Peptide cyclizationcatalysed by the thioesterase domain of tyrocidine synthetase. Nature 2000;407:215–218.
32. Sieber SA, Walsh CT, Marahiel MA. Loading peptidyl-coenzyme A onto peptidylcarrier proteins: a novel approach in characterizing macrocyclization by thioesterasedomains. J. Am. Chem. Soc. 2003;125:10862–10866.
33. Ehmann DE, Trauger JW, Stachelhaus T, Walsh CT. Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide syn-thetases. Chem. Biol. 2000;7:765–772.
34. Sieber SA, Tao J, Walsh CT, Marahiel MA. Peptidyl thiophenols as substrates fornonribosomal peptide cyclases. Angew. Chem. Int. Ed. Engl. 2004;43:493–498.
35. Grunewald J, Sieber SA, Mahlert C, Linne U, Marahiel MA. Synthesis and derivatiza-tion of daptomycin: a chemoenzymatic route to acidic lipopeptide antibiotics. J. Am.Chem. Soc. 2004;126:17025–17031.
FURTHER READING
Baltz RH. Molecular engineering approaches to peptide, polyketide and other antibiotics.Nat. Biotechnol. 2006;24:1533–1540.
Cane DE. Introduction: Polyketide and Nonribosomal Polypeptide Biosynthesis. FromCollie to Coli. Chem. Rev. 1997;97:2463–2464.
Du L, Shen B. Biosynthesis of hybrid peptide-polyketide natural products. Curr. Opin.Drug Discov. Devel. 2001;4:215–228.
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Grunewald J, Kopp F, Mahlert C, Linne U, Sieber SA, Marahiel MA. Fluorescenceresonance energy transfer as a probe of peptide cyclization catalyzed by nonribosomalthioesterase domains. Chem. Biol. 2005;12:873–881.
Hahn M, Stachelhaus T. Selective interaction between nonribosomal peptide synthetasesis facilitated by short communication-mediating domains. Proc. Natl. Acad. Sci. U.S.A.2004;101:15585–15590.
Salomon CE, Magarvey NA, Sherman DH. Merging the potential of microbial geneticswith biological and chemical diversity: an even brighter future for marine naturalproduct drug discovery. Nat. Prod. Rep. 2004;21:105–121.
Sieber SA, Linne U, Hillson NJ, Roche E, Walsh CT, Marahiel MA. Evidence fora monomeric structure of nonribosomal Peptide synthetases. Chem. Biol. 2002;9:955–956.
