JOURNAL OF BACTERIOLOGY, Dec. 2003, p. 7036–7043 Vol. 185, No. 240021-9193/03/$08.00�0 DOI: 10.1128/JB.185.24.7036–7043.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Learning from Nature’s Drug Factories: Nonribosomal Synthesis ofMacrocyclic Peptides

Stephan A. Sieber and Mohamed A. Marahiel*Fachbereich Chemie/Biochemie, Philipps-Universitat Marburg, 35032 Marburg, Germany

With the discovery of penicillin as an efficient antibacterialagent isolated from the fungus Penicillium notatum, microor-ganisms attracted considerable attention as a new source forpharmaceutical agents. Screening of microbial extracts re-vealed the large structural diversity of natural compounds withbroad biological activities, such as antimicrobial, antiviral, im-munosuppressive, and antitumor activities. Like penicillin,many of these products are small peptide molecules consistingof 3 to 22 residues with often unusual structural elements.These include heterocyclic elements, D-amino acids, and gly-cosylated and N-methylated residues, suggesting a nonriboso-mal origin of biosynthesis. Due to the potent pharmacologicalactivities of these compounds, there was an overwhelming in-terest in exploring their mechanism of synthesis. Lipmann et al.reported as early as the 1970s that the cyclic peptides grami-cidin S and tyrocidine from Bacillus spp. were produced in anucleic acid-independent way through the use of large enzymecomplexes similar to fatty acid synthases (17). Subsequently,other peptidic natural products were shown to be assembled bylarge enzymes, referred to as nonribosomal peptide syntheta-ses (NRPS), which utilize the multiple-carrier thiotemplatemechanism (34). A common feature of many nonribosomallyproduced peptides is their constrained structure, which ensuresa precise functionality important for a proper interaction withthe dedicated molecular target in the cell. Nature achieves thisrigidity in molecular structure through several strategies: themolecule can be oxidatively cross-linked, as in vancomycin,heterocyclized, as in penicillin or, more commonly, cyclized, asin fengycin (Fig. 1). Cyclization seems to be the predominantway of constraining nonribosomally synthesized peptides. Be-cause peptide cyclization from the point of view of chemicalsynthesis is difficult to achieve without protection of all of theside chains, there has been rapidly growing interest in explo-ration of the enzymatic cyclization mechanism for the devel-opment of new synthesis routes.

A SHORT WALK DOWN THE NRPS ASSEMBLY LINE

The nonribosomal machinery for peptide synthesis useslarge multienzyme complexes as an assembly line to catalyzestepwise peptide condensation. The substrates of these multi-

enzyme complexes are not restricted to the 20 amino acids,since hundreds of building blocks are now known to be inte-grated and modified by postsynthesis action. Common to thisassembly line is the incorporation of nonproteinogenic aminoacids, such as D-isomers, carboxy acids, and N-methylated res-idues, as well as the incorporation of heterocyclic rings andfatty acids (Fig. 1). Glycosylation and oxidative cross-linkingare common further postsynthetic modifications by enzymesthat are associated with the NRPS machinery.

In order to understand the principles of this enzymaticallydirected peptide synthesis, the mechanistic features of NRPSare briefly summarized with the surfactin synthetase from Ba-cillus subtilis as an example (see also references 18, 20, 25, and34). The surfactin synthetase is a large multienzyme complexconsisting of three enzymatic subunits, SrfA (402 kDa), SrfB(401 kDa), and SrfC (144 kDa), which consist of seven modulesthat comprise 24 catalytic domains (Fig. 2). Each module isresponsible for the specific incorporation of one dedicatedsubstrate into the growing heptapeptide chain (21). The N-terminal module of an assembly line, the initiation module,specifically recognizes and activates the N-terminal amino acidof the peptide product. All chemical reactions necessary toincorporate and modify each substrate are mediated by a cat-alytically independent set of domains incorporated within themodules. The first step in biosynthesis is the recognition andactivation of a dedicated substrate by the adenylation domain(A domain; about 550 amino acids) (4). By analogy to amino-acyl-tRNA synthetase, the A domain catalyzes the activation ofa substrate as aminoacyladenylate through the Mg2�-depen-dent hydrolysis of ATP and the release of pyrophoshate (7). Inthe next step, the aminoacyladenylate intermediate is trans-ferred to the free thio group of the cofactor phosphopanteth-eine, which is tethered to the thiolation domain (T domain, orpeptidyl carrier protein; about 80 amino acids) located down-stream of the A domain (29, 36). The phosphopantetheine armis attached to an invariant serine residue of the apo-T domainby a dedicated 4�-phosphopantetheine (ppan) transferase thatuses coenzyme A (CoA) as a substrate (15, 22). The interme-diates, tethered by the reactive thioester to the flexible cofactorphosphopantetheine (in each module), can be transferred toother domains for subsequent catalytic reactions. Peptide bondformation between two adjacent substrates is catalyzed by thecondensation domain (C domain; about 450 amino acids),which is located between the A and T domains of subsequentmodules (11). The C domain catalyzes the nucleophilic attackof the amino acid bound to the downstream T domain with its

* Corresponding author. Mailing address: Fachbereich Chemie/Bio-chemie, Philipps-Universitat Marburg, Hans-Meerwein-Str., 35032Marburg, Germany. Phone: 49 6421 2825722. Fax: 49 6421 2822191.E-mail: marahiel@chemie.uni-marburg.de.

7036

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

free �-amino group on the activated thioester of the upstreamT-domain-bound intermediate (1). For the surfactin initiationreaction, peptide bond formation occurs between modules 1and 2 by the nucleophilic attack of the �-amino group of

leucine on the thioester-activated carboxy group of glutamateto give a dipeptide which is then translocated to module 2.

Beside the A, T, and C domains, which are essential domainsof an elongation module, there are also other, optional do-

FIG. 1. Naturally occurring macrolactones and macrolactams.

VOL. 185, 2003 MINIREVIEW 7037

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

mains. A common structural motif of nonribosomal peptides isthe incorporation of D-amino acids, which is mediated by theepimerization domain (E domain; about 450 amino acids). TheE domain catalyzes the racemization of the T-domain-boundamino acid to form an equilibrium between the L and D con-formers. However, the C domain incorporates only the D-amino acid into the growing peptide chain (16). In the surfactinsynthetase, two epimerization domains in modules 3 and 6 areresponsible for the racemization of T-domain-bound L-Leu.The combination of L- and D-amino acids gives the peptide aunique conformation that is important for the specific interac-tion with its cellular target.

All catalytic domains discussed so far are repeating units ofthe enzymatic template and contribute to the synthesis of alinear peptide molecule tethered to the multienzyme. In orderto reactivate the multienzyme for a next synthesis cycle, themature peptide must be cleaved once it reaches the end of theassembly line. This reaction usually is accomplished by a thio-esterase domain (TE domain; about 280 amino acids) fused tothe C-terminal module. The peptide can be released either byhydrolysis as a linear acid or by an intramolecular reaction withan internal nucleophile to give a cyclic peptide. Hydrolyticrelease is observed, e.g., for vancomycin, whose peptide back-bone is constrained by further postsynthetic oxidative cross-

FIG. 2. NRPS assembly of surfactin. The surfactin synthetase consists of 24 individual domains responsible for the catalysis of 24 chemicalreactions. These domains catalyze activation (A domain), covalent binding (T domain), elongation (C domain), epimerization (E domain), andrelease (TE domain) by either cyclization or hydrolysis. The domains are organized in modules (indicated in blue and red), where each moduleincorporates one dedicated building block into the growing peptide chain. The peptide chain is covalently tethered to the multienzyme via thecofactor phosphopantetheine (ppan), and synthesis proceeds according to the enzymatic template from the N- to the C-terminal end. FA, fatty acidchain.

7038 MINIREVIEW J. BACTERIOL.

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

linking reactions (10). For surfactin, the peptide backbone isconstrained by the intramolecular nucleophilic attack of a�-hydroxyl group of the fatty acid moiety to give a branched-chain lipodepsipeptide (32) (Fig. 1). Many other cyclizationstrategies give rise to a large and diverse set of cyclic or cyclicbranched molecules with distinct biological activities. A TEdomain catalyzing a cyclization reaction also is referred to as apeptide cyclase.

CYCLIZATION STRATEGIES: NATURE’S WAY

All known NRPS macrocyclization strategies lead to cyclicor cyclic branched-chain peptides (Fig. 1). In macrolactones,the branch point can be either a hydroxylated amino acid sidechain or a hydroxylated fatty acid moiety. For the surfactinpeptide cyclase, the ring closure is enzymatically catalyzed be-tween an N-terminal �-hydroxyl fatty acid and the C-terminalpeptide end (32). Cyclization was observed only when the(R)-�-hydroxyl fatty acid was used, while the (S)-enantiomershowed only enzymatic hydrolysis, indicating stereoselectiverecognition (12). The lipodepsipeptide cyclic product is astrong detergent with antiviral, hemolytic, and antibacterialactivities (21). A major contribution to the detergent activity isprovided by the lipopeptide chain, which is believed to betransferred to the N-terminal residue during the initiation re-action by a fatty acid acyltransferase. In contrast, the syringo-mycin (Pseudomonas syringae) and fengycin (B. subtilis) li-popeptide cyclases accept serine and tyrosine side chains of thepeptide sequence as nucleophiles for cyclization, discriminat-ing the N-terminal �-hydroxyl group of the attached fatty acidmoiety (24, 26, 33) (Fig. 1). Moreover, these peptide cyclasesdisplay a very high level of regioselectivity by selecting only onespecific residue of the substrate from a large source of nucleo-philes for cyclization.

In addition to macrolactonization, natural product diversityalso is increased by various enzymatic macrolactamizationstrategies. Basic head-to-tail peptide macrolactamization is ob-served in the antibiotic tyrocidine from Bacillus brevis and thepotent immunosuppressive drug cyclosporin A from Tolypocla-dium niveum. In cyclosporin A, the final peptide bond isformed by a putative condensation domain (35) instead of apeptide cyclase, emphasizing that nature developed two en-zyme species capable of catalyzing product release by cycliza-tion. Besides head-to-tail cyclized lactams, branched-chain lac-tams also are observed. The peptide cyclase of the antifungallipopetide mycosubtilin from B. subtilis forms regioselectivelyan amide bond between an N-terminal �-amino fatty acid andthe peptide C-terminal end (Fig. 1). As in surfactin, in myco-subtilin a fatty acid chain is involved in the cyclization process,providing an amine as a nucleophile (8). While the precursorsin both cases seem to be �-keto fatty acid residues derivedfrom fatty acid synthases, nature processes these ketones indifferent ways. They are either reduced to a hydroxyl group, asobserved in surfactin, or they are reductively aminated in aprocess catalyzed by aminotransferases, as observed in myco-subtilin. In both cases, nature uses a common precursor motif,which is subsequently diversified by the application of differentsynthetic strategies to increase the product outcome. As infengycin and syringomycin, enzymatic amide bond formationalso can occur between an ornithine side chain and the C-

terminal peptide end, as observed in the antibiotic bacitracinfrom Bacillus licheniformis (14). The scope of cyclic lactamesand lactones can be further broadened by oligomerization ofpeptide monomers. This additional strategy enables the B.brevis gramicidin S peptide cyclase to cyclodimerize two linearpentapeptides by catalyzing two subsequent peptide bond for-mation steps to form the cyclic lactam antibiotic gramicidin S(12) (Fig. 1). Also, cyclotrimerization is observed for the sid-erophore-forming peptide cyclases of enterobactin and the ba-cillibactin synthetase from Escherichia coli and B. subtilis, re-spectively (9, 19). Three units of 2,3-dihydroxybenzoyl-serineare fused together by three subsequent ester bond formationsteps between the serine hydroxyl group of one molecule andthe C-terminal end of another molecule to give the cyclictrilactone enterobactin. This lactone displays iron-chelatingactivity, which is closely related to its structure. Three intramo-lecular catechol ligands provide electron donors required forthe coordination of iron, once more emphasizing the closerelationship between cyclic structural organization and biolog-ical activity.

NRPS peptide cyclases can generate diverse cyclic peptidemolecules ranging in size from very small, as in pristinamycin,with 7 residues, to very large, as in syringopeptin, with 22residues (6, 23). At the large end of the scale is another sourceof macrocyclic molecules observed in nature, referred to asnaturally occurring circular proteins (5). These proteins are ofbacterial origin and have a folded three-dimensional structure.In contrast to NRPS, they are produced by the translation ofgenes. Cyclization occurs posttranslationally only in a head-to-tail fashion to produce a seamless circle of peptide bonds. Incontrast to what is known for NRPS, not much is known aboutthe cyclization mechanism of the linear precursors.

The cyclization strategies reported here emphasize that na-ture has developed a large enzymatic tool set which allows theintroduction of diversity into linear peptide sequences by avariety of different cyclization steps. Selection from differentnucleophiles, enantiomers, and positions in the peptide se-quence makes peptide cyclases unique enzymatic tools withvery specific intrinsic stereo- and regioselective recognitionelements. Moreover, an understanding of the catalysis of one,two, or three subsequent condensation steps toward cyclizationrequires further studies of the structural and mechanistic as-pects of these enzymes.

CHEMOENZYMATIC CYCLIZATION

The great pharmacological potential of many cyclic peptidesemphasizes their role in drug discovery, as they show specificinteractions with cellular targets and a high level of resistanceto proteolytic enzymes. They are therefore most promisingscaffolds for pharmacophores. So far, synthetic chemistry facesseveral difficulties in the production of cyclic compounds pro-viding sufficiently good yields and regioselectivity. Althoughcyclization is an entropically favorable process, synthetic mac-rocyclization is difficult to achieve, since steric repulsion of ringresidues as well as the use of protecting groups to ensureproper regiochemistry decreases yields and makes chemicalsynthetic operations expensive and rather difficult. Since na-ture developed stereo- and regioselective peptide cyclizationenzymes, researchers have aimed to combine chemical linear

VOL. 185, 2003 MINIREVIEW 7039

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

peptide synthesis with enzymatically catalyzed cyclization. Thisapproach allows easy synthesis of linear peptide sequences byestablished solid-phase peptide chemistry, followed by selec-tive and efficient enzymatic cyclization without the use of pro-tecting groups and the formation of undesirable by-products.

In order to achieve chemoenzymology, translation betweenthe language of chemistry and the language of biology must beestablished by chemically mimicking the biological pathway asclosely as possible. This was first achieved by Trauger et al.,who cloned and overexpressed an excised peptide cyclase (28

kDa) from the tyrocidine synthetase (30). In order to prove theactivity of this isolated enzyme versus that of the natural en-zyme, which is embedded in a 724-kDa multienzyme complex,a short mimicked copy of the natural cofactor phospho-pantetheine, N-acetylcysteamine (SNAC), was attached to theC-terminal end of a chemically synthesized linear tyrocidinepeptide (Fig. 3A). SNAC represents a link between natural andartificial systems and is compatible with both. Incubation of themimicked substrate and the excised peptide cyclase revealedactivity with an observed cyclization/hydrolysis ratio of 6:1. The

FIG. 3. Chemoenzymatic cyclization strategies. (A) Peptidyl-SNAC. The natural cofactor phosphopantetheine T domain was mimicked forsynthetic substrates by attaching the last part of the cofactor, SNAC, to the C-terminal end of the tyrocidine linear peptide chain. This solublesubstrate was subsequently incubated with the excised tyrocidine cyclase (TE domain), and cyclic tyrocidine and a hydrolyzed product wereobserved. (B) Peptidyl-CoA. Synthetically made fengycin CoA is first loaded by the ppan transferase Sfp onto an invariant serine residue of theapo-T domain from an excised fengycin T-TE didomain. In the second step, the substrate is transferred to the peptide cyclase (TE domain) by thenucleophilic attack of an invariant serine residue of the TE domain active site on the peptidyl-ppan thioester. Subsequent cyclization and hydrolysisare catalyzed by the cyclase in the third step of the reaction.

7040 MINIREVIEW J. BACTERIOL.

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

turnover of 59 min�1 indicated a very rapid conversion of thelinear compound into the cyclic compound, indicating the use-fulness of this enzyme as an in vitro biocatalyst (30). Follow-upstudies with various peptidyl-SNAC substrates having variouslengths, stereochemical properties, and amino acid composi-tions revealed that the tyrocidine peptide cyclase recognizesonly C- and N-terminal residues of the substrate on the basis ofidentity and stereochemistry, leaving space for making longerand shorter substrates as well as for replacements of residueswithin the peptide backbone (12). A minimal recognitionmodel was postulated (31). This observed substrate toleranceof the excised tyrocidine peptide cyclase allowed the synthesisof diverse tyrocidine variants in which position 4 (D-Phe) wasreplaced by 1 of 96 natural and unnatural amino acids. Thislibrary of tyrocidine product analogs was subsequentlyscreened for improved or altered bioactivity. In contrast to thenatural antibiotic tyrocidine, which does not discriminate be-tween bacterial and eukaryotic cell membranes, the screenrevealed that the substitution of D-Phe at position 4 with apositively charged D-amino acid led to a 30-fold increase in theselective recognition of bacterial membranes (13).

In addition to their use with the tyrocidine peptide cyclase,SNAC substrates also were used to characterize the cyclizationof gramicidin S and surfactin (12, 32). In contrast to the tyro-cidine peptide cyclase, the surfactin peptide cyclase showedmuch less substrate tolerance, indicating differences in thebinding pockets of these enzymes. While the tyrocidine peptidecyclase was capable of cyclizing shorter and longer SNACsubstrates, the surfactin peptide cyclase showed only SNAChydrolysis for shorter and longer compounds. Moreover, achange in the N-terminal nucleophilic attacking group from anamine to a hydroxyl group in the tyrocidine sequence resultedin no change in the cyclization outcome, while an oppositechange from a hydroxyl fatty acid to an amino fatty acid groupin the surfactin sequence resulted in only hydrolysis and notcyclization. An important feature of cyclic peptides is the�-sheet content, which is high for molecules with (4n � 2)residues (12, 31). A peptide with a high �-sheet content, suchas the dekapeptide tyrocidine (n � 2), facilitates cyclizationthrough substrate preorganization by backbone-to-backbonehydrogen bonds. This intrinsic property of tyrocidine facilitateseasy cyclization, which was also reported to occur withoutcatalysis, but at a lower efficiency (3). In contrast, the hep-tapeptide surfactin displays fewer � sheets and therefore nosubstantial substrate preorganization. The �-sheet contents inthe peptide sequences of surfactin and tyrocidine thereforealso may contribute to the observed differences in substratetolerance.

To expand the set of cyclization catalysts, peptide cyclasesfrom other NRPS systems, such as mycosubtilin and fengycinfrom B. subtilis and syringomycin from P. syringae, recentlywere cloned and overexpressed. Contrary to the observationsfor the surfactin and tyrocidine cyclases, no activity was ob-served for the mycosubtilin, fengycin, and syringomycin cycla-ses with synthetically made peptidyl-SNAC substrates, indicat-ing a limitation in the chemoenzymatic potential of the lattercyclases. The inability to recognize or bind SNAC substrates inthe active site of the excised peptide cyclase could be affectedby the manner in which the short mimicked copy of SNAC ispresented to the enzyme. To overcome this limitation, the

cyclase domain (TE domain) was excised with the precedingcofactor-binding T domain as a T-TE didomain. Recombinantapo-T-TE cyclases then could be loaded in vitro with chemi-cally synthesized peptidyl-CoA by using the ppan transferaseSfp. The resulting peptidyl-ppan-T-TE holocyclase carried thecovalent cofactor-bound substrate in a way that mimicked thenatural substrate presentation in the NRPS assembly line (28)(Fig. 3B). Incubation of fengycin T-TE cyclase with fengycinCoA and Sfp revealed cyclization and hydrolysis activities,which were not observed with SNAC substrates alone. Theseresults indicate that the peptide needs to be directed into thepeptide cyclase active site by the cofactor ppan to ensure cor-rect enzyme recognition; in contrast, soluble substrates are notproperly directed into the active site by diffusion. Cyclaseswhich do not show activity with SNAC substrates seem torequire covalent binding of the peptide substrate to the ppanT-domain in order to catalyze cyclic product formation.

STRUCTURAL AND MECHANISTIC ASPECTS OF NRPSPEPTIDE CYCLASES

The three-dimensional organization of enzyme residues en-codes all information required to understand the principlesand general features of macrocyclization. Regiospecific selec-tion of only one nucleophile for cyclization and the exclusion ofwater to prevent undesired hydrolysis are features which areembedded in the structural fold. Moreover, the question ofhow thioesterases from different NRPS systems catalyze ter-mination in one case by cyclization and in another case byhydrolysis needs to be elucidated. Crystallographic data for theexcised surfactin peptide cyclase (TE domain) showed that thisenzyme is a member of the �,�-hydrolase family (2) (Fig. 4A).Since this was the first crystal structure determined for anNRPS peptide cyclase, it served as a prototype for detailedmechanistic investigations (32). The structural similarity toserine hydrolases suggested that an active-site catalytic triad isresponsible for the macrocyclization activity. This notion is inagreement with a recent structural model of the surfactin cy-clase which suggested that the ppan T-domain-bound peptidylchain is directed through a cleft into the active site of thepeptide cyclase and transferred to an invariant serine residue(Ser80), which is activated by histidine (His207) and aspartate(Asp107) (2) (Fig. 4B). The identity of this catalytic triad wasconfirmed by mutational analysis, which showed that all threeresidues were essential for enzyme activity (32). The hydro-phobic surfactin peptidyl chain of the acyl-O-enzyme interme-diate is accommodated in a predominantly hydrophobic bind-ing pocket with two cationic residues predicted to directcyclization through specific interactions with the substrate (Fig.4B). Mutation of these residues to alanine resulted in a dra-matic decrease in overall activity, indicating their relevance forpeptide recognition.

Further studies of enzymatic substrate recognition elementsfor the surfactin peptide cyclase were carried out by using adetailed substrate scan and cocrystallization analysis for en-zyme-bound inhibitor–substrate. The crystallization studies re-vealed well-defined binding pockets for the two C-terminalleucine residues in the enzyme, while the rest of the peptidesequence seems to be less well coordinated (32) (Fig. 4C). Inthe deacylation step of the reaction, the �-hydroxyl group of

VOL. 185, 2003 MINIREVIEW 7041

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

the fatty acid moiety is activated by the same histidine andaspartate to facilitate an intramolecular nucleophilic attack onthe acyl-enzyme ester bond to release the final lactone product.Many thioesterase domains from other NRPS systems, e.g.,vancomycin, as well as structurally related lipases only hydro-lyze and do not cyclize their products. A sequence alignmentbetween the surfactin peptide cyclase and other members ofthe �,�-hydrolase enzyme family that catalyze only hydrolysisrevealed the conservation among lipases of a glycine residueinvolved in the formation of the oxyanion hole. In the surfactinpeptide cyclase, a proline residue is located at this position. Amutation of proline to glycine resulted in a 12-fold change inthe product ratio in favor of hydrolysis, indicating that thechange from a rigid proline to a flexible glycine increases theconformational freedom in this region of the active site andcreates more access for water to capture the acyl-enzyme in-termediate (32). This residue seems to be a switch betweenhydrolysis and cyclization among �,�-hydrolases.

Detailed investigations of the surfactin peptide cyclase pro-vided insights into mechanistic and architectural features of an

enzyme which produces a branched cyclic lipodepsipeptide.Less is known about the mechanism and structure of othercyclases, particularly oligomerizing cyclases. A detailed massspectrometric analysis was carried out for the last module ofthe enterobactin assembly line (EntF), containing a C-terminalpeptide cyclase which catalyzes the cyclotrimerization of three2,3-dihydroxybenzoyl-serine (DHB-Ser) units to give the cyclictrilactone enterobactin (27). In order to localize acyl-enzymeintermediates, an active-site histidine-to-alanine mutant en-zyme with a very low substrate turnover was used. With thisapproach, it was possible to provide evidence for a covalentacyl-O-TE domain intermediate and demonstrate that the pep-tide cyclase is involved in two reactions: acyl-chain growth andcyclization. In the first steps of acyl-chain growth, DHB-Ser istransferred to the active-site serine of the peptide cyclase bythe nucleophilic attack of active-site serine on the acyl-thio-ester of the upstream holo-T domain. The second step requirescatalytic generation of a DHB-Ser alkoxide, which in turnallows nucleophilic attack on another DHB-Ser thioester-bound T domain to form a dimeric ester. The elongation step

FIG. 4. Structural data for surfactin cyclase SrfTE. (A) Surface (right) and ribbon (left) representations of surfactin cyclase (�,�-hydrolasefold). The peptide substrate and the ppan arm are modeled into a hydrophobic binding pocket and into a substrate channel, respectively. Modelingwith the T domain (yellow) revealed a putative T-TE didomain docking site. (B) Magnification of the binding pocket shows the catalytic triad(Ser80, His207, and Asp107) and several hydrophobic residues, with the exception of Lys111 and Arg120. (C) Cocrystallization study of SrfTE witha boronic acid inhibitor substrate revealed specific binding pockets for D-Leu6 and Leu7. Based on modeling experiments, the rest of the peptidesequence seems to loop out of the enzyme.

7042 MINIREVIEW J. BACTERIOL.

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

is repeated a third time before the final cyclic trilactone isreleased by the intramolecular nucleophilic attack of serine onthe acyl-O-TE domain ester bond. This mechanistic analysissuggests that the enterobactin cyclase serves as a “waitingroom” while the phosphopantetheinyl T domain is reacylated,a process which requires a stable ester bond and the exclusionof any water from the active site. Crystallographic data for thispeptide cyclase and others will provide more insight into theoverall mechanisms and allow for a comparison of their fea-tures.

CONCLUDING REMARKS

Research efforts of the past few years in the growing field ofenzymatic peptide cyclization in nonribosomal peptide synthe-sis, summarized in this article, have revealed substantial in-sights into the architectural, mechanistic, and functional orga-nization of NRPS peptide cyclases. Based on the diversity ofnatural cyclization strategies, chemoenzymatic approacheswere developed to allow cross talk between biology and chem-istry to reprogram natural peptide sequences by chemical pep-tide synthesis and subsequent enzymatic cyclization. Thismethod can serve as a new source of small cyclic peptidemolecules with altered or improved pharmacological activities.Since these enzyme catalysts are valuable tools for the synthe-sis of cyclic molecules, future research efforts also will concen-trate on in vitro protein evolution to generate custom-madecatalysts for cyclization of a given peptide sequence.

ACKNOWLEDGMENTS

This work was supported by Studienstiftung des Deutschen Volkes(SAS) and Deutsche Forschungsgemeinschaft (MAM).

REFERENCES

1. Belshaw, P. J., C. T. Walsh, and T. Stachelhaus. 1999. Aminoacyl-CoAs asprobes of condensation domain selectivity in nonribosomal peptide synthe-sis. Science 284:486–489.

2. Bruner, S. D., T. Weber, R. M. Kohli, D. Schwarzer, M. A. Marahiel, C. T.Walsh, and M. T. Stubbs. 2002. Structural basis for the cyclization of thelipopeptide antibiotic surfactin by the thioesterase domain SrfTE. Structure(Cambridge) 10:301–310.

3. Bu, X., X. Wu, G. Xie, and Z. Guo. 2002. Synthesis of tyrocidine A and itsanalogues by spontaneous cyclization in aqueous solution. Org. Lett. 4:2893–2895.

4. Conti, E., T. Stachelhaus, M. A. Marahiel, and P. Brick. 1997. Structuralbasis for the activation of phenylalanine in the non-ribosomal biosynthesis ofgramicidin S. EMBO J. 16:4174–4183.

5. Craik, D. J., N. L. Daly, I. Saska, M. Trabi, and K. J. Rosengren. 2003.Structures of naturally occurring circular proteins from bacteria. J. Bacteriol.185:4011–4021.

6. de Crecy-Lagard, V., W. Saurin, D. Thibaut, P. Gil, L. Naudin, J. Crouzet,and V. Blanc. 1997. Streptogramin B biosynthesis in Streptomyces pristi-naespiralis and Streptomyces virginiae: molecular characterization of the laststructural peptide synthetase gene. Antimicrob. Agents Chemother.41:1904–1909.

7. Dieckmann, R., Y. O. Lee, H. van Liempt, H. von Dohren, and H. Kleinkauf.1995. Expression of an active adenylate-forming domain of peptide syntheta-ses corresponding to acyl-CoA-synthetases. FEBS Lett. 357:212–216.

8. Duitman, E. H., 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. 1999. The mycosubtilin synthetase of Bacillus subtilisATCC6633: a multifunctional hybrid between a peptide synthetase, anamino transferase, and a fatty acid synthase. Proc. Natl. Acad. Sci. USA96:13294–13299.

9. Gehring, A. M., I. Mori, and C. T. Walsh. 1998. Reconstitution and charac-terization of the Escherichia coli enterobactin synthetase from EntB, EntE,and EntF. Biochemistry 37:2648–2659.

10. Hubbard, B. K., and C. T. Walsh. 2003. Vancomycin assembly: nature’s way.Angew. Chem. Int. Ed. Engl. 42:730–765.

11. Keating, T. A., C. G. Marshall, C. T. Walsh, and A. E. Keating. 2002. Thestructure of VibH represents nonribosomal peptide synthetase condensa-tion, cyclization and epimerization domains. Nat. Struct. Biol. 9:522–526.

12. Kohli, R. M., J. W. Trauger, D. Schwarzer, M. A. Marahiel, and C. T. Walsh.2001. Generality of peptide cyclization catalyzed by isolated thioesterasedomains of nonribosomal peptide synthetases. Biochemistry 40:7099–7108.

13. Kohli, R. M., C. T. Walsh, and M. D. Burkart. 2002. Biomimetic synthesisand optimization of cyclic peptide antibiotics. Nature 418:658–661.

14. Konz, D., A. Klens, K. Schorgendorfer, and M. A. Marahiel. 1997. Thebacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molec-ular characterization of three multi-modular peptide synthetases. Chem.Biol. 4:927–937.

15. Lambalot, R. H., A. M. Gehring, R. S. Flugel, P. Zuber, M. LaCelle, M. A.Marahiel, R. Reid, C. Khosla, and C. T. Walsh. 1996. A new enzyme super-family—the phosphopantetheinyl transferases. Chem. Biol. 3:923–936.

16. Linne, U., and M. A. Marahiel. 2000. Control of directionality in nonribo-somal peptide synthesis: role of the condensation domain in preventingmisinitiation and timing of epimerization. Biochemistry 39:10439–10447.

17. Lipmann, F., W. Gevers, H. Kleinkauf, and R. Roskoski, Jr. 1971. Polypep-tide synthesis on protein templates: the enzymatic synthesis of gramicidin Sand tyrocidine. Adv. Enzymol. Relat. Areas Mol. Biol. 35:1–34.

18. Marahiel, M. A., T. Stachelhaus, and H. D. Mootz. 1997. Modular peptidesynthetases involved in non-ribosomal peptide synthesis. Chem. Rev. 97:2651–2673.

19. May, J. J., T. M. Wendrich, and M. A. Marahiel. 2001. The dhb operon ofBacillus subtilis encodes the biosynthetic template for the catecholic sid-erophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibac-tin. J. Biol. Chem. 276:7209–7217.

20. Mootz, H. D., D. Schwarzer, and M. A. Marahiel. 2002. Ways of assemblingcomplex natural products on modular nonribosomal peptide synthetases.Chembiochem 3:490–504.

21. Peypoux, F., J. M. Bonmatin, and J. Wallach. 1999. Recent trends in thebiochemistry of surfactin. Appl. Microbiol. Biotechnol. 51:553–563.

22. Reuter, K., M. R. Mofid, M. A. Marahiel, and R. Ficner. 1999. Crystalstructure of the surfactin synthetase-activating enzyme Sfp: a prototype ofthe 4�-phosphopantetheinyl transferase superfamily. EMBO J. 18:6823–6831.

23. Scholz-Schroeder, B. K., J. D. Soule, and D. C. Gross. 2003. The sypA, sypS,and sypC synthetase genes encode twenty-two modules involved in the non-ribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv.syringae B301D. Mol. Plant-Microbe Interact. 16:271–280.

24. Scholz-Schroeder, B. K., J. D. Soule, S. E. Lu, I. Grgurina, and D. C. Gross.2001. A physical map of the syringomycin and syringopeptin gene clusterslocalized to an approximately 145-kb DNA region of Pseudomonas syringaepv. syringae strain B301D. Mol. Plant-Microbe Interact. 14:1426–1435.

25. Schwarzer, D., R. Finking, and M. A. Marahiel. 2003. Nonribosomal pep-tides: from genes to products. Nat. Prod. Rep. 20:275–287.

26. Segre, A., R. C. Bachmann, A. Ballio, F. Bossa, I. Grgurina, N. S. Iacobellis,G. Marino, P. Pucci, M. Simmaco, and J. Y. Takemoto. 1989. The structureof syringomycins A1, E and G. FEBS Lett. 255:27–31.

27. Shaw-Reid, C. A., N. L. Kelleher, H. C. Losey, A. M. Gehring, C. Berg, andC. T. Walsh. 1999. Assembly line enzymology by multimodular nonribosomalpeptide synthetases: the thioesterase domain of E. coli EntF catalyzes bothelongation and cyclolactonization. Chem. Biol. 6:385–400.

28. Sieber, S. A., C. T. Walsh, and M. A. Marahiel. 2003. Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in character-izing macrocyclization by thioesterase domains. J. Am. Chem. Soc. 125:10862–10866.

29. Stachelhaus, T., A. Huser, and M. A. Marahiel. 1996. Biochemical charac-terization of peptidyl carrier protein (PCP), the thiolation domain of mul-tifunctional peptide synthetases. Chem. Biol. 3:913–921.

30. Trauger, J., R. Kohli, H. Mootz, M. Marahiel, and C. Walsh. 2000. Peptidecyclization catalysed by the thioesterase domain of tyrocidine synthetase.Nature 407:215–218.

31. Trauger, J. W., R. M. Kohli, and C. T. Walsh. 2001. Cyclization of backbone-substituted peptides catalyzed by the thioesterase domain from the tyroci-dine nonribosomal peptide synthetase. Biochemistry 40:7092–7098.

32. Tseng, C. C., S. D. Bruner, R. M. Kohli, M. A. Marahiel, C. T. Walsh, andS. A. Sieber. 2002. Characterization of the surfactin synthetase C-terminalthioesterase domain as a cyclic depsipeptide synthase. Biochemistry41:13350–13359.

33. Vanittanakom, N., W. Loeffler, U. Koch, and G. Jung. 1986. Fengycin—anovel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29–3.J. Antibiot. (Tokyo) 39:888–901.

34. Walsh, C. T. 2003. Antibiotics: actions, origins, and resistance, p. 195–220.ASM Press, Washington, D.C.

35. Weber, G., and E. Leitner. 1994. Disruption of the cyclosporin synthetasegene of Tolypocladium niveum. Curr. Genet. 26:461–467.

36. Weber, T., R. Baumgartner, C. Renner, M. A. Marahiel, and T. A. Holak.2000. Solution structure of PCP, a prototype for the peptidyl carrier domainsof modular peptide synthetases. Struct. Fold Des. 8:407–418.

VOL. 185, 2003 MINIREVIEW 7043

on Decem

ber 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from