Peptidyl Carrier Protein Domain Research Articles

Domain Structure Characterization of the Multifunctional α-Aminoadipate Reductase from Penicillium chrysogenum by Limited Proteolysis: ACTIVATION OF α-AMINOADIPATE DOES NOT REQUIRE THE PEPTIDYL CARRIER PROTEIN BOX OR THE REDUCTION DOMAIN

The alpha-aminoadipate reductase (alpha-AAR) of Penicillium chrysogenum, an enzyme that activates the alpha-aminoadipic acid by forming an alpha-aminoadipyl adenylate and reduces the activated intermediate to alpha-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for alpha-aminoadipic acid, ATP, and NADPH were determined. Sequencing of the N-terminal end confirmed the 10 first amino acids deduced from the nucleotide sequence. Its domain structure has been investigated using limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within the primary structure was established by N-terminal sequence analysis. Initial proteolysis generated two fragments: an N-terminal fragment housing the adenylation and the peptidyl carrier protein (PCP) domains (116 kDa) and a second fragment containing most of the reductive domain (28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated. Time-dependent acylation of alpha-AAR and of fragments containing the adenylation domain with tritiated alpha-aminoadipate occurred in vitro in the absence of NADPH. Addition of NADPH to the labeled alpha-AAR released most of the radioactive substrate. A fragment containing the adenylation domain was labeled even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the stability of the acyl intermediate. Low intensity direct acylation of the PCP box has also been observed. A domain structure of this multienzyme is proposed.

The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI.

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia. Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase. Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera. We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products. In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role. It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.

Structural Basis for the Cyclization of the Lipopeptide Antibiotic Surfactin by the Thioesterase Domain SrfTE

Many biologically active natural peptides are synthesized by nonribosomal peptide synthetases (NRPS). Product release is accomplished by dedicated thioesterase (TE) domains, some of which catalyze an intramolecular cyclization to form macrolactone or macrolactam cyclic peptides. The excised 28 kDa SrfTE domain, a member of the α/β hydrolase enzyme family, exhibits a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and tolerates its folding to form a cyclic structure. A substrate analog confirms the substrate binding site and suggests a mechanism for substrate acylation/deacylation. Docking of the peptidyl carrier protein domain immediately preceding SrfTE positions the 4′-phosphopantheinyl prosthetic group that transfers the nascent acyl-peptide chain to SrfTE. The structure provides a basis for understanding the mechanism of acyl-PCP substrate recognition and for the cyclization reaction that results in release of the macrolactone cyclic heptapeptide.

Conversion of L-Proline to Pyrrolyl-2-Carboxyl- S-PCP during Undecylprodigiosin and Pyoluteorin Biosynthesis

Several medically and agriculturally important natural products contain pyrrole moieties. Precursor labeling studies of some of these natural products have shown that L-proline can serve as the biosynthetic precursor for these moieties, including those found in coumermycin A 1, pyoluteorin, and one of the pyrroles of undecylprodigiosin. This suggests a novel mechanism for pyrrole biosynthesis. The biosynthetic gene clusters for these three natural products each encode proteins homologous to adenylation (A) and peptidyl carrier protein (PCP) domains of nonribosomal peptide synthetases in addition to novel acyl-CoA dehydrogenases. Here we show that the three proteins from the undecylprodigiosin and pyoluteorin biosynthetic pathways are sufficient for the conversion of L-proline to pyrrolyl-2-carboxyl- S-PCP. This establishes a novel mechanism for pyrrole biosynthesis and extends the hypothesis that organisms use A/PCP pairs to partition an amino acid into secondary metabolism.

Epothilone biosynthesis: assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit

Background: Polyketides (PKs) and non-ribosomal peptides (NRPs) are therapeutically important natural products biosynthesized by multimodular protein assembly lines, termed the PK synthases (PKSs) and NRP synthetases (NRPSs), via a similar thiotemplate-mediated mechanism. The potential for productive interaction between these two parallel enzymatic systems has recently been demonstrated, with the discovery that PK/NRP hybrid natural products can be of great therapeutic importance. One newly discovered PK/NRP product, epothilone D from Sorangium cellulosum, has shown great potential as an anti-tumor agent. Results: The chain-initiating methylthiazole ring of epothilone has been generated in vitro as an acyl- S-enzyme intermediate, using five domains from two modules of the polymodular epothilone synthetase. The acyl carrier protein (ACP) domain, excised from the EpoA gene, was expressed in Escherichia coli, purified as an apo protein, and then post-translationally primed with acetyl-CoA using the phosphopantetheinyl transferase enzyme Sfp. The four-domain 150-kDa EpoB subunit (cyclization–adenylation–oxidase–peptidyl carrier protein domains: Cy–A–Ox–PCP) was also expressed and purified in soluble form from E. coli. Post-translational modification with Sfp and CoASH introduced the HS-pantP prosthetic group to the apo-PCP, enabling subsequent loading with L-cysteine to generate the Cys- S-PCP acyl enzyme intermediate. When acetyl- S-ACP (EpoA) and cysteinyl- S-EpoB were mixed, the Cy domain of EpoB catalyzed acetyl transfer from EpoA to the amino group of the Cys- S-EpoB, generating a transient N-Ac-Cys- S-EpoB intermediate that is cyclized and dehydrated to the five-membered ring methylthiazolinyl- S-EpoB. Finally, the FMN-containing Ox domain of EpoB oxidized the dihydro heterocyclic thiazolinyl ring to the heteroaromatic oxidation state, the methylthiazolylcarboxy- S-EpoB. When other acyl-CoAs were substituted for acetyl-CoA in the Sfp-based priming of the apo-CP domain, additional alkylthiazolylcarboxy- S-EpoB acyl enzymes were produced. Conclusions: These experiments establish chain transfer across a PKS and NRPS interface. Transfer of the acetyl group from the ACP domain of EpoA to EpoB reconstitutes the start of the epothilone synthetase assembly line, and installs and converts a cysteine group into a methyl-substituted heterocycle during this natural product chain growth.

Coumarin formation in novobiocin biosynthesis: β-hydroxylation of the aminoacyl enzyme tyrosyl- S-NovH by a cytochrome P450 NovI

Background: Coumarin group antibiotics, such as novobiocin, coumermycin A 1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a β-hydroxy-tyrosine (β-OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. Results: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl- S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl- S-NovH acyl enzyme. The product β-OH-tyrosyl- S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [ 3H]β-OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of β-OH-Tyr. The amino acid released was (3 R,2 S)-3-OH-Tyr by comparison with authentic standards. Conclusions: This work establishes that NovH and NovI are responsible for the formation of a β-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A–PCP/P450 pairs for amino acid β-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A 1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.

The mtaA gene of the myxothiazol biosynthetic gene cluster from Stigmatella aurantiaca DW4/3-1 encodes a phosphopantetheinyl transferase that activates polyketide synthases and polypeptide synthetases.

Myxothiazol is synthesized by the myxobacterium Stigmatella aurantiaca DW4/3-1 via a combined polyketide synthase/polypeptide synthetase. The biosynthesis of this secondary metabolite is also dependent on the gene product of mtaA. The deduced amino acid sequence of mtaA shows similarity to 4'-phosphopantetheinyl transferases (4'-PP transferase). This points to an enzyme activity that converts inactive forms of the acyl carrier protein domains of polyketide synthetases (PKSs) and/or the peptidyl carrier protein domains of nonribosomal polypeptide synthetases (NRPSs) of the myxothiazol synthetase complex to their corresponding holo-forms. Heterologous co-expression of MtaA with an acyl carrier protein domain of the myxothiazol synthetase was performed in Escherichia coli. The proposed function as a 4'-PP transferase was confirmed and emphasizes the significance of MtaA for the formation of a catalytically active myxothiazol synthetase complex. Additionally, it is shown that MtaA has a relaxed substrate specificity: it processes an aryl carrier protein domain derived from the enterobactin synthetase of E. coli (ArCP) as well as a peptidyl carrier protein domain from a polypeptide synthetase of yet unknown function from Sorangium cellulosum. Therefore, MtaA should be a useful tool for activating heterologously expressed PKS and NRPS systems.

Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase.

The 207-kDa polyketide synthase (PKS) module (residues 1-1895) and the 143-kDa nonribosomal peptidyl synthetase (NRPS) module (1896-3163) of the 350-kDa HMWP1 subunit of yersiniabactin synthetase have been expressed in and purified from Escherichia coli in soluble forms to characterize the acyl carrier protein (ACP) domain of the PKS module and the homologous peptidyl carrier protein (PCP(3)) domain of the NRPS module. The apo-ACP and PCP domains could be selectively posttranslationally primed by the E. coli ACPS and EntD phosphopantetheinyl transferases (PPTases), respectively, whereas the Bacillus subtilis PPTase Sfp primed both carrier protein domains in vitro or during in vivo coexpression. The holo-NRPS module but not the holo-PKS module was then selectively aminoacylated with cysteine by the adenylation domain embedded in the HMWP2 subunit of yersiniabactin synthetase, acting in trans. When the acyltransferase (AT) domain of HMWP1 was analyzed for its ability to malonylate the holo carrier protein domains, in cis acylation was first detected. Then, in trans malonylation of the excised holo-ACP or holo-PCP(3)-TE fragments by HMWP1 showed both were malonylated with a 3:1 catalytic efficiency ratio, showing a promiscuity to the AT domain.

Control of directionality in nonribosomal peptide synthesis: role of the condensation domain in preventing misinitiation and timing of epimerization.

Product assembly by nonribosomal peptide synthetases (NRPS) is initiated by starter modules that comprise an adenylation (A) and a peptidyl carrier protein (PCP) domain. Elongation modules of NRPS have in addition a condensation (C) domain that is located upstream of the A domain. They cannot initiate peptide bond formation. To understand the role of domain arrangements and the influence of the domains present upstream of the A domains of the elongation modules of TycB on the initiation and epimerization activities, we constructed a set of proteins derived from the tyrocidine synthetases of Bacillus brevis, which represent several N-terminal truncations of TycB and the first module of TycC. The latter was fused with the thioesterase domain (Te) to give TycC(1)-CAT-Te and to ensure product turnover. TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, lacking an N-terminal C domain, were capable of initiating peptide synthesis and epimerizing. In contrast, the corresponding constructs with a cognate N-terminal C domain, TycB(2)(-)(3)-T.CATE and TycB(3)-CATE, were strongly reduced in initiation and epimerization. Evidence is also provided that this reduction is due to substrate binding in an enantioselective binding pocket at the acceptor position of the C domains. By using TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, we were able to turn an elongation module into an initiation module, and to establish an in-trans system for the formation of new di- and tripeptides with recombinant NRPS modules. We also show that epimerization domains of elongation modules can in principle epimerize both aminoacyl-S-Ppant (TycB(3)-ATE) and peptidyl-S-Ppant (TycB(2)(-)(3)-AT.CATE) substrates, although the efficiency for epimerizing the noncognate aminoacyl-S-Ppant substrates appears to be lowered.

Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic.

The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with l-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with l-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins.

Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with l-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with l-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins.

The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates.

Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) and thioesterase protein, EntF PCP-TE. The adenylation domain activity of EntF C-A formed seryl-AMP but lost the ability to transfer the seryl moiety to the cognate EntF PCP-TE in trans. Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase, was observed at rates of 0.5 min(-1) and 0.01 min(-1), respectively. The possibility that these slow acylation rates reflected dissociation of acyl/aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PP(i)) released from the adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PP(i) release from EntF C-A or EntE. In this light, aminoacylations in trans between A and PCP domain fragments of NRPS assembly lines must be subjected to kinetic scrutiny to determine whether transfer is truly between protein domains or results from slow aminoacyl-AMP release and subsequent nonenzymatic thiol capture.

Selectivity of the yersiniabactin synthetase adenylation domain in the two-step process of amino acid activation and transfer to a holo-carrier protein domain.

The adenylation (A) domain of the Yersinia pestis nonribosomal peptide synthetase that biosynthesizes the siderophore yersiniabactin (Ybt) activates three molecules of L-cysteine and covalently aminoacylates the phosphopantetheinyl (P-pant) thiols on three peptidyl carrier protein (PCP) domains embedded in the two synthetase subunits, two in cis (PCP1, PCP2) in subunit HMWP2 and one in trans (PCP3) in subunit HMWP1. This two-step process of activation and loading by the A domain is analogous to the operation of the aminoacyl-tRNA synthetases in ribosomal peptide synthesis. Adenylation domain specificity for the first step of reversible aminoacyl adenylate formation was assessed with the amino acid-dependent [(32)P]-PP(i)-ATP exchange assay to show that S-2-aminobutyrate and beta-chloro-L-alanine were alternate substrates. The second step of A domain catalysis, capture of the bound aminoacyl adenylate by the P-pant-SH of the PCP domains, was assayed both by catalytic release of PP(i) and by covalent aminoacylation of radiolabeled substrates on either the PCP1 fragment of HMWP2 or the PCP3-thioesterase double domain fragment of HMWP1. There was little selectivity for capture of each of the three adenylates by PCP3 in the second step, arguing against any hydrolytic proofreading of incorrect substrates by the A domain. The holo-PCP3 domain accelerated PP(i) release and catalytic turnover by 100-200-fold over the leak rate (<1 min(-1)) of aminoacyl adenylates into solution while PCP1 in trans had only about a 5-fold effect. Free pantetheine could capture cysteinyl adenylate with a 25-50-fold increase in k(cat) while CoA was 10-fold less effective. The K(m) of free pantetheine (30-50 mM) was 3 orders of magnitude larger than that of PCP3-TE (10-25 microM), indicating a net 10(4) greater catalytic efficiency for transfer to the P-pant arm of PCP3 by the Ybt synthetase A domain, relative to P-pant alone.

Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily

The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4'-phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non-ribosomal peptide synthesis. The structure of the Sfp-CoA complex determined at 1.8 A resolution reveals a novel alpha/beta-fold exhibiting an unexpected intramolecular 2-fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA-protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

Structure/Function of Medium Chain Acyl-CoA Dehydrogenase: The Importance of Substrate Polarization

Several medically and agriculturally important natural products contain pyrrole moieties. Precursor labeling studies of some of these natural products have shown that L-proline can serve as the biosynthetic precursor for these moieties, including those found in coumermycin A1, pyoluteorin, and one of the pyrroles of undecylprodigiosin. This suggests a novel mechanism for pyrrole biosynthesis. The biosynthetic gene clusters for these three natural products each encode proteins homologous to adenylation (A) and peptidyl carrier protein (PCP) domains of nonribosomal peptide synthetases in addition to novel acyl-CoA dehydrogenases. Here we show that the three proteins from the undecylprodigiosin and pyoluteorin biosynthetic pathways are sufficient for the conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP. This establishes a novel mechanism for pyrrole biosynthesis and extends the hypothesis that organisms use A/PCP pairs to partition an amino acid into secondary metabolism.

Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003

Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a structurally diverse group of peptides by the multiple-carrier thiotemplate mechanism. All NRPSs known to date are exclusively type I modular enzymes that consist of domains, such as adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains, for individual enzyme activities. Although several A and PCP domains have been demonstrated to function independently, aminoacylation in trans has been successful only between PCPs and their cognate A domains. We have identified within the bleomycin-biosynthesis gene cluster from Streptomyces verticillus ATCC15003 the blmI gene that encodes a discrete PCP protein. We overexpressed the blmI gene in Escherichia coli, purified the BlmI protein, and demonstrated that apo-BlmI can be efficiently modified into holo-BlmI either in vivo or in vitro by PCP-specific 4'-phosphopantetheine transferases (PPTases). Unlike the PCP domains in type I NRPSs, BlmI lacks its cognate A domain and can be aminoacylated by Val-A, an A domain from a completely unrelated type I NRPS. BlmI represents the first characterized type II PCP. The BlmI type II PCP, like the PCP domains of type I NRPSs, can be 4'-phospho-pantetheinylated by PCP-specific PPTases but is biochemically distinct in that it can be aminoacylated by an A domain from a completely unrelated type I NRPS. Our results provide for the first time the genetic and biochemical evidence to support the existence of a type II NRPS, which might be useful in the combinatorial manipulation of NRPS proteins to generate novel peptides.

Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases.

The Bacillus subtilis Sfp protein is required for the non-ribosomal biosynthesis of the lipoheptapeptide antibiotic surfactin. It converts seven peptidyl carrier protein (PCP) domains of the surfactin synthetase SfrA-(A-C) to their active holo-forms by 4'-phosphopantetheinylation. The B. subtilis sfp gene was overexpressed in Escherichia coli and its gene product was purified to homogeneity and crystallized. Well diffracting single crystals were obtained from Sfp as well as from a selenomethionyl derivative, using sodium formate as a precipitant. The crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = b = 65.3, c = 150.5 A. They diffract beyond 2.8 A and contain one molecule in the asymmetric unit.

The nonribosomal peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of yersiniabactin, an iron-chelating virulence factor of yersinia pestis

Pathogenic Yersinia species have been shown to synthesize a siderophore molecule, yersiniabactin, as a virulence factor during iron starvation. Here we provide the first biochemical evidence for the role of the Yersinia pestis high molecular weight protein 2 (HMWP2), a nonribosomal peptide synthetase homologue, and YbtE in the initiation of yersiniabactin biosynthesis. YbtE catalyzes the adenylation of salicylate and the transfer of this activated salicyl group to the N-terminal aryl carrier protein domain (ArCP; residues 1-100) of HMWP2. A fragment of HMWP2, residues 1-1491, can adenylate cysteine and with the resulting cysteinyl-AMP autoaminoacylate the peptidyl carrier protein domain (PCP1; residues 1383-1491) either in cis or in trans. Catalytic release of hydroxyphenylthiazoline carboxylic acid (HPT-COOH) and/or N-(hydroxyphenylthiazolinylcarbonyl)cysteine (HPT-cys) is observed upon incubation of YbtE, HMWP2 1-1491, L-cysteine, salicylate, and ATP. These products presumably arise from nucleophilic attack by water or cysteine of a stoichiometric hydroxyphenylthiazolinylcarbonyl-S-PCP1-HMWP2 intermediate. Detection of the heterocyclization capacity of HMWP2 1-1491 implies salicyl-transferring and thiazoline-forming activity for the HMWP2 condensation domain (residues 101-544) and is the first demonstration of such heterocyclization ability in a nonribosomal peptide synthetase enzyme.

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis

Background: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. Results: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions ( irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. Conclusions: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

The nonribosomal peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of yersiniabactin, an iron-chelating virulence factor of Yersinia pestis.

Pathogenic Yersinia species have been shown to synthesize a siderophore molecule, yersiniabactin, as a virulence factor during iron starvation. Here we provide the first biochemical evidence for the role of the Yersinia pestis high molecular weight protein 2 (HMWP2), a nonribosomal peptide synthetase homologue, and YbtE in the initiation of yersiniabactin biosynthesis. YbtE catalyzes the adenylation of salicylate and the transfer of this activated salicyl group to the N-terminal aryl carrier protein domain (ArCP; residues 1-100) of HMWP2. A fragment of HMWP2, residues 1-1491, can adenylate cysteine and with the resulting cysteinyl-AMP autoaminoacylate the peptidyl carrier protein domain (PCP1; residues 1383-1491) either in cis or in trans. Catalytic release of hydroxyphenylthiazoline carboxylic acid (HPT-COOH) and/or N-(hydroxyphenylthiazolinylcarbonyl)cysteine (HPT-cys) is observed upon incubation of YbtE, HMWP2 1-1491, L-cysteine, salicylate, and ATP. These products presumably arise from nucleophilic attack by water or cysteine of a stoichiometric hydroxyphenylthiazolinylcarbonyl-S-PCP1-HMWP2 intermediate. Detection of the heterocyclization capacity of HMWP2 1-1491 implies salicyl-transferring and thiazoline-forming activity for the HMWP2 condensation domain (residues 101-544) and is the first demonstration of such heterocyclization ability in a nonribosomal peptide synthetase enzyme.