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Mohamed A. Marahiel - One of the best experts on this subject based on the ideXlab platform.
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Chapter 13. Nonribosomal Peptide synthetases mechanistic and structural aspects of essential domains.
Methods in Enzymology, 2020Co-Authors: Mohamed A. Marahiel, Lars-oliver EssenAbstract:A widespread class of therapeutically important natural products is of peptidic origin. They are produced Nonribosomally by large “assembly line”‐like multienzyme complexes, the Nonribosomal Peptide synthetases (NRPS). In contrast to ribosomal Peptide Synthesis, Nonribosomally assembled Peptides contain unique structural features such as d‐amino acids, N‐terminally attached fatty acid chains, N‐ and C‐methylated amino acids, N‐formylated residues, heterocyclic elements, glycosylated amino acids, and phosphorylated residues. In recent research using genetic, biochemical, and structural methods, experiments have revealed profound insights into the molecular mechanism of Nonribosomal Peptide Synthesis. Based on this, it was possible to alter existing Nonribosomally produced Peptides either by changing their biosynthetic templates or by the combined action of chemical Peptide Synthesis and subsequent enzyme catalysis. An overview of the structural aspects of the NRPS machinery with a focus on mechanistic and structural aspects of essential domains is presented.
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Mutational analysis of the C‐domain in Nonribosomal Peptide Synthesis
FEBS Journal, 2020Co-Authors: Veit Bergendahl, Uwe Linne, Mohamed A. MarahielAbstract:The initial condensation event in the Nonribosomal bioSynthesis of the Peptide antibiotics gramicidin S and tyrocidine␣A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a Peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and Peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in Peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-α-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP Synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by Peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.
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impact of epimerization domains on the intermodular transfer of enzyme bound intermediates in Nonribosomal Peptide Synthesis
ChemBioChem, 2006Co-Authors: Daniel B Stein, Uwe Linne, Martin Hahn, Mohamed A. MarahielAbstract:Abstract Assembly of bioactive natural compounds through the action of Nonribosomal Peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of Peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of Nonribosomal Peptides.
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Formylation Domain: An Essential Modifying Enzyme for the Nonribosomal BioSynthesis of Linear Gramicidin
Journal of the American Chemical Society, 2006Co-Authors: Georg Schoenafinger, Uwe Linne, Nadine Schracke, Mohamed A. MarahielAbstract:Formylation is an important part of ribosomal Peptide Synthesis of prokaryotes. In Nonribosomal Peptide Synthesis, however, N-formylation is rather unusual and therefore so far unexplored. In this work, the first module of the linear gramicidin Nonribosomal Peptide synthetase, LgrA1, consisting of a hypothetical formylation domain, an adenylation, and a peptidyl carrier protein domain was tested for formyltransferase activity in vitro. We demonstrate here that the putative formylation domain does indeed transfer the formyl group of formyltetrahydrofolate (fH4F) onto the first amino acid valine using both cofactors N10- and N5-fH4F, respectively. Most important, the necessity of the formylated starter unit formyl−valine for the initiation of the gramicidin bioSynthesis was tested by elongation assays with the bimodular system from LgrA. By omitting the formyl group donor, no condensation product of valine with the subsequent building block glycine was detected, whereas the diPeptide formyl−valyl−glycine wa...
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mutational analysis of a type ii thioesterase associated with Nonribosomal Peptide Synthesis
FEBS Journal, 2004Co-Authors: Uwe Linne, Dirk Schwarzer, Gunnar N Schroeder, Mohamed A. MarahielAbstract:Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and Nonribosomal Peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipoPeptide antibiotic surfactin (TEIIsrf), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEIIsrf were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEIIsrf belongs to the α/β-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a Nonribosomal Peptide synthetase TEII.
Uwe Linne - One of the best experts on this subject based on the ideXlab platform.
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Mutational analysis of the C‐domain in Nonribosomal Peptide Synthesis
FEBS Journal, 2020Co-Authors: Veit Bergendahl, Uwe Linne, Mohamed A. MarahielAbstract:The initial condensation event in the Nonribosomal bioSynthesis of the Peptide antibiotics gramicidin S and tyrocidine␣A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a Peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and Peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in Peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-α-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP Synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by Peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.
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impact of epimerization domains on the intermodular transfer of enzyme bound intermediates in Nonribosomal Peptide Synthesis
ChemBioChem, 2006Co-Authors: Daniel B Stein, Uwe Linne, Martin Hahn, Mohamed A. MarahielAbstract:Abstract Assembly of bioactive natural compounds through the action of Nonribosomal Peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of Peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of Nonribosomal Peptides.
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Formylation Domain: An Essential Modifying Enzyme for the Nonribosomal BioSynthesis of Linear Gramicidin
Journal of the American Chemical Society, 2006Co-Authors: Georg Schoenafinger, Uwe Linne, Nadine Schracke, Mohamed A. MarahielAbstract:Formylation is an important part of ribosomal Peptide Synthesis of prokaryotes. In Nonribosomal Peptide Synthesis, however, N-formylation is rather unusual and therefore so far unexplored. In this work, the first module of the linear gramicidin Nonribosomal Peptide synthetase, LgrA1, consisting of a hypothetical formylation domain, an adenylation, and a peptidyl carrier protein domain was tested for formyltransferase activity in vitro. We demonstrate here that the putative formylation domain does indeed transfer the formyl group of formyltetrahydrofolate (fH4F) onto the first amino acid valine using both cofactors N10- and N5-fH4F, respectively. Most important, the necessity of the formylated starter unit formyl−valine for the initiation of the gramicidin bioSynthesis was tested by elongation assays with the bimodular system from LgrA. By omitting the formyl group donor, no condensation product of valine with the subsequent building block glycine was detected, whereas the diPeptide formyl−valyl−glycine wa...
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mutational analysis of a type ii thioesterase associated with Nonribosomal Peptide Synthesis
FEBS Journal, 2004Co-Authors: Uwe Linne, Dirk Schwarzer, Gunnar N Schroeder, Mohamed A. MarahielAbstract:Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S- phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and Nonribosomal Peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipoPeptide antibiotic surfactin (TEIIsrf), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEIIsrf were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEIIsrf belongs to the α/β-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a Nonribosomal Peptide synthetase TEII.
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Regeneration of misprimed Nonribosomal Peptide synthetases by type II thioesterases
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Dirk Schwarzer, Henning D. Mootz, Uwe Linne, Mohamed A. MarahielAbstract:Nonribosomal Peptide synthetases (NRPSs) assemble structurally complex Peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in Nonribosomal Peptide Synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the Peptide antibiotics surfactin (TEIIsrf) and bacitracin (TEIIbac). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4′-phosphopantetheine (4′PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEIIsrf, a KM of 0.9 μM and a kcat of 95 min−1 was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of Nonribosomal Peptide Synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid Synthesis were also not significantly hydrolyzed, as investigated with TEIIsrf. These findings support a model in which the physiological role of TEIIs in Nonribosomal Peptide Synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4′PP transferases that use not only free CoA, but also acyl-CoAs as 4′PP donors.
Brett A Neilan - One of the best experts on this subject based on the ideXlab platform.
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cereulide the emetic toxin of bacillus cereus is putatively a product of Nonribosomal Peptide Synthesis
Journal of Applied Microbiology, 2004Co-Authors: Michelle C Moffitt, L Henrichsen, Mark J Raftery, Kevin D Barrow, Christopher P Marquis, Brett A NeilanAbstract:Aims: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a Nonribosomal Peptide synthetase (NRPS). Methods and Results: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to Peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. Conclusions: The results indicate that cereulide is produced by a NRPS complex. Significance and Impact of the Study: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.
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Nonribosomal Peptide Synthesis and toxigenicity of cyanobacteria
Journal of Bacteriology, 1999Co-Authors: Brett A Neilan, Elke Dittmann, Leo Rouhiainen, Amanda R Bass, Verena Schaub, Kaarina Sivonen, Thomas BornerAbstract:Nonribosomal Peptide Synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as Peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial bioSynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546–1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69–72, 1995). Most Nonribosomal Peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and Nonribosomal Peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of Nonribosomal Peptide Synthesis in diverse species of cyanobacteria. Amplification of Peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of Peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The Nonribosomal Peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.
Dennis C Gross - One of the best experts on this subject based on the ideXlab platform.
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the sypa sypb and sypc synthetase genes encode twenty two modules involved in the Nonribosomal Peptide Synthesis of syringopeptin by pseudomonas syringae pv syringae b301d
Molecular Plant-microbe Interactions, 2003Co-Authors: Brenda K Scholzschroeder, Jonathan D Soule, Dennis C GrossAbstract:Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to Peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each...
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Analysis of the syrB and syrC genes of Pseudomonas syringae pv. syringae indicates that syringomycin is synthesized by a thiotemplate mechanism.
Journal of Bacteriology, 1995Co-Authors: Jian-hua Zhang, Neil B. Quigley, Dennis C GrossAbstract:The syrB and syrC genes are required for Synthesis of syringomycin, a lipodepsiPeptide phytotoxin produced by Pseudomonas syringae pv. syringae, and are induced by plant-derived signal molecules. A 4,842-bp chromosomal region containing the syrB and syrC genes of strain B301D was sequenced and characterized. The open reading frame (ORF) of syrB was 2,847 bp in length and was predicted to encode an approximately 105-kDa protein, SyrB, with 949 amino acids. Searches of databases revealed that SyrB shares homology with members of a superfamily of adenylate-forming enzymes involved in Peptide antibiotic and siderophore Synthesis in a diverse spectrum of microorganisms. SyrB exhibited the highest degree of overall similarity (56.4%) and identity (33.8%) with the first amino acid-activating domain of pyoverdin synthetase, PvdD, of Pseudomonas aeruginosa. The N-terminal portion of SyrB contained a domain of approximately 600 amino acids that resembles the amino acid-activating domains of thiotemplate-employing Peptide synthetases. The SyrB domain contained six signature core sequences with the same order and spacing as observed in all known amino acid-activating domains involved in Nonribosomal Peptide Synthesis. Core sequence 6 of SyrB, for example, was similar to the binding site for 4'-phosphopantetheine, a cofactor required for thioester formation. The syrC ORF (1,299 bp) was located 175 bp downstream of the syrB ORF. Analysis of the transcriptional and translational relationship between the syrB and syrC genes demonstrated that they are expressed independently. The syrC ORF was predicted to encode an approximately 48-kDa protein product of 433 amino acids which is 42 to 48% similar to a number of thioesterases, including fatty acid thioesterases, haloperoxidases, and acyltransferases, that contain a characteristic GXS (C) XG motif. In addition, a zinc-binding motif was found near the C terminus of SyrC. The data suggest that SyrB and SyrC function as Peptide synthetases in a thiotemplate mechanism of syringomycin bioSynthesis.
Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N-Hydroxylase.
Biochemistry, 2014Co-Authors: Jeremy W. Setser, Christopher T Walsh, John R. Heemstra, Catherine L. DrennanAbstract:The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsiPeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via Nonribosomal Peptide Synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor....
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a protein interaction surface in Nonribosomal Peptide Synthesis mapped by combinatorial mutagenesis and selection
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Michael A Fischbach, Christopher T WalshAbstract:Nonribosomal Peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS bioSynthesis.
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dissection of the entf condensation domain boundary and active site residues in Nonribosomal Peptide Synthesis
Biochemistry, 2003Co-Authors: Eric D Roche, Christopher T WalshAbstract:Nonribosomal Peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively pro...
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in vitro reconstitution of the pseudomonas aeruginosa Nonribosomal Peptide Synthesis of pyochelin characterization of backbone tailoring thiazoline reductase and n methyltransferase activities
Biochemistry, 2001Co-Authors: Hiten M Patel, Christopher T WalshAbstract:During iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosamakes the Nonribosomal Peptide siderophore pyochelin by a four protein, 11 domain assembly line, involving a cascade of acyl-S-enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an NADPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzyme, regiospecifically converting one of the dihy- droheterocyclic thiazoline rings to a thiazolidine. The Km for the PchG protein is 1 IM, and the kcat for throughput to pyochelin is 2 min -1 . The nitrogen of the newly generated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl- S-PchF at the PCP domain. A presumed methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min -1 , at least 30-40-fold faster than in the absence of hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-enzyme. Thus, full reconstitution of the Nonribosomal Peptide synthetase assembly line by purified protein components has been obtained for production of this tandem bisheterocyclic siderophore.
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aminoacyl coas as probes of condensation domain selectivity in Nonribosomal Peptide Synthesis
Science, 1999Co-Authors: Peter J Belshaw, Christopher T Walsh, Torsten StachelhausAbstract:In Nonribosomal bioSynthesis of Peptide antibiotics by multimodular synthetases, amino acid monomers are activated by the adenylation domains of the synthetase and loaded onto the adjacent carrier protein domains as thioesters, then the formation of Peptide bonds and translocation of the growing chain are effected by the synthetase9s condensation domains. Whether the condensation domains have any editing function has been unknown. Synthesis of aminoacyl–coenzyme A (CoA) molecules and direct enzymatic transfer of aminoacyl-phosphopantetheine to the carrier domains allow the adenylation domain editing function to be bypassed. This method was used to demonstrate that the first condensation domain of tyrocidine synthetase shows low selectivity at the donor residue (d-phenylalanine) and higher selectivity at the acceptor residue (l-proline) in the formation of the chain-initiating d-Phe-l-Pro dipeptidyl-enzyme intermediate.
