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Yi Tang - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of long chain n acyl amide by a truncated polyketide synthase Nonribosomal Peptide Synthetase hybrid megasynthase in fungi
Journal of the American Chemical Society, 2018Co-Authors: Yang Hai, Yi TangAbstract:Truncated iterative polyketide synthase–Nonribosomal Peptide Synthetase (PKS-NRPS) megasynthases in which only the C domain is present are widespread in fungi, yet nearly all members have unknown functions. Bioinformatics analysis showed that the C domains of such PKS-C enzymes are noncanonical due to substitution at the second histidine in the active site HHxxxDG motif. Here, we used genome mining strategy to characterize a cryptic PKS-C hybrid from Talaromyces wortmanii and discovered the products are reduced long-chain polyketides amidated with a specific ω-amino acid 5-aminopentanoic acid (5PA). The wortmanamides resemble long-chain N-acyl-amide signaling lipids that target diverse receptors including GPCRs. The noncanonical C domain of this PKS-C hybrid was also demonstrated to be a bona fide condensation domain that specifically selects 5PA and catalyzes amidation to release polyketide chain.
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Biosynthesis of Long-Chain N‑Acyl Amide by a Truncated Polyketide Synthase–Nonribosomal Peptide Synthetase Hybrid Megasynthase in Fungi
2018Co-Authors: Yang Hai, Yi TangAbstract:Truncated iterative polyketide synthase–Nonribosomal Peptide Synthetase (PKS-NRPS) megasynthases in which only the C domain is present are widespread in fungi, yet nearly all members have unknown functions. Bioinformatics analysis showed that the C domains of such PKS-C enzymes are noncanonical due to substitution at the second histidine in the active site HHxxxDG motif. Here, we used genome mining strategy to characterize a cryptic PKS-C hybrid from Talaromyces wortmanii and discovered the products are reduced long-chain polyketides amidated with a specific ω-amino acid 5-aminopentanoic acid (5PA). The wortmanamides resemble long-chain N-acyl-amide signaling lipids that target diverse receptors including GPCRs. The noncanonical C domain of this PKS-C hybrid was also demonstrated to be a bona fide condensation domain that specifically selects 5PA and catalyzes amidation to release polyketide chain
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methylation dependent acyl transfer between polyketide synthase and Nonribosomal Peptide Synthetase modules in fungal natural product biosynthesis
Organic Letters, 2014Co-Authors: Yi Zou, Yuta Tsunematsu, Mancheng Tang, Kenji Watanabe, Yi TangAbstract:Biochemical studies of purified and dissected fungal polyketide synthase and Nonribosomal Peptide Synthetase (PKS-NRPS) hybrid enzymes involved in biosynthesis of pseurotin and aspyridone indicate that one α-methylation step during polyketide synthesis is a prerequisite and a key checkpoint for chain transfer between PKS and NRPS modules. In the absence of the resulting γ-methyl feature, the completed polyketide intermediate is offloaded as an α-pyrone instead of being aminoacylated by the NRPS domain. These examples illustrate that precisely timed tailoring domain activities play critical roles in the overall programming of the iterative PKS (and NRPS) functions.
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An Iterative, Bimodular Nonribosomal Peptide Synthetase that Converts Anthranilate and Tryptophan into Tetracyclic Asperlicins
Chemistry & biology, 2013Co-Authors: Xue Gao, Yi Tang, Wei Jiang, Gonzalo Jiménez-osés, Moon Seok Choi, Kendall N. Houk, Christopher T WalshAbstract:The bimodular 276 kDa Nonribosomal Peptide Synthetase AspA from Aspergillus alliaceus, heterologously expressed in Saccharomyces cerevisiae, converts tryptophan and two molecules of the aromatic β-amino acid anthranilate (Ant) into a pair of tetracyclic peptidyl alkaloids asperlicin C and D in a ratio of 10:1. The first module of AspA activates and processes two molecules of Ant iteratively to generate a tethered Ant-Ant-Trp-S-enzyme intermediate on module two. Release is postulated to involve tandem cyclizations, in which the first step is the macrocyclization of the linear tripeptidyl-S-enzyme, by the terminal condensation (CT) domain to generate the regioisomeric tetracyclic asperlicin scaffolds. Computational analysis of the transannular cyclization of the 11-membered macrocyclic intermediate shows that asperlicin C is the kinetically favored product due to the high stability of a conformation resembling the transition state for cyclization, while asperlicin D is thermodynamically more stable.
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a fungal Nonribosomal Peptide Synthetase module that can synthesize thiopyrazines
ChemInform, 2011Co-Authors: Kangjian Qiao, Wenjun Zhang, Hui Zhou, Neil K. Garg, Yi TangAbstract:A library of more than 60 different thiopyrazine compounds is prepared from amino acids and mercaptans.
Louis S Tisa - One of the best experts on this subject based on the ideXlab platform.
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inactivation of the major hemolysin gene influences expression of the Nonribosomal Peptide Synthetase gene swra in the insect pathogen serratia sp strain scbi
Journal of Bacteriology, 2017Co-Authors: Lauren M Petersen, Kaitlyn Lacourse, Tim A Schoner, Helge B Bode, Louis S TisaAbstract:Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a Nonribosomal Peptide Synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI.IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences in regulation of common virulence mechanisms between these two species. With the emergence of antibiotic-resistant microorganisms, there is a widespread need to understand the regulation of pathogenesis. The significance of this study is the presentation of evidence for cross-pathway regulation of virulence factors and how the elimination of one mechanism may be compensated for by the upregulation of others.
Alison Butler - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of amphi enterobactin siderophores by vibrio harveyi baa 1116 identification of a bifunctional Nonribosomal Peptide Synthetase condensation domain
Journal of the American Chemical Society, 2014Co-Authors: Hannah K Zane, Hiroaki Naka, Margo G Haygood, Federico Rosconi, Moriah Sandy, Alison ButlerAbstract:The genome of Vibrio harveyi BAA-1116 contains a Nonribosomal Peptide Synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl Peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like Synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.
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biosynthesis of amphi enterobactin siderophores by vibrio harveyi baa 1116 identification of a bifunctional Nonribosomal Peptide Synthetase condensation domain
Journal of the American Chemical Society, 2014Co-Authors: Hannah K Zane, Hiroaki Naka, Margo G Haygood, Federico Rosconi, Moriah Sandy, Alison ButlerAbstract:The genome of Vibrio harveyi BAA-1116 contains a Nonribosomal Peptide Synthetase (NRPS) gene cluster (aebA–F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl Peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like Synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-l-serine and acyl-l-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli a...
Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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An Iterative, Bimodular Nonribosomal Peptide Synthetase that Converts Anthranilate and Tryptophan into Tetracyclic Asperlicins
Chemistry & biology, 2013Co-Authors: Xue Gao, Yi Tang, Wei Jiang, Gonzalo Jiménez-osés, Moon Seok Choi, Kendall N. Houk, Christopher T WalshAbstract:The bimodular 276 kDa Nonribosomal Peptide Synthetase AspA from Aspergillus alliaceus, heterologously expressed in Saccharomyces cerevisiae, converts tryptophan and two molecules of the aromatic β-amino acid anthranilate (Ant) into a pair of tetracyclic peptidyl alkaloids asperlicin C and D in a ratio of 10:1. The first module of AspA activates and processes two molecules of Ant iteratively to generate a tethered Ant-Ant-Trp-S-enzyme intermediate on module two. Release is postulated to involve tandem cyclizations, in which the first step is the macrocyclization of the linear tripeptidyl-S-enzyme, by the terminal condensation (CT) domain to generate the regioisomeric tetracyclic asperlicin scaffolds. Computational analysis of the transannular cyclization of the 11-membered macrocyclic intermediate shows that asperlicin C is the kinetically favored product due to the high stability of a conformation resembling the transition state for cyclization, while asperlicin D is thermodynamically more stable.
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nrps substrate promiscuity diversifies the xenematides
Organic Letters, 2011Co-Authors: Jason M Crawford, Christopher T Walsh, Cyril Portmann, Renee Kontnik, Jon ClardyAbstract:Xenematide, a cyclic depsiPeptide antibiotic produced by Xenorhabdus nematophila, had a candidate Nonribosomal Peptide Synthetase (NRPS) with atypical features. Differential metabolite analysis between a mutant and wildtype validated that this stand-alone NRPS was required for xenematide production, and further analysis led to a series of new xenematide derivatives encoded by the same NRPS. Our results indicate that adenylation domain promiscuity and relaxed downstream processing in the X. nematophila NRPS provide a conduit for xenematide diversification.
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the Nonribosomal Peptide Synthetase enzyme ddad tethers nβ fumaramoyl l 2 3 diaminopropionate for fe ii α ketoglutarate dependent epoxidation by ddac during dapdiamide antibiotic biosynthesis
Journal of the American Chemical Society, 2010Co-Authors: Marie A Hollenhorst, Neil L Kelleher, Stefanie B Bumpus, Megan L Matthews, Martin J Bollinger, Christopher T WalshAbstract:The gene cluster from Pantoea agglomerans responsible for biosynthesis of the dapdiamide antibiotics encodes an adenylation−thiolation didomain protein, DdaD, and an Fe(II)/α-ketoglutarate-dependent dioxygenase homologue, DdaC. Here we show that DdaD, a Nonribosomal Peptide Synthetase module, activates and sequesters Nβ-fumaramoyl-l-2,3-diaminopropionate as a covalently tethered thioester for subsequent oxidative modification of the fumaramoyl group. DdaC catalyzes Fe(II)- and α-ketoglutarate-dependent epoxidation of the covalently bound Nβ-fumaramoyl-l-2,3-diaminopropionyl-S-DdaD species to generate Nβ-epoxysuccinamoyl-DAP (DAP = 2,3-diaminopropionate) in thioester linkage to DdaD. After hydrolytic release, Nβ-epoxysuccinamoyl-DAP can be ligated to l-valine by the ATP-dependent ligase DdaF to form the natural antibiotic Nβ-epoxysuccinamoyl-DAP-Val.
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the loading and initial elongation modules of rifamycin Synthetase collaborate to produce mixed aryl ketide products
Biochemistry, 2002Co-Authors: Suzanne J Admiraal, Chaitan Khosla, Christopher T WalshAbstract:Rifamycin Synthetase assembles the chemical backbone that members of the rifamycin family of antibiotics have in common. The Synthetase contains a mixed biosynthetic interface between its loading module, which uses a Nonribosomal Peptide Synthetase mechanism, and its initial elongation module, which uses a polyketide synthase mechanism. Biochemical studies of the loading and initial elongation modules of rifamycin Synthetase reveal that this bimodular protein (LM-M1) catalyzes the formation of the phenyl ketide 3-hydroxy-2-methyl-3-phenylpropionate via a series of reactions that require benzoate, Mg.ATP, methylmalonyl-CoA, and NADPH. The overall rate of phenyl ketide production appears to be determined by the covalent loading of benzoate onto LM-M1, rather than by subsequent steps such as intermodular transfer of benzoate or condensation of benzoate and methylmalonate. Substituted benzoates that have previously been shown to be substrates for the loading module alone can also be incorporated into the corresponding aryl ketides by LM-M1, suggesting that the bimodular protein has a broad substrate tolerance. Discrimination between the substituted benzoates appears to reside in the benzoate loading reaction, and preincubation of LM-M1 with substituted benzoates and Mg.ATP allows faster downstream reactions to be unmasked. LM-M1 may be a useful biochemical system for exploring interactions between Nonribosomal Peptide Synthetase and polyketide synthase modules.
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the thioesterase domain from a Nonribosomal Peptide Synthetase as a cyclization catalyst for integrin binding Peptides
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Rahul M Kohli, Junichi Takagi, Christopher T WalshAbstract:Nonribosomal Peptide Synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the Nonribosomal Peptide Synthetase responsible for the production of the cyclic decaPeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear Peptide thioester with the native sequence of tyrocidine A and can additionally cyclize Peptide analogs that incorporate limited alterations in the Peptide sequence. Here we show that TycC TE can catalyze macrocyclization of Peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several Peptide thioesters that retain a limited number of elements of the native Peptide sequence are shown to be substrates for TycC TE. These Peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the Peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic Peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and Nα-methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to αIIbβ3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic Peptide libraries that can be probed for novel biological function.
John D Lipscomb - One of the best experts on this subject based on the ideXlab platform.
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an unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway
Journal of the American Chemical Society, 2015Co-Authors: Thomas M Makris, Eckard Munck, Katlyn K Meier, Anna J Komor, Brent S Rivard, Lawrence Que, John D LipscombAbstract:Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a Nonribosomal Peptide Synthetase (NRPS)-based pathway to yield the nitroaryl gr...
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a family of diiron monooxygenases catalyzing amino acid beta hydroxylation in antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Thomas M Makris, Mrinmoy Chakrabarti, Eckard Munck, John D LipscombAbstract:The biosynthesis of chloramphenicol requires a β-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the Nonribosomal Peptide Synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with Nonribosomal Peptide Synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O2 is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
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a family of diiron monooxygenases catalyzing amino acid beta hydroxylation in antibiotic biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Thomas M Makris, Mrinmoy Chakrabarti, Eckard Munck, John D LipscombAbstract:The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the Nonribosomal Peptide Synthetase CmlP. EPR, Mossbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with Nonribosomal Peptide Synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.
