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Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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structural insights into nonribosomal peptide enzymatic assembly lines
Natural Product Reports, 2009Co-Authors: Alexander Koglin, Christopher T WalshAbstract:Nonribosomal peptides have a variety of medicinal activities including activity as antibiotics, antitumor drugs, immunosuppressives, and toxins. Their biosynthesis on multimodular assembly lines as a series of covalently tethered thioesters, in turn covalently attached on pantetheinyl arms on Carrier Protein way stations, reflects similar chemical logic and Protein machinery to fatty acid and polyketide biosynthesis. While structural information on excised or isolated catalytic adenylation (A), condensation (C), Peptidyl Carrier Protein (PCP) and thioesterase (TE) domains had been gathered over the past decade, little was known about how the NRPS catalytic and Carrier domains interact with each other both within and across elongation or termination modules. This Highlight reviews recent breakthrough achievements in both X-ray and NMR spectroscopic studies that illuminate the architecture of NRPS PCP domains, PCP-containing didomain-fragments and of a full termination module (C-A-PCP-TE).
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Protein assembly line components in prodigiosin biosynthesis characterization of piga g h i j
Journal of the American Chemical Society, 2006Co-Authors: Sylvie Garneautsodikova, Pieter C Dorrestein, Neil L Kelleher, Christopher T WalshAbstract:The red streptomycete metabolite prodigiosin has a unique tripyrrolic structure with two of the three pyrrolyl moieties in tandem. Five enzymes, PigA,G,H,I, and J, are involved in dipyrrole (rings A and B) formation. We have heterologously expressed and purified from Escherichia coli these five enzymes. At first, pyrrole ring A is formed on the Peptidyl Carrier Protein PigG by one of two possible ways: (i) by action of the adenylation domain PigI that transforms l-proline into l-prolyl-AMP and by the flavoProtein dehydrogenase PigA responsible for the four-electron oxidation reaction; (ii) by loading with the pyrrolyl-2-carboxyl-(S)-pantetheinyl moiety from synthetic pyrrolyl-CoA using the phosphopantetheinyl transferase Sfp. Subsequently, pyrrole ring B is constructed by PigH after the transfer of ring A to the ketosynthase of PigJ. PigH consists of three domains: two acyl Carrier Proteins (ACPs) and a seryltransferase (SerT). Using HPLC and nanospray-Fourier Transform Mass Spectrometry (nFTMS), we established that all three domains of PigH undergo post-translational modifications and gained insight into the machinery involved in 2,2-dipyrrole biosynthesis.
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Site-specific Protein labeling by Sfp phosphopantetheinyl transferase
Nature Protocols, 2006Co-Authors: David E. Golan, Christopher T WalshAbstract:Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the Peptidyl Carrier Protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific Protein labeling by Sfp-catalyzed Protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe–CoA conjugates, (iii) construction of target Protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target Protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe–CoA conjugates as the substrates of Sfp, 3 d for the cloning of target Protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged Proteins. Labeling of the PCP- or the ybbR-tagged Proteins in cell lysates or on cell surfaces should require only 15–30 min.
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excision of the epothilone synthetase b cyclization domain and demonstration of in trans condensation cyclodehydration activity
Biochemistry, 2005Co-Authors: Wendy L. Kelly, Nathan J. Hillson, Christopher T WalshAbstract:The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid involving Proteins EpoA-F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains: cyclization, adenylation, oxidation, and Peptidyl Carrier Protein (Cy-A-Ox-PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A-Ox-PCP) as subunit fragments to evaluate Cy as a free-standing domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its Protein-Protein interactions and mechanism of activity.
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excision of the epothilone synthetase b cyclization domain and demonstration of in trans condensation cyclodehydration activity
Biochemistry, 2005Co-Authors: Wendy L. Kelly, Nathan J. Hillson, Christopher T WalshAbstract:The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid involving Proteins EpoA-F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains: cyclization, adenylation, oxidation, and Peptidyl Carrier Protein (Cy -A- Ox-PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A-Ox-PCP) as subunit fragments to evaluate Cy as a free- standing domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its Protein-Protein interactions and mechanism of activity. Nonribosomal peptide synthetases (NRPSs) 1 and polyketide synthases (PKSs) are large, multimodular enzymes respon-
Moharned A. Marahiel - One of the best experts on this subject based on the ideXlab platform.
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functional dissection of surfactin synthetase initiation module reveals insights into the mechanism of lipoinitiation
Chemistry & Biology, 2010Co-Authors: Femke I Kraas, Verena Helmetag, Melanie Wittmann, Matthias Strieker, Moharned A. MarahielAbstract:Summary Although the N-terminally attached fatty acids are key structural elements of nonribosomally assembled lipopeptide antibiotics, little is known about the mechanism of lipid transfer during the initial step of biosynthesis. In this study, we investigated the activity of the dissected initiation module (C-A Glu -PCP) of surfactin synthetase SrfAA in vitro to gain further insights into the lipoinitiation reaction. The dissected condensation (C) domain catalyzes the transfer of CoA-activated 3-hydroxy fatty acid with high substrate specificity at its donor site to the Peptidyl Carrier Protein (PCP) bound amino acid glutamate (Glu 1 ). Additionally, biochemical studies on four putative acyl CoA ligases in Bacillus subtilis revealed that two of them activate 3-hydroxy fatty acids for surfactin biosynthesis in vitro and that the disruption of corresponding genes has a significant influence on surfactin production.
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structural and functional insights into a peptide bond forming bidomain from a nonribosomal peptide synthetase
Structure, 2007Co-Authors: Stefan A Samel, Georg Schoenafinger, Thomas A Knappe, Moharned A. Marahiel, Larsoliver EssenAbstract:Summary The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 A resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding Peptidyl Carrier Protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites ∼50 A apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis.
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structural and functional insights into a peptide bond forming bidomain from a nonribosomal peptide synthetase
Structure, 2007Co-Authors: Stefan A Samel, Georg Schoenafinger, Thomas A Knappe, Moharned A. Marahiel, Larsoliver EssenAbstract:The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 A resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding Peptidyl Carrier Protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites approximately 50 A apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis.
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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, Moharned 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 was found when assayed in the presence of either formyl donor. The proven formylation activity of the first domain of LgrA represents a novel tailoring enzyme in nonribosomal peptide synthesis.
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structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain srfte
Structure, 2002Co-Authors: Steven D. Bruner, Moharned A. Marahiel, Christopher T Walsh, Thomas Weber, Rahul M Kohli, Dirk Schwarzer, Milton T StubbsAbstract:Abstract 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.
Rolf Müller - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of the klebsiella oxytoca pathogenicity factor tilivalline heterologous expression in vitro biosynthesis and inhibitor development
ACS Chemical Biology, 2018Co-Authors: Alexander Von Tesmar, Antoine Abou Fayad, Viktoria Schmitt, Jennifer Herrmann, Michael J Hoffmann, Stephan Huttel, Rolf MüllerAbstract:Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxy-toca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a non-enzymatic indole incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the heterologous system was used to generate novel tilivalline derivatives by supplementation of respective anthranilate and indole precursors. Finally, it could be shown that salicylic and acetylsalicylic acid inhibit the biosynthesis of tilivalline in K. oxytoca liquid culture, presumably by blocking the Peptidyl Carrier Protein ThdA, pointing towards a potential application in combination therapy to prevent or alleviate symptoms of AAHC.
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Unusual Chemistry in the Biosynthesis of the Antibiotic Chondrochlorens
Chemistry & biology, 2009Co-Authors: Shwan Rachid, Maren Scharfe, Helmut Blöcker, Kira J. Weissman, Rolf MüllerAbstract:Summary The antibiotic chondrochlorens A and B from the myxobacterium Chondromyces crocatus Cm c5 incorporate several unusual structural features, notable among them a shared chloro-hydroxy-styryl functionality and the ethoxy group of chondrochloren B. Our analysis of the chondrochloren gene cluster by targeted gene inactivation coupled with assays in vitro has shed significant light on the biosynthesis of these metabolites. Chlorination of tyrosine occurs early in the pathway, likely on a Peptidyl Carrier Protein-bound intermediate, whereas decarboxylation to the styryl moiety appears to be accomplished by an unprecedented oxidative decarboxylase. We also show that the chondrochloren B ethoxy group arises from initial incorporation by the polyketide synthase of hydroxy malonate as an extender unit, methylation in cis by an O -methyltransferase, followed by a second methylation. This report therefore constitutes a direct demonstration of the involvement of a radical S -adenosylmethionine methylase in bacterial secondary metabolism.
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myxochelin biosynthesis direct evidence for two and four electron reduction of a Carrier Protein bound thioester
Journal of the American Chemical Society, 2008Co-Authors: Kira J. Weissman, Rolf MüllerAbstract:Microorganisms produce small molecules known as siderophores to scavenge iron from the environment. Insight into iron acquisition in myxobacteria has been provided recently by the sequencing of the gene cluster for the catecholate myxochelins A and B, from the myxobacterium Stigmatella aurantiaca Sg a15. The gene cluster contains enzymes (MxcCDEF) for assembly of 2,3-dihydroxybenzoic acid (DHBA), an amino transferase, MxcL, and a nonribosomal peptide synthetase (NRPS) subunit, MxcG. In the proposed pathway to the myxochelins, two molecules of DHBA are condensed with the two amino groups of lysine, which is itself tethered to the Peptidyl Carrier Protein domain (PCP) of MxcG. The resulting thioester is then reduced by the NADPH-dependent reductase (Red) domain of MxcG to generate an aldehyde intermediate; subsequent Red-catalyzed reduction yields myxochelin A, while transamination by MxcL produces myxochelin B. Although myxochelin A has been obtained successfully in vitro, it has not been possible to date ...
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in vitro reconstitution of the myxochelin biosynthetic machinery of stigmatella aurantiaca sg a15 biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Nikolaos Gaitatzis, Brigitte Kunze, Rolf MüllerAbstract:Abstract Microorganisms produce iron-chelating compounds to sequester the iron essential for growth from the environment. Many of these compounds are biosynthesized by nonribosomal peptide synthetases, some in cooperation with polyketide synthases. Myxochelins are produced by the myxobacterium Stigmatella aurantiaca Sg a15, and the corresponding gene cluster was cloned recently. We have undertaken to express heterologously the myxochelin biosynthetic machinery in Escherichia coli. To activate the involved Proteins posttranslationally, they were coexpressed with the phosphopantetheinyltransferase MtaA from the myxothiazol biosynthetic gene cluster. Phosphopantetheinylation of the Carrier Proteins could be verified by Protein mass analysis. Six active domains in Proteins MxcE, MxcF, and MxcG are capable of assembling myxochelin from ATP, NAD(P)H, lysine, and 2,3-dihydroxybenzoic acid in vitro. This fact demonstrates that the condensation domain of MxcG performs two condensation reactions, creating the aryl-capped α-amide and the aryl-capped γ-amide of the molecule. A previously unknown type of reductive release is performed by the reduction domain of MxcG, which alternatively uses NADPH and NADH to set free the Peptidyl-Carrier Protein-bound thioester as an aldehyde and further reduces it to the alcohol structure that can be found in myxochelin A. This type of reductive release seems to be a general mechanism in polyketide and nonribosomal peptide biosynthesis, because several systems with C-terminal similarity to the reductase domain of MxcG can be found in the databases. Alternatively, the aldehyde can be transaminated, giving rise to a terminal amine.
Ben Shen - One of the best experts on this subject based on the ideXlab platform.
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A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Shuangjun Lin, Steven G. Van Lanen, Ben ShenAbstract:Nonribosomal peptide synthetases (NRPSs) catalyze the biosynthesis of many biologically active peptides and typically are modular, with each extension module minimally consisting of a condensation, an adenylation, and a Peptidyl Carrier Protein domain responsible for incorporation of an amino acid into the growing peptide chain. C-1027 is a chromoProtein antitumor antibiotic whose enediyne chromophore consists of an enediyne core, a deoxy aminosugar, a benzoxazolinate, and a β-amino acid moiety. Bioinformatics analysis suggested that the activation and incorporation of the β-amino acid moiety into C-1027 follows an NRPS mechanism whereby biosynthetic intermediates are tethered to the Peptidyl Carrier Protein SgcC2. Here, we report the biochemical characterization of SgcC5, an NRPS condensation enzyme that catalyzes ester bond formation between the SgcC2-tethered (S)-3-chloro-5-hydroxy-β-tyrosine and (R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core. SgcC5 uses (S)-3-chloro-5-hydroxy-β-tyrosyl-SgcC2 as the donor substrate and exhibits regiospecificity for the C-2 hydroxyl group of the enediyne core mimic as the acceptor substrate. Remarkably, SgcC5 is also capable of catalyzing amide bond formation, albeit with significantly reduced efficiency, between (S)-3-chloro-5-hydroxy-β-tyrosyl-(S)-SgcC2 and (R)-2-amino-1-phenyl-1-ethanol, an alternative enediyne core mimic bearing an amine at its C-2 position. Thus, SgcC5 is capable of catalyzing both ester and amide bond formation, providing an evolutionary link between amide- and ester-forming condensation enzymes.
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characterization of the two component fad dependent monooxygenase sgcc that requires Carrier Protein tethered substrates for the biosynthesis of the enediyne antitumor antibiotic c 1027
Journal of the American Chemical Society, 2008Co-Authors: Shuangjun Lin, Steven G. Van Lanen, Ben ShenAbstract:C-1027 is a potent antitumor antibiotic composed of an apoProtein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an (S)-3-chloro-5-hydroxy-β-tyrosine moiety, the biosynthesis of which from l-α-tyrosine requires five Proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth Protein, SgcC5, catalyzes the incorporation of this β-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (Peptidyl Carrier Protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O2 and FADH2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted β-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but do...
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regiospecific chlorination of s β tyrosyl s Carrier Protein catalyzed by sgcc3 in the biosynthesis of the enediyne antitumor antibiotic c 1027
Journal of the American Chemical Society, 2007Co-Authors: Shuangjun Lin, Steven G. Van Lanen, Ben ShenAbstract:C-1027 is a potent antitumor antibiotic composed of an apo-Protein and a reactive enediyne chromophore. The chromophore consists of four different chemical subunits including an (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety, the biosynthesis of which from l-α-tyrosine is catalyzed by six Proteins, SgcC, SgcC1, SgcC2, SgcC3, SgcC4, and SgcC5. Biochemical characterization of SgcC3 unveiled the following: (i) SgcC3 is a flavin adenine dinucleotide (FAD)-dependent halogenase; (ii) SgcC3 acts only on the SgcC2 Peptidyl Carrier Protein-tethered substrates; (iii) SgcC3-catalyzed halogenation requires O2 and reduced FAD and either the C-1027 pathway-specific flavin reductase SgcE6 or E. coli flavin reductase (Fre) can support the SgcC3 activity; (iv) SgcC3 also efficiently catalyzes bromination but not fluorination or iodination; (v) SgcC3 can utilize both (S)- and (R)-β-tyrosyl-S-SgcC2 but not 3-hydroxy-β-tyrosyl-S-SgcC2 as a substrate. These results establish that SgcC3 catalyzes the third enzymatic transf...
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substrate specificity of the adenylation enzyme sgcc1 involved in the biosynthesis of the enediyne antitumor antibiotic c 1027
Journal of Biological Chemistry, 2006Co-Authors: Steven G. Van Lanen, Shuangjun Lin, Pieter C Dorrestein, Neil L Kelleher, Ben ShenAbstract:Abstract C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety that is derived from l-α-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-β-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any α-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[32P]PPi exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated β-amino acid substrate to the Peptidyl Carrier Protein SgcC2. The data establish (S)-β-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic β-amino acids such as β-phenylalanine, 3-chloro-β-tyrosine, and 3-hydroxy-β-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all α-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-β-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-β-tyrosyl-S-SgcC2 thioester substrate, and represents the first β-amino acid-specific adenylation enzyme characterized biochemically.
Milton T Stubbs - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of dlta implications for the reaction mechanism of non ribosomal peptide synthetase adenylation domains
Journal of Biological Chemistry, 2008Co-Authors: Huma Yonus, Piotr Neumann, Stephan Zimmermann, Jurgen J May, Milton T StubbsAbstract:Abstract DltA, the d-alanine:d-alanyl Carrier Protein ligase responsible for the initial step of lipoteichoic acid d-alanylation in Gram-positive bacteria, belongs to the adenylation domain superfamily, which also includes acetyl-CoA synthetase and the adenylation domains of non-ribosomal synthetases. The two-step reaction catalyzed by these enzymes (substrate adenylation followed by transfer to the reactive thiol group of CoA or the phosphopantheinyl prosthetic group of Peptidyl Carrier Proteins) has been suggested to proceed via large scale rearrangements of structural domains within the enzyme. The structures of DltA reported here reveal the determinants for d-Ala substrate specificity and confirm that the Peptidyl Carrier Protein-activating domains are able to adopt multiple conformational states, in this case corresponding to the thiolation reaction. Comparisons of available structures allow us to propose a mechanism whereby small perturbations of finely balanced metastable structural states would be able to direct an ordered formation of non-ribosomal synthetase products.
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structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain srfte
Structure, 2002Co-Authors: Steven D. Bruner, Moharned A. Marahiel, Christopher T Walsh, Thomas Weber, Rahul M Kohli, Dirk Schwarzer, Milton T StubbsAbstract:Abstract 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.
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structural basis for the cyclization of the lipopeptide antibiotic surfactin by the thioesterase domain srfte
Structure, 2002Co-Authors: Steven D. Bruner, Moharned A. Marahiel, Christopher T Walsh, Thomas Weber, Rahul M Kohli, Dirk Schwarzer, Milton T StubbsAbstract: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 alpha/beta 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.