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Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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a chemoenzymatic approach to glycopeptide antibiotics
Journal of the American Chemical Society, 2004Co-Authors: Christopher T WalshAbstract:Many biologically active natural products are constrained by macrocyclization and modified with carbohydrates. These two types of modifications are essential for their biological activities. Here we report a chemoenzymatic approach to make carbohydrate-modified cyclic peptide antibiotics. Using a thioesterase domain from the decapeptide Tyrocidine synthetase, 13 head-to-tail cyclized Tyrocidine derivatives were obtained with one to three propargylglycines incorporated at positions 3−8. These cyclic peptides were then conjugated to 21 azido sugars via copper(I)-catalyzed cycloaddition. Antibacterial and hemolytic assays showed that the two best glycopeptides, Tyc4PG-14 and Tyc4PG-15, have a 6-fold better therapeutic index than the natural Tyrocidine. We believe this method will also be useful for modifying other natural products to search for new therapeutics.
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chirality of peptide bond forming condensation domains in nonribosomal peptide synthetases the c5 domain of Tyrocidine synthetase is a dcl catalyst
Biochemistry, 2003Co-Authors: Susan L Clugston, Mohamed A Marahiel, Stephan A Sieber, Christopher T WalshAbstract:Nonribosomal peptides (NRP) such as the antibiotic Tyrocidine have d-amino acids, introduced by epimerase (E) domains embedded within modules of the enzymatic assembly lines. We predict that the peptide bond-forming condensation (C) domains immediately downstream of E domains are d-specific for the peptidyl donor and l-specific for the aminoacyl acceptor (DCL). To validate this prediction and establish that the C5 domain of Tyrocidine synthetase is indeed DCL, the apoT (thiolation) forms of module 4 (TycB3 AT4E) and module 5 (TycC1 C5AT5) were expressed. T5 was posttranslationally primed with CoASH to introduce the HS-pantetheinyl group and autoaminoacylated with radiolabeled l-Asn* or l-Asp*. Alternate donor substrates were introduced by priming apo AT4E with synthetically prepared tetrapeptidyl-CoA's differing in the chirality of Phe-4, d-Phe-l-Pro-l-Phe-l-Phe-CoA, and d-Phe-l-Pro-l-Phe-d-Phe-CoA. The tetrapeptidyl-S-T4 and l-Asp*-S-T5 were studied for peptide bond formation and chain translocation by C...
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biomimetic synthesis and optimization of cyclic peptide antibiotics
Nature, 2002Co-Authors: Rahul M Kohli, Christopher T Walsh, Michael D BurkartAbstract:Molecules in nature are often brought to a bioactive conformation by ring formation (macrocyclization)1. A recurrent theme in the enzymatic synthesis of macrocyclic compounds by non-ribosomal and polyketide synthetases is the tethering of activated linear intermediates through thioester linkages to carrier proteins, in a natural analogy to solid-phase synthesis2. A terminal thioesterase domain of the synthetase catalyses release from the tether and cyclization3,4. Here we show that an isolated thioesterase can catalyse the cyclization of linear peptides immobilized on a solid-phase support modified with a biomimetic linker, offering the possibility of merging natural-product biosynthesis with combinatorial solid-phase chemistry. Starting from the cyclic decapeptide antibiotic Tyrocidine A, this chemoenzymatic approach allows us to diversify the linear peptide both to probe the enzymology of the macrocyclizing enzyme, TycC thioesterase, and to create a library of cyclic peptide antibiotic products. We have used this method to reveal natural-product analogues of potential therapeutic utility; these compounds have an increased preference for bacterial over eukaryotic membranes and an improved spectrum of activity against some common bacterial pathogens.
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timing of epimerization and condensation reactions in nonribosomal peptide assembly lines kinetic analysis of phenylalanine activating elongation modules of Tyrocidine synthetase b
Biochemistry, 2002Co-Authors: Lusong Luo, Uwe Linne, Rahul M Kohli, Megumi Onishi, Christopher T WalshAbstract:The cyclic decapeptide antibiotic Tyrocidine has d-Phe residues at positions 1 and 4, produced during peptide chain growth from l-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2-3 bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (l-Phe-l-Phe-S-TycB3 ⇔ l-Phe-d-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over ti...
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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.
Moharned A. Marahiel - One of the best experts on this subject based on the ideXlab platform.
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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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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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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, Moharned 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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mutational analysis of the c domain in nonribosomal peptide synthesis
FEBS Journal, 2002Co-Authors: Veit Bergendahl, Uwe Linne, Moharned 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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generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases
Biochemistry, 2001Co-Authors: Rahul M Kohli, Moharned A. Marahiel, John W Trauger, Dirk Schwarzer, Christopher T WalshAbstract:The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the Tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the Tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.
Kuniki Kino - One of the best experts on this subject based on the ideXlab platform.
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production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class iii polyphosphate kinase 2 mediated atp regeneration
Journal of Bioscience and Bioengineering, 2018Co-Authors: Shin Suzuki, Ryotaro Hara, Kuniki KinoAbstract:An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported; however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of Tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of l -Trp- l -Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM l -Trp- l -Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ− putA−, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of l -Trp- l -Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only l -Trp- l -Pro, but also various other l -Xaa- l -Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.
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A chemoenzymatic process for amide bond formation by an adenylating enzyme-mediated mechanism
Scientific Reports, 2018Co-Authors: Ryotaro Hara, Shin Suzuki, Kengo Hirai, Kuniki KinoAbstract:Amide bond formation serves as a fundamental reaction in chemistry, and is practically useful for the synthesis of peptides, food additives, and polymers. However, current methods for amide bond formation essentially generate wastes and suffer from poor atom economy under harsh conditions. To solve these issues, we demonstrated an alternative synthesis method for diverse tryptophyl- N -alkylamides by the combination of the first adenylation domain of Tyrocidine synthetase 1 with primary or secondary amines as nucleophiles. Moreover, the physiological role of this domain is l -phenylalanine adenylation; however, we revealed that it displayed broad substrate flexibility from mono-substituted tryptophan analogues to even d -tryptophan. To the best of our knowledge, this is the first evidence for an adenylating enzyme-mediated direct amide bond formation via a sequential enzymatic activation of amino acids followed by nucleophilic substitution by general amines. These findings facilitate the design of a promising tool for biocatalytic straightforward amide bond formation with less side products.
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hydroxamate based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases
Analytical Biochemistry, 2015Co-Authors: Ryotaro Hara, Ryohei Suzuki, Kuniki KinoAbstract:Abstract We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of Tyrocidine synthetase (involved in Tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l -Trp–hydroxamate–Fe 3+ complex and the formation of l -Trp– l -Pro, where l -Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l -Pro formations was observed.
Rahul M Kohli - One of the best experts on this subject based on the ideXlab platform.
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biomimetic synthesis and optimization of cyclic peptide antibiotics
Nature, 2002Co-Authors: Rahul M Kohli, Christopher T Walsh, Michael D BurkartAbstract:Molecules in nature are often brought to a bioactive conformation by ring formation (macrocyclization)1. A recurrent theme in the enzymatic synthesis of macrocyclic compounds by non-ribosomal and polyketide synthetases is the tethering of activated linear intermediates through thioester linkages to carrier proteins, in a natural analogy to solid-phase synthesis2. A terminal thioesterase domain of the synthetase catalyses release from the tether and cyclization3,4. Here we show that an isolated thioesterase can catalyse the cyclization of linear peptides immobilized on a solid-phase support modified with a biomimetic linker, offering the possibility of merging natural-product biosynthesis with combinatorial solid-phase chemistry. Starting from the cyclic decapeptide antibiotic Tyrocidine A, this chemoenzymatic approach allows us to diversify the linear peptide both to probe the enzymology of the macrocyclizing enzyme, TycC thioesterase, and to create a library of cyclic peptide antibiotic products. We have used this method to reveal natural-product analogues of potential therapeutic utility; these compounds have an increased preference for bacterial over eukaryotic membranes and an improved spectrum of activity against some common bacterial pathogens.
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timing of epimerization and condensation reactions in nonribosomal peptide assembly lines kinetic analysis of phenylalanine activating elongation modules of Tyrocidine synthetase b
Biochemistry, 2002Co-Authors: Lusong Luo, Uwe Linne, Rahul M Kohli, Megumi Onishi, Christopher T WalshAbstract:The cyclic decapeptide antibiotic Tyrocidine has d-Phe residues at positions 1 and 4, produced during peptide chain growth from l-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2-3 bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (l-Phe-l-Phe-S-TycB3 ⇔ l-Phe-d-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over ti...
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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.
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cyclization of backbone substituted peptides catalyzed by the thioesterase domain from the Tyrocidine nonribosomal peptide synthetase
Biochemistry, 2001Co-Authors: John W Trauger, Rahul M Kohli, Christopher T WalshAbstract:The excised C-terminal thioesterase (TE) domain from the multidomain Tyrocidine nonribosomal peptide synthetase (NRPS) was recently shown to catalyze head-to-tail cyclization of a decapeptide thioester to form the cyclic decapeptide antibiotic Tyrocidine A [Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A., and Walsh, C. T. (2000) Nature 407, 215-218]. The peptide thioester substrate was a mimic of the TE domain's natural, synthetase-bound substrate. We report here the synthesis of modified peptide thioester substrates in which parts of the peptide backbone are altered either by the replacement of three amino acid blocks with a flexible spacer or by replacement of individual amide bonds with ester bonds. Rates of TE domain catalyzed cyclization were determined for these substrates and compared with that of the wild-type substrate, revealing that some parts of the peptide backbone are important for cyclization, while other parts can be modified without significantly affecting the cyclization rate. We also report the synthesis of a modified substrate in which the N-terminal amino group of the wild-type substrate, which is the nucleophile in the cyclization reaction, is replaced with a hydroxyl group and show that this compound is cyclized by the TE domain to form a macrolactone at a rate comparable to that of the wild-type substrate. These results demonstrate that the TE domain from the Tyrocidine NRPS can catalyze cyclization of depsipeptides and other backbone-substituted peptides and suggest that during the cyclization reaction the peptide substrate is preorganized for cyclization in the enzyme active site in part by intramolecular backbone hydrogen bonds analogous to those in the product Tyrocidine A.
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generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases
Biochemistry, 2001Co-Authors: Rahul M Kohli, Moharned A. Marahiel, John W Trauger, Dirk Schwarzer, Christopher T WalshAbstract:The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the Tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the Tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.
Michael D Burkart - One of the best experts on this subject based on the ideXlab platform.
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crosslinking studies of protein protein interactions in nonribosomal peptide biosynthesis
Chemistry & Biology, 2009Co-Authors: Gene H Hur, Jordan L Meier, Jeremy M Baskin, Julian A Codelli, Carolyn R Bertozzi, Michael D BurkartAbstract:Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the Tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.
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biomimetic synthesis and optimization of cyclic peptide antibiotics
Nature, 2002Co-Authors: Rahul M Kohli, Christopher T Walsh, Michael D BurkartAbstract:Molecules in nature are often brought to a bioactive conformation by ring formation (macrocyclization)1. A recurrent theme in the enzymatic synthesis of macrocyclic compounds by non-ribosomal and polyketide synthetases is the tethering of activated linear intermediates through thioester linkages to carrier proteins, in a natural analogy to solid-phase synthesis2. A terminal thioesterase domain of the synthetase catalyses release from the tether and cyclization3,4. Here we show that an isolated thioesterase can catalyse the cyclization of linear peptides immobilized on a solid-phase support modified with a biomimetic linker, offering the possibility of merging natural-product biosynthesis with combinatorial solid-phase chemistry. Starting from the cyclic decapeptide antibiotic Tyrocidine A, this chemoenzymatic approach allows us to diversify the linear peptide both to probe the enzymology of the macrocyclizing enzyme, TycC thioesterase, and to create a library of cyclic peptide antibiotic products. We have used this method to reveal natural-product analogues of potential therapeutic utility; these compounds have an increased preference for bacterial over eukaryotic membranes and an improved spectrum of activity against some common bacterial pathogens.
