The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
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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total biosynthesis in vitro reconstitution of polyketide and Nonribosomal Peptide pathways
ChemInform, 2008Co-Authors: Elizabeth S Sattely, Michael A Fischbach, Christopher T WalshAbstract:This review surveys efforts to reconstitute key steps in polyketide and Nonribosomal Peptide biosynthetic pathways with purified enzymes and substrates; 344 references are cited.
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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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Biosynthesis of Yersiniabactin, a Complex Polyketide-Nonribosomal Peptide, Using Escherichia coli as a Heterologous Host
Applied and Environmental Microbiology, 2003Co-Authors: Blaine A. Pfeifer, Christopher T Walsh, Clay C. C. Wang, Chaitan KhoslaAbstract:The medicinal value associated with complex polyketide and Nonribosomal Peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-Nonribosomal Peptide hybrid natural product, using Escherichia coli as a heterologous host. After introducing the biochemical pathway for Ybt into E. coli, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 ± 21 (mean ± standard deviation) mg/liter and a volumetric productivity of 1.1 ± 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, Nonribosomal Peptide, and mixed polyketide-Nonribosomal Peptide natural products using E. coli.
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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, Mohamed A. Marahiel, 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...
T. Martin Schmeing - One of the best experts on this subject based on the ideXlab platform.
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structures of a dimodular Nonribosomal Peptide synthetase reveal conformational flexibility
Science, 2019Co-Authors: J.m. Reimer, M Eivaskhani, I Harb, A Guarne, Martin Weigt, T. Martin SchmeingAbstract:Nonribosomal Peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent Peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a Peptide from a module-swapped dimodular NRPS.
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Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases
Chemistry & Biology, 2016Co-Authors: Kristjan Bloudoff, D A Alonzo, T. Martin SchmeingAbstract:Nonribosomal Peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in Nonribosomal Peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.
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Synthetic cycle of the initiation module of a formylating Nonribosomal Peptide synthetase
Nature, 2016Co-Authors: J.m. Reimer, Martin N. Aloise, Paul M. Harrison, T. Martin SchmeingAbstract:WebX-ray crystal structures are presented of each major step of the assembly-line synthesis by the initiation module of the Nonribosomal Peptide synthetase (NRPS) LgrA; the structures reveal large conformational changes, demonstrating a requirement for NRPSs to be very dynamic. Non-ribosomal Peptides, such as the antibiotic vancomycin and the immunosuppressant cyclosporin A, are peptidic secondary metabolites produced by microorganisms. Non-ribosomal Peptide synthetases (NRPSs) are a family of large enzymes that utilize multiple catalytic domains to catalyse sequential steps in the biosynthetic pathway of this family of 'natural products'. Two papers in this issue of Nature present X-ray crystal structures that indicate that NRPSs are substantially more dynamic than previously believed. Andrew Gulick and colleagues studied two holo-non-ribosomal Peptide synthetase modules, each revealing a distinct step in the catalytic cycle. Martin Schmeing and colleagues report several structures of LgrA, which is involved in the biosynthesis of the antibiotic gramicidin. Nonribosomal Peptide synthetases (NRPSs) are very large proteins that produce small Peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics1. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites2,3. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space3,4. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in Nonribosomal Peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase5,6. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.
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Crystal Structures of the First Condensation Domain of CDA Synthetase Suggest Conformational Changes during the Synthetic Cycle of Nonribosomal Peptide Synthetases
Journal of Molecular Biology, 2013Co-Authors: Kristjan Bloudoff, D Rodionov, T. Martin SchmeingAbstract:Abstract Nonribosomal Peptide synthetases (NRPSs) are large modular macromolecular machines that produce small Peptide molecules with wide-ranging biological activities, such as antibiotics and green chemicals. The condensation (C) domain is responsible for amide bond formation, the central chemical step in Nonribosomal Peptide synthesis. Here we present two crystal structures of the first condensation domain of the calcium-dependent antibiotic (CDA) synthetase (CDA-C1) from Streptomyces coelicolor, determined at resolutions 1.8 A and 2.4 A. The conformations adopted by CDA-C1 are quite similar in these two structures yet distinct from those seen in other NRPS C domain structures. HPLC-based reaction assays show that this CDA-C1 construct is catalytically active, and small-angle X-ray scattering experiments suggest that the conformation observed in these crystal structures could faithfully represent the conformation in solution. We have performed targeted molecular dynamics simulations, normal mode analyses and energy-minimized linear interpolation to investigate the conformational changes required to transition between the observed structures. We discuss the implications of these conformational changes in the synthetic cycle and of the observation that the “latch” that covers the active site is consistently formed in all studied C domains.
Yoshimitsu Hamano - One of the best experts on this subject based on the ideXlab platform.
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e poly l lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
Nature Chemical Biology, 2008Co-Authors: Kazuya Yamanaka, Chitose Maruyama, Hiroshi Takagi, Yoshimitsu HamanoAbstract:e-Poly- L -lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
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e poly l lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
Nature Chemical Biology, 2008Co-Authors: Kazuya Yamanaka, Chitose Maruyama, Hiroshi Takagi, Yoshimitsu HamanoAbstract:e-Poly-L-lysine (e-PL) consists of 25–35 L-lysine residues in isoPeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of e-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an e-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an e-PL–producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the Nonribosomal Peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase–like catalytic activity for Peptide bond formation.
Hiroshi Takagi - One of the best experts on this subject based on the ideXlab platform.
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e poly l lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
Nature Chemical Biology, 2008Co-Authors: Kazuya Yamanaka, Chitose Maruyama, Hiroshi Takagi, Yoshimitsu HamanoAbstract:e-Poly- L -lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
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e poly l lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
Nature Chemical Biology, 2008Co-Authors: Kazuya Yamanaka, Chitose Maruyama, Hiroshi Takagi, Yoshimitsu HamanoAbstract:e-Poly-L-lysine (e-PL) consists of 25–35 L-lysine residues in isoPeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of e-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an e-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an e-PL–producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the Nonribosomal Peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase–like catalytic activity for Peptide bond formation.
Chitose Maruyama - One of the best experts on this subject based on the ideXlab platform.
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e poly l lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
Nature Chemical Biology, 2008Co-Authors: Kazuya Yamanaka, Chitose Maruyama, Hiroshi Takagi, Yoshimitsu HamanoAbstract:e-Poly- L -lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
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e poly l lysine dispersity is controlled by a highly unusual Nonribosomal Peptide synthetase
Nature Chemical Biology, 2008Co-Authors: Kazuya Yamanaka, Chitose Maruyama, Hiroshi Takagi, Yoshimitsu HamanoAbstract:e-Poly-L-lysine (e-PL) consists of 25–35 L-lysine residues in isoPeptide linkages and is one of only two amino acid homopolymers known in nature. Elucidating the biosynthetic mechanism of e-PL should open new avenues for creating novel classes of biopolymers. Here we report the purification of an e-PL synthetase (Pls; 130 kDa) and the cloning of its gene from an e-PL–producing strain of Streptomyces albulus. Pls was found to be a membrane protein with adenylation and thiolation domains characteristic of the Nonribosomal Peptide synthetases (NRPSs). It had no traditional condensation or thioesterase domain; instead, it had six transmembrane domains surrounding three tandem soluble domains. These tandem domains iteratively catalyzed L-lysine polymerization using free L-lysine polymer (or monomer in the initial reaction) as acceptor and Pls-bound L-lysine as donor, directly yielding chains of diverse length. Thus, Pls is a new single-module NRPS having an amino acid ligase–like catalytic activity for Peptide bond formation.
