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Andrew M Gulick - One of the best experts on this subject based on the ideXlab platform.
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structures of a nonribosomal peptide synthetase module bound to mbth like proteins support a highly dynamic domain architecture
Journal of Biological Chemistry, 2016Co-Authors: Ce Shi, Courtney C Aldrich, Andrew M Gulick, Bradley R Miller, Eric J DrakeAbstract:Abstract Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein (PCP), an amino acid-loading Adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some Adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an Adenylation domain has been previously reported using a truncated Adenylation domain, precluding any insight that might derive from understanding the influence of the MLP on the intact Adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full length NRPS EntF bound to the MLPs from E. coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatic support, further elaborate the residues that define the MLP-Adenylation domain interface. Additionally the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the Adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.
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analysis of the linker region joining the Adenylation and carrier protein domains of the modular nonribosomal peptide synthetases
Proteins, 2014Co-Authors: Bradley R Miller, Jesse A Sundlov, Eric J Drake, Thomas A Makin, Andrew M GulickAbstract:Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS Adenylation and PCP domains identified several conserved proline residues that are not found in standalone Adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the Adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the Adenylation-PCP linker. The LPxP motif interacts with a region on the Adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C-terminal subdomain of the Adenylation domain may coordinate movement of the PCP with the conformational change of the Adenylation domain. Through this work, we extend the conserved A10 motif of the Adenylation domain and identify residues that enable proper Adenylation domain function.
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structure determination of the functional domain interaction of a chimeric nonribosomal peptide synthetase from a challenging crystal with noncrystallographic translational symmetry
Acta Crystallographica Section D-biological Crystallography, 2013Co-Authors: Jesse A Sundlov, Andrew M GulickAbstract:The nonribosomal peptide synthetases (NRPSs) are a family of modular proteins that contain multiple catalytic domains joined in a single protein. Together, these domains work to produce chemically diverse peptides, including compounds with antibiotic activity or that play a role in iron acquisition. Understanding the structural mechanisms that govern the domain interactions has been a long-standing goal. During NRPS synthesis, amino-acid substrates are loaded onto integrated carrier protein domains through the activity of NRPS Adenylation domains. The structures of two Adenylation domain–carrier protein domain complexes have recently been determined in an effort that required the use of a mechanism-based inhibitor to trap the domain interaction. Here, the continued analysis of these proteins is presented, including a higher resolution structure of an engineered di-domain protein containing the EntE Adenylation domain fused with the carrier protein domain of its partner EntB. The protein crystallized in a novel space group in which molecular replacement and refinement were challenged by noncrystallographic pseudo-translational symmetry. The structure determination and how the molecular packing impacted the diffraction intensities are reported. Importantly, the structure illustrates that in this new crystal form the functional interface between the Adenylation domain and the carrier protein domain remains the same as that observed previously. At a resolution that allows inclusion of water molecules, additional interactions are observed between the two protein domains and between the protein and its ligands. In particular, a highly solvated region that surrounds the carrier protein cofactor is described.
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structure of pa1221 a nonribosomal peptide synthetase containing Adenylation and peptidyl carrier protein domains
Biochemistry, 2012Co-Authors: Carter A Mitchell, Ce Shi, Courtney C Aldrich, Andrew M GulickAbstract:Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream Adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused Adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. F...
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structural and functional investigation of the intermolecular interaction between nrps Adenylation and carrier protein domains
Chemistry & Biology, 2012Co-Authors: Jesse A Sundlov, Ce Shi, Daniel J Wilson, Courtney C Aldrich, Andrew M GulickAbstract:Nonribosomal peptide synthetases (NRPSs) are modular proteins that produce peptide antibiotics and siderophores. These enzymes act as catalytic assembly lines where substrates, covalently bound to integrated carrier domains, are delivered to adjacent catalytic domains. The carrier domains are initially loaded by Adenylation domains, which use two distinct conformations to catalyze sequentially the Adenylation of the substrate and the thioesterification of the pantetheine cofactor. We have used a mechanism-based inhibitor to determine the crystal structure of an engineered Adenylation-carrier domain protein illustrating the intermolecular interaction between the Adenylation and carrier domains. This structure enabled directed mutations to improve the interaction between nonnative partner proteins. Comparison with prior NRPS Adenylation domain structures provides insights into the assembly line dynamics of these modular enzymes.
Mohamed A. Marahiel - One of the best experts on this subject based on the ideXlab platform.
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a synthetic Adenylation domain based trna aminoacylation catalyst
Angewandte Chemie, 2015Co-Authors: Tobias W Giessen, Florian Altegoer, Annika J Nebel, Roman M Steinbach, Gert Bange, Mohamed A. MarahielAbstract:The incorporation of non-proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20-22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion-protein-based design for synthetic tRNA-aminoacylation catalysts based on combining NRPS Adenylation domains and a small eukaryotic tRNA-binding domain (Arc1p-C). Using rational design, guided by structural insights and molecular modeling, the Adenylation domain PheA was fused with Arc1p-C using flexible linkers and achieved tRNA-aminoacylation with both proteinogenic and non-proteinogenic amino acids. The resulting aminoacyl-tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA-aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non-proteinogenic amino acids.
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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, Mohamed A. Marahiel, 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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Crystal Structure of the Termination Module of a Nonribosomal Peptide Synthetase
Science, 2008Co-Authors: Alan Tanovic, Stefan A Samel, Larsoliver Essen, Mohamed A. MarahielAbstract:Nonribosomal peptide synthetases (NRPSs) are modular multidomain enzymes that act as an assembly line to catalyze the biosynthesis of complex natural products. The crystal structure of the 144-kilodalton Bacillus subtilis termination module SrfA-C was solved at 2.6 angstrom resolution. The Adenylation and condensation domains of SrfA-C associate closely to form a catalytic platform, with their active sites on the same side of the platform. The peptidyl carrier protein domain is flexibly tethered to this platform and thus can move with its substrate-loaded 4'-phosphopantetheine arm between the active site of the Adenylation domain and the donor side of the condensation domain. The SrfA-C crystal structure has implications for the rational redesign of NRPSs as a means of producing novel bioactive peptides.
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aminoacyl coenzyme a synthesis catalyzed by Adenylation domains
FEBS Letters, 2007Co-Authors: Uwe Linne, Milton T Stubbs, Antje Schafer, Mohamed A. MarahielAbstract:Adenylate forming enzymes play an important role in nature as they are involved in a number of essential biochemical pathways. In this study, we investigated the ability of a set of structurally related recombinant bacterial adenylate forming enzymes derived from nonribosomal peptide synthetases for their ability to synthesize acyl-CoAs in vitro. Adenylation-domains normally transfer their reactive aminoacyl-adenylates onto the covalently attached 4′-phosphopantetheine moiety of small carrier proteins. In detail, DltA, DhbE, GrsA-A, TycB3-A, and TycC3-A were investigated for their ability to synthesize acyl-CoAs. As reference, acetyl-CoA-synthetase (Acs) of B. subtilis was utilized, which naturally synthesizes acetyl-CoA from acetate, CoA-SH and ATP. Interestingly, all enzymes were capable of producing acyl-CoAs, albeit with differing efficiencies. Surprisingly, both CoA-SH and ATP were observed to inhibit the Adenylation reaction at higher concentrations. Product quantification for kinetic determination was carried out by ESI-SIM-MS. Our results allow speculation as to evolutionary relationships within the large class of adenylate forming enzymes.
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exploitation of the selectivity conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics
Biochemistry, 2002Co-Authors: Katrin Eppelmann, Torsten Stachelhaus, Mohamed A. MarahielAbstract:Recently, the solved crystal structure of a phenylalanine-activating Adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonr...
Sylvie Garneautsodikova - One of the best experts on this subject based on the ideXlab platform.
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probing the limits of interrupted Adenylation domains by engineering a trifunctional enzyme capable of Adenylation n and s methylation
Organic and Biomolecular Chemistry, 2019Co-Authors: Taylor A Lundy, Shogo Mori, Sylvie GarneautsodikovaAbstract:The Adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M3S domain from TioS(A3aM3SA3b) between the a8-a9 region of a mono-interrupted A domain, TioN(AaMNAb), that already contained a side chain methylating MN domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AMNAM3SA) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.
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structural basis for backbone n methylation by an interrupted Adenylation domain
Nature Chemical Biology, 2018Co-Authors: Shogo Mori, Allan H Pang, Taylor A Lundy, Atefeh Garzan, Oleg V Tsodikov, Sylvie GarneautsodikovaAbstract:Interrupted Adenylation domains are enigmatic fusions, in which one enzyme is inserted into another to form a highly unusual bifunctional enzyme. We present the first crystal structure of an interrupted Adenylation domain that reveals a unique embedded methyltransferase. The structure and functional data provide insight into how these enzymes N-methylate amino acid precursors en route to nonribosomal peptides. The crystal structure of a methyltransferase domain embedded within an interrupted Adenylation domain provides insight into how a nonribosomal peptide synthetase N-methylates amino acid precursors for their incorporation into the peptide product.
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Adenylation and s methylation of cysteine by the bifunctional enzyme tion in thiocoraline biosynthesis
Journal of the American Chemical Society, 2014Co-Authors: Ahmad H Almestarihi, German Villamizar, Javier Fernandez, Olga E Zolova, Felipe Lombo, Sylvie GarneautsodikovaAbstract:The antitumor agent thiocoraline is a nonribosomally biosynthesized bisintercalator natural product, which contains in its peptidic backbone two S-methylated l-cysteine residues. S-Methylation occurs very rarely in nature, and is observed extremely rarely in nonribosomal peptide scaffolds. We have proposed that during thiocoraline biosynthesis, TioN, a stand-alone Adenylation domain interrupted by the S-adenosyl-l-methionine binding region of a methyltransferase enzyme, is capable of performing two functions: the Adenylation and S-methylation of l-cysteine. Herein, by preparation of knockouts of TioN and its MbtH-like protein partner TioT, we confirmed their role in thiocoraline biosynthesis. We also co-expressed recombinant TioN and TioT and biochemically investigated three potential pathways involving activation, methylation, and loading of l-cysteine onto the TioN partner thiolation domain, TioS(T4). The valuable insights gained into the pathway(s) followed for the production of S-Me-l-Cys-S-TioS(T4) will serve as a guide for the development of novel engineered interrupted Adenylation enzymes for combinatorial biosynthesis.
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a nonradioactive high throughput assay for screening and characterization of Adenylation domains for nonribosomal peptide combinatorial biosynthesis
Analytical Biochemistry, 2009Co-Authors: Thomas J Mcquade, Oleg V Tsodikov, Abbie D Shallop, Anita Sheoran, James Delproposto, Sylvie GarneautsodikovaAbstract:Abstract Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of Adenylation domains has been measured by radioactive ATP-[ 32 P]pyrophosphate (PP i ) exchange assays. To identify Adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P i ) concentrations after degradation by inorganic pyrophosphatase of the PP i released during aminoacyl-AMP formation by action of the Adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A 4 , one of the seven Adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[ 32 P]PP i exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.
Courtney C Aldrich - One of the best experts on this subject based on the ideXlab platform.
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structures of a nonribosomal peptide synthetase module bound to mbth like proteins support a highly dynamic domain architecture
Journal of Biological Chemistry, 2016Co-Authors: Ce Shi, Courtney C Aldrich, Andrew M Gulick, Bradley R Miller, Eric J DrakeAbstract:Abstract Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein (PCP), an amino acid-loading Adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some Adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an Adenylation domain has been previously reported using a truncated Adenylation domain, precluding any insight that might derive from understanding the influence of the MLP on the intact Adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full length NRPS EntF bound to the MLPs from E. coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatic support, further elaborate the residues that define the MLP-Adenylation domain interface. Additionally the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the Adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.
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measurement of nonribosomal peptide synthetase Adenylation domain activity using a continuous hydroxylamine release assay
Methods of Molecular Biology, 2016Co-Authors: Benjamin P Duckworth, Daniel J Wilson, Courtney C AldrichAbstract:Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS Adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered Adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.
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structure of pa1221 a nonribosomal peptide synthetase containing Adenylation and peptidyl carrier protein domains
Biochemistry, 2012Co-Authors: Carter A Mitchell, Ce Shi, Courtney C Aldrich, Andrew M GulickAbstract:Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream Adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused Adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. F...
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structural and functional investigation of the intermolecular interaction between nrps Adenylation and carrier protein domains
Chemistry & Biology, 2012Co-Authors: Jesse A Sundlov, Ce Shi, Daniel J Wilson, Courtney C Aldrich, Andrew M GulickAbstract:Nonribosomal peptide synthetases (NRPSs) are modular proteins that produce peptide antibiotics and siderophores. These enzymes act as catalytic assembly lines where substrates, covalently bound to integrated carrier domains, are delivered to adjacent catalytic domains. The carrier domains are initially loaded by Adenylation domains, which use two distinct conformations to catalyze sequentially the Adenylation of the substrate and the thioesterification of the pantetheine cofactor. We have used a mechanism-based inhibitor to determine the crystal structure of an engineered Adenylation-carrier domain protein illustrating the intermolecular interaction between the Adenylation and carrier domains. This structure enabled directed mutations to improve the interaction between nonnative partner proteins. Comparison with prior NRPS Adenylation domain structures provides insights into the assembly line dynamics of these modular enzymes.
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a continuous kinetic assay for Adenylation enzyme activity and inhibition
Analytical Biochemistry, 2010Co-Authors: Daniel J Wilson, Courtney C AldrichAbstract:Abstract Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PPi). In a second half-reaction, Adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for Adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule, leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatase–purine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate–MesG assay is especially useful for characterizing the activity and inhibition of Adenylation enzymes that acylate a protein substrate and/or fail to undergo rapid ATP–PPi exchange.
Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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activation of the pacidamycin pacl Adenylation domain by mbth like proteins
Biochemistry, 2010Co-Authors: Wenjun Zhang, Christopher T Walsh, John R Heemstra, Heidi ImkerAbstract:Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid Adenylation in some NRPS modules. We also demonstrate that Adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways.
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substrate recognition and selection by the initiation module pheate of gramicidin s synthetase
Journal of the American Chemical Society, 2001Co-Authors: Lusong Luo, Torsten Stachelhaus, Michael D Burkart, Christopher T WalshAbstract:The initiation module of non-ribosomal peptide synthetases (NRPS) selects and activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizing L-phenylalanine (L-Phe) and activating it (by Adenylation domain) as tightly bound L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), transferring it to the HS-phosphopantetheine arm of the holo-thiolation (holo-T) domain, and then epimerizing it (by epimerization domain) to the D-Phe-S-4'-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE Adenylation domain with a number of proteinogenic amino acids and observed that three additional amino acids, L-Tyr, L-Trp, and L-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantetheine arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMPs and/or aminoacyl-S-4'-Ppant-acyl enzymes by the enzyme was not observed by three different assays for Adenylation domain function. The microscopic reaction rates and thermodynamic equilibrium constants obtained from single-turnover studies of reactions of L-Phe, L-Trp, L-Tyr, and L-Leu with holoPheATE allowed us to construct free energy profiles for the reactions, revealing the kinetic and thermodynamic basis for substrate recognition and selection. In particular, the rates of epimerization of the L-aminoacyl-S-enzyme to the D-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300-, and 540-fold for L-Trp, L-Tyr, and L-Leu respectively, suggesting that the epimerization domain is an important gatekeeper for generation of the D-Phe-S-enzyme that starts gramicidin S chain growth.
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the loading module of rifamycin synthetase is an Adenylation thiolation didomain with substrate tolerance for substituted benzoates
Biochemistry, 2001Co-Authors: Suzanne J Admiraal, Christopher T Walsh, Chaitan KhoslaAbstract:The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the Adenylation and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for covalent arylation of the loading module were measured directly for the unnatural substrates benzoate and 3-hydroxybenzoate. This analysis was extended through competition experiments to determine the relative rates of incorporation of a series of substituted benzoates. Our results show that the loading module can accept a variety of substituted benzoates, although it exhibits a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most closely resemble its biological substrate. The considerable substrate tolerance of the loading module of rifamycin synthetase suggests that the module has potential as a tool for generating substituted derivatives of natural products.
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the entf and ente Adenylation domains of escherichia coli enterobactin synthetase sequestration and selectivity in acyl amp transfers to thiolation domain cosubstrates
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: David E Ehmann, Cathryn A Shawreid, Heather C Losey, Christopher T WalshAbstract:Enterobactin, the tris-(N-(2,3-dihydroxybenzoyl)serine) trilactone siderophore of Escherichia coli, is synthesized by a three-protein (EntE, B, F) six-module nonribosomal peptide synthetase (NRPS). In this work, the 142-kDa four-domain protein EntF was bisected into two double-domain fragments: a 108-kDa condensation and Adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein (PCP) and thioesterase protein, EntF PCP-TE. The Adenylation domain activity of EntF C-A formed seryl-AMP but lost the ability to transfer the seryl moiety to the cognate EntF PCP-TE in trans. Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase, was observed at rates of 0.5 min(-1) and 0.01 min(-1), respectively. The possibility that these slow acylation rates reflected dissociation of acyl/aminoacyl-AMP followed by adventitious thiolation by the heterologous PCPs in solution was addressed by measuring catalytic turnover of pyrophosphate (PP(i)) released from the Adenylation domain. The holo SrfB1 PCP protein as well as Ybt PCP1 did not stimulate an increase in PP(i) release from EntF C-A or EntE. In this light, aminoacylations in trans between A and PCP domain fragments of NRPS assembly lines must be subjected to kinetic scrutiny to determine whether transfer is truly between protein domains or results from slow aminoacyl-AMP release and subsequent nonenzymatic thiol capture.
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aminoacyl coas as probes of condensation domain selectivity in nonribosomal peptide synthesis
Science, 1999Co-Authors: Peter J. Belshaw, Christopher T Walsh, Torsten StachelhausAbstract:In nonribosomal biosynthesis of peptide antibiotics by multimodular synthetases, amino acid monomers are activated by the Adenylation domains of the synthetase and loaded onto the adjacent carrier protein domains as thioesters, then the formation of peptide bonds and translocation of the growing chain are effected by the synthetase9s condensation domains. Whether the condensation domains have any editing function has been unknown. Synthesis of aminoacyl–coenzyme A (CoA) molecules and direct enzymatic transfer of aminoacyl-phosphopantetheine to the carrier domains allow the Adenylation domain editing function to be bypassed. This method was used to demonstrate that the first condensation domain of tyrocidine synthetase shows low selectivity at the donor residue (d-phenylalanine) and higher selectivity at the acceptor residue (l-proline) in the formation of the chain-initiating d-Phe-l-Pro dipeptidyl-enzyme intermediate.