Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

Trauger, J. W.

doi:10.1073/pnas.040560597

Cited by 10 publications

(8 citation statements)

References 3 publications

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“…In M2, M4, and M5, there are three epimerases (E domain) that convert L-βHt 2 , L-Hpg 4 , and L-Hpg 5 into the corresponding D type amino acids. The N-terminal amino acid of vancomycin is D-methylleucine [ 47 ]. However, neither an epimerase nor a dual condensation/epimerization domain [ 48 , 49 ] was observed in M1 or the adjacent C domain in M2.…”

Section: Resultsmentioning

confidence: 99%

Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis

Huang

Wei³

et al. 2014

BMC Genomics

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BackgroundAmycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.ResultsThe complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.ConclusionsThis study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-363) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis

Huang

Wei³

et al. 2014

BMC Genomics

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“…The assumption is that the A domain of the first module activates a D-amino acid. For the cep cluster, however, Trauger and Walsh [42] show that the A domain of M1 prefers L-Leu over D-Leu in a 6:1 ratio; but on the other hand, they could not show which stereoisomer is processed further. This suggests the existence of an unknown E domain that acts on the L-Leu activated by M1.…”

Section: Resultsmentioning

confidence: 99%

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

et al. 2007

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BackgroundNon-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An LCL domain catalyzes a peptide bond between two L-amino acids, a DCL domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β-hydroxy-carboxylic acid (typically a β-hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.ResultsIn this paper, we report on the reconstruction of the phylogenetic relationship of NRPS C domain subtypes and analyze in detail the sequence motifs of recently discovered subtypes (Dual E/C, DCL and Starter domains) and their characteristic sequence differences, mutually and in comparison with LCL domains. Based on their phylogeny and the comparison of their sequence motifs, LCL and Starter domains appear to be more closely related to each other than to other subtypes, though pronounced differences in some segments of the protein account for the unequal donor substrates (amino vs. β-hydroxy-carboxylic acid). Furthermore, on the basis of phylogeny and the comparison of sequence motifs, we conclude that Dual E/C and DCL domains share a common ancestor. In the same way, the evolutionary origin of a C domain of unknown function in glycopeptide (GP) NRPSs can be determined to be an LCL domain. In the case of two GP C domains which are most similar to DCL but which have LCL activity, we postulate convergent evolution.ConclusionWe systematize all C domain subtypes including the novel Starter C domain. With our results, it will be easier to decide the subtype of unknown C domains as we provide profile Hidden Markov Models (pHMMs) for the sequence motifs as well as for the entire sequences. The determined specificity conferring positions will be helpful for the mutation of one subtype into another, e.g. turning DCL to LCL, which can be a useful step for obtaining novel products.

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“…Inspection of the CepABC subunit domain composition predicts epimerase (E) domains in module 2, 4, and 5 to convert l -aminoacyl-/peptidyl-S-enzyme intermediates to the d -isomers. The first module lacks an epimerase domain but can select and activate d -Leu directly . All told there are 24 domains in the seven-module CepABC assembly line, each with a well-defined function.…”

Section: Biosynthetic Strategiesenzymatic Assembly Lines and Tailori...mentioning

confidence: 99%

Glycopeptide and Lipoglycopeptide Antibiotics

et al. 2005

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received her B.S. in Chemistry in 2001. Upon graduation, she attended Princeton University, where she received her M.A. under the direction of Daniel Kahne. She has worked on a chemoenyzmatic synthesis of novel glycopeptide antibiotics. She is currently pursuing her Ph.D. with Daniel Kahne at Harvard University. Wei Lu received his B.E. degree from East China University of Chemical Technology in 1992. He did his Ph.D. in Philip Cole's lab at Johns Hopkins University. His Ph.D. research focused on the regulation of protein tyrosine phosphatases. Since 2002, he has been pursuing his postdoctoral research in Christopher Walsh's group at Harvard Medical School. His current research interest lies in functional analysis of glycosyltransferases involved in natural product biosynthesis. He is currently a Ruth L. Kirschstein Nation Institute of Health postdoctoral fellow.

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Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

Cited by 10 publications

References 3 publications

Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis

Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Glycopeptide and Lipoglycopeptide Antibiotics

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