Dataset Information

Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine.

ABSTRACT: Four proteins, DpgA-D, required for the biosynthesis by actinomycetes of the nonproteinogenic amino acid monomer (S)-3,5-dihydroxyphenylglycine (Dpg), that is a crosslinking site in the maturation of vancomycin and teicoplanin antibiotic scaffolds, were expressed in Escherichia coli, purified in soluble form, and assayed for enzymatic activity. DpgA is a type III polyketide synthase, converting four molecules of malonyl-CoA to 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) and three free coenzyme A (CoASH) products. Almost no turnover was observed for DpgA until DpgB was added, producing a net k(cat) of 1-2 min(-1) at a 3:1 ratio of DpgB:DpgA. Addition of DpgD gave a further 2-fold rate increase. DpgC had the unusual catalytic capacity to convert DPA-CoA to 3,5-dihydroxyphenylglyoxylate, which is a transamination away from Dpg. DpgC performed a net CH(2) to C=O four-electron oxidation on the Calpha of DPA-CoA and hydrolyzed the thioester linkage with a k(cat) of 10 min(-1). Phenylacetyl-CoA was also processed, to phenylglyoxylate, but with about 500-fold lower k(cat)/K(M). DpgC showed no activity in anaerobic incubations, suggesting an oxygenase function, but had no detectable bound organic cofactors or metals. A weak enoyl-CoA hydratase activity was detected for both DpgB and DpgD.

SUBMITTER: Chen H

PROVIDER: S-EPMC64956 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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Publications

Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine.

Chen H H Tseng C C CC Hubbard B K BK Walsh C T CT

Proceedings of the National Academy of Sciences of the United States of America 20011201 26

Four proteins, DpgA-D, required for the biosynthesis by actinomycetes of the nonproteinogenic amino acid monomer (S)-3,5-dihydroxyphenylglycine (Dpg), that is a crosslinking site in the maturation of vancomycin and teicoplanin antibiotic scaffolds, were expressed in Escherichia coli, purified in soluble form, and assayed for enzymatic activity. DpgA is a type III polyketide synthase, converting four molecules of malonyl-CoA to 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) and three free coenzyme A (Co ...[more]

PMID: 11752437

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Project description:Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains - known as condensation domains - is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

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Dataset Information

Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine.

Publications

Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine.

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