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

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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 kcat 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 CH2 to C⩵O four-electron oxidation on the Cα of DPA-CoA and hydrolyzed the thioester linkage with a kcat of 10 min−1. Phenylacetyl-CoA was also processed, to phenylglyoxylate, but with about 500-fold lower kcat/KM. 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.