Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine.
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.
Chen
H
H
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Tseng
C C
CC
Hubbard
B K
BK
Walsh
C T
CT
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins; D001483:Base Sequence; D003066:Coenzyme A Ligases / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D006020:Glycopeptides; D007700:Kinetics; D010084:Oxidation-Reduction; D012118:Resorcinols / Q000737:chemistry / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017931:DNA Primers; C439058:DpgA protein, Amycolatopsis mediterranei; D006020:Glycopeptides; D012118:Resorcinols; C079215:3,5-dihydroxyphenylglycine; D003066:Coenzyme A Ligases; D005998:Glycine