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 sol...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2001-12, Vol.98 (26), p.14901-14906
Main Authors: Chen, Huawei, Tseng, Claire C., Hubbard, Brian K., Walsh, Christopher T.
Format: Article
Language:English
Subjects:
3,5-Dihydroxyphenylglycine
Amino acids
Anti-Bacterial Agents - biosynthesis
Antibiotics
Bacterial Proteins
Base Sequence
Biochemistry
Biological Sciences
Biosynthesis
Coenzyme A Ligases - isolation & purification
Coenzyme A Ligases - metabolism
DNA Primers
DpgA protein
DpgB protein
DpgC protein
DpgD protein
Enzymes
Escherichia coli
Glycine - analogs & derivatives
Glycine - chemistry
Glycine - metabolism
glycopeptide antibiotics
Glycopeptides
Kinetics
Molecules
Monomers
Oxidation-Reduction
Peptides
polyketide synthase
Polyketides
Resorcinols - chemistry
Resorcinols - metabolism
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Summary: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 kcatof 1-2 min-1at 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 CH2to C=O four-electron oxidation on the Cα of DPA-CoA and hydrolyzed the thioester linkage with a kcatof 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.221582098