A polyketide synthase in glycopeptide biosynthesis: the biosynthesis of the non-proteinogenic amino acid (S)-3,5-dihydroxyphenylglycine.
Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20-30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.
Pfeifer
V
V
Universität Tübingen, Institut für Mikrobiologie and Biotechnologie, Auf der Morgenstelle, Germany.
Nicholson
G J
GJ
Ries
J
J
Recktenwald
J
J
Schefer
A B
AB
Shawky
R M
RM
Schröder
J
J
Wohlleben
W
W
Pelzer
S
S
D000596:Amino Acids / Q000096:biosynthesis; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry*; D017353:Gene Deletion; D005998:Glycine / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000737:chemistry*; D006020:Glycopeptides / Q000096:biosynthesis; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D012118:Resorcinols / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology; D014640:Vancomycin / Q000031:analogs & derivatives* / Q000096:biosynthesis
D000596:Amino Acids; D001426:Bacterial Proteins; C439058:DpgA protein, Amycolatopsis mediterranei; D018691:Excitatory Amino Acid Antagonists; D006020:Glycopeptides; D009097:Multienzyme Complexes; D012118:Resorcinols; C087127:balhimycin; C079215:3,5-dihydroxyphenylglycine; D014640:Vancomycin; D003066:Coenzyme A Ligases; D005998:Glycine