balhimycin and 3-5-dihydroxyphenylglycine

In the mutasynthetic approach, the DeltadpgA mutant of the vancomycin-type glycopeptide antibiotic producer Amycolatopsis balhimycina, which is deficient in the synthesis of 3,5-dihydroxyphenylglycine (DPg), was supplemented with synthetic DPg analogues to obtain the corresponding modified glycopeptides. Sterically more demanding 3,5-disubstituted methoxy derivatives as well as monosubstituted DPg analogues were accepted as substrates. These facts indicate that steric and electronic requirements suffice in several cases for the oxidative closure of the AB ring, thus leading to the generation of novel antibiotically active glycopeptide derivatives. The results represent a further step in evaluating the potential of mutasynthesis for peptidic secondary metabolites.

Topics: Actinobacteria; Anti-Bacterial Agents; Culture Media; Glycine; Glycopeptides; Molecular Conformation; Mutation; Resorcinols; Spectrometry, Mass, Electrospray Ionization; Vancomycin

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.

Topics: Amino Acids; Bacterial Proteins; Chromatography, High Pressure Liquid; Coenzyme A Ligases; Excitatory Amino Acid Antagonists; Gene Deletion; Glycine; Glycopeptides; Models, Chemical; Models, Genetic; Molecular Sequence Data; Multienzyme Complexes; Mutagenesis, Site-Directed; Plasmids; Resorcinols; Sequence Analysis, DNA; Streptomyces; Vancomycin