Probing a Proposed Hydroxylation in the Biosynthesis of Bleomycin by Streptomyces verticillus

Bleomycin is a non‐ribosomal peptide natural product produced by the bacterium Streptomyces verticillus . Non‐ribosomal peptides are produced by non‐ribosomal peptide synthetases (NRPS), which are megaenzymes composed of sequences of modules that incorporate individual amino acids into the non‐ribosomal peptide product. Because amino acids are added to the chain in the order of the modules, we can predict which modules incorporate which amino acids. Bleomycin contains a pyrimidine ring within its structure, the origins of which have not yet been determined. Comparison of the module sequence within the bleomycin NRPS and the structure of bleomycin suggests that asparagine is a precursor to the pyrimidine ring and that it undergoes an oxidation during ring formation. We hypothesize that the oxidation is achieved by hydroxylation of the asparagine to form β‐hydroxyasparagine. This oxidation could occur either on free asparagine or asparagine that had been loaded onto the corresponding NRPS module. We heterologously overexpressed Orf3, a predicted non‐heme iron‐dependent monooxygenases encoded within the bleomycin biosynthetic gene cluster, in E. coli . We examined Orf3's ability to hydroxylate free asparagine and asparagine loaded onto the appropriate NRPS module in vitro via HPLC. We determined that Orf3 did not hydroxylate asparagine in either context. However, we confirmed that asparagine, and not hydroxyasparagine, is loaded onto the NRPS module, confirming that asparagine is a precursor to the pyrimidine ring and strongly suggesting that the as‐yet‐unidentified hydroxylase acts on asparagine after it has been loaded onto the NRPS.