Bleomycin is a hybrid peptide-polyketide natural product produced by the bacterium <i>Streptomyces verticillus</i>. The backbone of the molecule contains a pyrimidine moiety predicted to originate from the non-proteinogenic amino acid β-hydroxyasparagine. An examination of the bleomycin megasynthetase gene cluster shows the presence of several hydroxylases as well as an asparagine synthetase, indicating that the β-hydroxyasparagine is generated locally. In order to understand the pathway leading to this amino acid, we attempted to synthesize these enzymes and characterize them by carrying out substrate selectivity assays using HPLC. Expression vectors containing the relevant gene sequences were cloned, successfully expressed in <i>E. coli </i>and the resulting enzymes purified . Significant hydroxylation of L-Asp by the hydroxylase ORF15 provides evidence that β-hydroxyasparagine is synthesized from L-Asp through a two-step pathway.

Understanding the generation of the non-proteinogenic amino acid β-hydroxyasparagine in bleomycin biosynthesis

255th National Meeting (2018)

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