The proposed research plan will focus on enzymology of peptide halogenases responsible for the biosynthesis of vancomycin group antibiotics. Two proposed halogenases will be cloned and expressed by standard biochemical techniques, and these gene products will be characterized for their halogenase activity. Inconsistencies in the current theories of haloperoxidase mechanism elicit careful investigation of the exact chemical and electronic mechanism of action. A detailed study of enzyme substrate specificity will allow an understanding of enzymatic timing of the biosynthetic gene products in vivo and inform further application of the enzymes to the synthesis of novel chlorinated peptides. With mechanistic and substrate information in hand, the application of these enzymes to the chlorination of yersiniabactin, a non-ribosomal peptide of the virulent bacteria, Yersinia pestis, will be attempted to verify the cross-application of peptide tailoring enzymes to other natural products. If successful, the halogenation of biosynthetic products will serve as a powerful method to introduce novel functionality into small molecules and have application to combinatorial biosynthetic approaches in drug discovery.
