Chemical methods for studying the biosynthesis of lantibiotics and prostaglandins

Lantibiotics are peptide antibiotics that are ribosomally synthesized and post-translationally modified by a multienzyme complex to their biologically active forms. One such compound, nisin, has been used effectively as a commercial food preservative, while other antibiotics show promising activity against bacterial infections. The first step of biosynthetic modification involves the transformation of specific threonines and serines in the precursor peptide into dehydro amino acids, followed by the cyclization of cysteine residues within the peptide onto these dehydro amino acids in a Michael-type addition. In an effort to study the mechanism by which lantibiotics are biosynthesized, we have cloned, purified, and characterized the aggregation state and metal content of the cyclase enzymes involved in the synthesis of nisin and subtilin. We have shown that each of these proteins exists as a monomer in solution and contains a stoichiometric zinc atom, perhaps involved in the Michael addition by activation of the nucleophile. We developed and tested a facile, chemoselective method to synthesize peptides containing dehydroalanine residues by oxidative elimination from (Se)-phenylselenocysteine, and used it to synthesize substrate analogs for the subtilin cyclase enzyme. Tests for activity with this truncated lantibiotic dehydro prepeptide have shown that this substrate is not sufficient for cyclase activity.; Prostaglandin H Synthase (PGHS) converts arachidonic acid (AA) into the first intermediate in the synthesis of prostaglandins via a radical mechanism. Prostaglandins play a number of roles in the body, including the regulation of blood flow to certain organs, controlling transport across membranes, and the stimulation of inflammation. A tyrosine radical is proposed to initiate atom at position 13 of AA to form a fatty acid radical, proposed to be a pentadienyl radical. Site-specifically deuterated AAs can contribute to corroboration of the proposed structure, as their reaction with the enzyme should lead to predictable changes in the hyperfine pattern observed in the EPR spectrum. We therefore designed syntheses for and prepared 15-[2H]-AA and four more deuterated compounds as structural probes. The EPR spectra obtained provide strong support for the assignment of the radical signal to a pentadienyl radical.