ENZYMATIC REACTION MECHANISMS

Harvard University (Medical School), Boston MA

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Abstract

The goals of this proposal are the study of enzymatic reaction mechanisms. In this project period the focus is on the reaction mechanisms of two kinds of enzyme systems involved in the biosynthesis of peptide antibiotics. The first is the enzymatic heterocyclization machinery involved in the conversion of the 69aa Microcin A protein, an antibiotic precursor, to Microcin B17, an antibiotic targetted against E.coli DNA gyrase, in which 14 residues (six gly, four ser, four cys) have been posttranslationally modified to four thiazole and four oxazole rings, essential for antibiotic activity. The second goal is analysis of the enzymatic strategies used by multimodular enzymes that make peptide bonds nonribosomally, e.g. in the biosynthesis of peptide antibiotics and iron-chelatina siderophores. The example to be studied is the four enzyme system, Ent B,D,E,F, responsible for formation of the E. coli iron chelator enterobactin. In particular the EntF enzyme has four domains (condensation, adenylation, peptidyl carrier protein and thioesterase) whose functions in assembly of the (dihydroxybenzoyl)-serine trilactone, enterobactin, will be analyzed in terms of covalent priming, initiation, elongation, and termination strategies.