Structural and mechanistic studies of the pantocin A biosynthesis

Pantocin A is a peptide-derived antibiotic produced by the nonpathogenic bacterium Pantoea aggomerans to inhibit the "fire blight" pathogen Erwinia amylovora . It is synthesized ribosomally as a 30-residue peptide precursor (PaaP) and posttranslationally modified to its mature form, a bicyclic small molecule of only 309 Daltons. This process breaks down the peptide backbone connectivity and involves the self-cyclization of two glutamate side-chains. Previous work has indicated that two enzymes, PaaA and PaaB, may be responsible for the biosynthesis of pantocin A. This thesis attempts to utilize tools from both biochemistry and structural biology to elucidate the biosynthesis of pantocin A. I have been able to reconstitute the activity of PaaA in vitro and discover that PaaA alone causes a loss of 80 daltons in the mass of the peptide substrate. The finding suggests that PaaA, a predicted adenylation enzyme, cyclizes the two glutamate side-chains in PaaP and forms the bicyclic moiety of the pantocin A. Based on the reconstitution experiments, the detailed mechanism of this remarkable posttranslational modification activity of PaaA was proposed and investigated. In order to further understand the complex enzymatic mechanism of PaaA, the crystal structure of PaaA was determined. Structure-function studies based on the crystal structure suggest that PaaA functions as a dimer.