The reaction kinetics, crystal structures and novel inhibitors of bacterial OSB-CoA synthetase.

摘要

There is an increasing need to develop novel antibiotics against bacteria such as Bacillus anthracis, a human pathogen that causes the lethal disease anthrax. The menaquinone biosynthetic pathway is an essential pathway for the growth of many bacteria and the pathway is absent in human cells. Therefore, enzymes in the menaquinone pathway are hypothesized to be ideal targets for the discovery of novel antibiotics.;OCS (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA. In this dissertation, the first comprehensive study on the kinetic mechanism of B. anthracis OCS is reported to be an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The first X-ray crystal structures for an OCS from Bacillus subtilis in its apo, SO42- bound, and OSB-AMP bound forms are presented. The active site structure reveals the nucleotide and the OSB binding pockets as well as the roles of conserved residues within the family. Conformational changes on the C-terminal domains are observed in transforming from the apo form to the OSB-AMP bound form of the enzyme, suggesting that conformational isomerization may occur during the adenylation reaction. Based on the B. subtilis OCS crystal structures, we propose an inline nucleophilic attack mechanism for the adenylation reaction.;An HTS assay for OCS was developed and the first HTS of a diverse library of 100,000 small molecules against B. anthracis OCS was performed to detect inhibitors. Nine compounds were found to be specific inhibitors to OCS. These molecules ranged in potency (IC50) from 5 to 100 muM against the enzyme. Two of the nine compounds showed Minimal Inhibitory Concentration (MIC) values around 50 muM against the B. anthracis DeltaANR strain and may represent starting points for new small molecule therapeutics directed against B. anthracis.