Cloning and Characterization of Catalytic Subunit of Acetohydroxyacid Synthase from Shigella sonnei

Abstract

Acetohydroxy acid synthase (AHAS, EC 2. 2. 1. 6) is the ThDP and FAD dependent important plant and microbial enzyme catalyzing the first common step in the biosynthesis of essential amino acid such as leucine, isoleucine and valine. AHAS is the attractive target site for many commercial class of herbicides, includes sulfonylureas, imidazolinones, and triazolpyrimidines. In this study with an aim to develop potent antishigolosis drug we have cloned and expressed in competent Escherichia coli BL21 (DE3) the AHAS gene coding for catalytic subunit of Shigella sonnei, a causative agent of shigellosis. The purified S. sonnei AHAS catalytic subunit size was observed approximately 65kDa on SDS-PAGE. The optimum pH and temperature for S.sonnei AHAS was at 7.5 and 37oCThe enzyme catalyzes condensation of two molecules of pyruvate to form acetolactate or 2-ketobutyrate to give 2-aceto-2-hydroxybutyrate, an intermediate product formed in the synthesis of essential amino acid with a Km of 8.01mM and specific activity of 0.117U/mg. The cofactor activation constant (KS) for ThDP and Mg2+ is 0.01mM and 0.18mM respectively. The dissociation constant (Kd) for ThDP determined through tryptophan fluorescence quenching was found to be 0.14mM. Through chemical library screening We found two potent inhibitors of S. sonnei AHAS KHG20612 and KHG25240 inhibiting 87-93% AHAS activity at 100uM of inhibitor concentration with an IC50 value of 9.3 and 12.1uM respectively. The inhibition kinetics of KHG20612 reveals Non-competitive inhibition mode with a Kii 2.65mM and kis whereas KHG25240 was Uncompetitive with kii 5.2mM and Kis 1.62mM. Docking of KHG20612 inhibitor with S. sonnei AHAS yields the lowest binding energy, -9.8kcal/mol and -7.4Kcal/mol respectively where KHG20612 shown a single hydrogen bond at 1.7A0 distance. This current study would give an impetus in the development of novel strong antibacterial agent targeting AHAS based on the scaffold of found potent inhibitors of S. sonnei AHAS.