Mechanistic studies of acetohydroxyacid synthase from pathogenic bacteria: Bacillus anthracis and Mycobacterium tuberculosis

Abstract

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6; formerly known as acetolactate synthase, ALS) is a thiamin-and FAD-dependent enzyme which catalyzes the first common step in the biosynthesis of the branched-chain amino acids (BCAA) isoleucine, leucine and valine. AHAS is capable of catalyzing the synthesis of either (2S)-acetolactate (AL) from two molecules of pyruvate or (2S)-2-aceto-2-hydroxybutyrate (AHB) from pyruvate and 2-ketobutyrate. AHAS and the pathway for biosynthesis of the BCAA are found in bacteria, fungi, algae and higher plants, but not in animals. Indeed, several classes of widely and safely used commercial herbicides, such as the sulfonylureas and the imidazolinones families which act as potent inhibitors. AHAS inhibitors are considered to be the most potent commercial herbicides in use today. Recently, it has been shown that branched amino acids auxotrophic strain of mycobacterium failed to proliferate because of the inability to use amino acids from their hosts, indicating that inhibitors for the branched-chain amino acid biosynthesis could be used as anti-mycobacterium agents. These observations suggested that AHAS could be a potential target of new anti-TB drugs. The aim of this study was to create a tool that could be used in the search for new anti- bacterial drugs that inhibit branched chain amino acid biosynthesis, as there are essential amino acids that are not available to bacteria during growth in an effect organism. So this pathway for synthesis of particular amino acids can be another potential target for anti-bacterial agents. For the study of searching a new target protein for anti- bacterial agents, we have cloned, expressed, purified and characterized acetohydroxyacid synthase from Bacillus anthracis and Mycobacterium tuberculosis. Twenty six commercial herbicides of the sulfonylureas and imidazoliones classes were tested for their influence on catalytic subunit protein. Two of the sulfonylurea classes and one imidazoliones class were potent inhibitors of the enzyme with IC50 values of 7.01?0.81, 6.97?0.44, and 10.02?1.42 ?M, respectively. CE and IQ were noncompetitive and uncompetitive inhibitors of B. anthracis AHAS with Kii values of 3.77 and 2.02 ?M, respectively. In vivo inhibition of AHAS enzyme activity by selected inhibitors and binding sites of the B. anthracis AHAS?CE complex was evaluated. Besides, we also expressed, purified and characterized recombinant AHAS protein from M. tuberculosis, a micro plate based enzyme assay was developed to screen the inhibition of M. tuberculosis catalytic subunit AHAS and reconstituted enzyme with chemical library from Korea Chemical Bank and Korea Institute of Science and Technology and identified four AVS chemicals (AVS 2087, AVS 2093, AVS 2236, AVS 2397) that inhibited more than 95% of the M. tuberculosis AHAS activity. These compounds with IC50 values of 0.28 ? 3.88 ?M (catalytic subunit) and 0.26 -1.18 ?M (reconstituted enzyme), emphasizing the advantage of using a mycobacterial enzyme as a tool in the search for new anti-TB drugs.