Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development

Abstract

Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential for de novo synthesis of branched-chain amino acids. The genome sequence of Mycobacterium tuberculosis revealed genes encoding four catalytic subunits, ilvB1 (Rv3003c), ilvB2 (Rv3470c), ilvG (Rv1820) and ilvX (Rv3509c), and one regulatory subunit, ilvN (Rv3002c), of AHAS. All these genes were found to be expressed in M. tuberculosis growing in vitro. Each AHAS subunit gene was cloned and expressed in Escherichia coli. AHAS activity of IlvB1 and IlvG was found in cell-free lysates and with recombinant purified proteins. Kinetic studies with purified IlvG revealed positive cooperativity towards substrate and cofactors. To understand the role of the catalytic subunits in the biology of M. tuberculosis, expression of AHAS genes was analysed in different physiological conditions. ilvB1, ilvB2 and ilvG were differentially expressed. The role of ilvB1 in persistence is known, but the upregulation of ilvB2 and ilvG in extended stationary phase, ex vivo, and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival of M. tuberculosis by functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.