The 138^th residue of acetohydroxyacid synthase in Corynebacterium glutamicum is important for the substrate binding...

Corynebacterium glutamicum acetohydroxyacid synthase (AHAS), composed of two subunits IlvB and IlvN, catalyzes the first reaction in the biosynthetic pathway of branched-chain amino acids. It either condenses two pyruvates to yield acetolactate, leading to the biosynthesis of L-valine and L-leucine, or condenses pyruvate with 2-ketobutyrate to yield acetohydroxybutyrate, leading to L-isoleucine biosynthesis. However, the mechanism for the substrate specificity of C. glutamicum AHAS remains unknown. In this study, AHASs from an L-valine-producing C. glutamicum VWB-1 and an L-isoleucine-producing C. glutamicum IWJ001 were analyzed. The amino acid sequence of IlvN from both strains are the same, but the 138^th and 404^th residues of IlvB from the two strains are different; they are alanine and valine in IWJ001 (IlvB^138A404V), but valine and alanine in VWB-1 (IlvB^138V404A). When IlvB^138A404V and IlvB^138V404A were overexpressed in wild type C. glutamicum ATCC14067 and its △alr△aceE△ilvA△leuA mutant YTW-104, the latter led to much more L-valine production than the former. AHAS activity studies also showed that the 138^th valine was important for binding the 2^nd substrate pyruvate but not the 404^th alanine. YTW-104/pJYW4-ilvB^138V404A-ilvNCE could produce 25.93 g/L L-valine. The results indicate that the 138^th valine of IlvB in AHAS could play an important role, leading to the increased L-valine biosynthesis in C. glutamicum.