Purified recombinant Escherichia coli ketol-acid reductoisomerase is unsuitable for use in a coupled assay of acetohydroxyacid synthase activity due to an unexpected side reaction

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-ace to-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction b y NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzym e was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme purified by metal-ligand affinity chromatography. The p ure enzyme was tested for its ability to provide a sensitive and continuous coupled assay for acetohydroxyacid synthase (EC 4.1.3.18), the preceding e nzyme in the pathway of branched-chain amino acid biosynthesis. An unexpect ed side reaction of ketol-acid reductoisomerase was observed in which it ca talyzes the reduction of pyruvate. Although relatively slow, this side reac tion is high enough to prohibit the use of this enzyme in a coupled assay f or acetohydroxyacid synthase. (C) 1999 Academic Press.