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  • Title: 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.
    Author: Hill CM, Duggleby RG.
    Journal: Protein Expr Purif; 1999 Feb; 15(1):57-61. PubMed ID: 10024470.
    Abstract:
    Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme purified by metal-ligand affinity chromatography. The pure enzyme was tested for its ability to provide a sensitive and continuous coupled assay for acetohydroxyacid synthase (EC 4.1.3.18), the preceding enzyme in the pathway of branched-chain amino acid biosynthesis. An unexpected side reaction of ketol-acid reductoisomerase was observed in which it catalyzes the reduction of pyruvate. Although relatively slow, this side reaction is high enough to prohibit the use of this enzyme in a coupled assay for acetohydroxyacid synthase.
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