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  • Title: Facile crystallization of Escherichia coli ketol-acid reductoisomerase.
    Author: McCourt JA, Tyagi R, Guddat LW, Biou V, Duggleby RG.
    Journal: Acta Crystallogr D Biol Crystallogr; 2004 Aug; 60(Pt 8):1432-4. PubMed ID: 15272168.
    Abstract:
    Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+ -dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP+, Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 A resolution; attempts to phase them are currently in progress.
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