Facile crystallization of Escherichia coli ketol-acid reductoisomerase

Acta Crystallogr D Biol Crystallogr. 2004 Aug;60(Pt 8):1432-4. doi: 10.1107/S0907444904012247. Epub 2004 Jul 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alcohol Oxidoreductases / biosynthesis
  • Alcohol Oxidoreductases / chemistry*
  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / isolation & purification
  • Crystallization
  • Crystallography, X-Ray
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Gene Expression
  • Ketol-Acid Reductoisomerase

Substances

  • Alcohol Oxidoreductases
  • Ketol-Acid Reductoisomerase