Acta Crystallographica Section D

Biological Crystallography

Volume 60, Part 8 (August 2004)

Acta Cryst. (2004). D60, 1432-1434 [doi:10.1107/S0907444904012247]

Facile crystallization of Escherichia coli ketol-acid reductoisomerase

J. A. McCourt, R. Tyagi, L. W. Guddat, V. Biou and R. G. Duggleby

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 Å resolution; attempts to phase them are currently in progress.

Online 21 July 2004


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