Facile crystallization of Escherichia coli ketol-acid reductoisomerase.
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.
Department of Biochemistry and Molecular Biology, University of Queensland, Brisbane 4072, Australia.
Jennifer A
JA
McCourt
Rajiv
R
Tyagi
Luke W
LW
Guddat
Valérie
V
Biou
Ronald G
RG
Duggleby
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D050540:Ketol-Acid Reductoisomerase
D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase