Facile crystallization of Escherichia coli ketol-acid reductoisomerase

McCourt, J. A., Tyagi, R., Guddat, L. W., Biou, V. and Duggleby, R. G. (2004) Facile crystallization of Escherichia coli ketol-acid reductoisomerase. Acta Crystallographica Section D: Biological Crystallography, 60 1432-1434. doi:10.1107/S0907444904012247

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Author McCourt, J. A.
Tyagi, R.
Guddat, L. W.
Biou, V.
Duggleby, R. G.
Title Facile crystallization of Escherichia coli ketol-acid reductoisomerase
Formatted title
Facile crystallization of Escherichia coli ketol-acid reductoisomerase
Journal name Acta Crystallographica Section D: Biological Crystallography   Check publisher's open access policy
ISSN 0907-4449
Publication date 2004-01-01
Sub-type Article (original research)
DOI 10.1107/S0907444904012247
Open Access Status File (Publisher version)
Volume 60
Start page 1432
End page 1434
Total pages 3
Place of publication Malden, MA, United States
Publisher Wiley-Blackwell
Collection year 2004
Language eng
Subject C1
270108 Enzymes
780105 Biological sciences
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 Angstrom resolution; attempts to phase them are currently in progress.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
Biophysics
Crystallography
Reaction-intermediate Analogs
Molecular Replacement
Crystal-structure
Isomeroreductase
Diffraction
Resolution
Synthase
Mad
Q-Index Code C1

 
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Created: Wed, 15 Aug 2007, 13:45:14 EST