Conformational changes in a plant ketol-acid reductoisomerase upon Mg2+ and NADPH binding as revealed by two crystal structures

Leung, Eleanor W. W. and Guddat, Luke W. (2009) Conformational changes in a plant ketol-acid reductoisomerase upon Mg2+ and NADPH binding as revealed by two crystal structures. Journal of Molecular Biology, 389 1: 167-182. doi:10.1016/j.jmb.2009.04.012


Author Leung, Eleanor W. W.
Guddat, Luke W.
Title Conformational changes in a plant ketol-acid reductoisomerase upon Mg2+ and NADPH binding as revealed by two crystal structures
Formatted title
Conformational changes in a plant ketol-acid reductoisomerase upon Mg2+ and NADPH binding as revealed by two crystal structures
Journal name Journal of Molecular Biology   Check publisher's open access policy
ISSN 0022-2836
Publication date 2009-04-09
Sub-type Article (original research)
DOI 10.1016/j.jmb.2009.04.012
Open Access Status
Volume 389
Issue 1
Start page 167
End page 182
Total pages 16
Editor Peter Wright
Jonathan Karn
Place of publication United Kingdom
Publisher Academic Press
Language eng
Subject C1
070303 Crop and Pasture Biochemistry and Physiology
820402 Rice
Abstract Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions-alkyl migration and reduction-and requires Mg and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg and rice KARI-Mg-NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg complex are disordered. In the rice KARI-Mg-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release. Crown Copyright
Keyword Branched-chain amino acids
Protein dynamics
Ketol-acid reductoisomerase
NADPH
X-ray crystallography
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
School of Chemistry and Molecular Biosciences
 
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Created: Thu, 03 Sep 2009, 17:59:28 EST by Mr Andrew Martlew on behalf of School of Chemistry & Molecular Biosciences