title: Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol-Acid Reductoisomerase.
abstract: Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. It catalyses a complex two-part reaction: an alkyl migration followed by a NADPH-dependent reduction. Both reactions occur within the one active site, but in particular, the mechanism of the isomerisation step is poorly understood. Here, using a combination of kinetic, thermodynamic and spectroscopic techniques, the reaction mechanisms of both Escherichia coli and rice KARI have been investigated. We propose a conserved mechanism of catalysis, whereby a hydroxide, bridging the two Mg(2+) ions in the active site, initiates the reaction by abstracting a proton from the C2 alcohol group of the substrate. While the μ-hydroxide-bridged dimetallic centre is pre-assembled in the bacterial enzyme, in plant KARI substrate binding leads to a reduction of the metal-metal distance with the concomitant formation of a hydroxide bridge. Only Mg(2+) is capable of promoting the isomerisation reaction, likely to be due to non-competent substrate binding in the presence of other metal ions.
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Sonya
authors.initials: S
authors.lastname: Tadrowski
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Marcelo M
authors.initials: MM
authors.lastname: Pedroso
authors.affiliation: Straubing Center of Science, Technische Universität München, Straubing, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
authors.affiliation: Department of Chemistry and Biochemistry, Middlebury College, Middlebury, VT, 05753, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Larrabee
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia. luke.guddat@uq.edu.au.
authors.forename: Luke W
authors.initials: LW
authors.lastname: Guddat
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia. schenk@uq.edu.au.
authors.forename: Gerhard
authors.identifier: 0000-0001-8619-0631
authors.initials: G
authors.lastname: Schenk
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D050540:Ketol-Acid Reductoisomerase / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D009249:NADP / Q000378:metabolism
chemical_list: D007477:Ions; D009249:NADP; D050540:Ketol-Acid Reductoisomerase; D008274:Magnesium
keywords: enzyme catalysis; enzymes; ketol-acid reductoisomerase; magnesium; metal ions