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.
Ketol‐acid reductoisomerase is an enzyme of the branched chain amino acid biosynthesis pathway. It has promise as a target for herbicides and antimicrobial agents, and as a catalyst for biofuel production. The enzyme requires specifically a dinuclear Mg2+ centre to activate a μ‐hydroxide that initiates catalysis. In presence of Co2+ the substrate binds in a non‐competent mode. More information can be found in the Full Paper by G. Schenk et al. on page 7427 ff.
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