Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase

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.