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Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase
Author(s) -
Tadrowski Sonya,
Pedroso Marcelo M.,
Sieber Volker,
Larrabee James A.,
Guddat Luke W.,
Schenk Gerhard
Publication year - 2016
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201600620
Subject(s) - chemistry , catalysis , hydroxide , active site , metal ions in aqueous solution , isomerization , metal , substrate (aquarium) , stereochemistry , reaction mechanism , organic chemistry , biology , ecology
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.