Mechanism and Inhibitor Exploration with Binuclear Mg Ketol‐Acid Reductoisomerase: Targeting the Biosynthetic Pathway of Branched‐Chain Amino Acids

Binuclear Mg ketol‐acid reductoisomerase (KARI), which converts ( S )‐2‐acetolactate into ( R )‐2,3‐dihydroxyisovalerate, is responsible for the second step of the biosynthesis of branched‐chain amino acids in plants and microorganisms and thus serves as a key inhibition target potentially without effects on mammals. Here, through the use of density functional calculations and a chemical model, the KARI‐catalyzed reaction has been demonstrated to include the initial deprotonation of the substrate C2 hydroxy group, bridged by the two Mg ions, alkyl migration from the C2‐alkoxide carbon atom to the C3‐carbonyl carbon atom, and hydride transfer from a nicotinamide adenine dinucleotide phosphate [NAD(P)H] cofactor to C2. A dead‐end mechanism with a hydride transferred to the C3 carbonyl group has been ruled out. The nucleophilicity (migratory aptitude) of the migrating carbon atom and the provision of additional negative charge to the di‐Mg coordination sphere have significant effects on the steps of alkyl migration and hydride transfer, respectively. Other important mechanistic characteristics are also revealed. Inspired by the mechanism, an inhibitor (2‐carboxylate‐lactic acid) was designed and predicted by barrier analysis to be effective in inactivating KARI, hence probably enriching the antifungal and antibacterial library. Two types of slow substrate analogues (2‐trihalomethyl acetolactic acids and 2‐glutaryl lactic acid) were also found.