| dc.description.abstract | The conversion of 2-acetolactate (2-aceto-2-hydroxybutyrate) to 2,3-dihydroxy-isovalerate (2,3-dihydroxy-3-ethylbutyrate) is the second step in the biosynthesis of branched chain amino acids (BCAA) catalyzed by ketol-acid reductoisomerase (KARI). KARIs catalyze an alkyl migration of 2-acetolactate (2-aceto-2-hydroxybutyrate), followed by a ketol-acid reduction in terms of NAD(P)H. BCAA biosynthetic pathway is excellent target for the development of novel antibiotic and herbicide resistance as they are only present in bacteria, plants, and fungi; but absent in animals. However, the KARI reaction mechanism is remained unsolved; in particular, how the alkyl-migration catalyzed is still unknown. Here, we obtain the 3D structure of Sso-ilvC2 protein, a KARI from Sulfolobus solfataricus, from the cryo-EM (resolution = 3.38 Å) optimized by the iterative molecular dynamic flexible fitting (MDFF)-Rosetta technique. Based on the 3D structure of Sso-ilvC2 protein we obtained, we elucidate the catalytic mechanism of KARI reaction with density functional theory, hybrid quantum mechanical/molecular mechanical (QM/MM) theoretical calculations, and umbrella sampling QM/MM MD simulations. We observed that Glu233 plays a critical role in the catalytic reaction. Glu233 initiates the reaction in terms of proton abstraction on the coordinated water on the Mg2+ rather than on the hydroxyl of substrate directly. Proton shuttle by protonated Glu233 activated the methyl migration. The overall activation energy is good agreement with experimentally-measured value. In addition, the product in our mechanism has same structural features of active site of X-ray determined structure, where the Glu233 is bonded with the terminal oxygen of product and does not coordinated with Mg2+. Experimental activation energy and X-ray determined structure supports our catalytic mechanism. Our mechanism provides clues for rational design of de novo antibiotic and herbicide resistances. | en_US |