Double Proton Transfer during a Novel Tertiary α-Ketol Rearrangement in Ketol-Acid Reductoisomerase: A Water-Mediated, Metal-Catalyzed, Base-Induced Mechanism

Abstract

(KARI) catalyzes the conversion of (S)-2-acetolactate or (S)-2-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate, the second step in the biosynthesis of branched chain amino acids (BCAAs). Because the BCAA biosynthetic pathway is present in bacteria, plants, and fungi, but absent in animals, it is an excellent target for the development of new-generation antibiotics and herbicides. Nevertheless, the mechanism of the KARI-catalyzed reaction has not yet been fully solved. In this study, we used iterative molecular dynamics (MD) flexible fitting–Rosetta techniques to optimize the three-dimensional solution structure of archaea KARI from Sulfolobus solfataricus (Sso-KARI) determined from cryo-electron microscopy. On the basis of the structure of the Sso-KARI/2Mg2+/NADH/(S)-2-acetolactate complex, we deciphered the catalytic mechanism of the KARI-mediated reaction through hybrid quantum mechanics/molecular mechanics MD simulations in conjunction with umbrella sampling. With an activation energy of only 6.06 kcal/mol, a water-mediated, metal-catalyzed, base-induced (WMMCBI) mechanism was preferred for deprotonation of the tertiary OH group of (S)-2-acetolactate in Sso-KARI. The WMMCBI mechanism for double proton transfer occurred within a proton wire route with two steps involving the formation of hydroxide: (i) Glu233 served as a general base to deprotonate the Mg2+-bound water, forming a hydroxide-coordinated Mg2+ ion; (ii) this hydroxide ion acted as a strong base that rapidly deprotonated the ternary OH group of the substrate. In contrast, the direct deprotonation of the substrate by Glu233 was kinetically unfavorable. This mechanism suggests a novel approach for designing catalysts for deprotonation and provides clues for the development of new-generation antibiotics and herbicides.

Reduction of Np(vi) with hydrazinopropionitrileviawater-mediated proton transfer

“…Water-assisted proton transfer plays an essential role in lowering the reaction energy barrier in systems containing phosphotungstic acid, 44 amino acids, [45][46][47] and nucleic acid bases. 48 However, reports of reactions with water-assisted proton transfer are sparse for metal-bearing systems, [49][50][51][52][53] especially actinide metals. The mechanism identified here for Np(VI) reduction with HzPn involves a remarkable water-mediated proton transfer, which may contribute to further advances in separations of Np from U and Pu in spent fuel reprocessing.…”

Characterization of a class II ketol-acid reductoisomerase from Mycobacterium tuberculosis

“…There has been considerable interest in exploring the biocatalytic potential of NADH-dependent KARIs as they are desirable in industrial applications including anaerobic fermentation to produce branched-chain amino acid or biofuels. 19 The fact that MtKARI-II is a new NADH/NADPH bi-cofactor-dependent KARI suggested that this Mt variant may possess a considerable degree of evolutionary advantages because living cells usually contain higher levels of NADH.…”