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Mechanistic investigation of family 4 glycosidases : a novel redox-elimination mechanism in the hydrolysis of glycosides Yip, Vivian Ling Yee


The chemical mechanisms of glycoside hydrolases, which catalyze the hydrolysis of glycosidic linkages, are some of the most extensively studied amongst enzymatic reactions. Decades of research have led to the widespread acceptance of the two general glycosidase mechanisms--both involving nucleophilic displacement steps and oxocarbenium ion-like transition states--first proposed by Koshland in 1953. Glycoside hydrolase family 4 is an anomaly within this large class of enzymes. Not only does this family uniquely contain both α- and β-glycosidases, but members of family 4 also require both a divalent metal (Mn²⁺, Co²⁺, Ni²⁺) and a NAD⁺ cofactor for activity. The unusual cofactor requirement, coupled with the observation of solvent deuterium incorporation into C2 of the reaction product prompted the proposal of a mechanism involving key NAD⁺ -mediated redox steps as well as elimination of the glycosidic oxygen. Mechanistic and structural analyses of BglT (a 6-phospho-β-glucosidase from Thermotoga maritima) and GlvA (a 6-phospho-α-glucosidase from Bacillus subtilis ) were performed, revealing a common mechanism for both the α- and β-glycosidases in this family. The key steps include: (1) C3 hydride abstraction by the NAD⁺ cofactor and consequent oxidation of the C3 hydroxyl; (2) abstraction of the C2 proton via general base catalysis; (3) α,β-elimination of the aglycone; (4) 1,4-Michael-like addition of water to the α,β-unsaturated intermediate; (5) reprotonation at C2; and finally (6) reduction of the C3 carbonyl via oxidation of the "on-board" NADH cofactor. Primary kinetic isotope effects and Brønsted relationships reveal that the C3 hydride and C2 proton abstractions are both partially rate-limiting and that the C1-O1 linkage is cleaved rapidly. The evidence suggests that both BgIT and GlvA utilize an E1 cb -type mechanism. Currently, only family 4 glycosidases are known to utilize an elimination mechanism proceeding via anionic transition state(s). Therefore, they stand in stark contrast to other glycoside hydrolases that use "classical" nucleophilic displacement mechanisms and oxocarbenium ion-like transition states. Structural analyses suggest that a tyrosine residue found in close proximity to the C2 proton of the substrate is oriented ideally to act as the catalytic base for C2 deprotonation. Consistent with this data, mutants in which this tyrosine residue have been replaced by phenylalanine or alanine have significantly lower activity than the wild type enzyme. Direct evidence for the role of NAD⁺ was obtained by reduction in situ using NaBH₄ leading to an inactive enzyme that could be reactivated by the addition of excess NAD⁺. This was accompanied by the expected UV-vis spectrophotometric changes. Furthermore, in the BgIT Y241A mutant, the deprotonation/elimination step is slowed sufficiently that steady state accumulation of the reduced nicotinamide cofactor NADH is observed during catalysis. Some clues as to the origins of this unusual class of enzymes come from their structural similarities to α-hydroxy organic acid dehydrogenases. Further, the proposed mechanism shows striking resemblances to those of NAD⁺-dependent sugar dehydratases and SAH-hydrolases.

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