The enzymatic hydrolysis of O-glycosidic linkages is one of the most diverse and widespread reactions in nature and involves a classic "textbook" enzyme mechanism. A multidisciplinary analysis of a beta-glycoside hydrolase, the Cel5A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-intermediate, and product complexes have been determined and their interconversions analyzed kinetically, providing unprecedented insights into the mechanism of this enzyme class. Substrate is bound in a distorted 1S3 skew-boat conformation, thereby presenting the anomeric carbon appropriately for nucleophilic attack as well as satisfying the stereoelectronic requirements for an incipient oxocarbenium ion. Leaving group departure results in the trapping of a covalent alpha-glycosyl-enzyme intermediate in which the sugar adopts an undistorted 4C1 conformation. Finally, hydrolysis of this intermediate yields a product complex in which the sugar is bound in a partially disordered mode, consistent with unfavorable interactions and low product affinity.
Among the numerous well-characterized families of glycosidases, family 4 appears to be the anomaly, requiring both catalytic NAD+ and a divalent metal for activity. The unusual cofactor requirement prompted the proposal of a mechanism involving key NAD+-mediated redox steps as well as elimination of the glycosidic oxygen. Primary kinetic isotope effects for the 2- and 3-deutero substrate analogues, isotopic exchange with solvent, and structural analysis of a 6-phospho-beta-glucosidase, BglT (E.C. 3.2.1.6), provided evidence in support of the proposed mechanism, which has striking resemblances to that of the sugar dehydratases. Furthermore, analysis of the stereochemical outcome indicated that family 4 enzymes are retaining glycosidases.
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