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Mechanism of Class 1 (Glycosylhydrolase Family 47) α-Mannosidases Involved in N-Glycan Processing and Endoplasmic Reticulum Quality Control*

Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER α-mannosidase I and subsequent recognition by the ER degradation-enhancing α-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER α-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel 3S1 sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the 1C4 conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the –1 subsite. A novel 3H4 conformation is proposed as the exploded transition state.