Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-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 alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-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 alpha-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 (3)S(1) sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the (1)C(4) 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 (3)H(4) conformation is proposed as the exploded transition state.
Karaveg
Khanita
K
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Siriwardena
Aloysius
A
Tempel
Wolfram
W
Liu
Zhi-Jie
ZJ
Glushka
John
J
Wang
Bi-Cheng
BC
Moremen
Kelley W
KW
D019943:Amino Acid Substitution; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013997:Time Factors; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism*
D011134:Polysaccharides; D043323:alpha-Mannosidase