Structure of Penicillium citrinum α1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes

Abstract

Class I α1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) α1,2-mannosidases that primarily trim Man9GlcNAc2 to Man8GlcNAc2 isomer B whereas subgroup 2 includes mammalian Golgi α1,2-mannosidases IA, IB, and IC that trim Man9GlcNAc2 to Man5GlcNAc2 via Man8GlcNAc2 isomers A and C. The structure of the catalytic domain of the subgroup 2 α1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-Å resolution. The fungal α1,2-mannosidase is an (αα)7-helix barrel, very similar to the subgroup 1 yeast (Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) and human (Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287-41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two α1,2mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal α1,2-mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man8GlcNAc2 isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER α1,2-mannosidases.