Class I ␣1,2-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) ␣1,2-mannosidases that primarily trim Man 9 GlcNAc 2 to Man 8 GlcNAc 2 isomer B whereas subgroup 2 includes mammalian Golgi ␣1,2-mannosidases IA, IB, and IC that trim Man 9 GlcNAc 2 to Man 5 GlcNAc 2 via Man 8 GlcNAc 2 isomers A and C. The structure of

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... e catalytic domain of the subgroup 2 ␣1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-Å resolution. The fungal ␣1,2-mannosidase is an (␣␣) 7 -helix barrel, very similar to the subgroup 1 yeast (Vallé ) 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 ␣1,2mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal ␣1,2-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 Man 8 GlcNAc 2 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 ␣1,2-mannosidases.