title: Structure of Penicillium citrinum alpha 1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes.
abstract: Class I alpha1,2-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) alpha1,2-mannosidases that primarily trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B whereas subgroup 2 includes mammalian Golgi alpha1,2-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 the catalytic domain of the subgroup 2 alpha1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-A resolution. The fungal alpha1,2-mannosidase is an (alphaalpha)(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 alpha1,2-mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal alpha1,2-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 alpha1,2-mannosidases.
authors.affiliation: Program in Structural Biology and Biochemistry, Research Institute, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.
authors.forename: Yuri D
authors.initials: YD
authors.lastname: Lobsanov
authors.forename: Francois
authors.initials: F
authors.lastname: Vallée
authors.forename: Anne
authors.initials: A
authors.lastname: Imberty
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Patrick
authors.initials: P
authors.lastname: Yip
authors.forename: Annette
authors.initials: A
authors.lastname: Herscovics
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
mesh_terms: D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D008361:Mannosidases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000470:Alkaloids; D004220:Disulfides; D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; C065629:kifunensine; D017485:1-Deoxynojirimycin; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D002118:Calcium