W0184

Structural Basis for Substrate Specificity Among Class I α1,2-Mannosidases. Wolfram Tempel¹, Khanita Karaveg^1,2, Zhi-Jie Liu,¹ John P. Rose¹, Kelly W. Moremen^1,2, and Bi-Cheng Wang¹, ¹Dept.of Biochemistry and Molecular Biology, and ²Complex Carbohydrate Research Center², Univ. of Georgia, Athens, GA 30602.

Mammalian processing and catabolic α -mannosidases have been organized into two multigene families, Class 1 and Class 2, which differ in primary sequence and biochemical characteristics. Three Class 1 subfamilies have been identified: (1) ER mannosidase I subfamily cleaves a single residue from Man₉GlcNAc₂ to generate a specific Man₈GlcNAc₂ isomer, (2) Golgi mannosidase I subfamily cleaves Man_9-8GlcNAc₂ structures to Man₅GlcNAc₂ and (3) mannosidase-related proteins that do not appear to have an intrinsic hydrolase activity, but appear to be required for degradation of terminally unfolded glycoproteins in the ER.

In an effort to define the structural basis for substrate recognition, we chose to study a recombinant murine Golgi mannosidase IA, a representative member of the Golgi sub-family. Crystals belonging to space group P2₁2₁2₁ (a = 55.29Å, b = 72.16Å, c = 129.57Å) were obtained and the structure was solved by molecular replacement using the human ER mannosidase I as a search model. The structure at 1.5Å resolution revealed an (αα)₇ barrel that is similar to other Class I mannosidases. A Man₅GlcNac₂ oligosaccharide attached to Asn⁵¹⁵ was found to extend into the active site of an adjoining protein unit in a presumed enzyme-product complex analogous to the structure of S. cerevisiae ER mannosidase I. A comparison of the observed protein-carbohydrate interactions with related glycosidases will be reported.

Work is supported in part with funds from the National Institute of Health (GM47533 and RR05351), The Georgia Research Alliance, and The University of Georgia Research Foundation.