Structural and mechanistic insight into N-glycan processing by endo-?-mannosidase.

ABSTRACT: N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-?-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-?-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (?/?)(8) barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-Man. Using the bespoke substrate ?-Glc-1,3-?-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-?-mannosidase inhibitor ?-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, ?-Glc-1,3-isofagomine, and with the reducing-end product ?-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

SUBMITTER: Thompson AJ 

PROVIDER: S-EPMC3271872 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature