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Proc Natl Acad Sci U S A. 2012 Jan 17;109(3):781-6. doi: 10.1073/pnas.1111482109. Epub 2012 Jan 4.

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

Author information

1
Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom.

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-α-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-α-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (β/α)(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 α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-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.

PMID:
22219371
PMCID:
PMC3271872
DOI:
10.1073/pnas.1111482109
[Indexed for MEDLINE]
Free PMC Article

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