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

DOI: 10.1073/pnas.1111482109
PMID: 22219371

Authors: Andrew Thompson (University of York) , Rohan J. Williams (University of Melbourne) , Zalihe Hakki (University of Melbourne) , Dominic S. Alonzi (University of Oxford) , Tom Wennekes (Wageningen University) , Tracey Gloster (University of York) , Kriangsak Songsrirote (University of York) , Jane E. Thomas-oates (University of York) , Tanja M. Wrodnigg (Graz University of Technology) , Josef Spreitz (Graz University of Technology) , Arnold E. Stutz (Graz University of Technology) , Terry D. Butters (University of Oxford) , Spencer J. Williams (University of Melbourne) , Gideon J. Davies (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences

State: Published (Approved)
Published: January 2012

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 Glc1/3Man9GlcNAc2 yields Glc1/3-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.

Journal Keywords: Biocatalysis; Carbohydrate; Catalytic; Conserved; Humans; Kinetics; Ligands; Models; Molecular; Polysaccharides; Spectrometry; Mass; Matrix-Assisted; Static; alpha-Mannosidase

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)

Other Facilities: ESRF

Added On: 10/01/2012 11:04

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