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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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