Details

Autor(en) / Beteiligte

• Thompson, Andrew J
• Williams, Rohan J
• Hakki, Zalihe
• Alonzi, Dominic S
• Wennekes, Tom
• Gloster, Tracey M
• Songsrirote, Kriangsak
• Thomas-Oates, Jane E
• Wrodnigg, Tanja M
• Spreitz, Josef
• Stütz, Arnold E
• Butters, Terry D
• Williams, Spencer J
• Davies, Gideon J

Titel

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

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2012-01, Vol.109 (3), p.781-786

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2012

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 Glc1/3Man9GlcNAc2 yields Glc1/3-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.