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

TY - JOUR

T1 - Structural and mechanistic insight into N-glycan processing by endo-alpha-mannosidase

AU - Thompson, Andrew J.

AU - Williams, Rohan J.

AU - Hakki, Zalihe

AU - Alonzi, Dominic S.

AU - Wennekes, Tom

AU - Gloster, Tracey M.

AU - Songsrirote, Kriangsak

AU - Thomas-Oates, Jane E.

AU - Wrodnigg, Tanja M.

AU - Spreitz, Josef

AU - Stuetz, Arnold E.

AU - Butters, Terry D.

AU - Williams, Spencer J.

AU - Davies, Gideon J.

PY - 2012/1/17

Y1 - 2012/1/17

N2 - 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 read-ornment steps. One unusual enzyme, endo-alpha-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-alpha-mannosidase. Structures solved at resolutions 1.7-2.1 angstrom reveal a (beta/alpha)(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 alpha-Glc-1,3-alpha-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-alpha-mannosidase inhibitor alpha-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, alpha-Glc-1,3-isofagomine, and with the reducing-end product alpha-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.

AB - 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 read-ornment steps. One unusual enzyme, endo-alpha-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-alpha-mannosidase. Structures solved at resolutions 1.7-2.1 angstrom reveal a (beta/alpha)(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 alpha-Glc-1,3-alpha-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-alpha-mannosidase inhibitor alpha-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, alpha-Glc-1,3-isofagomine, and with the reducing-end product alpha-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.

UR - http://www.scopus.com/inward/record.url?scp=84863069740&partnerID=8YFLogxK

U2 - 10.1073/pnas.1111482109

DO - 10.1073/pnas.1111482109

M3 - Article

SN - 0027-8424

VL - 109

SP - 781

EP - 786

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 3

ER -