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

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

Research output: Contribution to journalArticlepeer-review

55 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)781-786
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number3
DOIs
Publication statusPublished - 17 Jan 2012

Keywords

  • 3D structure
  • enzyme inhibition
  • enzyme mechanism
  • glycobiology
  • glycosidase
  • ASPARAGINE-LINKED OLIGOSACCHARIDES
  • GLUCOSIDASE-II-DEFICIENT
  • GLYCOPROTEIN-BIOSYNTHESIS
  • ENDOMANNOSIDASE PATHWAY
  • ENDOPLASMIC-RETICULUM
  • GLYCOSIDE HYDROLASES
  • QUALITY-CONTROL
  • INHIBITORS
  • CELLS
  • CLASSIFICATION

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