Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase.
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 Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-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.
Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom.
Andrew J
AJ
Thompson
Rohan J
RJ
Williams
Zalihe
Z
Hakki
Dominic S
DS
Alonzi
Tom
T
Wennekes
Tracey M
TM
Gloster
Kriangsak
K
Songsrirote
Jane E
JE
Thomas-Oates
Tanja M
TM
Wrodnigg
Josef
J
Spreitz
Arnold E
AE
Stütz
Terry D
TD
Butters
Spencer J
SJ
Williams
Gideon J
GJ
Davies
D001439:Bacteroides / Q000201:enzymology*; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity; D043323:alpha-Mannosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D008024:Ligands; D011134:Polysaccharides; D043323:alpha-Mannosidase