Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation
Author
Klontz, E.H.Li, C.
Kihn, K.
Fields, J.K.
Beckett, D.
Snyder, G.A.
Wintrode, P.L.
Deredge, D.
Wang, L.-X.
Sundberg, E.J.
Date
2020-12-04Journal
Nature CommunicationsPublisher
Nature ResearchType
Article
Metadata
Show full item recordAbstract
Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N-glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen–deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants. Copyright 2020, The Author(s).Sponsors
This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.Keyword
alpha-L-FucosidaseIdentifier to cite or link to this item
http://hdl.handle.net/10713/14543ae974a485f413a2113503eed53cd6c53
10.1038/s41467-020-20044-z