Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation
Abstract
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. In this work, 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.
- Authors:
-
[2];
[2];
[1];
[1]
- Univ. of Maryland, Baltimore, MD (United States)
- Univ. of Maryland, College Park, MD (United States)
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1736274
- Grant/Contract Number:
- AC02-06CH11357; AC02-76SF00515; P41GM103393; T32 AI095190; R01 GM080374; R01 GM127578; R21AI154232
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; enzyme mechanisms; gylcosides; hydrolases; x-ray crystallography
Citation Formats
Klontz, Erik H., Li, Chao, Kihn, Kyle, Fields, James K., Beckett, Dorothy, Snyder, Greg A., Wintrode, Patrick L., Deredge, Daniel, Wang, Lai-Xi, and Sundberg, Eric J.. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. United States: N. p., 2020.
Web. doi:10.1038/s41467-020-20044-z.
Klontz, Erik H., Li, Chao, Kihn, Kyle, Fields, James K., Beckett, Dorothy, Snyder, Greg A., Wintrode, Patrick L., Deredge, Daniel, Wang, Lai-Xi, & Sundberg, Eric J.. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. United States. https://doi.org/10.1038/s41467-020-20044-z
Klontz, Erik H., Li, Chao, Kihn, Kyle, Fields, James K., Beckett, Dorothy, Snyder, Greg A., Wintrode, Patrick L., Deredge, Daniel, Wang, Lai-Xi, and Sundberg, Eric J.. Fri .
"Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation". United States. https://doi.org/10.1038/s41467-020-20044-z. https://www.osti.gov/servlets/purl/1736274.
@article{osti_1736274,
title = {Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation},
author = {Klontz, Erik H. and Li, Chao and Kihn, Kyle and Fields, James K. and Beckett, Dorothy and Snyder, Greg A. and Wintrode, Patrick L. and Deredge, Daniel and Wang, Lai-Xi and Sundberg, Eric J.},
abstractNote = {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. In this work, 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.},
doi = {10.1038/s41467-020-20044-z},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {2020},
month = {12}
}
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