GH18 ENDO-β-N-ACETYLGLUCOSAMINIDASES USE DISTINCT MECHANISMS TO PROCESS HYBRID-TYPE N-LINKED GLYCANS
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Trastoy, BeatrizDu, Jonathan J
Li, Chao
García-Alija, Mikel
Klontz, Erik H
Roberts, Blaine R
Donahue, Thomas C
Wang, Lai-Xi
Sundberg, Eric J
Guerin, Marcelo E
Date
2021-07-26Journal
Journal of Biological ChemistryPublisher
Elsevier Ltd.Type
Article
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N-glycosylation is one of the most abundant post-translational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology, but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid type (Hy-type) glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for Hy-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.Rights/Terms
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.Keyword
antibody glycoengineeringcarbohydrate active enzymes
endo-β-N-acetylglucosaminidases
enzyme specificity
glycoprotein bioengineering
glycoside hydrolases
gut microbiome
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http://hdl.handle.net/10713/16288ae974a485f413a2113503eed53cd6c53
10.1016/j.jbc.2021.101011
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