GH18 endo-β-n-acetylglucosaminidases use distinct mechanisms to process hybrid-type n-linked glycans

DOI: 10.1016/j.jbc.2021.101011

Authors: Beatriz Trastoy (Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA)) , Jonathan J. Du (Emory University School of Medicine) , Chao Li (University of Maryland, College Park) , Mikel García-Alija (Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA)) , Erik H. Klontz (University of Maryland School of Medicine) , Blaine R. Roberts (Emory University School of Medicine) , Thomas C. Donahue (University of Maryland) , Lai-Xi Wang (University of Maryland) , Eric J. Sundberg (Emory University School of Medicine) , Marcelo E. Guerin (Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA); Ikerbasque, Basque Foundation for Science)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 69

State: Published (Approved)
Published: July 2021
Diamond Proposal Number(s): 20113

Abstract: 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.

Journal Keywords: endo-β-N-acetylglucosaminidases; glycoside hydrolases; glycoprotein bioengineering; antibody glycoengineering; enzyme specificity; carbohydrate active enzymes; gut microbiome

Diamond Keywords: Enzymes; Gut Microbiota; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: BL13-XALOC at ALBA

Added On: 28/07/2021 08:30

Documents:
jacs.1c06133.pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)