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Instruments / Technical Area	Publication Details	Published	Actions
I24-Microfocus Macromolecular Crystallography	GH18 endo-β-n-acetylglucosaminidases use distinct mechanisms to process hybrid-type n-linked glycans Beatriz Trastoy , Jonathan J. Du , Chao Li , Mikel García-Alija , Erik H. Klontz , Blaine R. Roberts , Thomas C. Donahue , Lai-Xi Wang , Eric J. Sundberg , Marcelo E. Guerin Journal Of Biological Chemistry 69 DOI: 10.1016/j.jbc.2021.101011 Diamond Proposal Number(s): [20113] Open Access Show Abstract ▼ 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.	Jul 2021
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides Beatriz Trastoy , Jonathan J. Du , Erik H. Klontz , Chao Li , Javier O. Cifuente , Lai-Xi Wang , Eric J. Sundberg , Marcelo E. Guerin Nature Communications 11 DOI: 10.1038/s41467-020-14754-7 Diamond Proposal Number(s): [20113] Open Access Show Abstract ▼ Abstract: The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-β-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.	Feb 2020

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