title: Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism.
abstract: A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Megumi
authors.initials: M
authors.lastname: Takeuchi
authors.affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Jamie
authors.initials: J
authors.lastname: LeBarron
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Kantharia
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Erwin
authors.initials: E
authors.lastname: London
authors.affiliation: Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Bakker
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haltiwanger
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA.|Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeuchi
mesh_terms: D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014994:Xylose / Q000737:chemistry* / Q000378:metabolism; D000097608:UDP Xylose-Protein Xylosyltransferase
chemical_list: D004815:Epidermal Growth Factor; D010430:Pentosyltransferases; D051881:Receptor, Notch1; D011994:Recombinant Proteins; D014994:Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase; C497762:Notch1 protein, mouse