title: Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism.
abstract: O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the -1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.
authors: From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8W 3P6 and.
authors: Katie J
authors: KJ
authors: Gregg
authors: From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8W 3P6 and.
authors: Michael D L
authors: MD
authors: Suits
authors: the Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors: Lehua
authors: L
authors: Deng
authors: the Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors: David J
authors: DJ
authors: Vocadlo
authors: From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, V8W 3P6 and boraston@uvic.ca.
authors: Alisdair B
authors: AB
authors: Boraston
mesh_terms: D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D006026:Glycoside Hydrolases
keywords: Streptococcus pneumoniae; crystal structure; enzyme catalysis; glycobiology; glycoside hydrolase; mucin