title: Mechanisms of the sialidase and trans-sialidase activities of bacterial sialyltransferases from glycosyltransferase family 80.
abstract: Many important biological functions are mediated by complex glycan structures containing the nine-carbon sugar sialic acid (Sia) at terminal, non-reducing positions. Sia are introduced onto glycan structures by enzymes known as sialyltransferases (STs). Bacterial STs from the glycosyltransferase family GT80 are a group of well-studied enzymes used for the synthesis of sialylated glycan structures. While highly efficient at sialyl transfer, these enzymes also demonstrate sialidase and trans-sialidase activities for which there is some debate surrounding the corresponding enzymatic mechanisms. Here we propose a mechanism for STs from the glycosyltransferase family GT80 in which sialidase and trans-sialidase activities occur through reverse sialylation of CMP. The resulting CMP-Sia is then enzymatically hydrolyzed or used as a donor in subsequent ST reactions resulting in sialidase and trans-sialidase activities, respectively. We provide evidence for this mechanism by demonstrating that CMP is required for sialidase and trans-sialidase activities and that its removal with phosphatase ablates activity. We also confirm the formation of CMP-Sia using a coupled enzyme assay. A clear understanding of the sialidase and trans-sialidase mechanisms for this class of enzymes allows for more effective use of these enzymes in the synthesis of glycoconjugates.
authors.affiliation: Department of Chemistry and Centre for High-throughput Biology, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada.
authors.forename: Kevin
authors.initials: K
authors.lastname: Mehr
authors.affiliation: Department of Chemistry and Centre for High-throughput Biology, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada withers@chem.ubc.ca.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001419:Bacteria / Q000201:enzymology; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D016695:Glycosyltransferases; D012799:Sialyltransferases; C069715:trans-sialidase; D009439:Neuraminidase
keywords: glycoside synthesis; mechanism; sialoconjugates; sialyltransferase; trans-sialidase