title: Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis.
abstract: Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His(284) in the active site replaced by Asn, Asp or Tyr showed up to 10(4)-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His(284) in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.
authors.affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schmölzer
authors.affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Christiane
authors.initials: C
authors.lastname: Luley-Goedl
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria.
authors.forename: Tibor
authors.initials: T
authors.lastname: Czabany
authors.affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Ribitsch
authors.affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.affiliation: Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria.
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Weber
authors.affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria. Electronic address: bernd.nidetzky@tugraz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D055162:Biocatalysis; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010325:Pasteurella / Q000201:enzymology*; D011487:Protein Conformation; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases
keywords: Bacterial sialyltransferase; Donor substrate hydrolysis; Glycosyltransferase family GT-80; His(284) mutant; In situ(1)H NMR analysis; Inverting mechanism