title: Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading beta-N-acetylgalactosaminidase from Paenibacillus sp. TS12.
abstract: We report here the molecular cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase (β-NGA) from Paenibacillus sp. TS12 (NgaP). Consisting of 1034 putative amino acid residues, NgaP shares no sequence similarity with known proteins. Recombinant NgaP, expressed in Escherichia coli, cleaved the nonreducing terminal β-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-β-N-acetylgalactosaminide ∼100 times faster than para-nitrophenyl-β-N-acetylglucosaminide. GalNAc thiazoline, an analog of the oxazolinium intermediate and potent inhibitor for enzymes adopting substrate-assisted catalysis, competitively inhibited the enzyme. The K(i) of the enzyme for GalNAc thiazoline was 1.3 nM, whereas that for GlcNAc thiazoline was 46.8 μM. Comparison of the secondary structure with those of known enzymes exhibiting substrate-assisted catalysis and point mutation analysis indicated that NgaP adopts substrate-assisted catalysis in which Glu-608 and Asp-607 could function as a proton donor and a stabilizer of the 2-acetamide group of the β-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a β-NGA showing substrate-assisted catalysis. This is the first report describing the molecular cloning of a β-NGA adopting substrate-assisted catalysis.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Sumida
authors.forename: Ken
authors.initials: K
authors.lastname: Fujimoto
authors.forename: Makoto
authors.initials: M
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D006028:Glycosphingolipids / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D048828:beta-N-Acetyl-Galactosaminidase / Q000737:chemistry*
chemical_list: D006028:Glycosphingolipids; D011994:Recombinant Proteins; D048828:beta-N-Acetyl-Galactosaminidase