Molecular cloning and catalytic mechanism of a novel glycosphingolipid- degrading β-N-acetylgalactosaminidase from Paenibacillus sp. TS12

Tomomi Sumida, Ken Fujimoto, Makoto Ito

Research output: Contribution to journalArticle

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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 substrateassisted catalysis. This is the first report describing the molecular cloning of a β-NGA adopting substrate-assisted catalysis.

Original languageEnglish
Pages (from-to)14065-14072
Number of pages8
JournalJournal of Biological Chemistry
Volume286
Issue number16
DOIs
Publication statusPublished - Apr 22 2011

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Paenibacillus
Glycosphingolipids
Cloning
Molecular Cloning
Catalysis
Substrates
Enzymes
Enzyme Inhibitors
Point Mutation
Escherichia coli
Protons
Catalytic Domain
Amino Acids
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Molecular cloning and catalytic mechanism of a novel glycosphingolipid- degrading β-N-acetylgalactosaminidase from Paenibacillus sp. TS12. / Sumida, Tomomi; Fujimoto, Ken; Ito, Makoto.

In: Journal of Biological Chemistry, Vol. 286, No. 16, 22.04.2011, p. 14065-14072.

Research output: Contribution to journalArticle

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