Journal of Biological Chemistry
Volume 286, Issue 16, 22 April 2011, Pages 14065-14072
Journal home page for Journal of Biological Chemistry

Enzymology
Molecular Cloning and Catalytic Mechanism of a Novel Glycosphingolipid-degrading β-N-Acetylgalactosaminidase from Paenibacillus sp. TS12*

https://doi.org/10.1074/jbc.M110.182592Get rights and content
Under a Creative Commons license
open access

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 Ki 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.

Enzyme Catalysis
Enzyme Mechanisms
Enzyme Structure
Glycolipids
Protein Sequence

Cited by (0)

*

This work was supported in part by Grant-in-aid for Basic Research 21380066 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.