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 language | English |
|---|---|
| Pages (from-to) | 14065-14072 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 286 |
| Issue number | 16 |
| DOIs | |
| Publication status | Published - Apr 22 2011 |
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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 journal › Article
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TY - JOUR
T1 - Molecular cloning and catalytic mechanism of a novel glycosphingolipid- degrading β-N-acetylgalactosaminidase from Paenibacillus sp. TS12
AU - Sumida, Tomomi
AU - Fujimoto, Ken
AU - Ito, Makoto
PY - 2011/4/22
Y1 - 2011/4/22
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=79954614002&partnerID=8YFLogxK
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U2 - 10.1074/jbc.M110.182592
DO - 10.1074/jbc.M110.182592
M3 - Article
C2 - 21297160
AN - SCOPUS:79954614002
VL - 286
SP - 14065
EP - 14072
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 16
ER -