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.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Escherichia coli strains DH5␣ and BL21(DE3) and Pyrobest DNA polymerase were purchased from Takara Bio Inc
-Hexosaminidase (-HEX; EC 3.2.1.52) is an enzyme that hydrolyzes nonreducing terminal -GlcNAc and -GalNAc residues in oligosaccharides, glycoproteins, glycosaminoglycans, and glycosphingolipids (GSLs)
-HEX is considered a retaining hydrolase, it hydrolyzes substrates through substrate-assisted catalysis in which the carbonyl oxygen of the C-2 acetamide group of the substrate behaves as a catalytic nucleophile, and only one amino acid is required as a proton donor from the protein side [9]
Summary
Materials—Escherichia coli strains DH5␣ and BL21(DE3) and Pyrobest DNA polymerase were purchased from Takara Bio Inc. Expression Cloning of the Gene Encoding -NGA—E. coli DH5␣ cells transformed with the plasmids containing Paenibacillus DNA fragments were seeded (ϳ400 colonies/9.2-cm plate) on LB agar plates supplemented with 100 g/ml ampicillin and incubated at 37 °C for 16 h. Following incubation at 37 °C for 16 h with shaking, cells were harvested by centrifugation, suspended in 500 l of 10 mM sodium acetate buffer (pH 5.5), and lysed by sonication. In the first assay with pNP--GalNAc as the substrate, the reaction mixture contained 100 nmol of pNP--GalNAc and an appropriate amount of the enzyme in 100 l of 25 mM sodium acetate buffer (pH 6.0). In the second assay with GA2 or Gb4Cer as the substrate, the reaction mixture contained 5 nmol of GA2 or Gb4Cer and an appropriate amount of the enzyme in 20 l of 25 mM sodium acetate buffer (pH 6.0) containing 0.2% (w/v) taurodeoxycholate. Determination of Ki Values—The Ki values of NgaP for GalNAc thiazoline and GlcNAc thiazoline were determined by the Morrison equation [18] and Dixon plots [19], respectively
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