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

Abstract

We report here the molecular cloning, characterization, and catalytic mechanism of a novel glycosphingolipid-degrading ␤-N-acetylgalactosaminidase (␤-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 ␤-GalNAc residues of gangliotriaosylceramide and globotetraosylceramide. The enzyme hydrolyzed para-nitrophenyl-␤-N-acetylgalactosaminide ϳ100 times faster than para-nitrophenyl-␤-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 M. 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 ␤-GalNAc at the active site, respectively. These results clearly indicate that NgaP is a ␤-NGA showing substrateassisted catalysis. This is the first report describing the molecular cloning of a ␤-NGA adopting substrate-assisted catalysis.

Tandem Mass Spectrometry Has a Larger Analytical Range than Fluorescence Assays of Lysosomal Enzymes: Application to Newborn Screening and Diagnosis of Mucopolysaccharidoses Types II, IVA, and VI

“…In subsequent assays we used 2 mmol/L (Z)-Pugnac to ensure complete hexosaminidase A inhibition. To generate the aglycone from the desulfated glycoside, we explored the bacterial enzyme β - N -acetylgalactosaminidase ( β -NGA) (15). The data in online Supplemental Tables 6 and 7 show that β -NGA did not generate aglycones from the sulfated substrates, and β -NGA catalyzed hydrolysis of the non-sulfated GALNS and ARSB products were not sensitive to (Z)-Pugnac.…”

Fluorimetric assays for N-acetylgalactosamine-6-sulfatase and arylsulfatase B based on the natural substrates for confirmation of mucopolysaccharidoses types IVA and VI

“…In our MS/MS study we found that hexosaminidase A present in DBS hydrolyzes the glycosidic linkage of GalNAc-6-sulfate conjugates, therefore it is necessary to include (Z)-Pugnac as an inhibitor of this enzyme so that 4MU release does not occur independently of GALNS. To release 4MU from the GALNS product we used the bacterial enzyme β-N-acetylgalactosaminidase (β-NGA) [5] since we showed in our mass spectrometry study that this enzyme does not act on the GALNS substrate 4MU-GalNAc-6-S and is not inhibited by (Z)-Pugnac [4]. …”

Acid-catalysed carboxymethylation, methylation and dehydration of alcohols and phenols with dimethyl carbonate under mild conditions

“…Previous studies of GH123 members, initially NagP from Paenibacillus sp., 6 and CpNga123 from C. perfringens (whose structure was recently solved) 7 have led to the proposal of a two-step neighbouring group participation mechanism for catalysis ( Figure 1). According to such a mechanism, the enzyme possesses two key active site residues acting in the role of acid/base and transition state stabilization.…”