Glycoside Hydrolase Family 123

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• Author: ^^^Tomomi Sumida^^^
• Responsible Curator: ^^^Tomomi Sumida^^^

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Glycoside Hydrolase Family GH123
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/GH123.html

Substrate specificities

The glycoside hydrolases family 123 contains β-N-acetylgalactosaminidases (EC 3.2.1.53).

These enzymes specifically hydrolyze the non-reducing terminal β-GalNAc linkage, but not β-GlcNAc linkage. The β-N-acetylgalactosaminidase (EC 3.2.1.53) is distinguished from β-hexosaminidase (EC 3.2.1.52) or β-N-acetylglucosaminidase (EC 3.2.1.52). Because the β-N-acetylgalactosaminidase is specific to β-GalNAc linkage while β-N-acetylglucosaminidase is specific to β-GlcNAc linkage. β-Hexosaminidase hydrolyzes both β-GlcNAc and β-GalNAc linkages at non-reducing terminal. NgaP, N-acetylgalactosaminidase from Paenibacillus sp., is the first cloned β-N-acetylgalactosaminidase and its primary structure is not similar to any glycohydrolases reported so far [1], and, thus, this family is created.

The recombinant NgaP hydrolyzes pNP-β-GalNAc but not pNP-β-GlcNAc, pNP-β-Gal, pNP-α-GalNAc or other pNP-glycosides, indicating that NgaP is a typical β-N-acetylgalactosaminidase.

Kinetics and Mechanism

Glycoside hydrolases belonging to GH18, GH20 and GH85 cleave the sugar containing C2-acetamide group such as β-GlcNAc and β-GalNAc through substrate-assisted catalysis involving neighboring group participation. Since NgaP hydrolyzes the β-GalNAc linkage, NgaP is proposed to use substrate-assisted catalysis. A comparison of secondary structure of NgaP with that of other enzymes that utilize substrate-assisted catalysis suggested that Glu608 of NgaP functions as a proton donor. Point mutation analysis confirmed that Glu608 is integral for the activity of NgaP. GalNAc-thiazoline, a structural analog of the oxazolinium intermediate of neighboring group participation, was found to competitively inhibit the activity of NgaP. These results indicate that NgaP hydrolyzes the terminal β-GalNAc linkage through substrate-assisted catalysis.

Catalytic Residues

Point mutation analysis suggested that Glu608 functions as a proton donor in NgaP.

Three-dimensional structures

Unknown

Family Firsts

First stereochemistry determination

First catalytic nucleophile identification

The carbonyl oxygen of the C-2 acetamide group of the substrate behaves as a catalytic nucleophile.

First general acid/base residue identification

Site-directed mutagenesis indicated that Glu608 is an essential amino acid for the catalytic reaction in NgaP.

First 3-D structure

Not known

References

1. Sumida T, Fujimoto K, and Ito M. (2011). Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading beta-N-acetylgalactosaminidase from Paenibacillus sp. TS12. J Biol Chem. 2011;286(16):14065-72. DOI:10.1074/jbc.M110.182592 |