Glycoside Hydrolase Family 123

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

Glycoside Hydrolase Family GH123
Clan	none
Mechanism	probably retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH123.html

Substrate specificities

Glycoside hydrolase family 123 contains β-N-acetylgalactosaminidases (EC 3.2.1.53), which degrade glycosphingolipids. These enzymes hydrolyze the non-reducing terminal β-GalNAc linkage, but not β-GlcNAc linkages. β-N-Acetylgalactosaminidases (EC 3.2.1.53) are distinguished from β-hexosaminidases (EC 3.2.1.52) or β-N-acetylglucosaminidases (EC 3.2.1.52) because β-N-acetylgalactosaminidases are selective for a β-GalNAc linkage while β-N-acetylglucosaminidases are selective for a β-GlcNAc linkage; β-hexosaminidases hydrolyze both β-GlcNAc and β-GalNAc linkages at a non-reducing terminus. NgaP, N-acetylgalactosaminidase from Paenibacillus sp., is the founding member of this family [1]. 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 highly specific β-N-acetylgalactosaminidase. CpNga123 from Clostridium perfringens (CpNga123) is also a β-N-acetylgalactosaminidase with activity on the GA2 glycan [2].

Kinetics and Mechanism

The stereochemical outcome of substrate hydrolysis catalyzed by GH123 enzymes has not been determined. However, NgaP was proposed to be a retaining enzyme and to use substrate-assisted catalysis [1], based on its inhibition by Gal-thiazoline, a structural analog of the oxazolinium intermediate of a neighboring group participation mechanism [1]. In this proposed mechanism, the C2-acetamido group of the substrate is proposed to act as a nucleophile, with a mechanism proceeding through a oxazolinium ion intermediate. Other families of glycoside hydrolases that operate through neighboring group participation mechanisms include families GH18, GH20, GH56, GH84 and GH85 are retaining. A comparison of secondary structure of NgaP with that of other enzymes that utilize substrate-assisted catalysis suggested that Glu608 and Asp607 of NgaP functions as a general acid/base and a stabilizer of the 2-acetamide group of the β-GalNAc at the transition state, respectively. Point mutation analysis confirmed that Glu608 and Asp607 are integral for the activity of NgaP.

Catalytic Residues

Point mutation analysis suggested that Glu608 and Asp607 of NagP function as general acid/base and a transition state stabilizer of the 2-acetamido group [1].

Three-dimensional structures

The three-dimensional structure of a GH123 enzyme, CpNga123 from Clostridium perfringens (CpNga123) has been determined [2]. The crystal structures of CpNga123 (apo form and complex forms with β-GalNAc (product), GalNAc-F2, GA2 trisaccharide and Gb4 disaccharide) were determined. CpNga123 has a catalytic (β/α)₈-barrel domain and an N-terminal β-sandwich domain. It was also revealed that a structural change of the active site was essential for forming interactions to the substrate and the substrate-assisted catalytic mechanism. Furthermore, the difference of the hydrolysis activity of the enzyme toward GA2 and Gb4 glycosphingolipids was explained by the structural difference of the complex structures.

Family Firsts

First stereochemistry determination: None reported.
First catalytic nucleophile identification: Unknown. It has been proposed that the carbonyl oxygen of the C-2 acetamido group of the substrate behaves as a nucleophile [1].
First general acid/base residue identification: Site-directed mutagenesis indicated that Glu608 is an essential amino acid for the catalytic reaction in NgaP [1].
First 3-D structure: CpaNga123 from Clostridium perfringens [2].

References

All Medline abstracts: PubMed