Glycoside Hydrolase Family 123

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

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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 specific to β-GalNAc linkage while β-N-acetylglucosaminidases are specific to 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]. 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 highly specific β-N-acetylgalactosaminidase.

Kinetics and Mechanism

Glycoside hydrolases belonging to families GH18, GH20, GH56, GH84 and GH85 are retaining enzymes that cleave sugar residues containing C2-acetamide group such as β-GlcNAc and β-GalNAc through a mechanism in which the 2-acetamido group of the substrate assists catalysis (neighboring group participation). GalNAc-thiazoline, a structural analog of the oxazolinium intermediate of neighboring group participation, is a potent competitive inhibitor of NgaP [1]. 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 GalNAc-thiazoline. 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

CpaNga123 from Clostridium perfringens [2].

Family Firsts

First stereochemistry determination

None reported.

First catalytic nucleophile identification

Unknown. It has been proposed that the carbonyl oxygen of the C-2 acetamide 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

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 |
2. Noach I, Pluvinage B, Laurie C, Abe KT, Alteen MG, Vocadlo DJ, and Boraston AB. (2016). The Details of Glycolipid Glycan Hydrolysis by the Structural Analysis of a Family 123 Glycoside Hydrolase from Clostridium perfringens. J Mol Biol. 2016;428(16):3253-3265. DOI:10.1016/j.jmb.2016.03.020 |