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Glycoside Hydrolase Family 123

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Glycoside Hydrolase Family GH123
Clan none
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

Glycoside hydrolase family 123 contains N-acetyl-β-galactosaminidases (EC, which degrade glycosphingolipids. These enzymes hydrolyze the non-reducing terminal β-GalNAc linkage, but not β-GlcNAc linkages. N-Acetyl-β-galactosaminidases (EC are distinguished from β-hexosaminidases (EC or N-acetyl-β-glucosaminidases (EC because N-acetyl-β-galactosaminidases are selective for a β-GalNAc linkage while N-acetyl-β-glucosaminidases are selective for a β-GlcNAc linkage; β-hexosaminidases hydrolyze both β-GlcNAc and β-GalNAc linkages at a non-reducing terminus. NgaP N-acetyl-β-galactosaminidase 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 N-acetyl-β-galactosaminidase. CpNga123 from Clostridium perfringens (CpNga123) is also a N-acetyl-β-galactosaminidase with activity on the GA2 glycan [2]. Recombinant BvGH123 from Bacteroides vulgatus displayed a 23-fold preference for the hydrolysis of pNP-β-GalNAc versus pNP-β-GlcNAc, in terms of kcat/KM [3].

Kinetics and Mechanism

Family GH123 N-acetyl-β-galactosaminidases are retaining enzymes, as first shown by 1H NMR analysis of the hydrolysis of p-nitrophenyl N-acetyl-β-galactosaminide by Bacteroides vulgatus BvGH123 [3]. NgaP and BvGH123 are strongly inhibited by Gal-thiazoline, a mimic of an oxazolinium ion; on this basis family GH123 enzymes are proposed to act through a neighboring group participation mechanism involving an oxazolinium ion intermediate [1, 3]. 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 provide support that Glu608 of NgaP functions as general acid/base and Asp607 as a transition state stabilizer of the 2-acetamido group [1].

Three-dimensional structures

The three-dimensional structure of CpNga123 from Clostridium perfringens [2] and BvGH123 from Bacteroides vulgatus [3] have been solved . 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, with some similarity to so-called BACON domains. It was also revealed that a structural change of the active site occurred upon binding the substrate, and to order the active site residues for the proposed 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. An X-ray structure of BvGH123 in complex with Gal-thiazoline revealed movement of several active-site residues compared with the 'apo' structure. Residues Asp361 and Glu362 (equivalent to Asp607 and Glu608 in NgaP), were located in positions consistent with their proposed roles as transition state stabilizer and general acid/base, respectively [3].

Family Firsts

First stereochemistry determination
Bacteroides vulgatus GH123 N-acetyl-β-galactosaminidase by 1H NMR [3].
First catalytic nucleophile identification
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 supports Glu608 acting as general acid/base for NgaP [1].
First 3-D structure
CpNga123 from Clostridium perfringens [2].


  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 | PubMed ID:21297160 [SumidaJBC2011]
  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 | PubMed ID:27038508 [Noach2016]
  3. Roth C, Petricevic M, John A, Goddard-Borger ED, Davies GJ, and Williams SJ. (2016). Structural and mechanistic insights into a Bacteroides vulgatus retaining N-acetyl-β-galactosaminidase that uses neighbouring group participation. Chem Commun (Camb). 2016;52(74):11096-9. DOI:10.1039/c6cc04649e | PubMed ID:27546776 [Roth2016]

All Medline abstracts: PubMed