Glycoside Hydrolase Family 20

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• Author: ^^^Ian Greig^^^
• Responsible Curator: ^^^David Vocadlo^^^

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Glycoside Hydrolase Family GH20
Clan	GH-K
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH20.html

Substrate specificities

In addition to exo-acting β-N-acetylglucosaminidases, β-N-acetylgalactosamindase and β-6-SO₃-N-acetylglucosaminidases, GH20 also contains lacto-N-biosidases cleaving β-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides.

Kinetics and Mechanism

Neighbouring group participation has long been established as a reasonable mechanism for glycoside hydrolysis in solution[1, 2, 3, 4] and originally outlined as a possible (though subsequently refuted) mechanism for the hen egg-white lysozyme-catalyzed cleavage of β-aryl di-N-acetylchitobiosides[5]. The earliest kinetic evidence supporting a mechanism involving neighbouring group participation in an enzyme-catalyzed hydrolysis[6, 7] can be found for an N-acetyl-β-D-glucosaminidase isolated from Aspergillus oryzae[8], likely a GH20 enzyme. This work used free energy relationships to infer neighbouring group participation although complete Michaelis-Menten kinetic parameters were not determined. Such kinetic parameters were determined for a β-N-acetylglucosaminidase from Aspergillus niger and a similar free energy relationship-based analysis carried out to infer neighbouring group participation for this (likely GH20) enzyme.[9] The potency of "NAG -thiazoline" as a competitive inhibitor of the jack bean N-acetyl-β-D-hexosaminidase (K_i = 280 nM) has also been used to infer a mechanism of neighbouring group participation although the only retaining hexosaminidases reported currently (November 2010) reported in the CAZy database for the genus Canavalia are found in GH18. A comparative analysis of the activity of Streptomyces plicatus β-hexosaminidase (SpHex, GH20) and Vibrio furnisii β-hexosaminidase (ExoII, GH3) towards p-nitrophenyl N-acyl glucosaminides highlights contrasting reactivity trends expected for families of b-glucosaminidase utilizing a mechanism of substrate-assisted catalysis (GH20) and those which do not (GH3): sharp decreases in activity with increasing N-acyl fluorination are observed in the case of the SpHex enzyme whereas negligible changes in activity are observed for ExoII.REF Loss of activity upon non-reducing end deacatylation [10].

Catalytic Residues

Residues near the catalytically critical Asp and Glu residues show a conserved pattern of The catalytic N-acetyl residue is bound in a hydrophobic pocket defined by three conserved tryptophan residues. These three tryptophan residues define a compact pocket which does not accommodate (non-native) extented N-acyl side-chains as readily as the elongated hydrophobic pocket found in GH84 enzymes.[11] This residue is oriented for nucleophilic attack by hydrogen bonding to a conserved aspartate residue (Asp313, SpHex numbering). Kinetic and crystallographic analyses of Asp313 mutants of Streptomyces plicatus β-hexosaminidase show that it plays a critical role in orienting and polarising the substrate's N-acetyl group to act as a nucleophile towards the anomeric centre.

Three-dimensional structures

The first GH20 enzyme to have its structure determined was the Serratia marscescens chitobiase.[12] This enzyme's active site is located at the C-terminal end of the third of four protein domains, an (βα)₈-barrel. On the basis of these crystallographic studies the invariant Glu540 was identified as the likely catalytic general acid; a bound chitobiose molecule was found to have the oxygen atom of the N-acetamido group belonging to the non-reducing residue suitably positioned to act as the nucleophile.

Family Firsts

First sterochemistry determination

The stereochemistry of hydrolysis of three different hexosaminidases (human placenta, jack bean, and bovine kidney) was shown by the Withers group in 1994 [13] and it is (now) assumed that (some of) these are GH20 enzymes. The first stereochemical determination for a fully sequences GH20 was on the Serratia marscescens enzyme [10].

First catalytic nucleophile identification

This is a neighboring-group participation enzyme with the mechanism suggested both from 3-D structure [12], by analogy with GH18 enzymes and through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity [10].

First general acid/base residue identification

Inferred from the 3-D structure [12] and by analogy with closely related GH18 chitinases.

First 3-D structure

The 3-D structure of the Serratia marscescens chitobiase [12].

References

1. Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620.

2. Piszkiewicz, D, Bruice, T (1967) Glycoside Hydrolysis. I. Intramolecular Acetamido and Hydroxyl Group Catalysis in Glycoside Hydrolysis. J. Am. Chem. Soc. 89, 6237-6243.

3. Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. II. Intramolecular Carboxyl and Acetamido Group Catalysis in β-Glycoside Hydrolysis. J. Am. Chem. Soc. 90, 2156-2163.

4. Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. III. Intramolecular Acetamido Group Participation in the Specific Acid Catalyzed Hydrolysis of Methyl-2-Acetamido-2-deoxy-β-D-glucopyranoside. J. Am. Chem. Soc. 90, 5844-5848.

5. Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) Lysozyme-Catalysed Hydrolysis of some 'β-Aryl Di-N-acetylchitobiosides. Biochem J. 104(3), 893-899.

6. Yamamoto, K, (1973) N-Acyl Specificity of Taka-N-acetyl-β-D-glucosaminidase Studied by Synthetic Substrate Analogs II. Preparation of Some p-Nitrophenyl 2-Halogenoacetylamino-2-deoxy-β-D-glucopyranoside and Their Susceptibility to Enzymic Hydrolysis. J. Biochem. 73, 749-753.

7. Yamamoto, K, (1974) A Quantitative Approach to the Evaluation of β-Acetamide Substituent Effects on the Hydrolysis by Taka-N-acetyl-β-D-glucosaminidase. Role of the Substrate 2-Acetamide Group in the N-Acyl Specificity of the Enzyme J. Biochem. 76, 385-390.

8. Mega, T, Ikenaka, T, Matsushima, Y, (1970) Studies on N-Acetyl-β-D-glucosaminidase of Aspergillus oryzae. J. Biochem. 68, 109-117.

9. Jones, CS, Kosman, DJ (1980) Purification, Properties, Kinetics, and Mechanism of β-N-Acetylglucosaminidase from Aspergillus niger. J. Biol. Chem. 255(24), 11861-11869.

10. Drouillard S, Armand S, Davies GJ, Vorgias CE, and Henrissat B. (1997). Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. Biochem J. 1997;328 ( Pt 3)(Pt 3):945-9. DOI:10.1042/bj3280945 |
11. Macauley MS, Whitworth GE, Debowski AW, Chin D, and Vocadlo DJ. (2005). O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem. 2005;280(27):25313-22. DOI:10.1074/jbc.M413819200 |
12. Tews I, Perrakis A, Oppenheim A, Dauter Z, Wilson KS, and Vorgias CE. (1996). Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nat Struct Biol. 1996;3(7):638-48. DOI:10.1038/nsb0796-638 |
13. Lai EC and Withers SG. (1994). Stereochemistry and kinetics of the hydration of 2-acetamido-D-glucal by beta-N-acetylhexosaminidases. Biochemistry. 1994;33(49):14743-9. DOI:10.1021/bi00253a012 |
14. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n |
15. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 |

17. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006