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

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This is an example of how to make references to a journal article [1]. (See the References section below). Multiple references can go in the same place like this [1, 2]. You can even cite books using just the ISBN [3]. References that are not in PubMed can be typed in by hand [4].

Kinetics and Mechanism

Neighbouring group participation has long been established as a reasonable mechanism for glycoside hydrolysis in solution,[5, 6, 7, 8] and originally outlined as a possible (though subsequently refuted) mechanism for the hen egg-white lysozyme-catalyzed cleavage of β-aryl di-N-acetylchitobiosides[9]. The earliest kinetic evidence supporting a mechanism involving neighbouring group participation in an enzyme-catalyzed hydrolysis[10, 11] can be found for an N-acetyl-β-D-glucosaminidase isolated from Aspergillus oryzae[12], 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.[13] A comparative analysis of the activity of Streptomyces plicatus b-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 [14].

Catalytic Residues

Kinetic and crystallographic analyses of Asp313 mutants of Streptomyces plicatus b-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

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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 [15] 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 [14].

First catalytic nucleophile identification

This is a neighboring-group participation enzyme with the mechanism suggested both from 3-D structure [16], by analogy with GH18 enzymes and through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity [14].

First general acid/base residue identification

Inferred from the 3-D structure [16] and by analogy with closely related GH18 chitinases.

First 3-D structure

The 3-D structure of the Serratia marscescens chitobiase [16].

References

1. 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 |
2. 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 |

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

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

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

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

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

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

14. 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 |
15. 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 |
16. 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 |