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. Human isoenzymes hexosaminidase A (a heterodimer of α and β subunits) and B (a homodimer of β subunits) are responsible for the hydrolysis of the terminal GalNAc residue from the G_M2 ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB) with thiazoline competive inhibitors of these enzymes being developed as chaperones for the partial restoration of enzyme activity in vivo.

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.[10] Deacetylation of the non-reducing end of a series of chito-oligosaccharides results in a loss of activity of Serratia marscescens chitobiase (a verified GH20 enzyme) towards these compounds (which act as competitive inhibitors).[11]

Competitive Inhibitors of hexosaminidases. "NAG-thiazoline" (upper panel) and non-reducing end deacetylated chito-oligosaccharides are competitive inhibitors of hexosamindase employing neighbouring group participation

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 β-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.[12]

Catalytic Residues

The key catalytic residues of GH20 enzymes are found in a conserved Asp-Glu motif. The glutamate residue functions as the catalytic acid/base. As these enzymes employ neighbouring group participation the preceding aspartate is not a nucleophile. Rather kinetic and crystallographic studies have shown that this residue orients and polarizes the catalytic N-acetyl residue.[13] It may function as a general base (deprotonating the N-acetyl group in the intermediate and forming a neutral oxazoline intermediate) or alternatively it may electrostatically stabilize a positively charge oxazolinium ion intermediate. The catalytic N-acetyl group of the substarte 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.[14] Numerous other hydrogen-bonding residues make contact with

Three-dimensional structures

The first GH20 enzyme to have its structure determined was the Serratia marscescens chitobiase.[15] 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.[13]

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 [16] and it is (now) assumed that (some of) these are GH20 enzymes. The first stereochemical determination for a fully sequenced GH20 was on the Serratia marscescens enzyme [11].

First catalytic nucleophile identification

These enzymes employ neighbouring group participation. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) β-N-hexosaminidases from Aspergillus oryzae[8] and Aspergillus niger[9] supported such a mechanism. This mechanism is further suggested by both the 3-D structure of Serratia marcescens chitobiase [15] (by analogy with GH18 enzymes) and through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity [11].

First general acid/base residue identification

Inferred from the 3-D structure [15] and by analogy with closely related GH18 chitinases.

First 3-D structure

The 3-D structure of the Serratia marscescens chitobiase [15].

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. Knapp, S, Vocadlo, DJ, Gao, Z, Kirk, B, Lou, J, Withers, SG (1996) NAG-thiazoline, An N-Acetyl-β-hexosaminidase Inhibitor That Implicates Acetamido Participation. J. Am. Chem. Soc. 118, 6804-6805.

11. 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 |
12. Vocadlo DJ and Withers SG. (2005). Detailed comparative analysis of the catalytic mechanisms of beta-N-acetylglucosaminidases from families 3 and 20 of glycoside hydrolases. Biochemistry. 2005;44(38):12809-18. DOI:10.1021/bi051121k |
13. Williams SJ, Mark BL, Vocadlo DJ, James MN, and Withers SG. (2002). Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state. J Biol Chem. 2002;277(42):40055-65. DOI:10.1074/jbc.M206481200 |
14. 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 |
15. 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 |
16. 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 |
17. 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 |
18. 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 |
19. Robert V. Stick and Spencer J. Williams. (2009) Carbohydrates. Elsevier Science.

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