Glycoside Hydrolase Family 3

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• Authors: ^^^Geoff Fincher^^^ and ^^^Brian Mark^^^
• Responsible Curator: ^^^Bernard Henrissat^^^

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Glycoside Hydrolase Family GHnn
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/fam/GHnn.html

Substrate specificities

         Normal.dotm  0  0  1  249  1420  Uiversity of Manitoba  11  2  1743  12.0         0  false      18 pt  18 pt  0  0    false  false  false

There are a very large number of enzymes in this family and most originate from microorganisms. Their classification is based largely on nucleotide and amino acid sequence similarities of the corresponding genes. Relatively few members of the enzyme family have been purified and characterised in detail.

The family 3 enzymes have been classified as β-d-glucosidases, α-l-arabinofuranosidases, β-d-xylopyranosidases and N-acetyl-β-d-glucosaminidases [1]. In many cases the enzymes have dual or broad substrate specificities with respect to monosaccharide residues, linkage position and chain length of the substrate. For example, there are several well characterized ‘bifunctional’ enzymes in the family that have both α-l-arabinofuranosidase and β-d-xylopyranosidase activity [2]. In another example, the family 3 β-d-glucosidases from barley, which are more precisely referred to as β-d-glucan glucohydrolases, are broad specificity exo-hydrolases that remove single glucosyl residues from the non-reducing ends of a range of β-d-glucans, β-d-oligoglucosides and aryl b-d-glucosides, including (1,3)-β-d-glucans, (1,4)-β-d-glucans, (1,3;1,4)-β-d-glucans and (1,6)-β-d-glucans, 4nitrophenyl-β-d-glucoside, certain cyanogenic β-d-glucosides and some β-d-oligoxyloglucosides [3].

In contrast, family 3 N-acetyl-β-d-glucosaminidases (NagZ) are ‘monofunctional’ glycoside hydrolases that remove N-acetyl-β-d-glucosamine (GlcNAc) from glycoconjugates [4]. Highly conserved in Gram-negative bacteria, NagZ enzymes play an important role in peptidoglycan recycling by removing GlcNAc from 1,6-anhydroMurNAc-peptides [5], and this activity has been shown to mediate the induction of chromosomal AmpC beta-lactamase [6,7].

Kinetics and Mechanism

         Normal.dotm  0  0  1  89  508  Uiversity of Manitoba  4  1  623  12.0         0  false      18 pt  18 pt  0  0    false  false  false

Family 3 enzymes remove single glycosyl residues from the non-reducing ends of their substrates. Catalysis occurs via a double displacement mechanism and the b-anomeric configuration of the released glucose molecule is retained. The stereochemistry of the reaction has been determined experimentally for some family 3 enzymes. Detailed kinetic analyses are available for two purified barley β-d-glucan glucohydrolases and two barley ‘bifunctional’ α-l-arabinofuranosidase/β-d-xylopyranosidases [2, 3]. Detailed kinetic data are also available for a N-acetyl-β-d-glucosaminidase from Vibrio furnisii (ExoII) [8]

Catalytic Residues

         Normal.dotm  0  0  1  137  781  Uiversity of Manitoba  6  1  959  12.0         0  false      18 pt  18 pt  0  0    false  false  false

The catalytic amino acid residues for the barley β-d-glucan glucohydrolases have been identified by chemical and three-dimensional structural procedures [9]. The substrate-binding site consists of two glucosyl-binding subsites and the catalytic amino acid residues are located between these two subsites. In the plant family 3 β-d-glycosidases the catalytic nucleophile is Asp285, which is located in a highly conserved GFVISDW motif. The catalytic acid, E491, is highly conserved in plant family 3 enzymes but is more difficult to locate in more distantly related members of the family [1]. The reaction sequence and mechanism have been defined for this enzyme using a range of synthetic inhibitors [10].

The catalytic nucleophile for Vibrio furnisii ExoII (a NagZ) has been identified chemically as Asp242, which is conserved thought the family 3 NagZ enzymes [8]. A catalytic acid residue has not been identified for family 3 NagZ enzymes.

Three-dimensional structures

The family 3 β-d-glycosidases are globular monomeric enzymes of molecular mass around 60-70 kDa. The 3D structure of the β-d-glucan glucohydrolase isoenzyme ExoII from barley, determined by X-ray crystallography to 2.2 Å resolution, shows a two-domain, globular protein of 605 amino acid residues that is N-glycosylated at three sites [9]. The two domains are connected by a 16-amino acid helix-like linker. The first 357 residues constitute an (α/β)8 TIM barrel domain. The second domain consists of residues 374 to 559 arranged in a six-stranded β-sandwich, which contains a β-sheet of five parallel β-strands and one antiparallel β-strand, with 3 α-helices on either side of the sheet. A long antiparallel loop of 42 amino acid residues is found at the COOH-terminus of the enzyme. In some bacterial GH3 enzymes the order of the domains is reversed [1].

The active site of the barley β-d-glucan glucohydrolase consists of a relatively shallow substrate-binding pocket that is located at the interface of the two domains of the enzyme [9]. The active site pocket can accommodate the two glucosyl residues at the non-reducing terminus of the substrate and aligns the non-reducing terminal glycosidic linkage of the substrate with the catalytic amino acid residues Asp285 and Glu491. Thus, the catalytic amino acid residues are located on domains 1 and 2, respectively.

The broad specificity of the barley β-d-glucan glucohydrolase can be rationalized from the X-ray crystallographic data and from molecular modelling of enzyme-substrate complexes [9,11]. The glucosyl residue occupying binding subsite –1 is tightly locked into a relatively fixed position through interactions with six amino acid residues near the bottom of the shallow active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two tryptophan residues at the entrance of the pocket, where it is less tightly constrained. The flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme’s surface, means that the overall active site is largely independent of substrate conformation and will therefore accommodate a range of substrates in which the spatial dispositions of adjacent b-d-glucosyl residues vary as a result of glycosidic linkages between different C atoms of the adjacent b-d-glucosyl residues [11].

File:GH3 Fig1.png

Family Firsts

First 3D Structure

Barley [9].

First Catalytic Residues

Barley [9].

References

         Normal.dotm  0  0  1  517  2947  Uiversity of Manitoba  24  5  3619  12.0         0  false      18 pt  18 pt  0  0    false  false  false

1. Harvey, A.J., Hrmova, M., De Gori, R., Varghese, J.N. and Fincher, G.B. (2000) Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases. Proteins: Struct. Funct. Genet. 41:257-269.

2. Lee, R.C., Hrmova, M., Burton, R.A., Lahnstein, J. and Fincher, G.B. (2003) Bifunctional Family 3 Glycoside Hydrolases from Barley with α-L-Arabinofuranosidase and β-D-Xylosidase Activity: Characterization, Primary Structures and COOH-terminal processing. J. Biol. Chem. 278, 5377-5387.

3. Hrmova, M. and Fincher, G.B. (1998) Barley ß-D-glucan exohydrolases. Substrate specificity and kinetic properties. Carbohydr. Res. 305, 209-221.

4. Chitlaru, E. & Roseman, S. (1996). Molecular cloning and characterization of a novel beta-N-acetyl-D- glucosaminidase from Vibrio furnissii. J Biol Chem 271, 33433-9.

5. Cheng, Q., Li, H., Merdek, K. & Park, J. T. (2000). Molecular characterization of the beta-N-acetylglucosaminidase of Escherichia coli and its role in cell wall recycling. J Bacteriol 182, 4836-40.

6. Votsch, W. & Templin, M. F. (2000). Characterization of a beta-N-acetylglucosaminidase of Escherichia coli and elucidation of its role in muropeptide recycling and beta-lactamase induction. J Biol Chem 275, 39032-8

7. Asgarali, A., Stubbs, K. A., Oliver, A., Vocadlo, D. J. & Mark, B. L. (2009). Inactivation of the glycoside hydrolase NagZ attenuates antipseudomonal beta-lactam resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 53, 2274-82.

8. Vocadlo, D. J., Mayer, C., He, S. & Withers, S. G. (2000). Mechanism of action and identification of Asp242 as the catalytic nucleophile of Vibrio furnisii N-acetyl-beta-D-glucosaminidase using 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosyl fluoride. Biochemistry 39, 117-26.

9. Varghese, J.N., Hrmova, M. and Fincher, G.B. (1999) Three-dimensional structure of a barley b-D-glucan exohydrolase; a family 3 hydrolase. Structure 7,179-190.

10. Hrmova, M., Varghese, J.N., De Gori, R., Smith, B.J., Driguez, H. and Fincher, G.B. (2001) Catalytic Mechanisms and Reaction Intermediates along the Hydrolytic Pathway of a Plant β-d-Glucan Glucohydrolase. Structure 9, 1005-1016.

11. Hrmova M, De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, Fincher GB (2002) Structural basis for broad substrate specificity in higher plant β-d-glucan glucohydrolases. The Plant Cell 14, 1033-1052.

12. Stubbs, K. A., Balcewich, M., Mark, B. L. & Vocadlo, D. J. (2007). Small molecule inhibitors of a glycoside hydrolase attenuate inducible AmpC-mediated beta-lactam resistance. J Biol Chem 282, 21382-91.

13. Balcewich, M. D., Stubbs, K. A., He, Y., James, T. W., Davies, G. J., Vocadlo, D. J. & Mark, B. L. (2009). Insight into a strategy for attenuating AmpC-mediated beta-lactam resistance: structural basis for selective inhibition of the glycoside hydrolase NagZ. Protein Sci 18, 1541-51.

14. Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S. & Vorgias, C. E. (1996). Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nat Struct Biol 3, 638-48.

15. Mark, B. L., Vocadlo, D. J., Knapp, S., Triggs-Raine, B. L., Withers, S. G. & James, M. N. (2001). Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase. J Biol Chem 276, 10330-7.

16. Dennis, R. J., Taylor, E. J., Macauley, M. S., Stubbs, K. A., Turkenburg, J. P., Hart, S. J., Black, G. N., Vocadlo, D. J. & Davies, G. J. (2006). Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity. Nat Struct Mol Biol 13, 365-71.