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 GH3
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH3.html

Substrate specificities

The family 3 glycoside hydrolases 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, 4-nitrophenyl 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

Family 3 glycoside hydrolases remove single glycosyl residues from the non-reducing ends of their substrates. Catalysis occurs via a classical Koshland double-displacement mechanism with the β-anomeric configuration of the released glycose being 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]. The reaction sequence and mechanism of a barley β-glucosidase was further outlined using a range of synthetic inhibitors and crystallographic analysis [8]. Detailed kinetic data are also available for a N-acetyl-β-D-glucosaminidase from Vibrio furnisii (ExoII) [9]

Catalytic Residues

The catalytic amino acid residues of GH3 members was first informed by the three-dimensional structure of a barley β-D-glucan glucohydrolase, solved in complex with glucose bound in the -1 subsite [10]. Whereas the catalytic nucleophile is well conserved among GH3 members from diverse species, the catalytic acid/base has been difficult to generally identify. In many cases, this residue is borne on a flexible loop structure, which exhibits significant sequence divergence and conformational flexibility. The following sections detail the definitive assignments of the catalytic residues in a growing number of GH3 enzymes.

Catalytic nucleophile

In plant family 3 β-D-glycosidases, typified by the barley enzyme, active site consists of two glucosyl-binding subsites (-1 and +1) and the catalytic amino acid residues are located between these two subsites [8, 10]. The catalytic nucleophile, as identified in the crystal structure of a trapped 2-fluoroglycosyl enzyme, is Asp285, which is located in a highly conserved GFVISDW motif [8].

The catalytic nucleophile for Vibrio furnisii ExoII (a NagZ) has been identified chemically as the corresponding Asp242, which is conserved throughout the family 3 NagZ enzymes [9].

Catalytic acid/base

The general 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].

A general acid residue has not been identified for family 3 NagZ enzymes.

Three-dimensional structures

Figure 1. Ribbon representation of barley β-glucan exohydrolase isoenzyme ExoI. Domain 1, domain 2, and the linker region of the enzyme are coloured in magenta, cyan, and yellow, respectively. Figure from [1].

Figure 2. NagZ from Vibrio cholerae in complex with PUGNAc (PDB ID: 2OXN) [11]. NagZ enzymes are single domain proteins that adopt a TIM barrel fold. Active site residues are located within the loops that extend from the C-termini of the strands of the β-barrel.

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 ExoI 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 [10]. The two domains are connected by a 16-amino acid helix-like linker. The first 357 residues constitute a (β/α)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 [10]. 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 [10, 12]. 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 β-D-glucosyl residues vary as a result of glycosidic linkages between different C atoms of the adjacent β-D-glucosyl residues [12].

Family 3 NagZ enzymes are also globular, yet have a mass of ~ 36 kDa, which is a distinctive feature of NagZ enzymes from others within the family. NagZ from Vibrio cholerae has been determined in complex with GlcNAc (PDB ID: 1Y65) and with the N-acetyl-β-glucosaminidase inhibitor PUGNAc [11] and NagZ selective PUGNAc derivatives [13]. The enzyme is comprised of 340 amino acids and adopts a (β/α)₈ TIM barrel fold. The active site pocket is shallow and accommodates the 2-N-acetyl group of a terminal GlcNAc sugar in a solvent accessible groove alongside the binding site for the pyranose ring. This active site architecture is different from the active site architectures the functionally-related GH20 N-acetyl-β-hexosaminidases and GH84 O-GlcNAcases. These latter families, which also remove β-1,4-linked GlcNAc residues from glycoconjugates, use a mechanism involving neighboring group participation wherein the carbonyl oxygen of the 2-acetamido group of the terminal GlcNAc acts as a nucleophile, yielding an oxazolinium ion intermediate [14, 15, 16]. Thus, unlike family 3 NagZ enzymes, GH20 and GH84 do not possess an enzymic catalytic nucleophile; however, they do have an appropriately positioned catalytic acid residue. Together, these mechanistic differences have allowed for the development of 2-N-acyl derivatives of PUGNAc that are selective for family 3 NagZ over GH20 and GH84 enzymes [11, 13].

Family Firsts

First 3D Structure

Barley β-D-glucan glucohydrolase [10].

First Catalytic Residues

Barley β-D-glucan glucohydrolase [10].

References

1. Harvey AJ, Hrmova M, De Gori R, Varghese JN, and Fincher GB. (2000). Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases. Proteins. 2000;41(2):257-69. DOI:10.1002/1097-0134(20001101)41:2<257::aid-prot100>3.0.co;2-c |
2. Lee RC, Hrmova M, Burton RA, Lahnstein J, and Fincher GB. (2003). Bifunctional family 3 glycoside hydrolases from barley with alpha -L-arabinofuranosidase and beta -D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing. J Biol Chem. 2003;278(7):5377-87. DOI:10.1074/jbc.M210627200 |

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 and Roseman S. (1996). Molecular cloning and characterization of a novel beta-N-acetyl-D-glucosaminidase from Vibrio furnissii. J Biol Chem. 1996;271(52):33433-9. DOI:10.1074/jbc.271.52.33433 |
5. Cheng Q, Li H, Merdek K, and Park JT. (2000). Molecular characterization of the beta-N-acetylglucosaminidase of Escherichia coli and its role in cell wall recycling. J Bacteriol. 2000;182(17):4836-40. DOI:10.1128/JB.182.17.4836-4840.2000 |
6. Vötsch W and Templin MF. (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. 2000;275(50):39032-8. DOI:10.1074/jbc.M004797200 |
7. Asgarali A, Stubbs KA, Oliver A, Vocadlo DJ, and Mark BL. (2009). Inactivation of the glycoside hydrolase NagZ attenuates antipseudomonal beta-lactam resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2009;53(6):2274-82. DOI:10.1128/AAC.01617-08 |
8. Hrmova M, Varghese JN, De Gori R, Smith BJ, Driguez H, and Fincher GB. (2001). Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase. Structure. 2001;9(11):1005-16. DOI:10.1016/s0969-2126(01)00673-6 |
9. Vocadlo DJ, Mayer C, He S, and Withers SG. (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. 2000;39(1):117-26. DOI:10.1021/bi991958d |
10. Varghese JN, Hrmova M, and Fincher GB. (1999). Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure. 1999;7(2):179-90. DOI:10.1016/s0969-2126(99)80024-0 |
11. Stubbs KA, Balcewich M, Mark BL, and Vocadlo DJ. (2007). Small molecule inhibitors of a glycoside hydrolase attenuate inducible AmpC-mediated beta-lactam resistance. J Biol Chem. 2007;282(29):21382-91. DOI:10.1074/jbc.M700084200 |
12. Hrmova M, De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, and Fincher GB. (2002). Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases. Plant Cell. 2002;14(5):1033-52. DOI:10.1105/tpc.010442 |
13. Balcewich MD, Stubbs KA, He Y, James TW, Davies GJ, Vocadlo DJ, and Mark BL. (2009). Insight into a strategy for attenuating AmpC-mediated beta-lactam resistance: structural basis for selective inhibition of the glycoside hydrolase NagZ. Protein Sci. 2009;18(7):1541-51. DOI:10.1002/pro.137 |
14. 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 |
15. Mark BL, Vocadlo DJ, Knapp S, Triggs-Raine BL, Withers SG, and James MN. (2001). Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase. J Biol Chem. 2001;276(13):10330-7. DOI:10.1074/jbc.M011067200 |
16. Dennis RJ, Taylor EJ, Macauley MS, Stubbs KA, Turkenburg JP, Hart SJ, Black GN, Vocadlo DJ, and Davies GJ. (2006). Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity. Nat Struct Mol Biol. 2006;13(4):365-71. DOI:10.1038/nsmb1079 |