Glycoside Hydrolase Family 8

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• Authors: ^^^Motomitsu Kitaoka^^^ and ^^^Shinya Fushinobu^^^
• Responsible Curator: ^^^Shinya Fushinobu^^^

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Glycoside Hydrolase Family 8
Clan	GH-M
Mechanism	inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/GH8.html

Substrate specificities

Glycoside hydrolases of family 8 cleave β-1,4 linkages of β-1,4 glucans, xylans (or xylooligosaccharides), chitosans, and lichenans (1,3-1,4-β-D-glucan). All of GH8 members have been found from bacteria, and there are no members from Eukaryotic or Archaeal origin. The majority of the enzymes are endo-acting enzymes, but one member has an exo-activity that releases β-D-xylose residues from the reducing end of xylooligosaccharides. The substrate specificities found in GH8 are: chitosanase (EC 3.2.1.132), cellulase (EC 3.2.1.4), licheninase (EC 3.2.1.73), endo-1,4-β-xylanase (EC 3.2.1.8) and reducing-end-xylose releasing exo-oligoxylanase (EC 3.2.1.156).

Kinetics and Mechanism

Enzymes of glycoside hydrolase family 8 are inverting enzymes, as first shown by Fierobe et al. who monitored the reaction of endoglucanase C from Clostridium cellulolyticum (CelCCC) using proton NMR spectroscopy [1]. Hydrolysis by CelA is computationally simulated with QM/MM metadynamics [2].

Catalytic Residues

The general acid (proton donor to the leaving group) was first identified in CelA from C. thermocellum as Glu95 [3]. The general base (proton acceptor from the nucleophilic water) of GH8a subfamily was first identified in CelA from C. thermocellum as Asp278 [3]. The general base of GH8b subfamily was first identified in chitosanase from Bacillus sp. K17 as Glu309 based on its crystal structure and by making E309Q mutant [4].

Subfamilies

GH8 enzymes are divided into at least three subfamilies, depending on the position of the general base [4]. GH8a has the general base (Asp) at the N-terminal end of α8 helix. GH8a contains cellulases, xylanases and other enzymes. In GH8b enzymes, the Asp residue is replaced by Asn, and the general base is a Glu residue located in a long loop inserted between α7 and α8 helices. GH8b contains chitosanases, licheninases, cellulases and other enzymes. The position of the general base in GH8c is unknown.

Three-dimensional structures

Several three-dimensional structures of GH8 members from bacterial origin have been solved. The first solved 3-D structure was endoglucanase CelA from Clostridium thermocellum (PDB ID 1cem) in 1996 [3]. As members of Clan GH-M they have a (α/α)₆ fold similar to Glycoside Hydrolase Family 48. The general acid residue is located at the N-terminal end of α4 helix. Position of the general base differ among #Subfamilies. Atomic (0.94 Å) resolution structure of CelA in complex with substrate (PDB ID 1kwf) has been determined [5].

Glycosynthase

Reducing-end-xylose releasing exo-oligoxylanase from Bacillus halodurans C-125 is the first inverting GH that was converted to glycosynthase by mutating the general base residue [6].

Family Firsts

First sequence identification

Cellulase (celA) from Clostridium thermocellum [7]

First sterochemistry determination

Endoglucanase C from Clostridium cellulolyticum (CelCCC) [1]

First general acid residue identification

Cellulase (CelA) from Clostridium thermocellum [3]

First general base residue identification of GH8a

Cellulase (CelA) from Clostridium thermocellum [3]

First general base residue identification of GH8b

Chitosanase from Bacillus sp. K17 by crystal structure and a mutant [4].

First 3-D structure

Endoglucanase CelA from Clostridium thermocellum by X-ray crystallography (PDB ID 1cem) [3].

References

1. Fierobe HP, Bagnara-Tardif C, Gaudin C, Guerlesquin F, Sauve P, Belaich A, and Belaich JP. (1993). Purification and characterization of endoglucanase C from Clostridium cellulolyticum. Catalytic comparison with endoglucanase A. Eur J Biochem. 1993;217(2):557-65. DOI:10.1111/j.1432-1033.1993.tb18277.x |
2. Petersen L, Ardèvol A, Rovira C, and Reilly PJ. (2009). Mechanism of cellulose hydrolysis by inverting GH8 endoglucanases: a QM/MM metadynamics study. J Phys Chem B. 2009;113(20):7331-9. DOI:10.1021/jp811470d |
3. Alzari PM, Souchon H, and Dominguez R. (1996). The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum. Structure. 1996;4(3):265-75. DOI:10.1016/s0969-2126(96)00031-7 |
4. Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, Yatsunami R, Nakamura S, and Takénaka A. (2004). Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17. J Mol Biol. 2004;343(3):785-95. DOI:10.1016/j.jmb.2004.08.028 |
5. Guérin DM, Lascombe MB, Costabel M, Souchon H, Lamzin V, Béguin P, and Alzari PM. (2002). Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate. J Mol Biol. 2002;316(5):1061-9. DOI:10.1006/jmbi.2001.5404 |
6. Honda Y and Kitaoka M. (2006). The first glycosynthase derived from an inverting glycoside hydrolase. J Biol Chem. 2006;281(3):1426-31. DOI:10.1074/jbc.M511202200 |
7. Béguin P, Cornet P, and Aubert JP. (1985). Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J Bacteriol. 1985;162(1):102-5. DOI:10.1128/jb.162.1.102-105.1985 |