Glycoside Hydrolase Family 8

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

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

---

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). GH8 was one of the first glycoside hydrolase families classified by hydrophobic cluster analysis, and was previously known as "Cellulase Family D" [1, 2].

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 [3]. Hydrolysis by CelA was computationally simulated with QM/MM metadynamics [4].

Glycosynthase engineering

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 [5].

Catalytic Residues

The general acid (proton donor to the leaving group) was first identified in CelA from C. thermocellum as Glu95 [6]. The general base (proton acceptor from the nucleophilic water) of GH8a subfamily was first identified in CelA from C. thermocellum as Asp278 [6]. 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 [7].

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 [6]. 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 [8].

Subfamilies

GH8 enzymes are divided into at least three subfamilies, depending on the position of the general base [7]. 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.

Family Firsts

First sequence identification

Cellulase (celA) from Clostridium thermocellum [9]

First sterochemistry determination

Endoglucanase C from Clostridium cellulolyticum (CelCCC) [3]

First general acid residue identification

Cellulase (CelA) from Clostridium thermocellum [6]

First general base residue identification of GH8a

Cellulase (CelA) from Clostridium thermocellum [6]

First general base residue identification of GH8b

Chitosanase from Bacillus sp. K17 by crystal structure and a mutant [7].

First 3-D structure

Endoglucanase CelA from Clostridium thermocellum by X-ray crystallography (PDB ID 1cem) [6].

References

1. Henrissat B, Claeyssens M, Tomme P, Lemesle L, and Mornon JP. (1989). Cellulase families revealed by hydrophobic cluster analysis. Gene. 1989;81(1):83-95. DOI:10.1016/0378-1119(89)90339-9 |
2. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, and Warren RA. (1991). Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991;55(2):303-15. DOI:10.1128/mr.55.2.303-315.1991 |
3. 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 |
4. 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 |
5. 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 |
6. 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 |
7. 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 |
8. 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 |
9. 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 |