Glycoside Hydrolase Family 8

• Author: 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
http://www.cazy.org/fam/GH8.html

Substrate specificities

GH8 enzymes 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 is 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

GH8 enzymes are inverting enzymes, as first shown by Fiebrobe et al. on endoglucanase C from Clostridium cellulolyticum (CelCCC) [1].

Catalytic Residues

The general acid (proton donor to the leaving group) was first suggested/identified in ... as Glu... The general base (proton acceptor from the nucleophilic water) of GH8a subfamily was first suggested/identified in ... as Asp... 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 [2].

Subfamilies

GH8 enzymes are divided into at least three subfamilies, depending on the position of the general base [2]. 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 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 1kwf) has been determined [4].

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 [5].

Family Firsts

First gene cloning

Cellulase (celA) from Clostridium thermocellum [6]

First sterochemistry determination

Cite some reference here, with a short explanation .

First general acid residue identification

First general base residue identification of GH8a

First general base residue identification of GH8b

Chitosanase from Bacillus sp. K17 by crystal structure and a mutant [2].

First 3-D structure

Endoglucanase CelA from Clostridium thermocellum by X-ray crystallography (PDB 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. 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 |
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. 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 |
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. 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 |