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Glycoside Hydrolase Family 94
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|Glycoside Hydrolase Family 94|
|Clan||none (similar to GH-L)|
|Active site residues||known|
|CAZy DB link|
This family of glycoside hydrolases exclusively contains phosphorylases that cleave β-glycosidic bonds. The substrate specificities found in GH94 are: cellobiose (Glc-β1,4-Glc) phosphorylase (EC 184.108.40.206), cellodextrin ((Glc-β1,4-)n-1Glc; n ≥ 3) phosphorylase (EC 220.127.116.11), and N,N’-diacetyl chitobiose (GlcNAc-β1,4-GlcNAc) phosphorylase. Moreover, a phosphorylase domain belonging to this family is found in cyclic β-1,2-glucan synthase, a modular protein that also contains a glycosyltransferase domain from Glycosyltransferase Family 84 . The GH94 domain is thought to phosphorolyze protein-bound β-1,2-glucans synthesized from UDP-glucose by the GT84 domain.
GH94 enzymes were initially classified in Glycosyltransferase Family 36 because none of them show hydrolytic activity. However because of the evolutionary, structural and mechanistic relatedness with clan GH-L glycoside hydrolases, the family was re-assigned to family GH94 .
Kinetics and Mechanism
Phosphorolysis by GH94 enzymes proceeds with inversion of anomeric configuration, as first shown by Sih and McBee  on cellobiose phosphorylase from Clostridium thermocellum, i.e. cellobiose (Glc-β1,4-Glc) + Pi ↔ α-glucose 1-phosphate + glucose; these are therefore inverting enzymes. Considering the topology of the active site structure, the reaction mechanism for inverting phosphorylases is proposed to be similar to that for inverting GHs . With the aid of a general acid residue, enzymatic phosphorolysis begins with direct nucleophilic attack by phosphate on the anomeric C-1 carbon, instead of the water molecule activated by a general base residue, as in inverting GH reaction.
The general acid residue was first elucidated by superimposing the active site structure of chitobiose phosphorylase from Vibrio proteolyticus with a Glycoside Hydrolase Family 15 enzyme, glucoamylase from Thermoanaerobacterium thermosaccharolyticum . Considering the similarities of the active site structure, Asp492 was identified as the general acid residue. D492A/N mutants of this enzyme showed no detectable activity. A general base residue is not required in the reaction catalyzed by glycoside hydrolase-like inverting phosphorylases.
The first solved 3-D structure was chitobiose phosphorylase from Vibrio proteolyticus (PDB [PDB ID 1v7x in complex with GlcNAc and sulfate ) . The enzyme has a (α/α)6 barrel fold that is remarkably similar to clan GH-L. The position of the catalytic general acid is superimposable with Clan GH-L. It should be noted that GH94 enzymes act on β-bonds, whereas clan GH-L enzymes (Glycoside Hydrolase Family 15 and Glycoside Hydrolase Family 65) act on α-bonds.
- First sterochemistry determination
Cellobiose phosphorylase from Clostridium thermocellum 
- First gene cloning
Cellobiose phosphorylase and a cellodextrin phosphorylase from Clostridium stercorarium 
- First general acid residue identification
Vibrio proteolyticus chitobiose phosphorylase by kinetic studies with mutants 
- First 3-D structure
Vibrio proteolyticus chitobiose phosphorylase .
- Ciocchini AE, Guidolin LS, Casabuono AC, Couto AS, de Iannino NI, and Ugalde RA. (2007) A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides. Proc Natl Acad Sci U S A. 104, 16492-7. DOI:10.1073/pnas.0708025104 |
- Hidaka M, Honda Y, Kitaoka M, Nirasawa S, Hayashi K, Wakagi T, Shoun H, and Fushinobu S. (2004) Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold. Structure. 12, 937-47. DOI:10.1016/j.str.2004.03.027 |
- Sih CJ, and McBee RH. A cellobiose phosphorylase in Clostridium thermocellum. Proc Montana Acad Sci 1955, 15, 21-22.
- Reichenbecher M, Lottspeich F, and Bronnenmeier K. (1997) Purification and properties of a cellobiose phosphorylase (CepA) and a cellodextrin phosphorylase (CepB) from the cellulolytic thermophile Clostridium stercorarium. Eur J Biochem. 247, 262-7.