Glycoside Hydrolase Family 94

• Author: Masafumi Hidaka
• Responsible Curator: Shinya Fushinobu

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Glycoside Hydrolase Family 94
Clan	none (similar to GH-L)
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH94.html

Substrate specificities

This family exclusively contains Phosphorylases that cleave β-glycosidic bond. The substrate specificities found in GH94 are: cellobiose (Glc-β1,4-Glc) phosphorylase (EC 2.4.1.20), cellodextrin ((Glc-β1,4-)_n-1Glc; n ≥ 3) phosphorylase (EC 2.4.1.29), 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 [1]. 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 [2].

Kinetics and Mechanism

Phosphorolysis by GH94 enzymes proceeds with inversion of anomeric configuration, as first shown by Sih and McBee [3] on cellobiose phosphorylase from Clostridium thermocellum, i.e. cellobiose (Glc-β1,4-Glc) + Pi ↔ α-glucose 1-phosphate + glucose. Considering the topology of the active site structure, the reaction mechanism for inverting phosphorylase is proposed to be similar to that for inverting GH [2]. With the aid of general acid residue, the 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 in inverting GH reaction.

Catalytic Residues

The catalytic residue was firstly estimated by superimposing the active site structure of chitobiose phosphorylase from Vibrio proteolyticus with a Glycoside Hydrolase Family 15 enzyme, glucoamylase from Thermoanaerobacterium thermosaccharolyticum [2]. Considering the similarities of the active site structure, Asp492 was estimated as the general acid residue. D492A/N mutants of this enzyme showed no detectable activity. General base residue is not required for the reaction of glycoside hydrolase-like inverting phosphorylases.

Three-dimensional structures

The first solved 3-D structure was chitobiose phosphorylase from Vibrio proteolyticus (PDB 1V7V, 1V7W, 1V7X) [2]. 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.

Family Firsts

First sterochemistry determination

Cellobiose phosphorylase from Clostridium thermocellum [3]

First gene cloning

Cellobiose phosphorylase and a cellodextrin phosphorylase from Clostridium stercorarium [4]

First catalytic nucleophile identification

The inverting phosphorolytic reaction does not require catalytic general base residue, but inorganic phosphate act as a nucleophile.

First general acid residue identification

Vibrio proteolyticus chitobiose phosphorylase by kinetic studies with mutants [2]

First 3-D structure

Vibrio proteolyticus chitobiose phosphorylase [2].

References

1. 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. 2007;104(42):16492-7. DOI:10.1073/pnas.0708025104 |
2. 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. 2004;12(6):937-47. DOI:10.1016/j.str.2004.03.027 |

3. Sih CJ, and McBee RH. A cellobiose phosphorylase in Clostridium thermocellum. Proc Montana Acad Sci 1955, 15, 21-22.

4. 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. 1997;247(1):262-7. DOI:10.1111/j.1432-1033.1997.00262.x |
5. Sprogøe D, van den Broek LA, Mirza O, Kastrup JS, Voragen AG, Gajhede M, and Skov LK. (2004). Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis. Biochemistry. 2004;43(5):1156-62. DOI:10.1021/bi0356395 |
6. Egloff MP, Uppenberg J, Haalck L, and van Tilbeurgh H. (2001). Crystal structure of maltose phosphorylase from Lactobacillus brevis: unexpected evolutionary relationship with glucoamylases. Structure. 2001;9(8):689-97. DOI:10.1016/s0969-2126(01)00626-8 |
7. Hidaka M, Nishimoto M, Kitaoka M, Wakagi T, Shoun H, and Fushinobu S. (2009). The crystal structure of galacto-N-biose/lacto-N-biose I phosphorylase: a large deformation of a TIM barrel scaffold. J Biol Chem. 2009;284(11):7273-83. DOI:10.1074/jbc.M808525200 |