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 contains phosphorolytic enzymes (usually named using a combination of “the substrate” and “phosphorylase”) that cleave beta 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), (N.N’-diacetyl)chitobiose (GlcNAc-β1,4;-GlcNAc) phosphorylase, and a domain phosphorolyzing protein-bound β-1,2-glucan accompanied by cyclic β1,2-glucan synthase(EC 2.4.1.-) belonging to GT84.

Phosphorylases

Phosphorylases catalyze the phosphorolysis of glycosidic bonds to generate glycosyl-phosphate. The reaction is reversible due to the energy of the glycosyl-phosphate bond. Therefore, phosphorylases are categorized as “transferase” among enzyme nomenclature (EC 2.4.1.-). Together with the fact that none of GH94 enzymes showed hydrolytic activity, GH94 enzymes were formally classified in GlycosylTransferase Family 36. By revealing the evolutionary, structural and mechanistic relationship of GH94 pshophorylases with glycoside hydrolase of clan GH-L, the family is re-assigned to a GH family [1].
Today, phosphorylases are categorized based on the evolutionary origins. GH type phosphorylases are classified in Glycoside Hydrolase Family 13, Glycoside Hydrolase Family 65, GH94, and Glycoside Hydrolase Family 112. GH13 sucrose phosphorylase from Bifidobacterium adolescentis has a TIM barrel fold catalytic domain like other GH13 hydorolytic enzymes (PDB 1R7A) [2]. GH65 maltose phorphorylase from Lactobacillus brevis (PDB 1H54) [3] and GH94 enzymes share clan GH-L like (α/α)₆ barrel fold domain. GH112 galacto-N-biose/lacto-N-biose I phosphorylase from Bifidobacterium longum (PDB 2ZUS, 2ZUT, 2ZUU, 2ZUV, 2ZUW, ), which catalyzes phosphorolysis of β-galactosidic bond, has a TIM barrel fold domain similar with that of GH42 β-galactosidase, hydrolase for β-galactosidic bond [4]. GT-type phosphorylases are classified in GT4 and GT35. GT35 pyridoxal phosphate-dependent glycogen phosphorylases share structural and mechanistic similarities with typical NDP-dependent GTs.

Kinetics and Mechanism

Phosphorolysis by GH94 enzymes proceeds with inversion of anomeric configuration, as first shown by Sih and McBee [5] 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 [1]. 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.

Family Firsts

First sterochemistry determination

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

Cite some reference here, with a short explanation [1].

References

1. 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 |