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This family of glycoside hydrolases contains phosphorylases; beta-galactoside phosphorylase, β-1,3-D-galactosyl-D-hexososamine phosphorylase (EC 2.4.1.211) [1] and β-1,4-D-galactosyl-L-rhamnose phosphorylase (EC 2.4.1.-) [2]. The former enzymes are subcategorized into galacto-N-biose phosphorylase, (GNBP) [3], lacto-N-biose I phosphorylase (LNBP) [4], and galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) [1, 4, 5] based on the substrate preference on galacto-N-biose (GNB, Gal-β1,3-GalNAc) and lacto-N-biose I (LNB, Gal-β1,3-GlcNAc) [4].
Kinetics and Mechanism
Phosphorolysis by GH112 enzymes proceeds with inversion of anomeric configuration, as first shown by Derensy-Dron et al. [1] on β-1,3-D-galactosyl-D-hexososamine phosphorylase from Bifidobacterium bifidum, i.e. LNB + Pi ↔ α-galactose 1-phosphate + GlcNAc. Considering the topology of the active site structure, the reaction mechanism for inverting phosphorylase is proposed to be similar to that for inverting glycoside hydrolases. With the aid of general acid residue, the enzymatic phosphorolysis occurs through direct nucleophilic attack by phosphate on the anomeric C-1 carbon, instead of the water molecule activated by a general base residue in the inverting glycoside hydrolase reaction.
Catalytic Residues
The general acid residue was firstly estimated by mutational analysis on β-1,3-D-galactosyl-D-hexososamine phosphorylase from Bifidobacterium longum [6].
Three-dimensional structures
Figure 1: Movement of the catalytic TIM barrel domain induced by substrate binding. The TIM barrel domain is in blue, an IG-like domain is in green , an α/β domain is in yellow and a C-terminal domain is in red .
The first solved 3-D structure was β-1,3-D-galactosyl-D-hexososamine phosphorylase from Bifidobacterium longum (PDB 2ZUV in complex with GlcNAc, ethylene glycol, and nitrate) [7]. The catalytic domain consists of a partially broken TIM barrel fold that is structurally similar to a β-galactosidase of Glycoside Hydrolase Family 42, supporting the classification of these phosphorolytic enzymes as one of the GH families. A large conformational change in TIM barrel scaffold was observerd with substrate-binding (Figure 1).
Family Firsts
First sterochemistry determination
β-1,3-D-galactosyl-D-hexososamine phosphorylase from Bifidobacterium bifidum [1].
First sequence identification
β-1,3-D-galactosyl-D-hexososamine phosphorylase from Bifidobacterium longum [5].
