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This family of glycoside hydrolases was recently discovered following an effort to isolate enzymes for the selective and efficient removal of the terminal α-galactose residue on B antigen for the enzyme conversion of blood group B to universal group O [1]. The activity of several enzymes has been studied and they display narrow substrate specificity for the branched blood group B oligosaccharide (Galα1–3(Fucα1–2)Gal). Interestingly and in contrast to many cases where glycosidases display a higher apparent activity on pNP-substrates than on oligosaccharides, the enzyme from B. fragilis NCTC 9343 (BfGal110A) exhibited a lower activity with pNP-α-Gal (0.3 U/mg) compared to the blood group B substrate (6.6 U/mg). The substrate specificity was remarkably stringent for α-1,3-linked galactose in the branched blood group B structure. The enzyme cleaved neither α4Gal linkages found in P1 and Pk blood group antigens nor the α3Gal linkage in the linear B structure without fucose (the so-called Galili antigen)[1]. In a subsequent study Liu et al. have shown the existence of two distinct subfamilies, one subfamily being exclusively active on the branched blood group B structures, whereas the other subfamily contained linkage specific α1,3-galactosidases that act equally well on both branched blood group B and linear α-1,3-Gal structures [2].
Kinetics and Mechanism
The enzymes of this family are classified as inverting enzymes. The stereochemistry of hydrolysis has been monitored by 1H NMR using p-nitrophenyl α-galactoside as the substrate and B. fragilisBfGal110B as the enzyme. The results showed that the enzyme initially produces β-galactose, i.e. operates with overall inversion of the anomeric configuration, which is in striking contrast to all other known α-galactosidases that use a retaining mechanism (see Glycoside Hydrolase Family 4, Glycoside Hydrolase Family 27, Glycoside Hydrolase Family 36 and Glycoside Hydrolase Family 57) [2].
