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Glycoside Hydrolase Family 95
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|Glycoside Hydrolase Family GH95|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolases of GH95 includes 1,2-α-L-fucosidases (EC 220.127.116.11) that hydrolyze α-Fuc-1,2-Gal linkages attached at the non-reducing ends of oligosaccharides [1, 2], and 1,2-α-L-galactosidases that hydrolyze L-galactoside linkages in arabinoxylans . α-Fuc-1,2-Gal linkages are found in human milk oligosaccharides (2'-fucosyllactose; Fucα1-2Galβ1-4Glc) and blood group glycoconjugates (ABO and Lewis antigens) and are also found as branching residues on the plant polysaccharide xyloglucan (see below) . α-L-Gal-1,2-Xyl linkages are present on side branches on corn glucuronoarabinoxylan . An 1,2-α-L-fucosidase from Bifidobacterium bifidum (BbAfcA) cannot hydrolyze the fucosyl linkage when the Gal residue is further modified, i.e. the enzyme does not act on blood group A- and B-trisaccharides (see  for structures). 3-Fucosyllactose, Galβ1-4(Fucα1-3)Glc, is slightly hydrolyzed by the BbAfcA. 1,2-α-L-Fucosidases from Arabidopsis thaliana and Lilium longiflorum (lily) can liberate L-fucose from xyloglucan fragment XXFG [Xylα1-6Glcβ1-4(Xylα1-6)Glcβ1-4(Fucα1-2Galβ1-2Xylα1-6)Glcβ1-4Glc] as well as 2'-fucosyllactose, but does not liberate L-fucose from 3-fucosyllactose. Both BbAfcA and the plant enzymes do not act on other linkages and artificial substrates such as 4'-nitrophenyl-α-L-fucoside.
Kinetics and Mechanism
Hydrolysis catalyzed by this family of the enzymes proceeds via an inverting mechanism, as first shown by Katayama et al. using 1H-NMR . The best characterized member of this family is the 1,2-α-L-fucosidase from Bifidobacterium bifidum (BbAfcA). The kcat and Km values of BbAfcA for 2'-fucosyllactose, Fucα1-2Galβ1-4Glc, were determined to be 0.091 mM and 160 s-1, respectively .
GH95 enzymes are considered to employ a unique reaction mechanism, in which Asp-activated Asn acts as a general base catalyst while the role of the general acid catalyst is played by a canonical carboxylic residue, Glu. In BbAfcA, Glu566 and Asn423 have been identified as the general acid and general base residues, respectively . Glu566 is hydrogen-bonded with Asn421, and this hydrogen bond is thought to be important in orienting the side chain of Glu566 toward the oxygen atom (O2) of the departing Gal. Asn423 is activated by the neighboring Asp766, and consequently activates a nucleophilic water molecule. This model (carboxylic acid-mediated activation of amido group) bears some analogy to the neighboring group participation mechanism employed by members of GH18, GH20, GH25, GH56, GH84 and GH85. These four residues are invariable in the members of this family, and substitution with alanine or glycine diminishes activities by 1,000- to 10,000-fold.
The first solved 3D structure was of the catalytic domain (aa. 577-1474 of 1959) of BbAfcA (PDB ID 2eab WT in apo form, PDB ID 2eac WT in compex with deoxyfuconojirimycin, PDB ID 2ead E566A in complex with 2'-fucosyllactose, PDB ID 2eae D766A in complex with fucose and lactose) . The catalytic domain adopts an (α/α)6-barrel fold that is quite similar to those of clan GH-L (GH15, GH65, and GH125) and GH94. The members of clan GH-L and GH95 act on α-linkages, whereas GH94 acts on β-linkage. An X-ray structure of a Bacteroides ovatus α-L-galactosidase in complex with L-galactose highlighted Thr370 as a key residue interacting with the 6-hydroxyl group, which is a His residue in 1,2-α-L-fucosidases such as Bb, and which may determine specificity for L-galactose versus L-fucose .
- First stereochemistry determination
- 1,2-α-L-Fucosidase from Bifidobacterium bifidum, determined by 1H-NMR using 2'-fucosyllactose as a substrate .
- First molecular cloning
- 1,2-α-L-Fucosidase from Bifidobacterium bifidum, by expression cloning using a genomic library conctructed in Escherichia coli .
- First general base residue identification
- 1,2-α-L-Fucosidase from Bifidobacterium bifidum, by kinetic analysis and chemical rescue of the mutants .
- First general acid residue identification
- 1,2-α-L-Fucosidase from Bifidobacterium bifidum, by kinetic analysis of the mutant .
- First 3-D structure
- The catalytic domain of 1,2-α-L-fucosidase from Bifidobacterium bifidum, wild-type enzyme in apo-form, wild-type enzyme in complex with deoxyfuconojirimycin, E566A in complex with 2'-fucosyllactose, D766A in complex with fucose and lactose .
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- Nagae M, Tsuchiya A, Katayama T, Yamamoto K, Wakatsuki S, and Kato R. (2007) Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-L-fucosidase from Bifidobacterium bifidum. J Biol Chem. 282, 18497-509. DOI:10.1074/jbc.M702246200 |