Glycoside Hydrolase Family 95

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• Author: ^^^Takane Katayama^^^
• Responsible Curator: ^^^Takane Katayama^^^

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Glycoside Hydrolase Family GHnn
Clan	none, (α/α)6
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GHnn.html

Substrate specificities

This family exclusively contains 1,2-α-L-fucosidases (EC 3.2.1.63) that hydrolyze Fucα1-2Gal linkages attached at the non-reducing ends of glycoconjugates [1][2]. The disaccharide structures constitute the core of ABO blood group substances of human and are also found as constituents of plant xyloglucan [2]. The enzymes cannot hydrolyze the linkage when the Gal residue is modified, i.e. they do not act on blood group A- and B-trisaccharides. 3-Fucosyllactose, Galβ1-4(Fucα1-3)Glc, is slightly hydrolyzed by the enzyme, but the other linkages and artificial substrates such as pNP-Fuc are completely resistant.

Kinetics and Mechanism

Hydrolysis catalyzed by this family of the enzymes proceeds via an inverting mechanism, as first shown by Katayama et al. [1]. The most characterized member of this family is 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.

Catalytic Residues

1,2-α-L-Fucosidases are considered to employ a unique reaction mechanism, in which Asn423 is activated by the neighboring Asp766 to act as a general base residue.

Three-dimensional structures

The first solved 3-D structure was the catalytic domain (aa. 577-1474 of 1959) of 1,2-α-L-fucosidase from Bifidobacterium bifidum (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)[3]. The catalytic domain adopts (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.

Family Firsts

First stereochemistry determination

1,2-α-L-Fucosidase from Bifidobacterium bifidum, determined by 1H-NMR using 2'-fucosyllactose as a substrate.[1].

First molecular cloning

1,2-α-L-Fucosidase from Bifidobacterium bifidum, by expression cloning using a genomic library conctructed in Escherichia coli[1].

First catalytic base identification

1,2-α-L-Fucosidase from Bifidobacterium bifidum, kinetic analysis and chemical rescue of the mutants [3].

First catalytic acid residue identification

1,2-α-L-Fucosidase from Bifidobacterium bifidum, kinetic analysis of the mutant [3].

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 [3].

References

<biblio>

1. Katayama2004 pmid=15262925
2. Altmann2008 pmid=18495185
3. Nagae2007 pmid=17459873