Glycoside Hydrolase Family 95

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

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Glycoside Hydrolase Family GH95
Clan	none, (α/α)6
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/GH95.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 oligosaccharides [1, 2]. Such structures 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) [2]. 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 do not act on blood group A- and B-trisaccharides. 3-Fucosyllactose, Galβ1-4(Fucα1-3)Glc, is slightly hydrolyzed by the enzyme. 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 the other linkages and artificial substrates such as pNP-Fuc.

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 best characterized member of this family is the 1,2-α-L-fucosidase from Bifidobacterium bifidum (BbAfcA). The k_cat and K_m values of BbAfcA for 2'-fucosyllactose, Fucα1-2Galβ1-4Glc, were determined to be 0.091 mM and 160 s-1, respectively [1].

Catalytic Residues

GH95 enzymes are considered to employ a unique reaction mechanism, in which Asp-activated Asn acts as a base residue while the role of an acid residue is played by a canonical carboxylic residue Glu. In BbAfcA, Glu566 and Asn423 are identified to be the general acid and base residues, respectively[3]. Glu566 is hydrogen-bonded with Asn421, and this hydrogen bond is thought to be important for the side chain of Glu566 to be suitable oriented towards the oxygen atom (O2) of the leaving Gal. Asn423 is activated by the neighboring Asp766, and consequently activates a nucleophilic water molecule. This model (carboxylic acid-mediated activation of amido group) invokes the substrate-assisted catalysis employed by GH18, 20, and 85. The four residues are invariable in the members of this family, and the substitution of alanine or glycine diminished the activities by 1,000- to 10,000-fold[3].

Three-dimensional structures

The first solved 3-D structure was 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)[3]. 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.

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

1. Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, and Yamamoto K. (2004). Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 2004;186(15):4885-93. DOI:10.1128/JB.186.15.4885-4893.2004 |
2. Léonard R, Pabst M, Bondili JS, Chambat G, Veit C, Strasser R, and Altmann F. (2008). Identification of an Arabidopsis gene encoding a GH95 alpha1,2-fucosidase active on xyloglucan oligo- and polysaccharides. Phytochemistry. 2008;69(10):1983-8. DOI:10.1016/j.phytochem.2008.03.024 |
3. 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. 2007;282(25):18497-18509. DOI:10.1074/jbc.M702246200 |