Glycoside Hydrolase Family 72

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Authors: ^^^Ramon Hurtado-Guerrero^^^ and ^^^Thierry Fontaine^^^
Responsible Curator: ^^^Bernard Henrissat^^^

Glycoside Hydrolase Family GH72
Clan	none, (βα)8 fold
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH72.html

Substrate specificities

The GH72 family is formed exclusively by transglycosylases of the fungal kindgom whose activity was firstly characterized in Aspergillus fumigatus [1] and yeasts [2, 3, 4]. These GPI-anchored plasma membrane enzymes elongate and remodel the β-1,3 glucan of the cell wall [4, 5, 6, 7, 8, 9]. This activity is due to their catalytic domain is located in the external part of the plasma membrane. Two sub-families have been described for GH72 family members depending on the presence or absence of a C-terminal cysteine rich domain (carbohydrate binding domain, CBM43) in addition to the catalytic domain [10].

Kinetics and Mechanism

The catalysis by GH72 family enzymes occurs via a classical Koshland retaining mechanism, which leads to net retention of the β-anomeric configuration of the final product. Enzymatic kinetics were determined by HPLC and showed that these enzymes are transglycosidases rather than glycoside hydrolases. These enzymes cleave internally a β-1,3-glucan molecule and transfer the newly generated reducing end to the non-reducing end of a second β-1,3-glucan molecule through a β-1,3-linkage, resulting in the elongation of the chain. The minimum size of the donor and acceptor substrates described in few fungal species are laminaridecaose and laminaripentaose, respectively [1, 11]. Despite that the overall mechanism of hydrolysis and transglycosylation is well known, it is still unclear how transglycosylases can favor transglycosylation in a 55 M water medium. By structural studies with different laminarioligosaccharides and enzymatic activity assays, the “Base occlusion mechanism” was proposed to explain why these enzymes favor transglycosylation versus hydrolysis. In this mechanism, the acceptor sugar blocks the entrance of water molecules and thus avoids hydrolysis, favouring transglycosylation [12].

Catalytic Residues

Multiple sequence alignments have highlighted conserved amino acid between GH72 family members [13]. Hydrophobic cluster analysis allowed to identify two highly conserved glutamate residues in the region comparable to the C-terminal end of strands β-4 and β-7 of the endoglucanase A (GH5 member) of Clostridium cellulolyticum [2]. Site-direct mutagenesis of these two glutamate residues in A. fumigatus Gel1p and S. cerevisiae Gas1p have shown their essentiality for the transglycosidase activity [3, 13] and support that these residues are the acid-base and nucleophilic residues responsible for the catalytic mechanism. The identity of these residues were further confirmed by the resolution of the crystal structure of S. cerevisiae Gas2 (ScGas2) (see below) [12].

Three-dimensional structures

The only three-dimensional structure available is that of ScGas2. The enzyme folds as a (ba8 barrel similar to that prevailing in other families constituting Clan GH-A [12] (Figure 1). The full length enzyme also harbors a CBM43 module at the C-terminus. The crystal structure also showed that both domains share extensive contacts [12] (Figure 1).

Family Firsts

First stereochemistry determination

β-1,3-glucanosyltransglycosilase (Gel1p) from Aspergillus fumigatus [1]

First catalytic nucleophile identification

Shown in the β-1,3-glucanosyltransglycosilase (Gel1p) from Aspergillus fumigatus [13]

First general acid/base residue identification

Shown in the β-1,3-glucanosyltransglycosilase (Gel1p) from Aspergillus fumigatus [13]

First 3-D structure: ScGas2 crystal structure [12]

References

All Medline abstracts: PubMed