Glycoside Hydrolase Family 72

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

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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

Glycoside hydrolase family GH72 is formed exclusively by transglycosylases of the fungal kingdom 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 transglycosylases 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 mechanisms of hydrolysis and transglycosylation are well known, it is still unclear how transglycosylases limit or prevent hydrolysis in aqueous media, where the concentration of water is 55 M. By structural studies with different laminarioligosaccharides and enzymatic activity assays, a “base occlusion mechanism”, in which the acceptor sugar blocks the entrance of water molecules, was proposed to explain this phenomenon [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 Clostridium cellulolyticum endoglucanase A (a GH5 member) [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 was further confirmed by the resolution of the crystal structure of S. cerevisiae Gas2 (ScGas2) (see below) [12].

Three-dimensional structures

Figure 1. Crystal structure of ScGas2 (PDB ID 2w62).

The first three-dimensional structures available for a GH72 member are that of ScGas2 in free form (PDB ID 2w61) and in complex with carbohydrates (PDB ID 2w62, PDB ID 2w63) (Figure 1). The enzyme folds as a (beta/alpha)₈ barrel similar to that prevailing in other families constituting Clan GH-A [12]. 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].

Family Firsts

First stereochemistry determination

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

First catalytic nucleophile identification

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

First general acid/base residue identification

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

First 3-D structure

ScGas2 crystal structure [12]

References

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