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Glycoside hydrolase family GH72 is formed exclusively of transglycosylases of the fungal kingdom. Their activity was first characterized in Aspergillus fumigatus [1] and yeasts [2, 3, 4]. GH72 transglycosidases are GPI-anchored plasma membrane enzymes that elongate and remodel the 1,3-β-glucan of the cell wall [4, 5, 6, 7, 8, 9]. The 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
Catalysis by GH72 family enzymes occurs through 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. Fungal GH72 enzymes internally cleave a 1,3-β-glucan molecule and form a glycosyl enzyme which reacts with the non-reducing end of a second β-1,3-glucan molecule, forming a new β-1,3-glucosidic linkage, resulting in the truncation of the first chain and elongation of the second. The minimum size of the donor and acceptor substrates so far described are laminaridecaose and laminaripentaose, respectively [1, 11].
Despite the fact 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 identification of 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 the assignment of these residues as the acid-base and nucleophilic residues (Glu-160 and Glu-261, respectively, of Gel1p from C. albicans). 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 were that of S. cerevisiae 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].
