A cysteine-enriched motif related to the plant carbohydrate binding module 43 is essential for the functionality of family GH72+ glucanosyltransferases

Gas/Gel/Phr protein families from Saccharomyces cerevisiae, Aspergillus fumigatus and Candida albicans belong to a broad family of proteins from fungal and yeast species that catalyze the hydrolysis and transfer of a (1,3)-glucan resulting in the elongation of linear chains. The glucanosyltransferase (GT) activity is required for the correct assembly of the cell wall. 70 proteins with this activity are gathered in family 72 of Glycoside Hydrolases. These proteins share an N-terminal domain containing two glutamate residues essential for the catalysis. A subset (subfamily 72+) is endowed with a C-ternimal cysteine-enriched module (Cys-box) whereas the other subset lacks this module (subfamily 72-). The Cys-box shares similarity with a module appended to (1,3)-glucanases or lectins of plant origin that defined a new family of Carbohydrate-Binding Modules. In this work we applied a combined approach based on expression of truncated proteins, bioinformatics, spectroscopical, biochemical and phylogenetic analysis to investigate on the structure of Gas1p, a model of 72+ proteins. The Cys-box resulted to be essential for (1,3)-GT activity and to contribute to the stabilization of the structure of Gas1p. A comparison with olive pollen (1,3)-glucanase Ole e 9, indicated that Cys-box does not play a non-catalytic CBM role. The N and C-terminal regions of 72+ enzymes appear to be interdependent and could constitute two lobes of a globule entity thus reflecting distinctive structural features of the active sites of yeast and fungal (1,3)-GT with respect to (1,3)-glucanases. Work partially supported by COFIN 2005 and CanTrain RTN