Cys-enriched domain related to a novel plant carbohydrate-binding module is essential for the glucan-transferase activity of a PHR/GEL/GAS protein subfamily glucan-transferase activity of a PHR/GEL/GAS protein subfamily.

The Gas/Gel/Phr proteins from Saccharomyces cerevisiae, Aspergillus fumigatus and Candida albicans belong to a broader Family of proteins from fungal and yeast species. Gel1, Gas1 and Phr1-2 proteins have been shown to catalyze the hydrolysis and transfer of a beta(1,3)-glucan resulting in the elongation of linear chains. The glucanosyltransferase activity is required for correct assembly of the cell wall, for morphogenesis and for the virulence of fungal pathogens. A total of 70 proteins with this activity was gathered in Family 72 of the Glycoside Hydrolase classification. All these proteins share an N-terminal globular domain containing two conserved glutamate residues essential for the catalysis. Moreover a subset (subfamily 72+) is endowed with a cysteine-enriched module, named Cys-box, whereas the other subset lacks this module (subfamily 72-). The Cys-box shares similarity with the non-catalytic glucan-binding domain present in lectins or associated with several (1,3)-glucanases of plant origin that recently defined a new Family of Carbohydrate-Binding Module (CBM43). In this work we applied a combined approach based on expression of truncated proteins, bioinformatics, spectroscopical and biochemical analysis to investigate on the domain organization of Gas1p of Saccharomyces cerevisiae, a model GH72+ protein. The Cys-box resulted to be essential for (1,3)-glucanosyltransferase activity and contributed to the stabilization of the entire structure of Gas1p. A direct comparison between the properties of Gas1p and olive pollen (1,3)-glucanase Ole e 9, indicated that the Cys-box does not play a role as a non-catalytic CBM. Differently from plant glucanases, the N and C-terminal domains of GH72+ enzymes appear to be interdependent and could constitute two lobes of a single globule entity thus reflecting distinctive structural features of the active sites of yeast and fungal beta(1,3)-glucanosyltransferases with respect to beta(1,3)-glucanases.