The structure of the exo-beta-(1,3)-glucanase from Candida albicans in native and bound forms: Relationship between a pocket and groove in family 5 glycosyl hydrolases

A group of fungal exo-beta-(1,3)-glucanases, including that from the human pathogen Candida albicans (Exg), belong to glycosyl hydrolase family 5 that also includes many bacterial cellulases (endo-beta-1,4-glucanases). Family members, despite wide sequence variations, share a common mechanism and ar e characterised by possessing eight invariant residues making up the active site. These include two glutamate residues acting as nucleophile and acid/ base, respectively. Exg is an abundant secreted enzyme possessing both hydr olase and transferase activity consistent with a role in cell wall glucan m etabolism and possibly morphogenesis. The structures of Exg in both free an d inhibited forms have been determined to 1.9 Angstrom resolution. A distor ted (beta/alpha)(8) barrel structure accommodates an active site which is l ocated within a deep pocket, formed when extended loop regions close off a cellulase-like groove. Structural analysis of a covalently bound mechanism- based inhibitor (2-fluoroglucosylpyranoside) and of a transition-state anal ogue (castanospermine) has identified the binding interactions at the -1 gl ucose binding site. in particular the carboxylate of Glu27 serves a dominan t hydrogen-bonding role. Access by a 1,3-glucan chain to the pocket in Exg can be understood in terms of a change in confirmation of the terminal gluc ose residue from chair to twisted boat. The geometry of the pocket is not, however, well suited for cleavage of 1,4-glycosidic linkages. A second gluc ose site was identified at the entrance to the pocket, sandwiched between t wo antiparallel phenylalanine side-chains. This aromatic entrance-way must not only direct substrate into the pocket but also may act as a clamp for a n acceptor molecule participating in the transfer reaction. (C) 1999 Academ ic Press.