Abstract
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 are 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 hydrolase and transferase activity consistent with a role in cell wall glucan metabolism and possibly morphogenesis. The structures of Exg in both free and inhibited forms have been determined to 1.9 Angstrom resolution. A distorted (beta/alpha)(8) barrel structure accommodates an active site which is located 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 analogue (castanospermine) has identified the binding interactions at the -1 glucose binding site. in particular the carboxylate of Glu27 serves a dominant 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 glucose 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 glucose site was identified at the entrance to the pocket, sandwiched between two antiparallel phenylalanine side-chains. This aromatic entrance-way must not only direct substrate into the pocket but also may act as a clamp for an acceptor molecule participating in the transfer reaction. (C) 1999 Academic Press.
Original language | English |
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Pages (from-to) | 771-783 |
Number of pages | 13 |
Journal | Journal of Molecular Biology |
Volume | 294 |
Issue number | 3 |
Publication status | Published - 3 Dec 1999 |
Keywords
- Candida albicans
- crystal structure
- exoglucanase
- mechanism-based inhibitors
- ACID-SEQUENCE SIMILARITIES
- ACTIVE-SITE
- MACROMOLECULAR STRUCTURES
- CRYSTAL-STRUCTURE
- PROTEIN MODELS
- IDENTIFICATION
- CLASSIFICATION
- INTERMEDIATE
- SPECIFICITY
- GLUCOSIDASE