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

被引:86
作者
Cutfield, SM
Davies, GJ
Murshudov, G
Anderson, BF
Moody, PCE
Sullivan, PA
Cutfield, JF
机构
[1] Univ Otago, Dept Biochem, Sch Med Sci, Dunedin, New Zealand
[2] Univ York, Dept Chem, York YO1 5DD, N Yorkshire, England
[3] Massey Univ, Inst Mol Biosci, Palmerston North, New Zealand
关键词
Candida albicans; crystal structure; exoglucanase; mechanism-based inhibitors;
D O I
10.1006/jmbi.1999.3287
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:771 / 783
页数:13
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