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PDBsum entry 1eqc
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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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.
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Authors
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S.M.Cutfield,
G.J.Davies,
G.Murshudov,
B.F.Anderson,
P.C.Moody,
P.A.Sullivan,
J.F.Cutfield.
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Ref.
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J Mol Biol, 1999,
294,
771-783.
[DOI no: ]
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PubMed id
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Abstract
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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 A 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 conformation 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.
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Figure 1.
Figure 1. Enzyme glycosylation mechanism and two inhibitors of exo-b-(1,3)-glucanase. (a) Formation of the
covalent glycosyl-enzyme intermediate is presumed to proceed through an oxo-carbenium ion-like transition state
and involve nucleophile Glu292 and proton donor Glu192, which act on the glycosidic bond at the non-reducing end
of a b-1,3-glucan chain. The chemical structures of the glucosidase inhibitor, castanospermine, and of the mechanism-
based inactivator 2 ,4 -dinitrophenyl-2-deoxy-2-fluoro-b-D-glucopyranoside are labelled (b) and (c) respectively.
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Figure 6.
Figure 6. GRASP electrostatic surface representation of the binding site of Exg with the two bound saccharides,
following reaction of Exg crystals with the mechanism-based inhibitor DNP-DFG (see Figure 1(c)). Covalently bound
DFG (green spheres) is at the bottom of the pocket (shown left) while a second DFG (yellow spheres) is held between
two phenylalanyl side-chains at the pocket entrance (shown right).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1999,
294,
771-783)
copyright 1999.
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