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, 181 (15), 4611-6

Functional Analysis of the Carbohydrate-Binding Domains of Erwinia Chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia Coli Putative Chitinase

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Functional Analysis of the Carbohydrate-Binding Domains of Erwinia Chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia Coli Putative Chitinase

H D Simpson et al. J Bacteriol.

Abstract

The Cel5 cellulase (formerly known as endoglucanase Z) from Erwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBD(Cel5) has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBD(Cel5) exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBD(Cel5) bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

Figures

FIG. 1
FIG. 1
(a) Three-dimensional structure of CBDCel5. The residues that were mutated have been highlighted. (b) Backbone structure of CBDCel5. The highlighted residues represent the region that was deleted in the mutant Cel5ΔD17-P23.
FIG. 2
FIG. 2
Binding isotherms on BC at 4°C in 20 mM Tris buffer at pH 7.5 for wild-type (w.t.) Cel5 and single mutants of Cel5 (a), double (W43AY44A) and triple (W18AW43AY44A) mutants of Cel5 (b), the deletion mutant Cel5ΔD17-P23 (c), and pure CBDCel5(His)6 (BC and chitin) (d). [B] and [F] stand for bound and free, respectively.
FIG. 3
FIG. 3
The upper blots show the binding of wild-type Cel5 and Cel5ΔAsp17-Pro23 to BC (1 mg/ml) and chitin (10 mg/ml). The lower blots show the binding of CBDCel5(His)6 and Yheb(His)6 to BC (1 mg/ml), Avicel (15 mg/ml), and chitin (10 mg/ml). Binding at 4°C for 2 h was analyzed on gels and by immunoblotting as described in Materials and Methods. Lanes: 1 and 4, starting sample; 2 and 5, unbound; 3 and 6, bound.

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