Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (endoglucanase Z) and an Escherichia coli putative chitinase

The Ce15 cellulase (formerly known as endoglucanase Z) from Erwinia chrysan themi is a multidomain enzyme consisting of a catalytic domain, a linker re gion, and a cellulose binding domain (CBD), A three-dimensional structure o f the CBDCe15 has previously been obtained by nuclear magnetic resonance. I n order to define the role of individual residues in cellulose binding, sit e-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 imp ortant for the stability of Ce15. A deletion mutant lacking the residues As p17 to Pro23 bound only weakly to cellulose. The sequence of CBDCe15 exhibi ts homology to a series of five repeating domains of a putative large prote in, referred to as Yheb, from Escherichia coli. One of the repeating domain s (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromo some and purified by metal chelating chromatography. While CBDCe15 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorl y to cellulose. The Yheb protein contains a region that exhibits sequence h omology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.