Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101.
Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBD(CI) and ChBD(CII) and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBD(CII) or ChBD(CICII). Polysaccharide-binding studies revealed that ChBD(CICII) not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBD(CII), the binding affinities of ChBD(CICII) are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD(CI) and ChBD(CII). In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD(CICII) and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding.
Chang
Ming Chung
MC
Department of Biochemistry, Medical College, National Cheng Kung University, Tainan 701, Taiwan.
Lai
Pe Lin
PL
Wu
Mei Li
ML
D000327:Adsorption; D016980:Aeromonas hydrophila / Q000201:enzymology*; D024322:Amino Acids, Aromatic / Q000502:physiology; D001665:Binding Sites; D002482:Cellulose / Q000378:metabolism; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003102:Colloids / Q000378:metabolism; D004252:DNA Mutational Analysis; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011134:Polysaccharides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
D024322:Amino Acids, Aromatic; D003102:Colloids; D011134:Polysaccharides; D014990:Xylans; D002686:Chitin; D002482:Cellulose; D002688:Chitinases