title: Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae.
abstract: Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae. Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the Km values were different. The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.
authors.affiliation: Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung. Fplin@ntou66.ntou.edu.tw
authors.forename: F P
authors.initials: FP
authors.lastname: Lin
authors.forename: H C
authors.initials: HC
authors.lastname: Chen
authors.forename: C S
authors.initials: CS
authors.lastname: Lin
mesh_terms: D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism; D014468:Umbelliferones / Q000378:metabolism
chemical_list: C092930:4-methylumbelliferyl-N-acetylchitotriose; D017931:DNA Primers; D011994:Recombinant Proteins; D014312:Trisaccharides; D014468:Umbelliferones; D002688:Chitinases