Mutation of active site residues in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: use of a green fluorescent protein binding assay.
A fluorescent binding assay was developed to investigate the effects of mutagenesis on the binding affinity and substrate specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. The chitin-binding domain was genetically fused to the N-terminus of a green fluorescent protein, and the polyhistidine-tagged hybrid protein was expressed in Escherichia coli. Residues likely to be involved in the binding site were mutated and their contributions to binding and substrate specificity were evaluated by affinity electrophoresis and depletion assays. The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation. Non-conservative substitution of tryptophan residue (W687) nearly abolished chitin-binding affinity and dramatically lowered chitosan binding while retaining the original level of curdlan binding. Double mutation E668K/P689A had altered specificity for several substrates and also impaired chitin binding significantly. Other substitutions in the binding site altered substrate specificity but had little effect on overall affinity for chitin. Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than the wildtype. Furthermore, the ChBD-GFP hybrid protein was tested for use in diagnostic staining of cell walls of fungi and yeast and for the detection of fungal infections in tissue samples.
Hardt
Markus
M
Department of Biological Sciences, Biochemistry and Molecular Biology Division, Louisiana State University, Baton Rouge, LA 70803, USA.
Laine
Roger A
RA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014018:Tissue Distribution
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D002686:Chitin; D049452:Green Fluorescent Proteins; D002688:Chitinases