Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase.
C-terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, k(cat)/K(M), of AcD1ChiAK606 with the 4MU-(GlcNAc)(2) and the 4MU-(GlcNAc)(3) chitin substrates was 15-26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble alpha-chitin substrate. Both fluorescence and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent on the putative C-terminal chitin-binding domain and the function-unknown A region.
Lin
Fu-Pang
FP
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan. fpl5505@mail.ntou.edu.tw
Chuang
Hsu-Han
HH
Liu
Yi-Hsuan
YH
Hsieh
Chia-Yu
CY
Lin
Pei-Wen
PW
Lin
Hsu-Yang
HY
D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D016296:Mutagenesis; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases