Role of the N-terminal polycystic kidney disease domain in chitin degradation by chitinase A from a marine bacterium, Alteromonas sp. strain O-7.
OBJECTIVE
The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin.


RESULTS
Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single- and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87.6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathione-Sepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule.


CONCLUSIONS
PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule.


CONCLUSIONS
The findings of this study provide important information on chitin degradation by microbial chitinases.
Orikoshi
H
H
Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.
Nakayama
S
S
Hanato
C
C
Miyamoto
K
K
Tsujibo
H
H
D000327:Adsorption; D020591:Alteromonas / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002686:Chitin / Q000235:genetics* / Q000378:metabolism; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media; D006868:Hydrolysis; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation; D007690:Polycystic Kidney Diseases / Q000235:genetics*; D011994:Recombinant Proteins; D016415:Sequence Alignment / Q000379:methods
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases