Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101.
The gene (chi92) encoding the extracellular chitinase of Aeromonas hydrophila JP101 has been cloned and expressed in Escherichia coli. The mature form of Chi92 is an 842-amino-acid (89.830-kDa) modular enzyme comprised of a family 18 catalytic domain, an unknown-function region (the A region), and three chitin-binding domains (ChBDs; Chi92-N, ChBD(CI), and ChBD(CII)). The C-terminally repeated ChBDs, ChBD(CI) and ChBD(CII), were grouped into family V of cellulose-binding domains on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminally truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to unprocessed and colloidal chitins. Further adsorption experiments with glutathione S-transferase (GST) fusion proteins (GST-CI and GST-CICII) demonstrated that a single ChBD (ChBD(CI)) could promote efficient chitin and cellulose binding. In contrast to the two C-terminal ChBDs, the Chi92-N domain is similar to ChiN of Serratia marcescens ChiA, which has been proposed to participate in chitin binding. A truncated derivative of Chi92 that contained only a catalytic domain and Chi92-N still exhibited insoluble-chitin-binding and hydrolytic activities. Thus, it appears that Chi92 contains Chi92-N as the third ChBD in addition to two ChBDs (ChBD(CI) and ChBD(CII)).
Wu
M L
ML
Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Republic of China.
Chuang
Y C
YC
Chen
J P
JP
Chen
C S
CS
Chang
M C
MC
D016980:Aeromonas hydrophila / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D011993:Recombinant Fusion Proteins; D002686:Chitin; D005982:Glutathione Transferase; D002688:Chitinases