Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101

Abstract

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)).

FIG. 1

(A) Restriction endonuclease maps of a plasmid clone harboring the gene for chitinase (Chi92). (B) Domain structures of Chi92 and various derivative recombinant proteins used in this study. The numbers refer to the positions of amino acids. Abbreviations: SP, signal peptide; Chi92-N, all-β-strand N-terminal region; GHF 18, catalytic domains of glycosyl hydrolase family 18; A, unknown-function region. ChBD, chitin-binding domain; GST, glutathione S-transferase.

FIG. 2

Optimal alignment of the putative ChBDs of A. hydrophila JP101 Chi92 with those of other proteins. The sequences listed are those of A. hydrophila JP101 chitinase 92, A. caviae chitinase A (29), S. coelicolor chitinase A (23), Vibrio harveyi chitinase A (32), S. griseus chitinase C (20), Aeromonas sp. ORF-1 to -4 (28), Aeromonas sp. chitinase II (37), J. lividum chitinase A (9), Alteromonas sp. strain O-7 chitinases 85 (35) and C (36), C. paraputrificum chitinase B (18), B. subtilis chitinase (accession no. AF069131), P. kodakaraensis KOD1 chitinase A (33), B. licheniformis chitinase (accession no. U71214), S. marcescens QMB1466 chitinase B (10), Cellulomonas pachnodae xylanase 10B (5), Bacillus sp. strain N-4 cellulases A and B (8), B. agaradhaerens cellulase 5A (7), and E. chrysanthemi endoglucanase Z (4). The amino acid numbers are listed on the right, and they are numbered from Met-1 of the proteins. The stop codon is indicated by an asterisk. The black background regions indicate highly conserved amino acid residues. Dashes represent gaps introduced during the alignment process.

FIG. 3

Chitin substrate-binding capacity of Chi92. The binding of Chi92 to various amounts of chitin was measured as described in Materials and Methods. The concentration of Chi92 was 50 μg/ml; the concentrations of colloidal and unprocessed chitin were in the range 0.1 to 10 mg/ml. Symbols: ♦, colloidal chitin; ●, unprocessed chitin.

FIG. 4

SDS-PAGE analysis of truncated Chi92 derivatives and GST fusion proteins. (A) Chi92ΔCII, Chi92ΔCICII, and Chi92ΔACICII purified by Q Sepharose and Sephacryl S-200 chromatography as described in Materials and Methods. Lanes: 1, Chi92 purified from E. coli JM109(pHX); 2, Chi92ΔCII purified from E. coli JM109(pChi87); 3, Chi92ΔCICII purified from E. coli JM109(pChi81); 4, Chi92ΔACICII purified from E. coli JM109(pChi60). (B) GST-CICII and GST-CI purified by glutathione Sepharose 4B chromatography. Lanes: 1, crude cell extract from E. coli XL1-Blue(pGEX-CI); 2, purified GST-CI; 3, crude cell extract from E. coli XL1-Blue(pGEX-CICII); 4, purified GST-CICII. The proteins were analyzed by SDS-PAGE, and the gel was stained with Coomassie brilliant blue R-250. Molecular mass standards were run in lanes M.