Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function.
Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.
Itoh
Yoshikane
Y
Department of Biosystem Science, Graduate School of Science and Technology, Niigata University, Japan.
Kawase
Tomokazu
T
Nikaidou
Naoki
N
Fukada
Harumi
H
Mitsutomi
Masaru
M
Watanabe
Takeshi
T
Itoh
Yoshifumi
Y
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002151:Calorimetry; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013305:Streptomyces griseus / Q000201:enzymology*
D000935:Antifungal Agents; D017931:DNA Primers; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases