Structure and Enzymatic Properties of a Two-Domain Family GH19 Chitinase from Japanese Cedar (Cryptomeria japonica) Pollen

• Published in: Journal of agricultural and food chemistry Vol. 66; no. 22; pp. 5699 - 5706
• Main Authors: Takashima, Tomoya, Numata, Tomoyuki, Taira, Toki, Fukamizo, Tamo, Ohnuma, Takayuki
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.06.2018
• Subjects: crystal structure; Cryptomeria japonica; cedar pollen; antifungal activity; Chitinase allergen; chitin
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.8b01140

CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc) n , polysaccharide substrates, glycol chitin, and β-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc) n hydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward β-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize.