Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, Watanabe T
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Japan.
J Bacteriol. 1996 Sep;178(17):5065-70. doi: 10.1128/jb.178.17.5065-5070.1996.
The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.
灰色链霉菌HUT 6037的几丁质酶C-1对部分乙酰化壳聚糖中β-1,4-糖苷键的水解特异性不同于其他微生物几丁质酶。为了研究这种独特几丁质酶的一级结构，克隆了编码几丁质酶C-1的chiC基因并测定了其核苷酸序列。该基因编码一个由294个氨基酸组成的多肽，计算大小为31.4 kDa。将推导多肽的氨基酸序列与其他蛋白质的序列进行比较，发现其C端催化结构域与形成糖基水解酶家族19的植物I类、II类和IV类几丁质酶的催化结构域有相当大的序列相似性。推导多肽的N端结构域与几种微生物几丁质酶和纤维素酶的底物结合结构域表现出序列相似性，但与植物几丁质酶的几丁质结合结构域不同。先前从灰色链霉菌中纯化的几丁质酶C-1被认为是通过蛋白水解去除N端几丁质结合结构域而产生的，并且与chiC基因编码的几丁质酶的催化结构域相对应。对N-乙酰壳四糖水解产物的高效液相色谱分析表明，几丁质酶C-1催化糖苷键的水解，同时异头构型发生反转，这与先前报道的植物I类几丁质酶的反转机制一致。这是首次在高等植物以外的生物体中发现糖基水解酶家族19的几丁质酶的报道。
