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The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the ␤-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.Chitinases (EC 3.2.1.14) are glycosyl hydrolases which catalyze the degradation of chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine. Recently, chitinases have been receiving renewed attention because of their possible application for the biological control of chitin-containing organisms and also for the exploitation of natural chitinous materials. They are present in a wide range of organisms including bacteria, insects, viruses, plants, and animals and play important physiological and ecological roles. Chitinases so far sequenced are classified in two different families (namely, families 18 and 19) of the classification of glycosyl hydrolases based on amino acid sequence similarities (13,14). Family 18 contains chitinases from bacteria, fungi, viruses, and animals and class III and V chitinases from plants. On the other hand, class I, II, and IV plant chitinases belong to family 19, and this family solely comprises chitinases of plant origin. The two different families of chitinases display no sequence similarities with each other and have different three-dimensional structures (5).Oligosaccharides produced from partially acetylated chitosan by microbial chitinases have been previously studied in an attempt to clarify their specificity for ␤-1,4-N-acetylglucosaminic versus ␤-1,4-glucosaminic linkages (26, 27, 30). Except for chitinase C-1 from Streptomyces griseus HUT 6037, all of the examined microbial chitinases hydrolyzed GlcNAc-GlcNA...

Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia marcescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and hydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline ␤-chitin and colloidal chitin. The substitution of Phe-232, which is located closest to the catalytic cleft, did not affect the binding activity. On the other hand, the hydrolyzing activity against ␤-chitin microfibrils was significantly reduced by the substitution of any one of the four aromatic residues including Phe-232. None of the mutations reduced the hydrolyzing activity against soluble substrates. These results clearly demonstrate that the four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appeared to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalline chitin by chitinase A is proposed.

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The third chitinase gene (chiC) of Serratia marcescens 2170, specifying chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial chitinase evolution. Ser. marcescens chitinase C1 is found in one subfamily, whereas chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens chitinase that has an Fn3 domain. The presence of multiple, divergent, chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.