The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the -conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc) 6 is predominantly cleaved into (GlcNAc) 2 and (GlcNAc) 4 , where the (GlcNAc) 2 group arises from the nonreducing end of the substrate and is formed as the -anomer. With time, transglycosylation occurs, generating (GlcNAc) 8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc) 5 and (GlcNAc) 4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc) 4 is 50 µM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc) 6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.

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Sugar recognition specificities of class III (OsChib1a) and class I (OsChia1cDeltaChBD) chitinases from rice, Oryza sativa L., were investigated by analyzing (1)H- and (13)C-nuclear magnetic resonance spectra of the enzymatic products from partially N-acetylated chitosans. The reducing end residue of the enzymatic products obtained by the class III enzyme was found to be exclusively acetylated, whereas both acetylated and deacetylated units were found at the nearest neighbor to the reducing end residue. Both acetylated and deacetylated units were also found at the nonreducing end residue and its nearest neighbor of the class III enzyme products. Thus, only subsite (-1) among the contiguous subsites (-2) to (+2) of the class III enzyme was found to be specific to an acetylated residue. For the class I enzyme, the reducing end residue was preferentially acetylated, although the specificity was not absolute. The nearest neighbor to the acetylated reducing end residue was specifically acetylated. Moreover, the nonreducing end residue produced by the class I enzyme was exclusively acetylated, although there was a low but significant preference for deacetylated units at the nearest neighbor to the nonreducing end. These results suggest that the three contiguous subsites (-2), (-1), and (+1) of the class I enzyme are specific to three consecutive GlcNAc residues of the substrate. In rice plants, the target of the class I enzyme might be a consecutive GlcNAc sequence probably in the cell wall of fungal pathogen, whereas the class III enzyme might act toward an endogenous complex carbohydrate containing GlcNAc residue.

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1c deltaCBD, class II) were produced by the Pichia expression system, and the hydrolytic mechanism toward N-acetylglucosamine hexasaccharide [(GlcNAc)6] was investigated by HPLC analysis of the reaction products. The profile of the time-course of (GlcNAc)6 degradation obtained by OsChia1c was identical to that obtained by OsChia1c deltaCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1c deltaCBD, the free energy changes of sugar residue binding were estimated to be -0.4, -4.7, +3.4, -0.5, -2.3, and -1.0 kcal/mol for the individual subsites of (-3), (-2), (-1), (+1), (+2), and (+3), respectively. The hexasaccharide substrate appears to bind to the enzyme through interactions at the high-affinity sites, (-2) and (+2), and the sugar residues at both ends more loosely bind to the corresponding subsites, (-3) and (+3). The docking study of (GlcNAc)6 with the modeled structure of OsChia1c deltaCBD supported the subsite structure estimated from the experimental time-course of hexasaccharide degradation. Since the class II chitinase from barley seeds was reported to possess a similar subsite structure from (-3) to (+3) and a similar free energy distribution, substrate-binding mode of plant chitinases of this class would be similar to each other.