Family 19 chitinase from rice (Oryza sativa L.):: substrate-binding subsites demonstrated by kinetic and molecular modeling studies

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1cDeltaCBD, 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 OsChia1cDeltaCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1cDeltaCBD, 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 OsChia1cDeltaCBD 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.