Untitled

Abstract

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.

Chitinases: An update

“…[27] Class I and class II chitinases use an inverting mechanism in order to hydrolyze β-glycosidic linkage while class III chitinases do this through a retaining mechanism. [2829] It has been established by Sasaki et al . (2006)?…”

Structure and function of chitinases from glycoside hydrolase family 19

“…However, it was obvious that when the two modules were linked together, activity towards the insoluble chitin polymer was enhanced (unpublished data). However, Sasaki et al 20 have observed that rice chitinase intact protein (CM + CtBM) has slightly higher activity towards (GlcNAc) 6 degradation than CM alone. Further, the CtBM plays a vital role in the effectiveness of antifungal activity but it is not necessary for either antifungal or catalytic activities 21, 22.…”

“…However, the CtBM does not possess catalytic function20 and it is not clear whether it has any activity other than anchoring the protein on chitin. The class IV chitinase from Norway spruce is known to inhibit Heterobasidion annosum growth better when the CtBM is attached to the CM 22 .…”

“…The catalytic clefts vary in length, probably accommodating three to seven GlcNAc units depending on the enzyme. Docking studies have revealed the enzyme from rice has six subsites (−3, − 2, − 1, + 1, + 2, + 3) where the − 2, − 1, + 1 and + 2 sites have high affinity towards the substrate enhancing tight binding 20. Binding of GlcNAc in the papaya chitinase catalytic cleft suggests the − 2 and + 1 substrate/product binding sites to be the strongest 37.…”

“…Therefore, chitin binding in these CMs is dominated by hydrogen bonding interactions. It has been noted that the pH of the medium affects substrate binding 20…”