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

Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by... A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1cΔCBD, 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 OsChia1cΔCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1cΔCBD, 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 OsChia1cΔCBD 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

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

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2003 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1023972007681
Publisher site
See Article on Publisher Site

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