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 (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.
Sasaki
Chiye
C
Laboratory of Enzyme System Science, Department of Food and Nutrition, Kinki University, 3327-204, Naka-machi, Nara 631-8505, Japan.
Itoh
Yoshifumi
Y
Takehara
Hideki
H
Kuhara
Satoru
S
Fukamizo
Tamo
T
D000117:Acetylglucosamine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors
D009844:Oligosaccharides; D011994:Recombinant Proteins; D002688:Chitinases; D000117:Acetylglucosamine