Role of the loop structure of the catalytic domain in rice class I chitinase.
In the three-dimensional structure of a rice class I chitinase (OsChia1b) determined recently, a loop structure (loop II) is located at the end of the substrate-binding cleft, and is thus suggested to be involved in substrate binding. In order to test this assumption, deletion of the loop II region from the catalytic domain of OsChia1b and replacement of Trp159 in loop II with Ala were carried out. The loop II deletion and the W159A mutation increased hydrolytic activity not only towards (GlcNAc)6 but also towards polysaccharide substrates. Similar results were obtained for kcat/Km values determined for substrate reduced-(GlcNAc)5. The two mutations shifted the splitting positions in (GlcNAc)6 to the reducing end side, but the shift was less intensive in the Trp mutant. Theoretical analysis of the reaction time course indicated that sugar residue affinity at the +3 subsite was reduced from -2 kcal/mol to +0.5 kcal/mol by loop II deletion. Reduced affinity at the +3 subsite might enhance the release of product fragments, resulting in higher turnover and higher enzymatic activities. Thus, we concluded that loop II is involved in sugar residue binding at the +3 subsite, but that Trp159 itself appears to contribute only partly to sugar residue interaction at the subsite.
Mizuno
Ryoji
R
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata, Japan.
Fukamizo
Tamo
T
Sugiyama
Shinichi
S
Nishizawa
Yoko
Y
Kezuka
Yuichiro
Y
Nonaka
Takamasa
T
Suzuki
Kazushi
K
Watanabe
Takeshi
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012275:Oryza / Q000201:enzymology*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013305:Streptomyces griseus / Q000201:enzymology
D017931:DNA Primers; D002688:Chitinases