Molecular cloning, functional expression, and mutagenesis of cDNA encoding rye (Secale cereale) seed chitinase-c.
We cloned a complete cDNA encoding rye seed chitinase-c, designated RSC-c, by rapid amplification of cDNA end and PCR procedures. The cDNA of RSC-c consists of 1,018 nucleotides and includes an open reading frame encoding a polypeptide of 266 amino acid residues. A recombinant RSC-c was produced by expression in Escherichia coli Origami(DE3) and purified. rRSC-c had almost the same chitinase activity toward glycolchitin and antifungal activity against Trichoderma sp. as the authentic RSC-c did. RSC-c mutants were subsequently constructed and characterized with respect to their chitinase and antifungal activities. Mutation of Glu67 to Gln completely abolished the chitinase activity and diminished the antifungal activity. Considerable decreases in both activities were observed in the mutations of Trp72 and Ser120 to Ala, and Glu89 to Gln. The roles of these residues in the catalytic event of RSC-c are discussed.
Ohnuma
Takayuki
T
Laboratory of Protein Chemistry and Engineering, Graduate School of Bioresource and Environmental Science, Kyushu University, Fukuoka, Japan.
Yagi
Mikako
M
Yamagami
Takeshi
T
Taira
Toki
T
Aso
Yoichi
Y
Ishiguro
Masatsune
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D012434:Secale / Q000196:embryology / Q000201:enzymology / Q000235:genetics*; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010940:Plant Proteins; D002688:Chitinases; C119825:chitinase C