Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy.
Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum. This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907. The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe. The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities. The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.
Back
K
K
Department of Genetic Engineering, Biotechnology Research Institute, Chonnam National University, Kwangju, Korea. kback@chonnam.chonnam.ac.kr
Nah
J
J
Lee
S B
SB
Song
J H
JH
Shin
D H
DH
Kim
H Y
HY
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002212:Capsicum / Q000201:enzymology*; D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase