Cinnamate 4-hydroxylase from Catharanthus roseus, and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli.
A PCR-based approach was used to isolate cDNAs for cinnamate 4-hydroxylase (C4H) from Catharanthus roseus cell cultures. The protein shared 75.9% identity with C4H from other plants, and the transcription was induced under various stress conditions. The cloned protein was used to investigate the functional expression of plant P450/P450-reductase fusions in E. coli. Fusions containing a modified N-terminal membrane anchor were located in the membrane and possessed C4H activity without solubilization or addition of other factors. The results indicate that the fusion protein strategy provides a useful tool to analyze the activities encoded in the rapidly increasing number of plant P450 sequences of uncertain or unknown function. We also discuss critical elements of the strategy: the choice of the E. coli host strain, the N-terminal membrane anchor, and the conditions for protein expression.
Hotze
M
M
Institut für Biologie II, Universität Freiburg, Germany.
Schröder
G
G
Schröder
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017385:Sequence Homology; D050564:Trans-Cinnamate 4-Monooxygenase
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase