CINNAMATE 4-HYDROXYLASE FROM CATHARANTHUS-ROSEUS, AND A STRATEGY FOR THE FUNCTIONAL EXPRESSION OF PLANT CYTOCHROME-P-450 PROTEINS AS TRANSLATIONAL FUSIONS WITH P-450 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 P-450/P-450-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 P-450 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.