Cinnamate 4‐hydroxylase from <i>Catharanthus roseus</i> and a strategy for the functional expression of plant cytochrome P₄₅₀ proteins as translational fusions with P₄₅₀ reductase in <i>Escherichia coli</i>

Abstract

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.