CHARACTERIZATION OF RECOMBINANT PLANT CINNAMATE 4-HYDROXYLASE PRODUCED IN YEAST - KINETIC AND SPECTRAL PROPERTIES OF THE MAJOR PLANT P450 OF THE PHENYLPROPANOID PATHWAY

Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 super-family which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae . The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisiae strains producing human P450 reduct ase or normal or overproduced amounts of yeast P450 reductase were tra nsformed to express recombinant CA4H. When grown on galactose, yeast c ells produced CA4H holoprotein bound to the endoplasmic reticulum memb rane as judged from the reduced iron/carbon monoxide difference spectr um centered at 452 nm and from typical cinnamate 4-hydroxylase activit y upon coupling with the different P450 reductases and NADPH. Some CA4 H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the y east-produced CA4H in different redox protein environments are present ed using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constitut ed by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-depe ndent P450 reaction. Based on spectral perturbation and inhibition stu dies, coumarate appeared to have no detectable affinity for the enzyme . A possible geometry of the substrate recognition pocket is discussed in the light of these data.