Characterization of recombinant plant cinnamate 4‐hydroxylase produced in yeast

Abstract

Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Succharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisk strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced ironkarbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted 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-dependent P450 reaction. Based on spectral perturbation and inhibition studies, 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.

Regulation of the Cinnamate 4-Hydroxylase (CYP73A1) in Jerusalem Artichoke Tubers in Response to Wounding and Chemical Treatments

“…CYP73A1 content was quantified by differential spectrophotometry (presence versus absence of saturating concentration of substrate) using an absorption coefficient ( E ) for the A,,, to A,,, difference of 128 mM-' (Urban et al, 1994).…”

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

“…[17]), and by the finding that a plant P45o-RED was able to support reactions of animal P450 [19]. It has also been shown that yeast reductase interacted successfully with plant P450 [4,7,36].…”

“…Expression of functionally identified plant P450 in other cells has been reported (e.g. yeast [4,7] and animal cells [8,9]), but each of the available systems suffer from certain limitations including ease of use or expense.…”

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple Are Cytochrome P450 736A Proteins

“…The turnover number (k cat ) and the catalytic efficiency (k cat /K m ) with 3-hydroxy-5-methoxybiphenyl were 0.342 min 21 and 2,375 M 21 s 21 , respectively (Table II). When SaCPR was incubated with cytochrome c as an artificial electron acceptor (Urban et al, 1994), the K m value for NADPH was 51 6 5.4 mM. No appreciable loss of SaB4H activity was observed upon storage of microsomal preparations in Tris-EDTA-glycerol buffer at 280°C for 6 months.…”

“…The CPR assay (1 mL) contained 40 mM cytochrome c (equine heart; SigmaAldrich; www.sigmaaldrich.com), 0.1 mM EDTA, and 12 mg of microsomal fraction in 0.1 M potassium phosphate buffer, pH 7.5 (Urban et al, 1990(Urban et al, , 1994. The reaction was started by the addition of NADPH.…”