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 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 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 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 iron/carbon 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.
Centre de Génétique Moléculaire du CNRS, Laboratoire Propre Associé à l'Université Pierre-Marie-Curie, Gif-sur-Yvette, France.
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D001483:Base Sequence; D002248:Carbon Monoxide / Q000737:chemistry; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006368:Helianthus / Q000201:enzymology*; D015151:Immunoblotting; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D050564:Trans-Cinnamate 4-Monooxygenase; D014158:Transcription, Genetic
D010940:Plant Proteins; D011994:Recombinant Proteins; D009249:NADP; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase