Multiple Forms of Plant Cytochromes P-450

Donaldson, Robert P.; Luster, Douglas G.

doi:10.1104/pp.96.3.669

Cited by 159 publications

(86 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In plants, P450s are further involved in secondary metabolisms such as the phenylpropanoid and terpenoid biosynthetic pathways and herbicide metabolism (Donaldson and Luster, 1991;Sanderman, 1992;Hallahan et al, 1993 ;Durst and Benveniste, 1993). Information concerning sequences of plant P4SOs were still recently relatively limited.…”

Section: Marie-curie F-91198 Gif-sur-yvette Francementioning

confidence: 99%

Characterization of recombinant plant cinnamate 4‐hydroxylase produced in yeast

Urban

Werck‐Reichhart

Teutsch

et al. 1994

European Journal of Biochemistry

155

118

View full text Add to dashboard Cite

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.

show abstract

Section: Marie-curie F-91198 Gif-sur-yvette Francementioning

confidence: 99%

Characterization of recombinant plant cinnamate 4‐hydroxylase produced in yeast

Urban

Werck‐Reichhart

Teutsch

et al. 1994

European Journal of Biochemistry

155

118

View full text Add to dashboard Cite

show abstract

“…The reductase, essential for the transfer of electrons from NADPH to Cyt P-450 plays a critical role in the catalytic activity of all these monooxygenases (7). This enzyme, re-' This work was supported by Ministere de la Recherche et de la Technologie grant No.…”

Section: Methodsmentioning

confidence: 99%

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Lesot

Benveniste²,

Hasenfratz³

et al. 1992

Plant Physiol.

View full text Add to dashboard Cite

Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPHcytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPHCyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.markable by the presence of both FAD2 and FMN prosthetic groups, exists as a single form in animals (23). From Jerusalem artichoke (Helianthus tuberosus) tubers we purified a plant NADPH-Cyt P-450 reductase (2). This enzyme preparation, which migrated as a single broad band (82 kD) on 12.5% SDS-PAGE, was used to raise polyclonal antibodies (3). Western blotting of the reductase after 7.5% SDS-PAGE showed that the reductase preparation was composed of three proteins of very close molecular mass: 80, 82, and 84 kD. To get insight into the function and immunological relationships among these proteins, we set out to produce mAbs against the reductase preparation. A similar approach had produced mAbs specific for hepatic Cyt P-450 isoforms from rats treated with different inducers (21).Here we present the characterization of two families of mAbs we have obtained, and we discuss the relationship between the isoforms of Jerusalem artichoke NADPH-Cyt P-450 reductase. MATERIALS AND METHODSPurification of NADPH-Cyt P-450 Reductase NADPH-Cyt P-450 reductase (fpl) from Jerusalem artichoke (Helianthus tuberosus) was purified from solubilized microsomes as described (2).The microsomal Cyt P-450-dependent monooxygenases are multienzyme complexes consisting essentially of the hemoprotein Cyt P-450 and the flavoprotein NADPH-Cyt P-450 reductase (24). In plants, different isoforms of Cyt P-450 are implicated in essential biosynthetic pathways such as the production of lignin, at the level of cinnamate hydroxylation (1) and ferulate hydroxylation (11); the biosynthe...

show abstract

“…The results is a duel operation of both plants and microorganisms, working together, as an in-situ bioremediation system. Donaldson and Luster (1991) have reported on the accumulating evidence indicating that there is a multiplicity of cytochrome P-450 enzymes in plants. These enzymes have been implicated in the metabolic degradation of sterols, terpenes, gibberellins, and xenobiotics.…”

Section: E Vegetative Uptakementioning

confidence: 99%

An evaluation of in-situ bioremediation processes

Cole¹,

Rashidi²

1996

View full text Add to dashboard Cite

Multiple Forms of Plant Cytochromes P-450

Cited by 159 publications

References 31 publications

Characterization of recombinant plant cinnamate 4‐hydroxylase produced in yeast

Characterization of recombinant plant cinnamate 4‐hydroxylase produced in yeast

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

An evaluation of in-situ bioremediation processes

Contact Info

Product

Resources

About