Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar

Abstract

NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa ϫ Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue.Cytochrome P450 monooxygenase enzymes (P450s) play key roles in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. In animals, xenobiotic detoxification and sterol biosynthesis are the major functions of P450s. However, P450-mediated reactions in plants encompass a much broader spectrum, including biosynthesis of plant hormones and signal molecules, defenserelated chemicals and structural secondary natural products, and herbicide detoxification (for review, see Werck-Reichhart et al., 2000;Feldmann, 2001). The Arabidopsis genome encodes 273 P450 genes (www.biobase.dk/P450), highlighting the important biochemical roles of P450-mediated reactions that have evolved in plants.Most eukaryotic P450s are not self-sufficient enzymes, and their catalytic activities strictly rely on an electron donor, NADPH:cytochrome P450 reductase (CPR; Lu et al., 1969). CPR (EC 1.6.2.4) transfers two electrons from NADPH to diverse P450s via two prosthetic groups, FAD and FMN, in sequence as electron entry and exit ports, respectively (Iyanagi and Mason, 1973;Vermilion et al., 1981). Both P450s and CPR are integral membrane-bound proteins, and the reducing equivalents are delivered mainly through transient electrostatic interactions on the endoplasmic reticulum (ER) membrane (Nisimoto, 1986;Shen and Kasper, 1995). CPR is also known to transfer electrons to cytochrome b 5 , which forms part of electron transport ch...

Reconstitution of the Entry Point of Plant Phenylpropanoid Metabolism in Yeast (Saccharomyces cerevisiae)

“…SDS-PAGE and Immunoblot Analysis-SDS-PAGE and immunoblot for C4H, and CPR was performed as previously described (24). For recombinant PAL2 detection, primary polyclonal antibody (31) at 1:5,000 dilution was used with horseradish peroxidase-conjugated antirabbit IgG antibody (Amersham Biosciences) at 1:2,500 dilution.…”

“…While PAL is operationally a cytosolic enzyme, the redox enzymes C4H and CPR have been shown in previous work to be ER-bound (23,24), where they could, in principle, help anchor a MEC that includes PAL. In previous studies from our laboratory, two closely related PAL genes, PtdPAL1 and PtdPAL2, were shown to be highly expressed in epidermal cells of young leaves and stems, but poorly expressed in developing secondary xylem of poplar (22,25 In this study, we describe the cloning of a cDNA for a divergent, xylem-expressed PAL gene that completes the set of entry point phenylpropanoid genes from poplar.…”

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

“…S2). The extended N-terminal sequence upstream of the membrane anchor identified the rowan enzyme as a class II CPR (Ro et al, 2002).…”

“…CPR catalyzes the transfer of two electrons from NADPH via FAD and FMN, as electron acceptor and donor flavins, respectively, to the prosthetic heme group of diverse CYP enzymes (Porter, 2004;Jensen and Møller, 2010). In contrast to animals, which contain a single CPR (Porter et al, 1990), plants have multiple CPR isoforms that fall into two classes structurally distinguishable by their different N termini (Urban et al, 1997;Ro et al, 2002). In Arabidopsis and Centaurium erythraea, the expression of class II CPR is induced by wounding, light treatment, and elicitation, whereas class I CPR is constitutively regulated (Nisimoto, 1986;Meijer et al, 1993;Schwarz et al, 2009).…”

“…MDP0000152900 and closely related apple unigenes as the most promising candidates for hydroxylation in biphenyl metabolism served to derive 59 and 39 primers for the amplification of the cds of the putative rowan B4H cDNA (Supplemental Table S3). To clone a CPR transcript from rowan cell cultures, the poplar (Populus trichocarpa) CPR3 sequence (AF302498.1), which encodes a class II CPR (Ro et al, 2002), was used as a query to search the GDR via the BLASTN server. The apple unigene MDP0000167955, which shared high similarity with the only CPR transcript fragment in the rowan SSH cDNA library, was selected as a candidate CPR gene, from which 59 and 39 primers were derived for the amplification of the CPR cds.…”