Plant Physiology and Biochemistry 82 (2014) 116e122
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Research article
Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum Ga-Young Lee 1, Hyun Min Kim 1, Sang Hoon Ma, Se Hee Park, Young Hee Joung**, Chul-Ho Yun* School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2014 Accepted 20 May 2014 Available online 4 June 2014
Two NADPH-cytochrome P450 reductase (CPR) genes (CaCPR1 and CaCPR2) were isolated from hot pepper (Capsicum annuum L. cv. Bukang). At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. It gradually increased during fruit ripening. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues. To investigate the enzymatic properties of CaCPR1, the cDNA of CaCPR1 was heterologously expressed in Escherichia coli without any modification of amino acid sequences, and CaCPR1 was purified. The enzymatic properties of CaCPR1 were confirmed using cytochrome c and cytochrome b5 as protein substrates. The CaCPR1 could support human CYP1A2-catalyzed reaction. It also reduced tetrazolium salts and ferricyanide. These results show that CaCPR1 is the major CPR in most hot pepper tissues. It is suggested that the CaCPR1 can be used a prototype for studying biological functions and biotechnological applications of plant CPRs. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Plant NADPH-cytochrome P450 reductase Heterologous expression Cytochrome P450 Tetrazolium salts Doxorubicin
1. Introduction NADPH-cytochrome P450 reductase (CPR) (EC 1.6.2.4) serves as the electron donor for most of eukaryotic cytochromes P450 (P450 or CYP). CPR shuttles electrons from NADPH through the FAD and FMN cofactors into the central heme iron of P450s. Although animals, insects, and yeast have only a single isoform of CPR to interact with various P450s, plants have one to four CPR genes depending on the plant species (Jensen and Møller, 2010; Rana et al., 2013). CPR is an essential redox partner for multiple P450s involved in primary and secondary metabolite biosynthesis. The presence of multiple CPRs in plants may accommodate the high demand for the supply of electrons to P450s under stress conditions or during plant developmental stages (Mizutani and Ohta, 1998; Ro et al., 2002). P450s have crucial roles in the metabolic pathways of all known species. In plants, P450-mediated oxidation reactions contribute to
Abbreviations: b5, cytochrome b5; CaCPR, Capsicum annuum NADPH-cytochrome P450 reductase; CPR, NADPH-cytochrome P450 reductase; CTC, 5-cyano-2,3-ditolyl tetrazolium chloride; 7-EC, 7-ethoxycoumarin; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; P450 or CYP, cytochrome P450; rCPR, rat CPR. * Corresponding author. Tel.: þ82 62 530 2194; fax: þ82 62 530 2199. ** Corresponding author. Tel.: þ82 62 530 5202; fax: þ82 62 530 2199. E-mail addresses: [email protected] (Y.H. Joung), [email protected], chyun49@ gmail.com (C.-H. Yun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2014.05.010 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
the biosynthesis of hormones, signal molecules, defence-related chemicals, and secondary metabolites (Mizutani, 2012). More than 7400 plant P450 genes have been reported. Numerous novel P450 genes with unknown functions continue to be identified as plant genomes are sequenced (http://drnelson.utmem.edu/ CytochromeP450.html). Information about plant CPRs will contribute to functional studies of P450s whose functions have not yet been characterized. Sequencing of the tomato (Solanum lycopersicum) genome revealed two putative CPR genes: SGN-U581827 (SlCPR1) and SGNU573215 (SlCPR2) (Sol Genomics Network, http://solgenomics.net). Blast searches of the tomato CPR genes against the hot pepper (Capsicum annuum) genome database led to the identification of two CPR genes: CA00g01310 (CaCPR1) and CA07g08650 (CaCPR2) (http://passport.pepper.snu.ac.kr/V.1.5). Genome sequencing of the hot pepper revealed that it has 447 genes encoding P450s (Kim et al., 2014). Tissue-specific transcriptome profiling of the hot pepper showed that several P450 genes were differentially expressed during fruit development. Several P450 such as CYP76B28 (CA03g36540) and CYP76B13 (CA07g01780) showed the highest expression in mature green fruit. Some P450 group such as CYP 71A2 (CA03g24280), CYP71AT2v3 (CA00g75340) and CYP71AT2v1 (CA03g00020) showed dramatically increased after mature green stage. It was suggested that these P450s may be involved in secondary metabolism, such as capsaicinoids and b-carotene synthesis in pepper fruits. Recently, it
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was shown that the Capsicum CPR activity has been linked with these biosynthetic pathways (Mazourek et al., 2009; Kim et al., 2014). The functional studies of CaCPR enzymes are essential for characterization of hot pepper P450s because the latter enzymes should accept electrons from CPR for their functions in vivo. In addition, CPR is an essential component of the P450 monooxygenase system in in vitro assays. Furthermore, CaCPR may support other P450s from closely related species. Common strategies for the functional expression of plant CPRs in Escherichia coli include N-terminal sequence modification and truncation of the N-terminal hydrophobic region responsible for the interaction with the membrane for expression (Hull and Celenza, 2000; Hotze et al., 1995). To our knowledge, there are no reports of plant CPR expression in bacteria without the conventional modification at the N-terminus. Here, CaCPR1 was cloned and functionally expressed in E. coli without any amino acid modification including N-terminal sequence. In this study, the expression patterns of the two CaCPR genes in various tissues of C. annuum were examined, and the enzymatic properties of CaCPR1 were investigated. CaCPR1 was expressed as a major transcript in all tissues analysed and its expression gradually increased during fruit ripening. Hot pepper CaCPR1 cDNA was heterologously expressed in E. coli and purified by affinity chromatography. The purified CaCPR1 catalysed the reduction of typical protein substrates such as cytochrome c and cytochrome b5 (b5). Reconstitution of CaCPR1 with human CYP1A2 confirmed that CaCPR1 could support the human CYP1A2-catalysed reaction. The CaCPR1 also catalysed the reduction of typical chemical substrates such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), and ferricyanide, which are useful for the development of continuous and sensitive assays to monitor plant CPR activity in vivo and in vitro. Furthermore, CaCPR1 showed higher reductase activity to doxorubicin, a specific anticancer drug, to generate the major metabolite, 7-deoxydoxorubicinone, and higher thermal stability when compared to rat CPR (rCPR).
2. Materials and methods 2.1. Materials MTT, CTC, doxorubicin, potassium ferricyanide, 7ethoxycoumarin (7-EC), cytochrome c, b-NADPH, b-NADPþ, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co (http://www.sigmaaldrich. com). 7-Deoxydoxorubicinone was purchased from Santa Cruz
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Biotechnology, Inc. (http://www.scbt.kr). All other chemicals were of analytical grade. Recombinant rCPR (Hanna et al., 1998) and human CYP1A2 (Kim et al., 2008) were expressed in E. coli as previously described. The b5 protein was purified from rabbit as described previously (Shimada et al., 1986). 2.2. RNA isolation and RT-PCR RNA was isolated from hot pepper (C. annuum L. cv. Bukang) leaves using the Qiagen RNeasy kit (Qiagen Inc., http://www. qiagen.com) and treated with RNase-free DNase (Roche, http:// www.roche.co.kr) to remove DNA. Reverse transcription was performed using 1 mg total RNA, 20 ml reaction mix from the iNtRON Power cDNA synthesis Kit (Intron Biotechnology, http://www. intronbio.com) and AMV reverse transcriptase (Intron Biotechnology) according to the manufacturer's directions. To isolate CaCPR genes from hot pepper, RT-PCR was performed with a specific primer set (Table 1). For RT-PCR, 2 ml of each primer (0.2 mM final concentration) and 2 ml of the cDNA preparation were used in the reaction mix (25 ml final volume). The RT-PCR products were cloned into a pGEM-T vector (Promega, http://www.promega.com) for sequencing. Real-time PCR amplification was performed with genespecific primers and the SYBR Green Master Mix kit (Qiagen) using a Rotor-Gene 6000 real-time amplification operator (Qiagen). The reaction mix (30 ml final volume) consisted of 15 ml of SYBR Green PCR Master Mix, 3 ml of each primer (0.2 mM final concentration), and 2 ml of cDNA. The actin gene of hot pepper was used as the reference gene. The samples were analysed three times in triplicate, and the relative amount of target RNA for each sample was calculated using the statistical analysis method (Pfaffl et al., 2002). Database searches were performed using the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Phylogenetic analyses were performed using the neighbour-joining method of MEGA4 software (Thompson et al., 1994). 2.3. Heterologous expression of CaCPR1 in E. coli and purification Following amplification by RT-PCR, the CaCPR1 gene was cloned into expression vector pET-28a(þ) and transformed into E. coli Rosetta(DE3)pLysS. CaCPR1 was expressed and purified as previously described (Hanna et al., 1998) with minor modifications. The transformed cells were grown overnight in LB broth with kanamycin (50 mg/ml) and chloramphenicol (30 mg/ml) selection at 37 C. The pre-culture cells were used to inoculate a 200-ml culture of TB broth containing an additive solution (final concentrations of 2 mg/ml glucose, 50 mg/ml kanamycin, and 100 ng/ml riboflavin, sterilised by filtration) and chloramphenicol (30 mg/ml). The cells
Table 1 Sequence of primers used in this study. Use
Primer
Sequence (50 to 30 )
PCR product size (bp)
cDNA cloninga
CaCPR1 BamH1 F CaCPR1 Sal1 R CaCPR2 Sac1 F CaCPR2 Sal1 R CaCPR1 50 -end F CaCPR1 30 -UTR R CaCPR2 50 -end F CaCPR2 30 -UTR R actin-F actin-R
GGA TCC ATG GAG TCG AGT TCG GAG GTC GAC TCA CCA CAC ATC CCT GAG GAG CTC ATG GAC TCT ACA TCA GAA GTC GAC TCA CCA CAC ATC ACG CAG GAA TGC GAA CTC ATC CAA AG CAA TGG AAG AGC AAT AAC CAA ATC AGG GAT CAC TTG ACA GC GAG CTG TGG CGA CAA GTA GTA CGG AAT CCA CGA GAC TAC AT GGG AAG CCA AGA TAG AGC CT
2061
Real-time PCRb
2148 219 206 230
a To isolate CaCPR genes from hot pepper, RT-PCR was performed with a specific forward primer containing the initiation ATG (F) and a specific primer containing the stop codon (R) for each CaCPR gene. Each primer contained specific restriction enzyme sequence (under lines): CaCPR1 (BamH1 F and Sal1 R); CaCPR2 (Sac1 F and Sal1 R). b Real-time PCR was performed with gene-specific primers: CaCPR1 (50 -end F and 30 -UTR R); CaCPR2 (50 -end F and 30 -UTR R); actin (actin-F and actin-R).
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were grown at 37 C with agitation to an optical density of 0.6e0.8 at 600 nm. Protein expression was then induced by adding 0.5 mM IPTG, and the cultures were grown at 32 C with agitation for 24 h. The cells were harvested by centrifugation (20 min, 5000 g, 4 C), and the cell pellet was sonicated. After the lysate was centrifuged at 100,000 g (2 h, 4 C), membrane fraction containing P450 reductase was homogenised and solubilised at 4 C in 100 mM TriseHCl buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 1 mM FMN, 0.1 mM PMSF, 1% CHAPS, and 0.5% Tergitol NP-10. The solubilised sample was centrifuged at 100,000 g for 1 h at 4 C and the supernatant was loaded onto a 20 ,50 -ADP Sepharose 4B column. The column was equilibrated with 100 mM TriseHCl buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 300 mM NaCl, 0.1% Tergitol NP-10, and 0.5% sodium cholate. The column was washed with equilibration buffer containing 5 mM adenosine and 1 mM FMN. The reductase was then eluted with 100 mM TriseHCl buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 500 mM NaCl, 0.1 mM DTT, 1 mM FMN, 0.5 mM PMSF, 0.2% Tergitol NP-10, 0.2% sodium cholate, and 10 mM NADPþ. The eluted reductase fractions were dialysed against 50 mM TriseHCl buffer (pH 7.7) containing 20% glycerol and 0.1 mM EDTA. The purity of the purified CaCPR1 was assessed by SDSePAGE. CaCPR1 was quantitated by flavin absorbance (ε ¼ 21 mM1cm1) (French and Coon, 1979).
The reduction of b5 by CaCPR1 was assayed by monitoring the spectral change from oxidised b5 to reduced b5 at 350e600 nm (Tamura et al., 1983). The assay medium (1.0 ml) contained 1.0 mM b5 and 20 nM CaCPR1 in 100 mM potassium phosphate buffer (pH 7.4). The 7-EC oxidation activity was measured by HPLC analysis to determine if CaCPR1 could interact with human P450 enzymes and to mediate electron transfer, as previously described (Kim et al., 2006). The 7-EC oxidation reaction included 400 nM CYP1A2 and 800 nM rCPR or 100e800 nM CaCPR1 or in 0.25 ml 100 mM potassium phosphate buffer (pH 7.4) with 0.1 mM substrate.
2.4. Enzymatic activity of CaCPR1 toward chemical substrates and doxorubicin
3. Results and discussion
2.6. Thermal stability of CaCPR1 Thermal stability of CaCPR1 and rCPR was examined using a PCR thermocycler as previously described (Park et al., 2010), with slight modification. To analyse the enzyme thermal stability, CaCPR1 and rCPR were incubated at temperatures between 37 C and 75 C for 10 min in a PCR thermocycler followed by cooling to 4 C for 5 min. After centrifugation, the protein solutions were used to measure the ability of CaCPR1 and rCPR to reduce MTT. The stability was estimated from the differences of MTT reduction rate as described above.
3.1. Isolation of CPR genes from hot pepper Absorption spectra were recorded using a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan) with standard 1-cm cuvettes in a total reaction volume of 1 ml. The ability of CaCPR1 to reduce MTT (Yim et al., 2005), CTC (Kim et al., 2009), and ferricyanide (Schellenberg and Hellerman, 1958) was determined as previously described. The ability of CaCPR1 to reduce MTT and ferricyanide was determined using 1e200 mM of each substrate and 10 nM CaCPR1 in 100 mM potassium phosphate (pH 7.6). The reaction mixture for CTC contained 1e200 mM substrate and 10 nM CaCPR1 in 100 mM potassium phosphate (pH 7.4). The change in absorbance at the indicated wavelength for each substrate was measured following the addition of 100 mM NADPH (at 610 nm for MTT, 450 nm for CTC, and 420 nm for ferricyanide). The reduction rates were calculated using extinction coefficients of 11.3 mM1cm1 (at 610 nm), 16.24 mM1cm1 (at 450 nm), and 1.02 mM1cm1 (at 420 nm) for reduced MTT, CTC, and ferricyanide, respectively. The kinetic parameters (Km and kcat) were determined by nonlinear regression using Graph-Pad Prism software (San Diego, CA). Activity assay of CaCPR1 toward doxorubicin was determined by HPLC as described previously (Mizutani et al., 2003), with a slight modification. For the assay of doxorubicin reduction, the reaction mixture contained 100 nM CPR (CaCPR1 or rCPR), 100 mM potassium phosphate buffer (pH 7.4), and 400 mM of the substrate (doxorubicin) in a total volume of 0.50 ml. Quantitation was done using peak areas estimated with reference to 7deoxydoxorubicinone standards. 2.5. Enzymatic activity of CaCPR1 toward protein substrates Cytochrome c reductase assays were performed as previously described (Vermilion et al., 1981) with a slight modification. Briefly, 10 nM CaCPR1 and 0.5e200 mM cytochrome c in 100 mM potassium phosphate (pH 7.6) were mixed in a 1-ml cuvette, and 100 mM of NADPH was added to start the reaction. The change in A550 was recorded for 1 min at 30 C. The rate of cytochrome c reduction was calculated using an extinction coefficient of 21 mM1 cm1 (at 550 nm) for the conversion of the oxidised cytochrome c to the reduced form. All concentration points were assayed in triplicate.
RT-PCR was performed with specific primers to amplify two fulllength CPR cDNAs, named CaCPR1 and CaCPR2 (GenBank Numbers: KF726118 and KF726119, respectively) based on their homology with previously reported CPRs in the tomato genome. The nucleotide sequences of CaCPR1 and CaCPR2 contain complete open reading frames of 2061 and 2148 bp, corresponding to 686 and 715 amino acids, respectively (Supplementary Fig. 1). Amino acid identity between CaCPR1 and CaCPR2 was 67%. When the amino acid sequences of CaCPR1 and CaCPR2 were compared to the corresponding tomato CPRs (SlCPR1 and SlCPR2), the identities of CPR1 and CPR2 were 94.5% and 92.3%, respectively (Supplementary Fig. 1). Phylogenetic analysis based on CPR amino acid sequences from several plant species revealed that CaCPR1 and CaCPR2 fell into distinct classes among plant CPR (Supplementary Fig. 2). This analysis suggests that each CaCPR may have a distinct and critical role to accommodate the large number of P450 isoforms in C. annuum, which has been estimated to contain 477 CYP genes (Kim et al., 2014). When the predicted amino acid sequences of the CaCPRs were compared to other CPRs from plants and mammals, high degrees of similarity were observed at each domain (Supplementary Fig. 3). 3.2. Expression levels of CaCPR1 and CaCPR2 in hot pepper The expression level of CaCPR1 was higher than that of CaCPR2 in all tested tissues (leaves, flowers, and fruits) and gradually increased during fruit ripening. At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues (Fig. 1). According to the transcriptome analysis, the expression level of CaCPR1 is much higher than that of CaCPR2 in leaves and during fruit ripening stages (Kim et al., 2014). It is known that depending on the species, plants contain one to three CPRs of which one is inducible (Ro et al., 2002; Jensen and Møller, 2010). CaCPR1 may be the inducible CPR in hot pepper. These results suggest that CaCPR1 may have a critical role during
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Fig. 1. Expression levels of the CaCPR1 and CaCPR2 genes in different tissues and stages of fruit ripening. The relative accumulation levels of the CaCPR1 and CaCPR2 transcripts were determined by real-time PCR and normalised against the actin gene as an internal control, as described in the Materials and Methods. EF-1: early fruit stage 1 (7DAP), EF-2: early fruit stage 2 (15DAP), MG: mature green stage (28DAP), BG: breaking green stage (30DAP), RB: Red-turning brown stage (42DAP), RR: red ripe stage (45DAP). DAP: Days after pollination. Each bar represents the mean and standard error of three replicates.
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Fig. 2. (A) SDS-PAGE of CaCPR1. A 10% gel was stained with Coomassie Brilliant Blue R250. Purified CaCPR1 was loaded with protein molecular weight markers. (B) The absorption spectrum of CaCPR1 was measured in 100 mM potassium phosphate buffer (pH 7.4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the fruit ripening stage; in other words, some P450 enzymes may require the electrons transferred by CaCPR1 to function during fruit ripening. The phylogeny tree shows that CaCPR1 and CaCPR2 belong to two major phylogenetic groups, named CPR1 and CPR2, from vascular plants (Jensen and Møller, 2010). The presence of multiple CPRs in some plants may imply the need for the proper interaction of a large number of P450s with CPR to exert optimal activity for the adaptation to environmental changes or during plant developmental stages (Eberle et al., 2009; Rana et al., 2013).
The 7-EC is metabolised by several P450s, typically CYP1A2, supported by mammalian CPR and NADPH to 7-OH coumarin and 3-OH,7-EC (Kim et al., 2006). Two major metabolites, 3-OH,7-EC and 7-OH coumarin, were produced upon reaction of CaCPR1 and rCPR with CYP1A2. The formation rates of the 7-OH coumarin and 3-OH,7-EC products by 0.80 mM CaCPR1 and 0.40 mM CYP1A2 were 0.011 (±0.001) and 0.036 (±0.004) nmol product per min per nmol P450, respectively (Fig. 3C). However, the formation rates of these products were 29% and 24% of those of the reaction with rCPR. When molar ratio of CYP1A2 to CaCPR1 has been increased from 1:0.25 to 1:2 at fixed concentration of CYP1A2 (0.10 mM), the 7-EC oxidation rate increased with a maximum at 1:2 (Fig. 3C). These results imply that CaCPR1 interacts with CYP1A2 to mediate electron transfer between the two proteins.
3.3. Heterologous expression and purification
3.5. Enzymatic properties of CaCPR1 toward chemical substrates
We found that membrane fraction and cytosolic fraction contained 75% and 25% of expressed CaCPR1 in E. coli, respectively. CaCPR1 in the membrane fraction was purified using affinity chromatography after solubilisation of the membrane fraction. The purified CaCPR1 protein yielded the expected single band of 76 kDa by SDS-PAGE (Fig. 2A). The absolute absorption spectrum of purified CaCPR1 was characteristic of flavoproteins, with prominent peaks at 380 and 452 nm (Fig. 2B). This spectral property of CaCPR1 suggests that the CaCPR1 cDNA encodes a functionally active CPR in hot pepper.
The kcat values for CaCPR1 were 942 min1 for MTT, 902 min1 for CTC, and 4800 min1 for ferricyanide (Fig. 4). The Km values for CaCPR1 were in the range of 30e47 mM. The catalytic efficiency of ferricyanide reduction was 5.1-fold and 5.3-fold higher than that for MTT and CTC, respectively. However, MTT and CTC have several advantages over ferricyanide as the CPR substrate. They are tetrazolium salts that are used extensively to measure cell viability, metabolic activity, and oxidative reactions (Altman, 1976; Bernas and Dobrucki, 1999). They can be used as substrates for a colorimetric high-throughput assay because both form highly coloured reduced products (formazans) and display obvious colour changes (from yellow to blue for MTT and colourless to red for CTC). Especially, the CTC formazan is fluorescent, thereby allowing a more sensitive spectrofluorometric assay.
3.4. Enzymatic properties of CaCPR1 toward protein substrates Cytochrome c has been used a typical substrate to measure the reduction activity of plant CPR enzymes (Benveniste et al., 1991). The kcat value of cytochrome c reduction by CaCPR1 was 2740 min1, and the Km value was 81 mM (Fig. 3A). When the oxidised b5 was incubated with CaCPR1 and 100 mM NADPH, it was rapidly reduced and exhibited an absolute absorption spectrum with typical Soret peaks 423, 526, and 556 nm, similar to those observed for the dithionite-reduced form (Fig. 3B). These results demonstrate that CaCPR1 is functionally active as an NADPH- b5 reductase. The magnitude of the spectrum of b5 reduced by CaCPR1 and NADPH in 10 min was approximately 90% of that of the b5 spectrum obtained by sodium hydrosulfite (Fig. 3B).
3.6. Reduction of doxorubicin by CPR1 We tested the ability of CaCPR1 to reduce doxorubicin, a specific anticancer drug, which is reported to be the substrate of CPR in the pH range of 6.0~9.0 (Cummings et al., 1992). CaCPR1 produced one major metabolite, 7-deoxydoxorubicinone (Fig. 5A). CaCPR1 showed much higher activity of 7-deoxydoxorubicinone formation than that of rCPR in most of pH range except 6.0 and 9.0 (Fig. 5B and C). Rate of 7-deoxydoxorubicinone formation by CaCPR1 at pH 7.5 was 413 min1, which is about 4-fold of the turnover catalysed by
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rCPR. This result suggests that CaCPR1 has much higher reductase activity toward doxorubicin than that of rCPR. 3.7. Thermal stability of the CaCPR1 To estimate the stability of the CaCPR1, we determined the thermal stability (Tm, C) of CaCPR1 and compared the Tm value to that of rCPR (Fig. 6). The Tm value of CaCPR1 was measured to be 56.1 ± 0.4 C, whereas the Tm value of the rCPR was 46.1 ± 0.1 C. This result indicates that the plant CaCPR1 has a high thermal stability compared to that of mammalian rCPR. The high thermal stability of CaCPR1 might be related to the high demand for the
Fig. 3. Reduction of protein substrates by CaCPR1. (A) The reduction of cytochrome c was measured spectrophotometrically in the presence of NADPH for 10 min as described in the Materials and Methods. (B) Absolute absorption spectra of b5. Solid line, oxidised b5; dashed line, b5 reduced by CaCPR with 100 mM NADPH. (C) HPLC analysis of the metabolites of 7-EC produced by human CYP1A2 with rCPR and CaCPR1. The reactions were performed with substrate (100 mM 7-EC), 400 nM human CYP1A2, and 800 nM rCPR or 100e800 nM CaCPR1 in 100 mM potassium phosphate buffer (pH 7.4) for 30 min at 37 C. The HPLC eluates were detected at each of the indicated wavelengths on the Y-axis. Each bar represents the mean and the standard error of three replicates. Fig. 4. Concentration-dependent reduction of MTT (A), CTC (B), and ferricyanide (C) by CaCPR1. Reduction was measured spectrophotometrically in the presence of NADPH for 30 s as described in the Materials and methods.
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supply of electrons to P450s under stress conditions (Mizutani and Ohta, 1998). 4. Conclusions In summary, two CaCPR genes were isolated from hot pepper, and their expression levels were found to vary depending on the tissue analysed. At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. It gradually increased during fruit ripening. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues. The cDNA of CaCPR1 was heterologously expressed in E. coli without any modification of amino acids, and the enzymatic properties of the purified CaCPR1 were characterized. CaCPR1 reduced typical the CPR protein substrates cytochrome c and b5. It also could transfer electrons to human CYP1A2. Furthermore, CaCPR1 catalysed the reduction of typical chemical substrates such as MTT, CTC, and ferricyanide, which are useful for continuous and sensitive assays to monitor plant CPR activity in vivo and in vitro. CaCPR1 showed much higher reductase activity to doxorubicin to generate 7-deoxydoxorubicinone and much higher thermal stability when compared to rCPR. These results suggest that the enzymatic properties of CaCPR1 are similar to those of mammalian CPR and that CaCPR1 can be used as a surrogate for other CPRs from other plants and mammals. The expression procedure of plant CPR without any amino acid modification in E. coli and the purification method described here may provide an alternative to previously reported expression systems for other plant CPR enzymes involving yeast and baculovirus. It is suggested that CaCPR1 can be used a prototype for studying biological functions and biotechnological applications of plant CPRs. Competing interest statement Fig. 5. Reduction of doxorubicin by CaCPR1. (A) HPLC analysis of doxorubicin by CaCPR1. Doxorubicin (400 mM) was incubated with CaCPR1 for 5 min at 37 C. The HPLC eluates were detected at 254 nm. The retention times for doxorubicin and the major metabolite, 7-deoxydoxorubicinone, are 10.5 min and 16.2 min, respectively. pH-dependent reduction of doxorubicin to 7-deoxydoxorubicinone by CaCPR1 (B) and rCPR (C) was compared.
The authors declare no competing financial interests. Acknowledgements This work was partially supported by a grant from the NextGeneration BioGreen 21 Program, Rural Development Administration, Republic of Korea (PJ009052) (Y.H. Joung), and the Bio-industry Technology Development Program (111052-04-3-SB010), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (C.-H. Yun). Authors' contributions Joung and Yun supervised the research design and wrote the paper; Lee performed all biochemical experiments, Kim performed all molecular biology experiments and bioinformatics analysis, Ma and Park performed molecular biology experiments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.05.010. References
Fig. 6. Thermal stability of CaCPR1. To analyse the enzyme thermal stability, CaCPR1 and rCPR were incubated at temperatures between 37 C and 75 C for 10 min in a PCR thermocycler followed by cooling to 4 C for 5 min before measuring MTT activity. After centrifugation, the protein solutions were used to measure the ability of CaCPR1 to reduce MTT. The stability was estimated from the differences of MTT reduction rate as described in the Materials and methods.
Altman, F.P., 1976. Tetrazolium salts and formazans. Prog. Histochem. Cytochem. 9, 1e56. Benveniste, I., Lesot, A., Hasenfratz, M.P., Kochs, G., Durst, F., 1991. Multiple forms of NADPH-cytochrome P450 reductase in higher plants. Biochem. Biophys. Res. Commun. 177, 105e112.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum Ga-Young Lee 1, Hyun Min Kim 1, Sang Hoon Ma, Se Hee Park, Young Hee Joung**, Chul-Ho Yun* School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 April 2014 Accepted 20 May 2014 Available online 4 June 2014
Two NADPH-cytochrome P450 reductase (CPR) genes (CaCPR1 and CaCPR2) were isolated from hot pepper (Capsicum annuum L. cv. Bukang). At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. It gradually increased during fruit ripening. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues. To investigate the enzymatic properties of CaCPR1, the cDNA of CaCPR1 was heterologously expressed in Escherichia coli without any modification of amino acid sequences, and CaCPR1 was purified. The enzymatic properties of CaCPR1 were confirmed using cytochrome c and cytochrome b5 as protein substrates. The CaCPR1 could support human CYP1A2-catalyzed reaction. It also reduced tetrazolium salts and ferricyanide. These results show that CaCPR1 is the major CPR in most hot pepper tissues. It is suggested that the CaCPR1 can be used a prototype for studying biological functions and biotechnological applications of plant CPRs. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Plant NADPH-cytochrome P450 reductase Heterologous expression Cytochrome P450 Tetrazolium salts Doxorubicin
1. Introduction NADPH-cytochrome P450 reductase (CPR) (EC 1.6.2.4) serves as the electron donor for most of eukaryotic cytochromes P450 (P450 or CYP). CPR shuttles electrons from NADPH through the FAD and FMN cofactors into the central heme iron of P450s. Although animals, insects, and yeast have only a single isoform of CPR to interact with various P450s, plants have one to four CPR genes depending on the plant species (Jensen and Møller, 2010; Rana et al., 2013). CPR is an essential redox partner for multiple P450s involved in primary and secondary metabolite biosynthesis. The presence of multiple CPRs in plants may accommodate the high demand for the supply of electrons to P450s under stress conditions or during plant developmental stages (Mizutani and Ohta, 1998; Ro et al., 2002). P450s have crucial roles in the metabolic pathways of all known species. In plants, P450-mediated oxidation reactions contribute to
Abbreviations: b5, cytochrome b5; CaCPR, Capsicum annuum NADPH-cytochrome P450 reductase; CPR, NADPH-cytochrome P450 reductase; CTC, 5-cyano-2,3-ditolyl tetrazolium chloride; 7-EC, 7-ethoxycoumarin; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; P450 or CYP, cytochrome P450; rCPR, rat CPR. * Corresponding author. Tel.: þ82 62 530 2194; fax: þ82 62 530 2199. ** Corresponding author. Tel.: þ82 62 530 5202; fax: þ82 62 530 2199. E-mail addresses: [email protected] (Y.H. Joung), [email protected], chyun49@ gmail.com (C.-H. Yun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2014.05.010 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
the biosynthesis of hormones, signal molecules, defence-related chemicals, and secondary metabolites (Mizutani, 2012). More than 7400 plant P450 genes have been reported. Numerous novel P450 genes with unknown functions continue to be identified as plant genomes are sequenced (http://drnelson.utmem.edu/ CytochromeP450.html). Information about plant CPRs will contribute to functional studies of P450s whose functions have not yet been characterized. Sequencing of the tomato (Solanum lycopersicum) genome revealed two putative CPR genes: SGN-U581827 (SlCPR1) and SGNU573215 (SlCPR2) (Sol Genomics Network, http://solgenomics.net). Blast searches of the tomato CPR genes against the hot pepper (Capsicum annuum) genome database led to the identification of two CPR genes: CA00g01310 (CaCPR1) and CA07g08650 (CaCPR2) (http://passport.pepper.snu.ac.kr/V.1.5). Genome sequencing of the hot pepper revealed that it has 447 genes encoding P450s (Kim et al., 2014). Tissue-specific transcriptome profiling of the hot pepper showed that several P450 genes were differentially expressed during fruit development. Several P450 such as CYP76B28 (CA03g36540) and CYP76B13 (CA07g01780) showed the highest expression in mature green fruit. Some P450 group such as CYP 71A2 (CA03g24280), CYP71AT2v3 (CA00g75340) and CYP71AT2v1 (CA03g00020) showed dramatically increased after mature green stage. It was suggested that these P450s may be involved in secondary metabolism, such as capsaicinoids and b-carotene synthesis in pepper fruits. Recently, it
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was shown that the Capsicum CPR activity has been linked with these biosynthetic pathways (Mazourek et al., 2009; Kim et al., 2014). The functional studies of CaCPR enzymes are essential for characterization of hot pepper P450s because the latter enzymes should accept electrons from CPR for their functions in vivo. In addition, CPR is an essential component of the P450 monooxygenase system in in vitro assays. Furthermore, CaCPR may support other P450s from closely related species. Common strategies for the functional expression of plant CPRs in Escherichia coli include N-terminal sequence modification and truncation of the N-terminal hydrophobic region responsible for the interaction with the membrane for expression (Hull and Celenza, 2000; Hotze et al., 1995). To our knowledge, there are no reports of plant CPR expression in bacteria without the conventional modification at the N-terminus. Here, CaCPR1 was cloned and functionally expressed in E. coli without any amino acid modification including N-terminal sequence. In this study, the expression patterns of the two CaCPR genes in various tissues of C. annuum were examined, and the enzymatic properties of CaCPR1 were investigated. CaCPR1 was expressed as a major transcript in all tissues analysed and its expression gradually increased during fruit ripening. Hot pepper CaCPR1 cDNA was heterologously expressed in E. coli and purified by affinity chromatography. The purified CaCPR1 catalysed the reduction of typical protein substrates such as cytochrome c and cytochrome b5 (b5). Reconstitution of CaCPR1 with human CYP1A2 confirmed that CaCPR1 could support the human CYP1A2-catalysed reaction. The CaCPR1 also catalysed the reduction of typical chemical substrates such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), and ferricyanide, which are useful for the development of continuous and sensitive assays to monitor plant CPR activity in vivo and in vitro. Furthermore, CaCPR1 showed higher reductase activity to doxorubicin, a specific anticancer drug, to generate the major metabolite, 7-deoxydoxorubicinone, and higher thermal stability when compared to rat CPR (rCPR).
2. Materials and methods 2.1. Materials MTT, CTC, doxorubicin, potassium ferricyanide, 7ethoxycoumarin (7-EC), cytochrome c, b-NADPH, b-NADPþ, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co (http://www.sigmaaldrich. com). 7-Deoxydoxorubicinone was purchased from Santa Cruz
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Biotechnology, Inc. (http://www.scbt.kr). All other chemicals were of analytical grade. Recombinant rCPR (Hanna et al., 1998) and human CYP1A2 (Kim et al., 2008) were expressed in E. coli as previously described. The b5 protein was purified from rabbit as described previously (Shimada et al., 1986). 2.2. RNA isolation and RT-PCR RNA was isolated from hot pepper (C. annuum L. cv. Bukang) leaves using the Qiagen RNeasy kit (Qiagen Inc., http://www. qiagen.com) and treated with RNase-free DNase (Roche, http:// www.roche.co.kr) to remove DNA. Reverse transcription was performed using 1 mg total RNA, 20 ml reaction mix from the iNtRON Power cDNA synthesis Kit (Intron Biotechnology, http://www. intronbio.com) and AMV reverse transcriptase (Intron Biotechnology) according to the manufacturer's directions. To isolate CaCPR genes from hot pepper, RT-PCR was performed with a specific primer set (Table 1). For RT-PCR, 2 ml of each primer (0.2 mM final concentration) and 2 ml of the cDNA preparation were used in the reaction mix (25 ml final volume). The RT-PCR products were cloned into a pGEM-T vector (Promega, http://www.promega.com) for sequencing. Real-time PCR amplification was performed with genespecific primers and the SYBR Green Master Mix kit (Qiagen) using a Rotor-Gene 6000 real-time amplification operator (Qiagen). The reaction mix (30 ml final volume) consisted of 15 ml of SYBR Green PCR Master Mix, 3 ml of each primer (0.2 mM final concentration), and 2 ml of cDNA. The actin gene of hot pepper was used as the reference gene. The samples were analysed three times in triplicate, and the relative amount of target RNA for each sample was calculated using the statistical analysis method (Pfaffl et al., 2002). Database searches were performed using the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Phylogenetic analyses were performed using the neighbour-joining method of MEGA4 software (Thompson et al., 1994). 2.3. Heterologous expression of CaCPR1 in E. coli and purification Following amplification by RT-PCR, the CaCPR1 gene was cloned into expression vector pET-28a(þ) and transformed into E. coli Rosetta(DE3)pLysS. CaCPR1 was expressed and purified as previously described (Hanna et al., 1998) with minor modifications. The transformed cells were grown overnight in LB broth with kanamycin (50 mg/ml) and chloramphenicol (30 mg/ml) selection at 37 C. The pre-culture cells were used to inoculate a 200-ml culture of TB broth containing an additive solution (final concentrations of 2 mg/ml glucose, 50 mg/ml kanamycin, and 100 ng/ml riboflavin, sterilised by filtration) and chloramphenicol (30 mg/ml). The cells
Table 1 Sequence of primers used in this study. Use
Primer
Sequence (50 to 30 )
PCR product size (bp)
cDNA cloninga
CaCPR1 BamH1 F CaCPR1 Sal1 R CaCPR2 Sac1 F CaCPR2 Sal1 R CaCPR1 50 -end F CaCPR1 30 -UTR R CaCPR2 50 -end F CaCPR2 30 -UTR R actin-F actin-R
GGA TCC ATG GAG TCG AGT TCG GAG GTC GAC TCA CCA CAC ATC CCT GAG GAG CTC ATG GAC TCT ACA TCA GAA GTC GAC TCA CCA CAC ATC ACG CAG GAA TGC GAA CTC ATC CAA AG CAA TGG AAG AGC AAT AAC CAA ATC AGG GAT CAC TTG ACA GC GAG CTG TGG CGA CAA GTA GTA CGG AAT CCA CGA GAC TAC AT GGG AAG CCA AGA TAG AGC CT
2061
Real-time PCRb
2148 219 206 230
a To isolate CaCPR genes from hot pepper, RT-PCR was performed with a specific forward primer containing the initiation ATG (F) and a specific primer containing the stop codon (R) for each CaCPR gene. Each primer contained specific restriction enzyme sequence (under lines): CaCPR1 (BamH1 F and Sal1 R); CaCPR2 (Sac1 F and Sal1 R). b Real-time PCR was performed with gene-specific primers: CaCPR1 (50 -end F and 30 -UTR R); CaCPR2 (50 -end F and 30 -UTR R); actin (actin-F and actin-R).
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were grown at 37 C with agitation to an optical density of 0.6e0.8 at 600 nm. Protein expression was then induced by adding 0.5 mM IPTG, and the cultures were grown at 32 C with agitation for 24 h. The cells were harvested by centrifugation (20 min, 5000 g, 4 C), and the cell pellet was sonicated. After the lysate was centrifuged at 100,000 g (2 h, 4 C), membrane fraction containing P450 reductase was homogenised and solubilised at 4 C in 100 mM TriseHCl buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 1 mM FMN, 0.1 mM PMSF, 1% CHAPS, and 0.5% Tergitol NP-10. The solubilised sample was centrifuged at 100,000 g for 1 h at 4 C and the supernatant was loaded onto a 20 ,50 -ADP Sepharose 4B column. The column was equilibrated with 100 mM TriseHCl buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 300 mM NaCl, 0.1% Tergitol NP-10, and 0.5% sodium cholate. The column was washed with equilibration buffer containing 5 mM adenosine and 1 mM FMN. The reductase was then eluted with 100 mM TriseHCl buffer (pH 7.7) containing 20% glycerol, 1 mM EDTA, 500 mM NaCl, 0.1 mM DTT, 1 mM FMN, 0.5 mM PMSF, 0.2% Tergitol NP-10, 0.2% sodium cholate, and 10 mM NADPþ. The eluted reductase fractions were dialysed against 50 mM TriseHCl buffer (pH 7.7) containing 20% glycerol and 0.1 mM EDTA. The purity of the purified CaCPR1 was assessed by SDSePAGE. CaCPR1 was quantitated by flavin absorbance (ε ¼ 21 mM1cm1) (French and Coon, 1979).
The reduction of b5 by CaCPR1 was assayed by monitoring the spectral change from oxidised b5 to reduced b5 at 350e600 nm (Tamura et al., 1983). The assay medium (1.0 ml) contained 1.0 mM b5 and 20 nM CaCPR1 in 100 mM potassium phosphate buffer (pH 7.4). The 7-EC oxidation activity was measured by HPLC analysis to determine if CaCPR1 could interact with human P450 enzymes and to mediate electron transfer, as previously described (Kim et al., 2006). The 7-EC oxidation reaction included 400 nM CYP1A2 and 800 nM rCPR or 100e800 nM CaCPR1 or in 0.25 ml 100 mM potassium phosphate buffer (pH 7.4) with 0.1 mM substrate.
2.4. Enzymatic activity of CaCPR1 toward chemical substrates and doxorubicin
3. Results and discussion
2.6. Thermal stability of CaCPR1 Thermal stability of CaCPR1 and rCPR was examined using a PCR thermocycler as previously described (Park et al., 2010), with slight modification. To analyse the enzyme thermal stability, CaCPR1 and rCPR were incubated at temperatures between 37 C and 75 C for 10 min in a PCR thermocycler followed by cooling to 4 C for 5 min. After centrifugation, the protein solutions were used to measure the ability of CaCPR1 and rCPR to reduce MTT. The stability was estimated from the differences of MTT reduction rate as described above.
3.1. Isolation of CPR genes from hot pepper Absorption spectra were recorded using a Shimadzu UV-1601 spectrophotometer (Tokyo, Japan) with standard 1-cm cuvettes in a total reaction volume of 1 ml. The ability of CaCPR1 to reduce MTT (Yim et al., 2005), CTC (Kim et al., 2009), and ferricyanide (Schellenberg and Hellerman, 1958) was determined as previously described. The ability of CaCPR1 to reduce MTT and ferricyanide was determined using 1e200 mM of each substrate and 10 nM CaCPR1 in 100 mM potassium phosphate (pH 7.6). The reaction mixture for CTC contained 1e200 mM substrate and 10 nM CaCPR1 in 100 mM potassium phosphate (pH 7.4). The change in absorbance at the indicated wavelength for each substrate was measured following the addition of 100 mM NADPH (at 610 nm for MTT, 450 nm for CTC, and 420 nm for ferricyanide). The reduction rates were calculated using extinction coefficients of 11.3 mM1cm1 (at 610 nm), 16.24 mM1cm1 (at 450 nm), and 1.02 mM1cm1 (at 420 nm) for reduced MTT, CTC, and ferricyanide, respectively. The kinetic parameters (Km and kcat) were determined by nonlinear regression using Graph-Pad Prism software (San Diego, CA). Activity assay of CaCPR1 toward doxorubicin was determined by HPLC as described previously (Mizutani et al., 2003), with a slight modification. For the assay of doxorubicin reduction, the reaction mixture contained 100 nM CPR (CaCPR1 or rCPR), 100 mM potassium phosphate buffer (pH 7.4), and 400 mM of the substrate (doxorubicin) in a total volume of 0.50 ml. Quantitation was done using peak areas estimated with reference to 7deoxydoxorubicinone standards. 2.5. Enzymatic activity of CaCPR1 toward protein substrates Cytochrome c reductase assays were performed as previously described (Vermilion et al., 1981) with a slight modification. Briefly, 10 nM CaCPR1 and 0.5e200 mM cytochrome c in 100 mM potassium phosphate (pH 7.6) were mixed in a 1-ml cuvette, and 100 mM of NADPH was added to start the reaction. The change in A550 was recorded for 1 min at 30 C. The rate of cytochrome c reduction was calculated using an extinction coefficient of 21 mM1 cm1 (at 550 nm) for the conversion of the oxidised cytochrome c to the reduced form. All concentration points were assayed in triplicate.
RT-PCR was performed with specific primers to amplify two fulllength CPR cDNAs, named CaCPR1 and CaCPR2 (GenBank Numbers: KF726118 and KF726119, respectively) based on their homology with previously reported CPRs in the tomato genome. The nucleotide sequences of CaCPR1 and CaCPR2 contain complete open reading frames of 2061 and 2148 bp, corresponding to 686 and 715 amino acids, respectively (Supplementary Fig. 1). Amino acid identity between CaCPR1 and CaCPR2 was 67%. When the amino acid sequences of CaCPR1 and CaCPR2 were compared to the corresponding tomato CPRs (SlCPR1 and SlCPR2), the identities of CPR1 and CPR2 were 94.5% and 92.3%, respectively (Supplementary Fig. 1). Phylogenetic analysis based on CPR amino acid sequences from several plant species revealed that CaCPR1 and CaCPR2 fell into distinct classes among plant CPR (Supplementary Fig. 2). This analysis suggests that each CaCPR may have a distinct and critical role to accommodate the large number of P450 isoforms in C. annuum, which has been estimated to contain 477 CYP genes (Kim et al., 2014). When the predicted amino acid sequences of the CaCPRs were compared to other CPRs from plants and mammals, high degrees of similarity were observed at each domain (Supplementary Fig. 3). 3.2. Expression levels of CaCPR1 and CaCPR2 in hot pepper The expression level of CaCPR1 was higher than that of CaCPR2 in all tested tissues (leaves, flowers, and fruits) and gradually increased during fruit ripening. At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues (Fig. 1). According to the transcriptome analysis, the expression level of CaCPR1 is much higher than that of CaCPR2 in leaves and during fruit ripening stages (Kim et al., 2014). It is known that depending on the species, plants contain one to three CPRs of which one is inducible (Ro et al., 2002; Jensen and Møller, 2010). CaCPR1 may be the inducible CPR in hot pepper. These results suggest that CaCPR1 may have a critical role during
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Fig. 1. Expression levels of the CaCPR1 and CaCPR2 genes in different tissues and stages of fruit ripening. The relative accumulation levels of the CaCPR1 and CaCPR2 transcripts were determined by real-time PCR and normalised against the actin gene as an internal control, as described in the Materials and Methods. EF-1: early fruit stage 1 (7DAP), EF-2: early fruit stage 2 (15DAP), MG: mature green stage (28DAP), BG: breaking green stage (30DAP), RB: Red-turning brown stage (42DAP), RR: red ripe stage (45DAP). DAP: Days after pollination. Each bar represents the mean and standard error of three replicates.
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Fig. 2. (A) SDS-PAGE of CaCPR1. A 10% gel was stained with Coomassie Brilliant Blue R250. Purified CaCPR1 was loaded with protein molecular weight markers. (B) The absorption spectrum of CaCPR1 was measured in 100 mM potassium phosphate buffer (pH 7.4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the fruit ripening stage; in other words, some P450 enzymes may require the electrons transferred by CaCPR1 to function during fruit ripening. The phylogeny tree shows that CaCPR1 and CaCPR2 belong to two major phylogenetic groups, named CPR1 and CPR2, from vascular plants (Jensen and Møller, 2010). The presence of multiple CPRs in some plants may imply the need for the proper interaction of a large number of P450s with CPR to exert optimal activity for the adaptation to environmental changes or during plant developmental stages (Eberle et al., 2009; Rana et al., 2013).
The 7-EC is metabolised by several P450s, typically CYP1A2, supported by mammalian CPR and NADPH to 7-OH coumarin and 3-OH,7-EC (Kim et al., 2006). Two major metabolites, 3-OH,7-EC and 7-OH coumarin, were produced upon reaction of CaCPR1 and rCPR with CYP1A2. The formation rates of the 7-OH coumarin and 3-OH,7-EC products by 0.80 mM CaCPR1 and 0.40 mM CYP1A2 were 0.011 (±0.001) and 0.036 (±0.004) nmol product per min per nmol P450, respectively (Fig. 3C). However, the formation rates of these products were 29% and 24% of those of the reaction with rCPR. When molar ratio of CYP1A2 to CaCPR1 has been increased from 1:0.25 to 1:2 at fixed concentration of CYP1A2 (0.10 mM), the 7-EC oxidation rate increased with a maximum at 1:2 (Fig. 3C). These results imply that CaCPR1 interacts with CYP1A2 to mediate electron transfer between the two proteins.
3.3. Heterologous expression and purification
3.5. Enzymatic properties of CaCPR1 toward chemical substrates
We found that membrane fraction and cytosolic fraction contained 75% and 25% of expressed CaCPR1 in E. coli, respectively. CaCPR1 in the membrane fraction was purified using affinity chromatography after solubilisation of the membrane fraction. The purified CaCPR1 protein yielded the expected single band of 76 kDa by SDS-PAGE (Fig. 2A). The absolute absorption spectrum of purified CaCPR1 was characteristic of flavoproteins, with prominent peaks at 380 and 452 nm (Fig. 2B). This spectral property of CaCPR1 suggests that the CaCPR1 cDNA encodes a functionally active CPR in hot pepper.
The kcat values for CaCPR1 were 942 min1 for MTT, 902 min1 for CTC, and 4800 min1 for ferricyanide (Fig. 4). The Km values for CaCPR1 were in the range of 30e47 mM. The catalytic efficiency of ferricyanide reduction was 5.1-fold and 5.3-fold higher than that for MTT and CTC, respectively. However, MTT and CTC have several advantages over ferricyanide as the CPR substrate. They are tetrazolium salts that are used extensively to measure cell viability, metabolic activity, and oxidative reactions (Altman, 1976; Bernas and Dobrucki, 1999). They can be used as substrates for a colorimetric high-throughput assay because both form highly coloured reduced products (formazans) and display obvious colour changes (from yellow to blue for MTT and colourless to red for CTC). Especially, the CTC formazan is fluorescent, thereby allowing a more sensitive spectrofluorometric assay.
3.4. Enzymatic properties of CaCPR1 toward protein substrates Cytochrome c has been used a typical substrate to measure the reduction activity of plant CPR enzymes (Benveniste et al., 1991). The kcat value of cytochrome c reduction by CaCPR1 was 2740 min1, and the Km value was 81 mM (Fig. 3A). When the oxidised b5 was incubated with CaCPR1 and 100 mM NADPH, it was rapidly reduced and exhibited an absolute absorption spectrum with typical Soret peaks 423, 526, and 556 nm, similar to those observed for the dithionite-reduced form (Fig. 3B). These results demonstrate that CaCPR1 is functionally active as an NADPH- b5 reductase. The magnitude of the spectrum of b5 reduced by CaCPR1 and NADPH in 10 min was approximately 90% of that of the b5 spectrum obtained by sodium hydrosulfite (Fig. 3B).
3.6. Reduction of doxorubicin by CPR1 We tested the ability of CaCPR1 to reduce doxorubicin, a specific anticancer drug, which is reported to be the substrate of CPR in the pH range of 6.0~9.0 (Cummings et al., 1992). CaCPR1 produced one major metabolite, 7-deoxydoxorubicinone (Fig. 5A). CaCPR1 showed much higher activity of 7-deoxydoxorubicinone formation than that of rCPR in most of pH range except 6.0 and 9.0 (Fig. 5B and C). Rate of 7-deoxydoxorubicinone formation by CaCPR1 at pH 7.5 was 413 min1, which is about 4-fold of the turnover catalysed by
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rCPR. This result suggests that CaCPR1 has much higher reductase activity toward doxorubicin than that of rCPR. 3.7. Thermal stability of the CaCPR1 To estimate the stability of the CaCPR1, we determined the thermal stability (Tm, C) of CaCPR1 and compared the Tm value to that of rCPR (Fig. 6). The Tm value of CaCPR1 was measured to be 56.1 ± 0.4 C, whereas the Tm value of the rCPR was 46.1 ± 0.1 C. This result indicates that the plant CaCPR1 has a high thermal stability compared to that of mammalian rCPR. The high thermal stability of CaCPR1 might be related to the high demand for the
Fig. 3. Reduction of protein substrates by CaCPR1. (A) The reduction of cytochrome c was measured spectrophotometrically in the presence of NADPH for 10 min as described in the Materials and Methods. (B) Absolute absorption spectra of b5. Solid line, oxidised b5; dashed line, b5 reduced by CaCPR with 100 mM NADPH. (C) HPLC analysis of the metabolites of 7-EC produced by human CYP1A2 with rCPR and CaCPR1. The reactions were performed with substrate (100 mM 7-EC), 400 nM human CYP1A2, and 800 nM rCPR or 100e800 nM CaCPR1 in 100 mM potassium phosphate buffer (pH 7.4) for 30 min at 37 C. The HPLC eluates were detected at each of the indicated wavelengths on the Y-axis. Each bar represents the mean and the standard error of three replicates. Fig. 4. Concentration-dependent reduction of MTT (A), CTC (B), and ferricyanide (C) by CaCPR1. Reduction was measured spectrophotometrically in the presence of NADPH for 30 s as described in the Materials and methods.
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supply of electrons to P450s under stress conditions (Mizutani and Ohta, 1998). 4. Conclusions In summary, two CaCPR genes were isolated from hot pepper, and their expression levels were found to vary depending on the tissue analysed. At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. It gradually increased during fruit ripening. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues. The cDNA of CaCPR1 was heterologously expressed in E. coli without any modification of amino acids, and the enzymatic properties of the purified CaCPR1 were characterized. CaCPR1 reduced typical the CPR protein substrates cytochrome c and b5. It also could transfer electrons to human CYP1A2. Furthermore, CaCPR1 catalysed the reduction of typical chemical substrates such as MTT, CTC, and ferricyanide, which are useful for continuous and sensitive assays to monitor plant CPR activity in vivo and in vitro. CaCPR1 showed much higher reductase activity to doxorubicin to generate 7-deoxydoxorubicinone and much higher thermal stability when compared to rCPR. These results suggest that the enzymatic properties of CaCPR1 are similar to those of mammalian CPR and that CaCPR1 can be used as a surrogate for other CPRs from other plants and mammals. The expression procedure of plant CPR without any amino acid modification in E. coli and the purification method described here may provide an alternative to previously reported expression systems for other plant CPR enzymes involving yeast and baculovirus. It is suggested that CaCPR1 can be used a prototype for studying biological functions and biotechnological applications of plant CPRs. Competing interest statement Fig. 5. Reduction of doxorubicin by CaCPR1. (A) HPLC analysis of doxorubicin by CaCPR1. Doxorubicin (400 mM) was incubated with CaCPR1 for 5 min at 37 C. The HPLC eluates were detected at 254 nm. The retention times for doxorubicin and the major metabolite, 7-deoxydoxorubicinone, are 10.5 min and 16.2 min, respectively. pH-dependent reduction of doxorubicin to 7-deoxydoxorubicinone by CaCPR1 (B) and rCPR (C) was compared.
The authors declare no competing financial interests. Acknowledgements This work was partially supported by a grant from the NextGeneration BioGreen 21 Program, Rural Development Administration, Republic of Korea (PJ009052) (Y.H. Joung), and the Bio-industry Technology Development Program (111052-04-3-SB010), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (C.-H. Yun). Authors' contributions Joung and Yun supervised the research design and wrote the paper; Lee performed all biochemical experiments, Kim performed all molecular biology experiments and bioinformatics analysis, Ma and Park performed molecular biology experiments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.05.010. References
Fig. 6. Thermal stability of CaCPR1. To analyse the enzyme thermal stability, CaCPR1 and rCPR were incubated at temperatures between 37 C and 75 C for 10 min in a PCR thermocycler followed by cooling to 4 C for 5 min before measuring MTT activity. After centrifugation, the protein solutions were used to measure the ability of CaCPR1 to reduce MTT. The stability was estimated from the differences of MTT reduction rate as described in the Materials and methods.
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