cDNA cloning, overproduction and characterization of rat adrenodoxin reductase1

Biochimica et Biophysica Acta 1434 (1999) 284^295 www.elsevier.com/locate/bba

cDNA cloning, overproduction and characterization of rat adrenodoxin reductase1 Yasuhiro Sagara

a;

*, Yoshiya Watanabe a , Hiroyuki Kodama b , Hironori Aramaki

c

a

c

Department of Medical Biology, Kochi Medical School, Oko-cho, Nankoku, Kochi 783-8505, Japan b Department of Chemistry, Kochi Medical School, Oko-cho, Nankoku, Kochi 783-8505, Japan Department of Molecular Biology, Daiichi College of Pharmaceutical Sciences, Tamagawa-cho, Fukuoka, Fukuoka 815-8511, Japan Received 17 May 1999; received in revised form 29 July 1999; accepted 29 July 1999

Abstract We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region. The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extinction coefficient was estimated to be 10.9 mM31 cm31 at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1. From the a208 value in the circular dichroism spectrum, the K-helix content in the rat AdR was calculated to be 30%. In NADPHcytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent Km value of rat AdR for NADPH was 0.32 WM, a value significantly lower than that of bovine AdR (1.4 WM). The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, Km = 9.3 nM) than to the native partner (rat Ad, Km = 16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, Km = 37.1 nM) than to the heterologous partner (rat Ad, Km = 46.8 nM). The Km values showed a reverse correlation to the difference of pI values between the redox partners. These results indicate that AdR binds to Ad mainly by ionic interaction. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Adrenodoxin; Adrenodoxin reductase; cDNA cloning; Electron transfer; Rat; Escherichia coli

1. Introduction Abbreviations: AdR, NADPH-adrenodoxin reductase; Ad, adrenodoxin; EDTA, ethylenediaminetetraacetic acid; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis * Corresponding author; E-mail: [email protected] 1 The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with accession number D63761.

NADPH-adrenodoxin oxidoreductase (adrenodoxin reductase, EC 1.18.1.2) (AdR) is a component of the electron transfer system for mitochondrial cytochromes P-450 [1]. AdR is synthesized as a largesized precursor by cytoplasmic free polysomes and is processed to the mature form after transfer into the mitochondria [2,3]. The mature AdR has a £avin

0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 8 0 - 6

BBAPRO 35993 30-9-99

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

adenine dinucleotide and provides reducing equivalents from NADPH to the mitochondrial cytochromes P-450 via (2Fe-2S) type ferredoxin, adrenodoxin (Ad), thus contributes to the production of steroid hormones and bile acids and to activate vitamin D3 in the steroidogenic tissues, liver and kidney of vertebrates. AdR has been puri¢ed mainly from bovine adrenal gland. Crystallization and X-ray analysis of bovine AdR has been investigated and the tertiary structure of bovine AdR was very recently published [4^9]. The primary structure of AdR has been determined by nucleotide sequencing of cDNAs cloned from bovine adrenal cortex [10] and human testis [11] mRNAs. The gene structure of AdR was also revealed for both species [12,13]. Recently, the presence of a homologue of AdR in yeast was reported [14,15]. The mitochondrial cytochrome P-450 has not been found in yeast and biological functions of the AdR homologue in yeast are unknown. Regarding the electron transfer mechanism of the mitochondrial cytochrome P-450 systems, two hypotheses have been proposed. One is a `shuttle mechanism' in which Ad functions as a mobile electron shuttle between AdR and cytochrome P-450 [16]. The other is a `cluster mechanism' in which AdR, Ad and cytochrome P-450 form a 1:1:1 complex for steroid hydroxylation [17]. Using chemically cross-linked AdR-Ad complexes, Hara and Kimura observed that cytochrome P-450scc was reduced by the AdR-Ad complex while the reduction was stimulated by addition of free Ad [18]. These investigations were performed using an in vitro system reconstituted with enzyme components puri¢ed from bovine adrenal cortex. After cDNA clonings of AdR, Ad and cytochromes P-450 from bovine and human tissues were performed, heterologous expression systems for the proteins have been developed and various information on the interaction site of the proteins was obtained, using site-directed mutants [19^26]. The electron transfer mechanism has remained controversial. It is important to investigate reaction mechanisms in vivo, using whole animal tissues, and the rat is one of the most suitable animals for the approach. In a series of our studies on the mitochondrial cytochrome P-450 system of rats, we puri¢ed a small amount of mature Ad from 2300 rat adrenal glands

285

[27], isolated a full-length cDNA of rat Ad [28] and developed a heterologous expression system for rat Ad with a precise mature form [29]. Considering the low content of AdR (about 1/10 of Ad in the case of bovine adrenal gland), expression of recombinant AdR in Escherichia coli seems to be the only practical strategy to obtain large amounts of rat AdR. In the present work, we isolated a full-length cDNA of rat AdR and developed a heterologous direct expression system to obtain the precise mature form of the £avoprotein. We report herein cDNA cloning, overproduction in E. coli, simple puri¢cation and characterization of rat AdR. 2. Materials and methods 2.1. Materials Restriction endonucleases and modifying enzymes were purchased from New England Biolabs. DE52 was from Whatman Paper. HiLoad 16/10 SP Sepharose FF, HiTrap SP, HiLoad 16/60 Superdex 75 pg and HiTrap NHS-activated were from Amersham Pharmacia Biotech. NADPH was from Oriental Yeast (Tokyo, Japan). Cytochrome c (horse heart) was from Sigma Chemical. Protein molecular mass standards were from Daiichi Pure Chemicals (Tokyo, Japan). The bovine AdR and bovine Ad used in this study were recombinant proteins, which correspond to the mature forms of bovine AdR and bovine Ad, respectively, and were puri¢ed from E. coli cells harboring pBAR1607 [30] and pBA1159 [20,26], respectively. The rat Ad used was also a recombinant protein, which corresponds to the mature form of rat Ad, and was puri¢ed from E. coli harboring pRA3813 [29]. Concentrations of these proteins were determined using the extinction coe¤cient of 11.3 mM31 cm31 at 450 nm for bovine AdR [6] and 9.8 mM31 cm31 at 414 nm for rat and bovine Ads [27,31], respectively. 2.2. cDNA cloning of rat AdR A VZAP cDNA library constructed from adrenal gland mRNA of an adult male Wistar rat [28] was used for isolation of the AdR cDNA clone. Approx-

BBAPRO 35993 30-9-99

286

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

imately 3U105 phages were screened by plaque hybridization, as described [32]. The probe used was a 1.6 kb HindIII-EcoRI cDNA fragment excised from pBAR16, which covers an entire coding region of bovine AdR [10]. Six positive clones were isolated. One of the longest cDNA clones, pRAR60, was subcloned into pUC9 [33] and sequenced using a DNA Sequencer Model 373A with the Dye Terminator Cycle Sequencing kit (Applied Biosystems). All the cDNA inserts were sequenced on both strands. Sequence data were analyzed using a personal computer program, GENETYX-MAC (Software Development). 2.3. Construction of expression plasmid for rat mature AdR The cDNA fragment encoding the amino-terminal region of rat mature AdR, from the NdeI site, including the initiation codon to 0.1 kb downstream of the ApaI(216) site, was prepared by polymerase chain reaction (PCR) [34]. pRAR60 linearized by EcoRI digestion was used as template. The primers used were 5P-AGCATATGAGCACTCAAGAAACAACCCCTCAGATCTGT-3P for the amino-terminal side and 5P-GTGGTACCGGGCCCGGGTGTGGTGCT-3P for the carboxy-terminal side. The NdeI and ApaI sites are underlined in the primer sequence, respectively. The primer for the amino-terminal side was designed to add the initiator methionine just prior to the predicted amino-terminal serine of the mature AdR and to introduce several silent mutations in the amino-terminal region to increase the expression of AdR in E. coli [35,36]. The silent mutations introduced are shown in bold letters in the primer sequence. The product of PCR was bluntended by T4 DNA polymerase and cloned into the SmaI site of pHSG399 [37]. This construct was designated pRAR6014. The nucleotide sequence of the insert in pRAR6014 was con¢rmed, as described above. The 1.3 kb ApaI(216)/ApaI(1512) fragment excised from pRAR60, which covers the residual region to the carboxy-terminus of AdR, was inserted into the ApaI site of pRAR6014 so that the cDNA encoding the entire region of mature AdR was constructed in pHSG399. The 1.4 kb NdeI/KpnI fragment encoding mature AdR was excised and ligated into the NdeI/KpnI site of an expression vector

pCWori+ [36]. The ¢nal construct was designated pRAR6018. 2.4. Expression and puri¢cation of recombinant rat AdR Overnight cultures of E. coli JM109 [38] harboring pRAR6018 in LB broth containing 50 Wg/ml ampicillin were 1% seeded into 200 ml of Terri¢c broth containing 50 Wg/ml ampicillin and incubated at 30³C until the absorbance at 600 nm reached about 0.5. Expression of AdR was induced by adding 1 mM isopropyl L-D-thiogalactopyranoside. The incubation was continued for 24 h at 30³C with gentle shaking. Thereafter, all the puri¢cation procedures were carried out at 0^4³C. The E. coli cells were harvested by centrifugation at 5200Ug for 5 min and washed once with 10 mM potassium phosphate (pH 7.5) containing 150 mM NaCl. The cells were resuspended in 100 ml of 10 mM potassium phosphate (pH 7.5) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitors (Complete Protease Inhibitor Cocktail Set, Boehringer Mannheim), sonicated with Cell Disrupter (Ultrasonics, Model W-225R) using 50% pulses for 10 min at level 8 and then centrifuged at 105 000Ug for 90 min. The supernatants were combined and passed through a DE52 column (2.5U20 cm) equilibrated with 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA. The column was further washed with three column volumes of 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA. The AdR-rich fractions were combined and applied to a HiLoad 16/10 SP Sepharose FF column. After washing with two column volumes of 10 mM potassium phosphate (pH 7.5) containing 100 mM NaCl and 0.5 mM EDTA, AdR was eluted with a linear gradient of 100^500 mM NaCl (total 10 column volumes) in 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA. The peak fractions, eluted at about 330 mM NaCl, were combined and diluted with two volumes of 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA and then concentrated on a HiTrap SP column using 1 M NaCl in 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA. The AdR was then subjected to gel ¢ltration on a HiLoad 16/60 Superdex 75 pg column equilibrated with 10 mM potassium phosphate (pH 7.5) containing 200

BBAPRO 35993 30-9-99

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

mM NaCl and 0.5 mM EDTA. The peak fractions were combined and diluted with an equal volume of 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA and then applied to an Ad-Sepharose column (v = 5 ml) equilibrated with 10 mM potassium phosphate (pH 7.5) containing 100 mM NaCl. The Ad-Sepharose column was prepared by coupling rat Ad to a HiTrap NHS-activated column. After washing with three column volumes of 10 mM potassium phosphate (pH 7.5) containing 200 mM NaCl and 0.5 mM EDTA, AdR was eluted with a linear gradient of 200^800 mM NaCl (total 12 column volumes) in 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA. The AdR was eluted showing a peak at about 350 mM NaCl. 2.5. Amino acid sequence The amino-terminal amino acid sequence of puri¢ed rat AdR was determined using a 491 cLC Protein Sequencer (PE Applied Biosystems) after electroblotting of the protein onto a PVDF membrane [39]. 2.6. Circular dichroism (CD) CD spectra of rat and bovine AdRs were measured for 5 WM AdR in 10 mM potassium phosphate (pH 7.5), using a Jasco 720A spectropolarimeter (Japan Spectroscopic) at 25³C under a constant £ow of nitrogen. 2.7. Enzyme assay NADPH-cytochrome c reductase activity was measured at 25³C by following the increase in absorbance at 550 nm, using a Hitachi U-3210 spectrophotometer. Standard assay mixture consisted of 100 WM NADPH, 20 nM AdR, 10^100 nM Ad and 20 WM cytochrome c in 1 ml of 10 mM potassium phosphate (pH 7.5) [31]. The concentration of cytochrome c was determined using the reduced minus oxidized extinction coe¤cient of 19.5 mM31 cm31 at 550 nm [40]. 2.8. Analytical methods Protein concentrations were determined by the

287

method of Lowry et al. [41], using bovine serum albumin as a standard. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was carried out by the method of Laemmli [42] using 10% acrylamide gels. 3. Results and discussion 3.1. Isolation of the full-length cDNA clone for rat AdR We screened a cDNA library constructed from rat adrenal gland mRNA, using bovine AdR cDNA as a probe, and isolated a full-length cDNA clone for rat AdR, pRAR60. The nucleotide sequence of the cDNA and the deduced amino acid sequence are shown in Fig. 1. The cDNA in pRAR60 consisted of 22 bp of the 5P-untranslated region, 1482 bp of a coding region, 282 bp of the 3P-untranslated region and 22 bp of poly(A)-tail. The nucleotide sequence surrounding the initiation codon, CAGCCATGG, is consistent with the consensus sequence of the active initiation codon in eukaryotes, CCA/GCCATGG [43]. A typical polyadenylation signal sequence, AATAAA [44], is present 15 bp upstream of the polyadenylation site. The open reading frame has 494 amino acid residues. Fig. 2 shows an alignment of the deduced amino acid sequence of rat AdR with those of bovine [10] and human [11] counterparts. Since authentic rat AdR has not been puri¢ed, we predicted serine-35 in the deduced amino acid sequence to be the amino-terminus of rat mature AdR, based on this alignment. Therefore, the precursor of rat AdR likely consists of 34 amino-terminal amino acid residues as the extrapeptide, which is considered to be necessary for transport into mitochondria, and the following 460 amino acid residues for the mature peptide region. In the extrapeptide region, homologies of amino acid sequences are 70.8% between rat and bovine and 61.8% between rat and human AdRs. In the mature peptide region, however, the values are high, 88.5%, in both cases. The FAD-binding motif (G-X-G-X-X-G-X-X-X-A) and the NADPHbinding motif (G-X-G-X-X-A-X-X-X-A) reported for bovine AdR [10,45] are conserved both in rat

BBAPRO 35993 30-9-99

288

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

Fig. 1. The nucleotide sequence of rat AdR cDNA in pRAR60 and the deduced amino acid sequence. The amino acid residues are shown in single letters. The arrowhead indicates the predicted processing point of AdR precursor. The single asterisk under the nucleotide sequence indicates the stop codon. The nucleotide sequence surrounding the initiation codon (CAGCCATGG) and the possible polyadenylation signal (AATAAA) are underlined.

BBAPRO 35993 30-9-99

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

289

Fig. 2. Alignment of rat AdR with bovine and human AdRs. The deduced amino acid sequence of rat AdR was aligned with those of bovine and human counterparts. Amino acid residues are shown in single letters. The consensus residues in the FAD-binding site (G-X-G-X-X-G-X-X-X-A) and that in the NADPH-binding site (G-X-G-X-X-A-X-X-X-A) are marked by four asterisks, respectively. The open arrowhead indicates the insertion point of the six amino acid residues, which was predicted from the cDNA sequence for minor mRNA species of bovine and human AdRs (see text). This ¢gure shows amino acid sequences derived from the cDNA for major mRNA species.

and human AdRs. In human AdR, arginine-239 and arginine-243 were reported to be direct interaction sites with Ad [22]. The corresponding amino acid residues, arginine-240 and arginine-244 in the rat and bovine AdRs, are also conserved in these species. In the case of bovine and human AdRs, the presence of minor mRNA species containing an 18 bp insert was found by cDNA cloning and the minor mRNA was considered to be produced by alternative splicing [10^13]. However, the putative minor form of human AdR expressed in E. coli, using cDNA with the 18 bp insert, did not exhibit the AdR activity [46]. pRAR60 in the present study does not contain a sequence corresponding to the 18 bp insert, suggesting that this cDNA derived from the major mRNA species of rat AdR. We have not isolated the minor type cDNA of rat AdR.

3.2. Expression and puri¢cation of recombinant rat AdR There are reports in which recombinant proteins, with some modi¢cation at the amino- and/or carboxy-terminal region, are used for analysis of enzymatic properties instead of the authentic protein. In the case of Ad, however, not only modi¢cation at the amino-terminal region but also that at the carboxyterminal region a¡ected the enzymatic properties [20,24,26]. Therefore, we constructed an expression system to produce a precise mature form of rat Ad [29]. In the present study also, to obtain a precise mature form of rat AdR, we constructed a direct expression system where the enzyme can be directly puri¢ed from the bacterial cell lysate, with a high yield.

BBAPRO 35993 30-9-99

290

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

The rat AdR was puri¢ed from the cell lysate by passing through an anion-exchanger, cation-exchange chromatography, gel ¢ltration and a¤nity chromatography on Ad-Sepharose [5]. The puri¢cation yield was 6.5 mg from the cell lysate from 200 ml culture, i.e. more than 30 mg/l culture, in a typical case. This value is 12- and 300-fold higher than those of bovine [30] and human [46] AdRs expressed in E. coli, respectively. Compared with the bovine counterpart, rat AdR showed unique behavior on ion-exchange chromatography. In the case of bovine AdR, the enzyme was adsorbed to an anion-exchanger, DEAE-cellulose, at pH 7.4 under low salt conditions [4]. Rat AdR, however, passed through a DE52 column but was adsorbed to a cation-exchanger, SPSepharose, under 10 mM potassium phosphate (pH 7.5). The step of passing through DE52 was e¡ective for puri¢cation. Rat AdR was eluted from the SPSepharose column showing a peak at about 330 mM NaCl in 10 mM potassium phosphate (pH 7.5) and became near homogeneous after the chromatography (Fig. 3). The following gel ¢ltration and Ad-Sepharose chromatography were carried out to eliminate minor contaminants. This di¡erent behavior of rat and bovine AdRs on ion-exchange chromatography can be explained by the pI values of these AdRs. The pI value of rat AdR calculated from the cDNA sequence is 8.84 whereas that of bovine AdR is 6.90. Fig. 3 shows a SDS-PAGE analysis of rat AdR preparation at each step of puri¢cation. The apparent molecular mass of rat AdR on the analysis was 51 000, being close to the value, 50 316, of the predicted mature peptide calculated from the cDNA sequence. Amino acid sequence analysis of the puri¢ed rat AdR revealed the sequence of the amino-terminal 20 residues to be S-T-Q-E-T-T-P-Q-I-X-V-V-G-S-GP-A-G-F-Y (data not shown), a sequence identical with that of residue 2^21 of the product de¢ned by the cDNA sequence in the expression plasmid, pRAR6018, if the unidenti¢ed residue X is cysteine. Hirel et al. reported that the initiator methionine was processed in E. coli when the second amino acid was serine [47]. The ¢ndings in our study also indicate that the amino-terminal methionine just prior to serine, which corresponds to the predicted amino-terminal residue of the mature AdR, was processed from the recombinant AdR in E. coli, as expected. Consequently, the amino-terminal amino acid sequence

Fig. 3. SDS-PAGE analysis of the preparation at each step of rat AdR puri¢cation. Lane 1, molecular mass standards; lane 2, cell lysate; lane 3, fraction obtained by passing through DE52; lane 4, fraction obtained by chromatography on SPSepharose; lane 5, fraction obtained by gel ¢ltration through Superdex 75 pg; lane 6, fraction obtained by a¤nity chromatography on Ad-Sepharose. Protein amounts analyzed were 20 Wg in lane 1 and 5 Wg each in lanes 2^6. Proteins were stained with Coomassie brilliant blue R-250. The molecular mass standards used were phosphorylase b (97.4 kDa), bovine serum albumin (66.3 kDa), aldolase (42.4 kDa), carbonic anhydrase (30.0 kDa), soybean trypsin inhibitor (20.1 kDa) and lysozyme (14.4 kDa).

of the puri¢ed recombinant AdR is probably identical with that of authentic rat AdR. 3.3. Spectral properties Fig. 4 shows the absorption spectrum of the puri¢ed AdR in the oxidized form. The spectrum has peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. This feature of the spectrum is characteristic of a £avoprotein and is comparable to that of native bovine AdR [6]. On a basis of the molecular mass value of 50 316, the extinction coef¢cient of rat AdR was estimated to be 10.9 mM31 cm31 at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1, being lower than those of recombinant bovine AdR, 7.8 [30], and recombinant human AdR, 7.9 [46]. As shown in Fig. 2, the mature rat AdR probably contains eight tyrosine and ¢ve tryptophan residues. The tryptophan content of rat AdR is one residue lower than those of bovine and human AdRs, while the tyrosine content is the same in these

BBAPRO 35993 30-9-99

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

291

Fig. 4. Absorption spectrum of the puri¢ed rat AdR. The absorption spectrum was measured for 12.5 WM AdR in 10 mM potassium phosphate (pH 7.5) containing 0.5 mM EDTA and 50 mM NaCl at 25³C. The inset shows the absorption spectrum expanded in the absorbance range.

three AdRs. This low tryptophan content may result in the 270 nm/450 nm ratio to be low in rat AdR. To obtain information on the secondary structure of rat AdR, the CD spectrum in the ultraviolet wavelength range was measured and compared with that of bovine AdR. The CD spectra of rat and bovine AdRs were similar (data not shown). The K-helix content in rat and bovine AdRs was estimated to be 30 and 34% from the a208 value, respectively, by the method of Green¢eld and Fasman [48]. 3.4. Enzymatic activities of the recombinant rat AdR To examine the enzymatic properties of rat AdR, NADPH-cytochrome c reductase activity was measured, using a system reconstituted with Ad, and the kinetic parameters were compared with those of bovine AdR. The apparent Km value of rat AdR for NADPH was 1/4 of that of bovine AdR, indicating that rat AdR has a higher a¤nity for NADPH than the bovine counterpart. The Vmax value of the rat AdR+rat Ad system was 2/3 of the bovine enzymes system (Fig. 5, Table 1). Next we measured NADPH-cytochrome c reduc-

Fig. 5. NADPH-cytochrome c reductase activity determined under NADPH-limiting conditions. NADPH-cytochrome c reduction was measured at 25³C in 1 ml of 10 mM K-phosphate (pH 7.5) containing 20 nM AdR, 200 nM Ad, 20 WM cytochrome c and 0.4^10 WM NADPH. The results are shown in [S]0 /v versus [S]0 plots. The combinations of AdR and Ad examined were rat AdR+rat Ad (closed circle) and bovine AdR+bovine Ad (open circle).

BBAPRO 35993 30-9-99

292

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

Table 1 Kinetic parameters of AdRs for NADPH-cytochrome c reductase activity in a reconstituted system, under NADPH-limiting conditions Rat AdR Bovine AdR

Km for NADPH (WM)

Vmax (s31 )a

0.32 þ 0.10 1.4 þ 0.3

10.1 þ 0.4 15.2 þ 1.3

The assay conditions are shown in the legend for Fig. 5. The values were calculated from the results of three independent experiments.

tase activity under Ad-limiting conditions. In this experiment, four combinations of AdR and Ad were examined, i.e. rat AdR+rat Ad, rat AdR+bovine Ad, bovine AdR+rat Ad and bovine AdR+bovine Ad (Fig. 6). The kinetic parameters obtained from the experiment are summarized in Table 2. Rat AdR showed the apparent Km value for rat Ad to be almost twice that for bovine Ad. The Vmax values were much the same between these two combinations. On the other hand, bovine AdR showed that the apparent Km value for bovine Ad is somewhat lower than that for rat Ad. The Vmax value was higher in the combination with bovine Ad than that with rat Ad. These results indicate that the a¤nity of

rat AdR is higher for the heterologous redox partner (bovine Ad) than for the native partner (rat Ad) while that of bovine AdR is higher for the native partner (bovine Ad) than for the heterologous partner (rat Ad), an unusual phenomenon. To explain the di¡erent a¤nities between AdR and Ad from rat and bovine, we directed attention to the pI value of these four proteins. The pI values calculated from the respective cDNA sequence are also shown in Table 2, the pI value of rat AdR is signi¢cantly higher than that of bovine AdR and the pI value of rat Ad is also higher than that of bovine Ad. The apparent Km value was small when the di¡erence of pI values between the redox partners was large. This tendency was consistent in the four combinations of AdR and Ad from rat and bovine, namely, the Km values showed a reverse correlation to the di¡erence in pI values between the redox partners. The in vitro interaction between AdR and Ad depends on the ionic strength of the assay condition [16,49]. Replacement of the charged residues by site-directed mutagenesis of AdR [22] and Ad [19] a¡ected the interaction. Our ¢ndings are consistent with these reports and also indicate that AdR binds to Ad mainly by ionic interaction.

Fig. 6. NADPH-cytochrome c reductase activity determined under Ad-limiting conditions. NADPH-cytochrome c reduction was measured at 25³C in 1 ml of 10 mM K-phosphate (pH 7.5) containing 20 nM AdR, 10^100 nM Ad, 20 WM cytochrome c and 100 WM NADPH. The results are shown in [S]0 /v versus [S]0 plots. In this experiment, four combinations for AdR and Ad were examined. (A) Closed circle, rat AdR+rat Ad; open circle, bovine AdR+rat Ad. (B) Closed circle, rat AdR+bovine Ad; open circle, bovine AdR+bovine Ad.

BBAPRO 35993 30-9-99

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295

293

Table 2 Kinetic parameters of AdRs for NADPH-cytochrome c reductase activity in a reconstituted system, under Ad-limiting conditions Rat Ad (pI = 4.34) Rat AdR (pI = 8.84) Bovine AdR (pI = 6.90)

Bovine Ad (pI = 4.22)

Km for rat Ad (nM)

Vmax (s31 )a

Km for bovine Ad (nM)

Vmax (s31 )a

16.7 þ 0.8 46.8 þ 2.1

13.4 þ 0.2 16.8 þ 0.4

9.3 þ 0.5 37.1 þ 0.6

12.8 þ 0.4 20.1 þ 0.4

The assay conditions are given in the legend for Fig. 6. The kinetic values were calculated from results of three independent experiments. The pI values were estimated for mature proteins from the cDNA sequences, respectively, using the GENETYX-MAC program. a Mol cytochrome c reduced/s/mol of £avin.

Summarizing, we isolated full-length cDNA of rat AdR, puri¢ed rat AdR using a heterologous expression of the cDNA and identi¢ed enzymatic properties of the enzyme. Rat AdR showed typical spectral properties of £avoprotein, being similar to those of bovine AdR. However, the high pI value of rat AdR, 8.84, is remarkable, the value being signi¢cantly higher than that of bovine AdR, 6.90. This feature lent an unique behavior on ion-exchange chromatography to rat AdR and facilitated a simple puri¢cation. The a¤nity of rat AdR was higher for the heterologous redox partner (bovine Ad) than was the native partner (rat Ad), probably due to the lower pI value of bovine Ad. The Km value for Ad in the NADPH-cytochrome c reduction reaction showed a reverse correlation to the di¡erence in pI values of the AdR and the Ad used. These results further support the notion that AdR binds to Ad mainly by ionic interaction in vivo. Acknowledgements We thank Prof. A. Tominaga (Kochi Medical School, Japan) for the use of FPLC. References [1] S. Takemori, T. Yamazaki and S. Ikushiro, Cytochrome P450-linked electron transport system in monooxygenase reaction, in: T. Omura, Y. Ishimuram and Y. Fujii-Kuriyama (Eds.), Cytochrome P-450, 2nd edn., Kodansha, Tokyo, 1993, pp. 44^63. [2] N. Nabi, T. Omura, In vitro synthesis of adrenodoxin and adrenodoxin reductase: Existence of a putative large precursor form of adrenodoxin, Biochem. Biophys. Res. Commun. 97 (1980) 680^686.

[3] R.E. Kramer, R.N. DuBois, E.R. Simpson, C.M. Anderson, K. Kashiwagi, J.D. Lambeth, C.R. Jefcoate, M.R. Waterman, Cell-free synthesis of precursor forms of mitochondrial steroid hydroxylase enzymes of the bovine adrenal cortex, Arch. Biochem. Biophys. 215 (1982) 478^485. [4] K. Suhara, Y. Ikeda, S. Takemori, M. Katagiri, The puri¢cation and properties of NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria, FEBS Lett. 28 (1972) 45^47. [5] T. Sugiyama, T. Yamano, Puri¢cation and crystallization of NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria, FEBS Lett. 52 (1975) 145^148. [6] A. Hiwatashi, Y. Ichikawa, N. Maruya, T. Yamano, K. Aki, Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase form bovine adrenocortical mitochondria. I. Physicochemical properties of holo- and apo-NADPH-adrenodoxin reductase and interaction between non-heme iron proteins and the reductase, Biochemistry 15 (1976) 3082^3090. [7] Y. Nonaka, S. Aibara, T. Sugiyama, T. Yamano, Y. Morita, A crystallographic investigation on NADPH-adrenodoxin oxidoreductase, J. Biochem. (Tokyo) 98 (1985) 257^260. [8] A. Lapko, A. Mu«ller, O. Heese, K. Ruckpaul, U. Heinemann, Preparation and crystallization of a cross-linked complex of bovine adrenodoxin and adrenodoxin reductase, Proteins 28 (1997) 289^292. [9] G.A. Ziegler, C. Vonrhein, I. Hanukoglu, G.E. Schulz, The structure of adrenodoxin reductase of mitochondrial P450 systems: Electron transfer for steroid biosynthesis, J. Mol. Biol. 289 (1999) 981^990. [10] Y. Sagara, Y. Takata, T. Miyata, T. Hara, T. Horiuchi, Cloning and sequence analysis of adrenodoxin reductase cDNA from bovine adrenal cortex, J. Biochem. (Tokyo) 102 (1987) 1333^1336. [11] B.S. Solish, J. Picado-Leonard, Y. Morel, R.W. Kuhn, T.K. Mohandas, I. Hanukoglu, W.L. Miller, Human adrenodoxin reductase: Two mRNAs encoded by a single gene on chromosome 17cen-q25 are expressed in steroidogenic tissues, Proc. Natl. Acad. Sci. USA 85 (1988) 7104^7108. [12] D. Lin, Y. Shi, W.L. Miller, Cloning and sequence of the human adrenodoxin reductase gene, Proc. Natl. Acad. Sci. USA 87 (1990) 8516^8520. [13] Y. Takata, Y. Sagara, A. Kono, K. Sekimizu, T. Horiuchi,

BBAPRO 35993 30-9-99

294

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295 Gene structure of bovine adrenodoxin reductase, Biol. Pharm. Bull. 16 (1993) 1200^1206. T. Lacour, B. Dumas, A gene encoding a yeast equivalent of mammalian NADPH-adrenodoxin oxidoreductases, Gene 174 (1996) 289^292. T. Lacour, T. Achstetter, B. Dumas, Characterization of recombinant adrenodoxin reductase homologue (Arh1p) from yeast: Implication in in vitro cytochrome P45011L monooxygenase system, J. Biol. Chem. 273 (1998) 23984^ 23992. J.D. Lambeth, D.W. Seybert, H. Kamin, Ionic e¡ects on adrenal steroidogenic electron transport: The role of adrenodoxin as an electron shuttle, J. Biol. Chem. 254 (1979) 7255^7264. T. Kido, T. Kimura, The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase-adrenodoxin complex: The implication in ACTH function, J. Biol. Chem. 254 (1979) 11806^ 11815. T. Hara, T. Kimura, Active complex between adrenodoxin reductase and adrenodoxin in the cytochrome P-450scc reduction reaction, J. Biochem. (Tokyo) 105 (1989) 601^605. V.M. Coghlan, L.E. Vickery, Site-speci¢c mutations in human ferredoxin that a¡ect binding to ferredoxin reductase and cytochrome P450scc, J. Biol. Chem. 266 (1991) 18606^ 18612. Y. Sagara, T. Hara, Y. Ariyasu, F. Ando, N. Tokunaga, T. Horiuchi, Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modi¢ed amino-terminal regions, FEBS Lett. 300 (1992) 208^212. A. Wada, M.R. Waterman, Identi¢cation by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding, J. Biol. Chem. 267 (1992) 22877^22882. M.E. Brandt, L.E. Vickery, Charge pair interactions stabilizing ferredoxin- ferredoxin reductase complexes: Identi¢cation by complementary site-speci¢c mutations, J. Biol. Chem. 268 (1993) 17126^17130. V. Beckert, R. Dettmer, R. Bernhardt, Mutations of tyrosine 82 in bovine adrenodoxin that a¡ect binding to cytochromes P45011A1 and P45011B1 but not electron transfer, J. Biol. Chem. 269 (1994) 2568^2573. H. Uhlmann, R. Kraft, R. Bernhardt, C-terminal region of adrenodoxin a¡ects its structural integrity and determines di¡erences in its electron transfer function to cytochrome P-450, J. Biol. Chem. 269 (1994) 22557^22564. H. Uhlmann, R. Bernhardt, The role of threonine 54 in adrenodoxin for the properties of its iron-sulfur cluster and its electron transfer function, J. Biol. Chem. 270 (1995) 29959^29966. Y. Sagara, T. Hara, Y. Ariyasu, A. Kajiyama, T. Yasukochi, T. Horiuchi, Di¡erent e¡ects of carboxy-terminal deletion in the adrenodoxin molecule on cytochrome c and acetylated cytochrome c reductions, Biol. Pharm. Bull. 19 (1996) 1401^ 1406. Y. Sagara, K. Matsunaga, K. Nakamura, H. Aramaki, Y.

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

Watanabe, T. Hara, K. Sekimizu, T. Horiuchi, Puri¢cation and characterization of rat adrenodoxin, Biochimie 77 (1995) 719^723. Y. Sagara, Y. Watanabe, K. Kawamura, T. Yubisui, Cloning and sequence analysis of a full-length cDNA of rat adrenodoxin, Biol. Pharm. Bull. 19 (1996) 39^41. Y. Sagara, H. Aramaki, Overproduction in Escherichia coli and characterization of the precise mature form of rat adrenodoxin, Biol. Pharm. Bull. 21 (1998) 1106^1109. Y. Sagara, A. Wada, Y. Takata, M.R. Waterman, K. Sekimizu, T. Horiuchi, Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization, Biol. Pharm. Bull. 16 (1993) 627^630. T. Hara, T. Kimura, Puri¢cation and catalytic properties of a cross-linked complex between adrenodoxin reductase and adrenodoxin, J. Biochem. (Tokyo) 105 (1989) 594^600. J. Sambrook, E.F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, New York, 1989. J. Vieira, J. Messing, The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers, Gene 19 (1982) 259^268. R.K. Saiki, D.H. Gelfand, S. Sto¡el, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis, H.A. Erlich, Primer-directed enzymatic ampli¢cation of DNA with a thermostable DNA polymerase, Science 239 (1988) 487^494. A.C. Looman, J. Bodlaender, L.J. Comstock, D. Eaton, P. Jhurani, H.A. deBoer, P.H. vanKnippenberg, In£uence of the codon following the AUG initiation codon on the expression of a modi¢ed lacZ gene in Escherichia coli, EMBO J. 6 (1987) 2489^2492. H. Barnes, M.P. Arlotto, M.R. Waterman, Expression and enzymatic activity of recombinant cytochrome P45017K-hydroxylase in Escherichia coli, Proc. Natl. Acad. Sci. USA 88 (1991) 5597^5601. S. Takeshita, M. Sato, M. Toba, W. Masahashi, T. Hashimoto-Gotoh, High- copy-number and low-copy-number plasmid vectors for lacZ K-complementation and chloramphenicol- or kanamycin-resistance selection, Gene 61 (1987) 63^74. C. Yanisch-Perron, J. Vieira, J. Messing, Improved M13 phage cloning vectors and host strains Nucleotide sequences of the M13mp18 and pUC19 vectors, Gene 33 (1985) 103^ 119. H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350^4354. B. Chance, G.R. Williams, The respiratory chain and oxidative phosphorylation, Adv. Enzymol. 17 (1956) 65^134. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265^275. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680^685.

BBAPRO 35993 30-9-99

Y. Sagara et al. / Biochimica et Biophysica Acta 1434 (1999) 284^295 [43] M. Kozak, Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs, Nucleic Acids Res. 12 (1984) 857^872. [44] N.J. Proudfoot, G.G. Brownlee, 3P Non-coding region sequences in eukaryotic messenger RNA, Nature 263 (1976) 211^214. [45] I. Hanukoglu, T. Gut¢nger, cDNA sequence of adrenodoxin reductase: Identi¢cation of NADP-binding sites in oxidoreductases, Eur. J. Biochem. 180 (1989) 479^484. [46] M.E. Brandt, L.E. Vickery, Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli, Arch. Biochem. Biophys. 294 (1992) 735^740.

295

[47] P.-H. Hirel, J.-M. Schmitter, P. Dessen, G. Fayat, S. Blanquet, Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid, Proc. Natl. Acad. Sci. USA 86 (1989) 8247^8251. [48] N. Green¢eld, G.D. Fasman, Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry 8 (1969) 4108^4116. [49] J.-W. Chu, T. Kimura, Studies on adrenal steroid hydroxylases: Complex formation of the hydroxylase components, J. Biol. Chem. 248 (1973) 5183^5187.

BBAPRO 35993 30-9-99