Tài liệu Báo cáo khoa học: A novel electron transport system for thermostable CYP175A1 from Thermus thermophilus HB27 doc

A novel electron transport system for thermostable CYP175A1 from Thermus thermophilus HB27 Takao Mandai, Shinsuke Fujiwara and Susumu Imaoka Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Gakuen, Sanda, Japan Cytochrome P450s are associated with a number of physiologically essential reactions, including drug metabolism, carbon source assimilation, and the bio- synthesis of steroids, vitamins, prostaglandins, and antibiotics [1]. Cytochrome P450s have great potential to perform numerous industrially important reactions. Indeed, cytochrome P450sca-2 from Streptomyces car- bophilus has already been used for the production of pravastatin, a cholesterol-lowering drug [2]. However, low tolerance to various solvents and high temperature has generally limited the usefulness of cyto- chrome P450s for industrial applications. Thermophilic cytochrome P450s possess extreme stability, and might be used to overcome such limitations. Recently, two thermophilic cytochrome P450s, CYP119 and CYP175A1, were identified in Sulfolobus solfataricus and Thermus thermophilus, respectively [3,4]. CYP119 is well characterized, and its crystal struc- ture has been determined in the ligand-free state and in several ligand-bound states [5,6]. As expected, CYP119 is highly resistant to both high temperatures (T m =91°C) and high pressures (up to 2 kbar) [7]. Keywords CYP175A1; ferredoxin; ferredoxin–NAD(P) + reductase; Thermus thermophilus; b-carotene hydroxylase Correspondence S. Imaoka, Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Japan Tel ⁄ Fax: +81 79 565 7673 E-mail: imaoka@kwansei.ac.jp (Received 30 January 2009, revised 15 February 2009, accepted 18 February 2009) doi:10.1111/j.1742-4658.2009.06974.x CYP175A1 from Thermus thermophilus is a thermophilic cytochrome P450 and has great potential for industrial applications. However, a native elec- tron transport system for CYP175A1 has not been identified. Here, an elec- tron transport system for CYP175A1 was isolated from T. thermophilus HB27 by multistep chromatography, and identified as comprising ferre- doxin (Fdx; locus in the genome, TTC1809) and ferredoxin–NAD(P) + reductase (FNR; locus in the genome, TTC0096) by N-terminal amino acid sequence analysis and MALDI-TOF-MS, respectively. Although TTC0096, which encodes the FNR, is annotated as a thioredoxin reductase in the T. thermophilus HB27 genome database, TTC0096 lacks an active-site dithi- ol ⁄ disulfide group, which is required to exchange reducing equivalents with thioredoxin. The FNR reduced ferricyanide, an artificial electron donor, in the presence of NADH and NADPH, but preferred NADPH as a cofactor (K m for NADH = 2440 ± 546 lm; K m for NADPH = 4.1 ± 0.2 lm). Furthermore, the FNR reduced cytochrome c in the presence of NADPH and Fdx. The T m value of the FNR was 99 °C at pH 7.4. With an electron transport system consisting of Fdx and FNR, CYP175A1 efficiently cata- lyzed the hydroxylation of b-carotene at the 3-position and 3¢-position at 65 °C, and the K m and V max values for b-carotene hydroxylation were 14.3 ± 1.6 lm and 18.3 ± 0.6 nmol b-cryptoxanthinÆmin )1 Ænmol )1 CYP175A1, respectively. This is the first report of a native electron trans- port system for CYP175A1. Abbreviations Fdx, ferredoxin; FNR, ferredoxin–NAD(P) + reductase; IPTG, isopropyl-thio-b-D-galactoside; OFOR, 2-oxoacid:ferredoxin oxidoreductase; ONFR, oxygenase-coupled NADH–ferredoxin reductase; SD, standard deviation; TR, thioredoxin reductase; UPLC, ultra-performance liquid chromatography. 2416 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS The structure of CYP119 exhibits the typical cyto- chrome P450 fold [5]. However, differences between CYP119 and other cytochrome P450s include a rela- tively high number of salt bridges, a low number of Ala residues and a high number of Ile residues in the interior of CYP119, and the presence of more exten- sive aromatic networks [8]. It has been suggested that these differences contribute to the thermostability of CYP119. In particular, aromatic networks appear to contribute significantly to the thermostability of CYP119 [9]. On the other hand, CYP175A1 has been only partially characterized, although its crystal struc- ture has been determined [4]. CYP175A1 shows high thermostability (T m =88°C), and its substrate-bind- ing region is highly similar to the substrate-binding region of cytochrome P450 BM-3, which catalyzes the hydroxylation of saturated fatty acids [4]. However, CYP175A1 catalyzes the hydroxylation of b-carotene at the 3-position and 3¢-position, but does not catalyze the hydroxylation of fatty acids [10,11]. To perform their oxidative reactions, cyto- chrome P450s require two electrons supplied primarily from NAD(P)H via electron transport systems, which are composed of one or more redox proteins and are divided into two main classes. Most bacterial and mammalian mitochondrial cytochrome P450s utilize the class I system, which is composed of an iron–sulfur protein and an FAD-containing NAD(P)H-dependent reductase [12]. Eukaryotic cytochrome P450s utilize the class II system, composed of an NADPH-dependent reductase containing both FAD and FMN [12]. How- ever, recent studies have revealed a number of unusual electron transport systems for cytochrome P450s that cannot be described as belonging to either class I or class II [1,12]. The electron transport system for CYP119 is a good example of such a system. In this case, the electron transport system is composed of ferredoxin (Fdx) and 2-oxoacid:Fdx oxidoreductase (OFOR), and utilizes pyruvate as an electron source rather than NAD(P)H [13,14]. On the other hand, the native electron transport system for CYP175A1 has not yet been identified, although the catalytic activity of CYP175A1 has been detected using an artificial electron transport system for CYP101 from the meso- philic bacterium Pseudomonas putida [11]. Most Thermus species are known to produce carot- enoid-like pigments. CYP175A1 catalyzes the hydrox- ylation of b-carotene at the 3-position and 3¢-position, producing zeaxanthin via b-cryptoxanthin [10]. The zeaxanthin produced by CYP175A1 is used as an inter- mediate for the synthesis of thermozeaxanthins and thermobiszeaxanthins, which are the main carotenoids of T. thermophilus [15]. The insertion of thermozeax- anthins and thermobiszeaxanthins into the cell mem- brane reduces membrane fluidity and reinforces the membrane [16], contributing to the survival of T. ther- mophilus at high temperatures. Thus, identification of the electron transport system for CYP175A1 is consid- ered important not only for developing industrial applications, but also for investigating the physiologi- cal characteristics associated with this system. A native electron transport system for CYP175A1 has not yet been identified, although CYP175A1 pos- sesses great potential for industrial applications. Thus, in this study, a native electron transport system for CYP175A1 was isolated from the cytosol of T. thermo- philus HB27, in order to reconstitute a high-tempera- ture CYP175A1 catalytic system. The electron transport system was composed of Fdx and Fdx– NAD(P) + reductase (FNR), and these components were characterized at high temperature. Results Isolation and identification of the components of the CYP175A1 electron transport system To find the electron donor of the electron transport system for CYP175A1, we initially measured the b-carotene hydroxylation activity in the presence of purified CYP175A1, the cytosol of T. thermophilus, and the electron donors NADH, NADPH, and pyru- vate (+CoA), which are generally used in cyto- chrome P450 systems. The catalytic activities of CYP175A1 in the presence of NADH and NADPH were 0.03 and 0.43 nmol b-cryptoxanthinÆmin )1 Ænmol )1 CYP175A1, respectively. NADPH was about 14-fold more effective than NADH in this system. Pyruvate (+CoA) is known to be used in the CYP119 system [13], but was not effective in the CYP175A1 system. Then, in order to identify electron transport proteins, the cytosol of T. thermophilus was separated into five fractions using an anion exchange column (DE52) by stepwise elution with KCl (50, 100, 200, 300, and 500 mm). b-Carotene hydroxylation activity was not detected in the presence of any single fraction, but was detected in the presence of both the 100 mm KCl and 300 mm KCl fractions with purified CYP175A1 and NADPH. These results suggest that the electron trans- port system for CYP175A1 was dependent on NADPH and composed of at least two proteins in the 100 mm KCl and 300 mm KCl fractions. The 300 mm KCl fraction from the DE52 column was further puri- fied using a butyl–Sepharose column and a Mono Q column. b-Carotene hydroxylation activity was detected in a major peak when it was reacted with T. Mandai et al. Thermostable electron transport system FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2417 purified CYP175A1, NADPH, and the 100 mm KCl fraction from the DE52 column (data not shown). The peak was subjected to SDS ⁄ PAGE, and a single band was observed (Fig. 1A). These purification steps are summarized in Table 1. The purified protein gave a UV–visible spectrum with a broad absorption peak at 400 nm and a peak at 280 nm (A 400 ⁄ A 280 = 0.63) (Fig. 1B). The absorption spectrum was very similar to that of Fdx from T. thermophilus, which contains two iron–sulfur clusters (one [4Fe–4S] cluster and one [3Fe–4S] cluster) [17–19]. The N-terminal amino acid sequence of the purified protein was Pro-His-Val-Ile- X-Glu-Pro-X-Ile, which corresponds to the N-terminal sequence of the seven-iron Fdx (locus in the genome, TTC1809). These results suggest that Fdx is a com- ponent of an electron transport system for CYP175A1. The 100 mm KCl fraction from the DE52 column was further purified using a 2¢,5¢-ADP–Sepharose column and a Mono Q column. b-Carotene hydroxylation activity was detected in a major peak when it was reacted with purified CYP175A1, NADPH, and the 300 mm KCl fraction from the DE52 column (data not shown). The peak was subjected to SDS ⁄ PAGE, and a single band was observed (Fig. 2A). These purification steps are summarized in Table 2. The purified protein was analyzed by MALDI-TOF-MS. Peptide mass fin- gerprinting was used to search the NCBInr database using mascot. The result of the mascot search suggested that the band was a protein encoded by TTC0096 (locus in the genome). The molecular mass estimated by SDS ⁄ PAGE was 33.2 kDa, which corre- sponds to that calculated from the amino acid sequence of the protein encoded by TTC0096 (36 176 Da). On the other hand, the molecular mass of the purified protein under nondenaturing conditions was determined to be 74.9 kDa by gel filtration on a Superdex-200HR column (data not shown), suggesting that the protein encoded by TTC0096 forms a homo- dimer under nondenaturing conditions. Furthermore, the protein encoded by TTC0096 gave a UV–visible spectrum with absorption peaks at 273, 392, and 473 nm, which is characteristic of flavoproteins (Fig. 2B). The FAD content of the protein was 0.70 mol FADÆmol )1 subunit, suggesting that the FAD was noncovalently bound to the protein. These results suggest that another component of an electron trans- port system for CYP175A1 is a protein encoded by TTC0096, which functions as an FNR. Thus, we concluded that the electron transport system for CYP175A1 belongs to class I. Characterization of recombinant FNR The FNR and Fdx were expressed in Escherichia coli and purified to homogeneity. The purified recombinant FNR and Fdx had the same chromatographic, photo- metric and catalytic properties as the native FNR and Fdx (data not shown). Although the FNR reduced ferri- cyanide, an artificial electron acceptor, at 25 °C and at pH 7.4 in the presence of NADH as well as NADPH, the K m value of the FNR for NADPH was about 600-fold lower than that for NADH, and the V max value of the FNR with NADPH was about 55-fold higher A 62 47.5 32.5 25 16.5 kDa 12 3 45 B Wavelength (nm) 0.0 1.0 0.5 300 400 500 600 700 Absorbance Fig. 1. Purification and characterization of Fdx from T. thermophilus HB27. (A) SDS ⁄ PAGE of fractions containing Fdx at each step of purification. SDS ⁄ PAGE was carried out on a 15% polyacrylamide gel. Lane 1: molecular mass markers. Lane 2: cytosol of T. thermo- philus HB27 (20 lg). Lane 3: 300 m M KCl fraction from a DE52 column (8.3 lg). Lane 4: fraction eluted from a butyl–Sepharose column (13.3 lg). Lane 5: fraction eluted from a Mono Q column (4.6 lg). (B) Absorption spectrum of native Fdx purified from T. thermophilus HB27. The absorption spectrum of purified Fdx (25 l M) was measured in buffer A (50 mM potassium phosphate buffer, pH 7.4, 10% glycerol). Thermostable electron transport system T. Mandai et al. 2418 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS than that with NADH (Table 3). Taken together, these results show that the FNR prefers NADPH over NADH. Furthermore, the FNR showed 4.2-fold greater ferricyanide reduction activity at 50 °C with saturating concentrations of NADPH (1 mm) and ferricyanide (1 mm) than at 25 °C (data not shown). To determine the optimal pH of the FNR, we mea- sured ferricyanide reduction activity at 50 °C and at a range of pH values from 4.0 to 8.0 (Fig. 3A). Although the intracellular pH of T. thermophilus is known to be maintained at 6.9–7.1 [20], the FNR unexpectedly exhibited maximal activity at pH 4.5–6.5. The thermostability of the FNR was evaluated by measuring the residual ferricyanide reduction activity after incubation of the FNR for 30 min at various temperatures (Fig. 3B). The T m values of the FNR at pH 7.4 and at pH 5.0 were 99 and 95 °C, respectively. These results indicate that the FNR is an extremely thermostable protein at both pH 7.4 and pH 5.0. The FNR reduced cytochrome c at 50 °C in the presence of NADPH and Fdx, and the activity was dependent on the concentration of Fdx (Table 4). These results also indicate that the FNR, which is encoded by TTC0096, transfers electrons from NADPH to Fdx. Characterization of the CYP175A1 system reconstituted from its recombinant components We attempted to reconstitute b-carotene hydroxylation activity with the excess purified recombinant CYP175A1, Fdx, and FNR. The reconstitution system did support NADPH-dependent b-carotene hydroxyl- ation, and two hydroxylated products were detected by HPLC (Fig. 4A). Using ultra-performance liquid chro- matography (UPLC)-MS, we confirmed that the two products were b-cryptoxanthin and zeaxanthin (data not shown). Furthermore, b-carotene hydroxyl- ation products were not detected in the absence of CYP175A1, Fdx, or FNR (data not shown). There- fore, these results clearly indicate that the electron transport system for CYP175A1 is composed of Fdx, FNR, and NADPH (Fig. 4B). All quantitative analyses were performed at 65 °C for 2 min, to limit the production of a second metabo- lite, zeaxanthin, and to inhibit the degradation of b-carotene by high temperatures. The b-carotene hydroxylation activity was determined from the production of b-cryptoxanthin, and the production of zeaxanthin was ignored. In order to determine the optimal conditions for the reconstitution system, the effects of pH, Fdx, and Tween 20 on b-carotene hydroxylation activity were assessed. The optimal pH for the reconstitution system was pH 5.0, which is con- sistent with the optimal pH of the FNR (Fig. 5A). The Fdx ⁄ CYP175A1 ratio was saturated at 8 : 1, and the turnover rate at an Fdx ⁄ CYP175A1 ratio of 8 : 1 was 4.9-fold greater than that at a ratio of 1 : 1 (Fig. 5B). The addition of appropriate detergents or phospholip- ids was required to obtain maximal turnover with other carotenoid oxygenases, such as carotenoid dioxygenases, because detergents and phospholipids presumably aid the solubilization of carotenoid and thus increase its ability to access the active site of carotenoid oxygenases [21–23]. Thus, we assessed the effect of Tween 20 on b-carotene hydroxylation acti- vity (Fig. 5C). Tween 20 stimulated b-carotene hydrox- ylation activity, with maximal activity at 0.6–0.8%. The turnover rate of the reconstitution system under the optimal conditions was 12.4 nmol b-cryptoxan- thinÆmin )1 Ænmol )1 CYP175A1. Furthermore, the K m and V max values for b-carotene hydroxylation by the reconstitution system were determined under the opti- mized conditions (Fig. 5D). The reaction followed Michaelis–Menten kinetics, and the K m and V max values were 14.3 ± 1.6 lm and 18.3 ± 0.6 nmol b-cryptoxanthinÆmin )1 Ænmol )1 CYP175A1, respectively. Discussion In the present study, we isolated an electron transport system for CYP175A1 from T. thermophilus HB27 by Table 1. Purification of Fdx from T. thermophilus HB27. Total activity is defined as b-carotene hydroxylation activity. Activities were measured with reaction mixtures (total volume, 200 lL) containing CYP175A1 (0.5 l M), b-carotene (20 lM), NADPH (1 mM), and the 100 mM KCl fraction (10 lg) from the DE52 column in buffer A (50 mM potassium phosphate buffer, pH 7.4, and 10% glycerol). The reactions were performed at 65 °C for 2 min. Purification steps Total protein (mg) Total activity (nmolÆmin )1 ) Specific activity (nmolÆmin )1 Æmg )1 ) Purification (fold) Yield (%) Crude extract 474.5 53.2 0.1 1 100 DE52 28.1 47.0 1.7 17 88 Butyl–Sepharose 1.7 26.4 15.4 154 50 Mono Q 0.7 24.1 34.8 348 45 T. Mandai et al. Thermostable electron transport system FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2419 multistep chromatography, and identified the electron transport proteins. The system utilized NADPH as a source of electrons, and was composed of Fdx (TTC1809) and FNR (TTC0096). Thus, the electron transport system for CYP175A1 belongs to class I, along with electron transport systems for other bacte- rial cytochrome P450s, and is very different from another thermophilic cytochrome P450 (CYP119) system. In the CYP119 system from the thermophilic archaeon S. solfataricus, electrons are transferred from pyruvate via OFOR and Fdx to CYP119 [13,14]. Inter- estingly, the electron transport system for CYP175A1 did not utilize OFOR, although the T. thermophilus HB27 genome contains the genes encoding OFOR (TTC1591 and TTC1592) [24]. An Fdx that contains seven irons (one [4Fe–4S] cluster and one [3Fe–4S] cluster) was discovered more than 20 years ago in T. thermophilus [19], but its function has remained unclear. Thus, this is the first report to demonstrate that a protein encoded by TTC0096 functions as an FNR in T. thermophilus, and that the seven-iron Fdx functions as a redox partner of CYP175A1. Further- more, we attempted to purify native CYP175A1, and measured reduced CO difference spectra in order to investigate whether or not CYP175A1 would be expressed under the culture conditions used in this study, but we could not purify native CYP175A1 and detect an absorption peak at 450 nm (data not shown). Nonetheless, very low b-carotene hydroxylation acti- vity was detected in the presence of Fdx, FNR, NADPH, and the cytosol of T. thermophilus (data not shown), suggesting that CYP175A1 was expressed at very low levels under the culture conditions used in this study. TTC0096, which actually encodes FNR, is anno- tated as a thioredoxin reductase (TR) in the T. thermo- philus HB27 genome database [24]. According to a comparison with genuine TRs, shown in Fig. 6A, the protein encoded by TTC0096 shows significant identity with the TRs from E. coli and Aeropyrum pernix (31% and 34%, respectively), and possesses conserved motifs responsible for the binding of FAD (GXGXXA and GXFAAGD) and the binding of NADPH (GXGXXA), whereas the protein encoded by TTC0096 lacks a redox-active site (CXXC), which par- ticipates in various redox reactions, such as the reduc- tion of thioredoxin. Thus, the protein encoded by TTC0096 will not actually function as a TR, and TTC0096 is misannotated in the T. thermophilus HB27 genome database. A blast analysis with the FNR from T. thermophilus revealed a high level of identity with YumC from Bacillus subtilis (45%) and FNR from Chlorobium tepidum (44%). Seo et al. [25,26] have reported that YumC from B. subtilis and FNR from C. tepidum form a homodimer, contain noncova- lently bound FAD, and function as a FNR. Further- more, Seo et al. [25,26] have reported that YumC from B. subtilis and FNR from C. tepidum share high sequence identity with genuine TRs from various A B 175 83 62 47.5 32.5 25 16.5 kDa 12 3 4 5 Wavelength (nm) 0.0 0.1 0.2 0.3 0.4 300 400 500 600 Absorbance Absorbance Wavelength (nm) 450 0.00 0.02 0.04 550350 Fig. 2. Purification and characterization of FNR from T. thermophi- lus HB27. (A) SDS ⁄ PAGE of fractions containing FNR at each step of purification. SDS ⁄ PAGE was carried out on a 15% polyacryl- amide gel. Lane 1: molecular mass markers. Lane 2: cytosol of T. thermophilus HB27 (20 lg). Lane 3: 100 m M KCl fraction from a DE52 column (14 lg). Lane 4: fraction eluted from a 2¢,5¢-ADP– Sepharose column (4.6 lg). Lane 5: fraction eluted from a Mono Q column (2.1 lg). (B) Absorption spectrum of native FNR purified from T. thermophilus HB27. The absorption spectrum of purified FNR (3.3 l M) was measured in buffer A. The inset shows the absorption spectrum between 350 and 600 nm. Thermostable electron transport system T. Mandai et al. 2420 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS species, but both lack the redox-active site, and that YumC from B. subtilis and FNR from C. tepidum con- stitute a new type of FNR. These characteristics are similar to those of our FNR, suggesting that our FNR belongs to this new type. A phylogenetic tree of FNRs from different sources was constructed (Fig. 6B). As noted by Aliverti et al. [27], FNRs could be grouped into two families: plant-type and glutathione reduc- tase-type FNRs. The phylogenetic analysis revealed that our FNR as well as YumC from B. subtilis and FNR from C. tepidum belong to a new type of FNR among the glutathione reductase-type FNRs. To the best of our knowledge, this is the first demonstration that an FNR of this new type is related to a cyto- chrome P450 system. The rate of turnover for the reconstitution system consisting of CYP175A1, Fdx and FNR was 12.4 nmol b-cryptoxanthinÆmin )1 Ænmol )1 CYP175A1 under the optimized conditions, with the exception of temperature. This was about 54-fold greater than the turnover rate (0.23 nmol b-cryptoxanthinÆmin )1 Ænmol )1 CYP175A1) reported by Momoi et al. [11], who car- ried out reconstitution using an artificial electron transport system, putidaredoxin and putidaredoxin reductase from the mesophilic bacterium P. putida. Although CYP97A4 from Oryza sativa also catalyzes the hydroxylation of b-carotene at the 3-position and 3¢-position in E. coli [28], the activity of CYP97A4 had not been characterized in vitro. Thus, this is the first report to characterize a cytochrome P450-type b-caro- tene hydroxylase with its native electron transport system. In this study, the turnover rate of b-carotene hydroxylation by the reconstitution system containing CYP175A1, Fdx and FNR was about 5000-fold lower than that of ferricyanide reduction by the FNR. The reason for this discrepancy is unclear, but general class I systems such as mitochondrial cytochrome P450 systems also show a turnover rate of substrates of cytochrome P450 that is much lower than the turnover rate of ferricyanide reduction by FNR [29–31]. As noted above, the CYP175A1 system produces thermozeaxanthins and thermobiszeaxanthins for rein- forcement of the cell membrane at high temperature [16]. Most enzymes, including CYP175A1, that are related to the carotenoid biosynthetic pathway are encoded on a megaplasmid, pTT27 [24]. However, the electron transport system components, Fdx and FNR, are encoded on a chromosome, suggesting that the chromosome controls the carotenoid biosynthetic pathway. In conclusion, we have found that electrons are transferred from NADPH via Fdx and FNR to CYP175A1. The CYP175A1 system is composed of extremely thermostable proteins (Fig. 4B), and the T m values of CYP175A1, Fdx and FNR are 88, 114, and 99 °C, respectively [4,32]. The thermostability of this system may facilitate the development of novel industrial applications of CYP175A1. In particular, the substrate-binding region of CYP175A1 was found to Table 2. Purification of FNR from T. thermophilus HB27. Total activity is defined as b-carotene hydroxylation activity. Activities were measured with reaction mixtures (total volume, 200 lL) containing CYP175A1 (0.5 l M), b-carotene (20 lM), NADPH (1 mM), and the 300 mM KCl fraction (10 lg) from the DE52 column in buffer A. The reactions were performed at 65 °C for 2 min. Purification steps Total protein (mg) Total activity (nmolÆmin )1 ) Specific activity (nmolÆmin )1 Æmg )1 ) Purification (fold) Yield (%) Crude extract 474.5 118.3 0.2 1 100 DE52 51.0 85.4 1.7 7 72 ADP–Sepharose 1.5 69.6 46.2 185 59 Mono Q 0.4 29.9 77.8 312 25 Table 3. Kinetic parameters for the ferricyanide reduction activity of FNR. Ferricyanide reduction activities were measured in 50 m M potassium phosphate buffer (pH 7.4) containing potassium ferri- cyanide (1 m M). The K m value for NADH was determined in the pre- sence of FNR (200 n M) and NADH (0.5–7.0 mM), and the K m value for NADPH was determined in the presence of FNR (20 n M) and NADPH (2–100 l M). NADH NADPH K m (lM) 2440 ± 546 4.1 ± 0.2 V max (nmolÆmin )1 Ænmol )1 of FAD) 152 ± 15 8318 ± 71 Table 4. Cytochrome c reduction activities. Cytochrome c reduc- tion activities were measured in 50 m M potassium phosphate buffer (pH 7.4) containing horse heart cytochrome c (0.1 m M), FNR (50 n M), Fdx (50–500 nM), and NADPH (0.5 mM)at50°C. Ratio (FNR : Fdx) 1:0 1:1 1:2 1:5 1:10 (nmolÆmin )1 Ænmol )1 of FAD) 105 ± 2 150 ± 4 186 ± 1 346 ± 6 544 ± 10 T. Mandai et al. Thermostable electron transport system FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2421 be highly similar to the substrate-binding region of cytochrome P450 BM-3 [4], whose substrates are long- chain fatty acids. Cytochrome P450 BM-3 has been engineered to improve activities towards substrates such as naphthalene, propranolol, and dioxins other than long-chain fatty acids [33–35]. Thus, this system with Fdx, FNR and CYP175A1 engineered by site- directed and random mutagenesis may exhibit activity towards industrially useful compounds other than b-carotene, even in the context of industrial envi- ronments. Experimental procedures Materials T. thermophilus HB27 was a gift from S. Kuramitsu (Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan). KOD Plus DNA poly- merase was purchased from Toyobo (Osaka, Japan). Emul- gen 911 was a gift from Kao Chemical (Tokyo, Japan). NADPH, NADH and NADP + were purchased from Oriental Yeast (Tokyo, Japan). a-Cyano-4-hydroxycinnamic acid was obtained from Bruker Daltonics GmbH (Bremen, Germany). Molecular mass standards for gel filtration (MW-GF-200), glucose 6-phosphate and cytochrome c were purchased from Sigma Chemical Co. (St Louis, MO, USA). b-Carotene, glucose-6-phosphate dehydrogenase from yeast, potassium ferricyanide, chloramphenicol, ampicillin, isopro- pyl-thio-b-d-galactoside (IPTG) and phenylmethanesulfonyl fluoride were obtained from Wako Pure Chemical indus- tries (Osaka, Japan). Tween 20 was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Cloning, expression and purification of CYP175A1 T. thermophilus HB27 was cultured at 70 °CinThermus medium (4 g of tryptone, 2 g of yeast extract and 1 g of NaCl per liter, pH 7.5). T. thermophilus HB27 genomic DNA was extracted using the Wizard Genomic DNA Puri- fication Kit (Promega, Madison, WI, USA). CYP175A1 (locus in the genome, TT_P0059) was amplified by PCR using genomic DNA as a template and two oligonucleotide primers, 5¢-GGAATTCCATATGAAGCGCCTTTCCCTG- 3¢ (forward primer) and 5¢-CCAAGCTTTCACGCCCGCA CCTCCTCCCTAG-3¢ (reverse primer). PCR was carried out at 94 °C for 5 min, and this was followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min, using KOD Plus DNA polymerase. After the PCR product had been digested with NdeI and HindIII, the fragment was ligated into the expression vector pET-21a (Novagen, Mad- ison, WI, USA), and the construct was designated as pET– CYP175A1. E. coli BL21 (DE3) Codon Plus cells were transformed with pET–CYP175A1. The transformant was grown in 2 · YT medium containing chloramphenicol and ampicillin at 37 °CuptoanD 600 of 1.0, and CYP175A1 expression was induced by treatment with 0.5 mm IPTG for 24 h at 25 °C. Cells were harvested by centrifugation at 5000 g for 20 min. The pellet was suspended in buffer A (50 mm potassium phosphate buffer, pH 7.4, and 10% glycerol) containing 1 mm phenylmethanesulfonyl fluoride, 0.1 mm EDTA, and 0.1% Emulgen 911. Lysozyme was added to a final concentration of 1 mgÆmL )1 , and the Ferricyanide reduction activity (µmol·min –1 ·nmol of FAD –1 ) pH 4 5 6 7 8 20 40 60 A B 0 Residual activity (%) 100 80 60 40 20 0 Temperature (°C) 40 60 80 100 Fig. 3. Characterization of FNR. (A) Effect of pH on the activity of FNR. The buffers used in this experiment were 50 m M potassium acetate buffer of pH range 4.0–6.0 (closed circles and solid line) and 50 m M potassium phosphate buffer of pH range 6.0–8.0 (open circles and dotted line). Ferricyanide reduction assays were performed in each buffer containing 1 m M potassium ferricyanide, FNR (30 n M) and 1 mM NADPH at 50 °C. The values represent the mean ± standard deviation (SD) of triplicate experiments. (B) Ther- mostability of FNR. FNR (60 n M) was incubated at various tempera- tures (40–110 °C) for 30 min at pH 7.4 (closed circles and solid line) or pH 5.0 (open circles and dotted line). The residual ferricya- nide reduction activity was measured in 50 m M potassium phos- phate buffer (pH 7.4) or 50 m M potassium acetate buffer (pH 5.0) containing 1 m M potassium ferricyanide, heat-treated FNR and 1m M NADPH at 25 °C. The values represent the mean ± SD of triplicate experiments. Thermostable electron transport system T. Mandai et al. 2422 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS solution was stirred at 4 °C for 30 min. The cell suspension was disrupted by sonication, and the cell debris was then removed by centrifugation at 50 000 g for 30 min at 4 °C. The cytosolic fraction was fractionated with ammonium sulfate as described previously [11]. The pellet was sus- pended in buffer A, and the solution was diluted two-fold with buffer A containing 2.0 m ammonium sulfate. The diluted sample was loaded onto a butyl-Sepharose 4 Fast Flow column (Amersham Biosciences, Chalfont St Giles, UK) equilibrated with buffer A containing 1.0 m ammo- nium sulfate. The column was washed, and CYP175A1 was eluted with a stepwise gradient of ammonium sulfate (0.5, 0.2, and 0 m) in buffer A. The fraction containing CYP175A1 was dialyzed against 50 mm potassium phos- phate buffer (pH 6.3) containing 10% glycerol. The dia- lyzed solution was loaded onto a Mono S HR5 ⁄ 5 column (Pharmacia). After the column had been washed with 50 mm potassium phosphate buffer (pH 6.3) containing 10% glycerol and 100 mm KCl, CYP175A1 was eluted with a linear gradient of 100–600 mm KCl in 50 mm potassium phosphate buffer (pH 6.3) containing 10% glycerol, at a flow rate of 1.0 mLÆmin )1 . Fractions exhibiting a ratio of absorbance at 418 ⁄ 280 nm above 1.3 were pooled, dialyzed against buffer A, and stored at )80 °C until use. The con- centration of purified CYP175A1 was determined with an extinction coefficient of 104 mm )1 Æcm )1 at 418 nm [4]. Approximately 5 mg of purified CYP175A1 was obtained per 1 L of culture, and a single band was observed on SDS ⁄ PAGE. Purification of an electron transport system for CYP175A1 from T. thermophilus HB27 T. thermophilus HB27 was cultured in Thermus medium (total volume: 6 L) at 70 °C overnight. T. thermophilus HB27 was harvested by centrifugation at 5000 g for 20 min. All purification steps were performed at room tem- perature. The pellet was suspended in buffer B (20 mm potassium phosphate buffer, pH 7.7, and 10% glycerol) containing 1 mm phenylmethanesulfonyl fluoride and 0.1 mm EDTA, and the cell suspension was disrupted by sonication. The cell debris was removed by centrifugation at 100 000 g for 90 min at 4 °C, and the cytosolic fraction was then loaded onto a DE52 (Whatman, Maidstone, UK) column (column volume: 30 mL) equilibrated with buffer B. The column was washed with buffer B, and the proteins bound to it were eluted with a stepwise gradient of KCl (50, 100, 200, 300, and 500 mm) in buffer B. The 300 mm KCl fraction from the DE52 column was diluted two-fold with buffer A containing 3.0 m ammonium sulfate. The diluted sample was loaded onto a butyl-Sepharose 4 Fast Flow column equilibrated with buffer A containing 1.5 m ammonium sulfate. After the column had been washed with buffer A containing 1.5 m ammonium sulfate, the proteins were eluted with a stepwise gradient of ammo- nium sulfate (1.0, 0.5, and 0 m) in buffer A. The 1.0 m ammonium sulfate fraction from the butyl–Sepharose col- umn was concentrated and desalted on a Bio-Gel P6 DG column (Bio-Rad Laboratories, Hercules, CA, USA) equili- brated with buffer D (20 mm potassium phosphate buffer, pH 6.5, 10% glycerol). The desalted solution was loaded onto a Mono Q HR5 ⁄ 5 column (Pharmacia) equilibrated with buffer D. After the column had been washed with buf- fer D containing 200 mm KCl, the proteins were eluted with a linear gradient of 200–600 mm KCl at a flow rate of 1.0 mL Æmin )1 . The purified protein was desalted on a Bio-Gel P6 DG column equilibrated with buffer A, and stored at )80 °C. The 100 mm KCl fraction from the DE52 column was dialyzed against buffer C (20 mm potassium phosphate buffer, pH 7.4, 10% glycerol, and 0.1 mM EDTA), and the dialyzed solution was then loaded onto a 2¢,5¢-ADP–Sepharose column (Amersham Biosciences) equilibrated with buffer C. After the column had been washed with buffer C containing 150 mm KCl, the proteins were eluted with buffer C containing 150 mm KCl and 1mm NADP + . The fraction eluted from the 2¢,5¢-ADP– Sepharose column was dialyzed against buffer B. The β-carotene β-cryptoxanthin Retention time (min) 10 20 300 A 454 0 20 40 60 80 100 A B Zeaxanthin NADPH NADP + e – FNR Fdx CYP175A1 heme FAD (99 °C) a (114 °C) b (88 °C) c β-carotene β-cryptoxanthin zeaxanthin Fig. 4. (A) HPLC profiles of the metabolites produced by the recon- stitution system consisting of excess CYP175A1, Fdx, and FNR. The reaction mixtures contained CYP175A1 (0.4 l M), Fdx (0.8 lM), FNR (0.4 l M) and b-carotene (30 lM) in buffer A (total volume, 200 lL). The reactions were performed at 65 °C for 5 min without (solid line) or with (dotted line) 1 m M NADPH, and the products were then extracted with ice-cold acetonitrile (1.0 mL). The extracted products were analyzed by RP-HPLC. The HPLC analysis was performed as described in Experimental procedures. (B) Scheme of the electron transport system for CYP175A1. The num- bers in parentheses indicate the T m value of each protein at neutral pH. a Data from this study. b Data from Griffin et al. [32]. c Data from Yano et al. [4]. T. Mandai et al. Thermostable electron transport system FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2423 dialyzed solution was loaded onto a Mono Q HR5 ⁄ 5 column equilibrated with buffer B, and the column was washed with buffer B containing 50 mm KCl. The proteins were eluted with a linear gradient of 50–200 mm KCl in buffer B at a flow rate of 1.0 mLÆmin )1 . The purified pro- tein was dialyzed against buffer A and stored at )80 °C. Identification of the purified electron transport proteins The electron transport protein purified from the 300 mm KCl fraction eluted from the DE52 column was identified by determining the N-terminal amino acid sequence of the puri- fied protein, which was analyzed by an automated amino acid sequencer (PPSQ-21A; Shimadzu, Kyoto, Japan), according to the manufacturer’s instructions. The electron transport protein purified from the 100 mm KCl fraction eluted from the DE52 column was identified by MALDI- TOF-MS. The purified protein was electrophoresed with an SDS ⁄ polyacrylamide gel, and stained with Coomassie Brilliant Blue R-250. The band containing the purified protein was excised from the gel, dehydrated, and then digested with Trypsin Gold (Promega), according to the method reported by Wang et al. [36]. The concentrated peptides were mixed with a-cyano-4-hydroxycinnamic acid in 60% acetonitrile and 0.1% trifluoroacetic acid, and analyzed 8 pH 45 67 0 1 2 3 4 5 A C B D Turnover rate (nmol·min –1 ·nmol of CYP175A1 –1 ) Turnover rate (nmol·min –1 ·nmol of CYP175A1 –1 ) Tween 20 (%) 0.0 0 5 10 15 0.5 1.0 1.5 2.0 Turnover rate (nmol·min –1 ·nmol of CYP175A1 –1 ) Turnover rate (nmol·min –1 ·nmol of CYP175A1 –1 ) Fdx (n M ) 0 300 600 900 1200 0 2 4 6 8 β-carotene (µ M ) 0 20406080100 0 5 10 15 20 Fig. 5. Characterization of the reconstitution system. (A) Effect of pH on b-carotene hydroxylation activity. The reactions were performed at the indicated pH value in the presence of CYP175A1 (30 n M), Fdx (60 nM), FNR (30 nM), 20 lM b-carotene (containing 0.1% Tween-20) and NADPH (1 m M)at65°C for 2 min. The buffers used in this experiment were 50 mM potassium acetate buffer containing 10% glycerol of pH range 4.0–6.0 (closed circles and solid line) and 50 m M potassium phosphate buffer containing 10% glycerol of pH range 6.0–7.4 (open circles and dotted line). (B) Effect of Fdx on b-carotene hydroxylation activity. The reaction mixtures contained CYP175A1 (30 n M), Fdx (30–960 n M), FNR (30 nM), 20 lM b-carotene (containing 0.1% Tween-20) and NADPH (1 mM)in50mM potassium acetate buffer (pH 5.0) containing 10% glycerol (total volume, 200 lL). The reactions were performed at 65 °C for 2 min. (C) Effect of Tween-20 on b-carotene hydroxylation activity. The reaction mixtures contained CYP175A1 (30 n M), Fdx (240 nM), FNR (30 nM), Tween-20 (0.1–1.6%), 20 lM b-caro- tene (containing 0.1% Tween-20) and NADPH (1 m M)in50mM potassium acetate buffer (pH 5.0) containing 10% glycerol (total volume, 200 lL). The reactions were performed at 65 °C for 2 min. (D) Kinetic analysis of b-carotene hydroxylation by the reconstitution system. The reaction mixtures contained CYP175A1 (30 n M), Fdx (240 nM), FNR (30 nM), 0.8% Tween-20, b-carotene (1–80 lM) and NADPH (1 mM)in 50 m M potassium acetate buffer (pH 5.0) containing 10% glycerol (total volume, 200 lL). The reactions were performed at 65 °C for 2 min. The reaction products were extracted with 25-fold volumes of ice-cold acetonitrile. In all cases, HPLC of the reaction products was carried out as described in Experimental procedures, and the values represent the mean ± SD of triplicate experiments. Thermostable electron transport system T. Mandai et al. 2424 FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS T. thermophilus 1 MAADHTDVLIVGAGPAGLFAGFYVGMRGLSFRFVDPLPEPGGQLTAL 47 E. coli 1 MGTTKHSKLLILGSGPAGYTAAVYAARANLQPVLITGM-EKGGQLTTT 47 A. pernix 1 MPLRLSAVRAPKIPRGEEYDTVIVGAGPAGLSAAIYTTRF-LMSTLIVSM-DVGGQLNLT 58 . :*:*:**** * *. * :: : : ****. 1 T. thermophilus 48 YPEKYIYDVAG-FPKVYAKDLVKGLVEQVAPFNPVYSLGERAETLE-REGDLFKVTTSQG 105 E. coli 48 T EVENWPGDPNDLTGPLLMERMHEHATKFETEIIFD-HINKVD-LQNRPFRLNGDNG 102 A. pernix 59 N WIDDYPG-MGGLEASKLVESFKSHAEMFGAKIVTGVQVKTVDRLDDGWFLVRGSRG 114 : : .* : . *:: : .:. * . . : :.:: :. * : * T. thermophilus 106 NAYTAKAVIIAAGVGAFEPRRIGAPGEREFEGRGVYYAVKSKA-EFQGK-RVLIVGGGDS 163 E. coli 103 -EYTCDALIIATGASA RYLGLPSEEAFKGRGVSACATCDG-FFYRNQKVAVIGGGNT 157 A. pernix 115 LEVKARTVILAVGSRR RKLGVPGEAELAGRGVSYCSVCDAPLFKGKDAVVVVGGGDS 171 ::*:*.* * :* *.* : **** . . * : * ::***:: 2 3 T. thermophilus 164 AVDWALNLLDTARRITLIHRRPQFRAHEASVKELMKAHEEGRLEVLTPYELRRVEGDER- 222 E. coli 158 AVEEALYLSNIASEVHLIHRRDGFRAEKILIKRLMDKVENGNIILHTNRTLEEVTGDQMG 217 A. pernix 172 ALEGALLLSGYVGKVYLVHRRQGFRAKPFYVEEARKK-PNIEFILDS IVTEIRGRDR- 227 * :: ** * . . .: *:*** ***. ::. . : .: : : : .: * : T. thermophilus 223 VRWAVVFHNQTQEELA-LEVDAVLILAGYITKLGPLANWGLALEKNKIK VDTTMA 276 E. coli 218 VTGVRLRDTQNSDNIESLDVAGLFVAIGHSPNT-AIFEGQLELENGYIKVQSGIHGNATQ 276 A. pernix 228 VESVVVKNKVTGEEKE-LRVDGIFIEIGSEPPK-ELFEA-IGLETDSMG NVVVDEWMR 282 * . : . :: * * .::: * . : : : ** : . T. thermophilus 277 TSIPGVYACGDIVTYPGKLPLIVLGFGEAAIAANHAAAYAN-PALKVNPGHSSEKAAPGT 335 E. coli 277 TSIPGVFAAGDVMDHI YRQAITSAGTGCMAALDAERYLD GLADAK 321 A. pernix 283 TSIPGIFAAGDCTSMWPGFRQVVTAAAMGAVAAYSAYTYLQEKGLYKPKPLTGLK 337 *****::*.** : . . :** * * : .* 1 C. tepidum FNR B. subtilis YumC T. thermophilus FNR M. tuberculosis FNR Pseudomonas sp. BphA4 P. putida PDR S. cerevisiae ADR M. tuberculosis FprA H. sapiens ADR Nostoc sp. PCC 7120 FNR Z. mays FNR S. oleracea FNR E .coli FNR R. capsulatus FNR A. vinelandii FNR Plant-type FNRs Plastidic-type Bacterial-type New type A B ADR-like ONFR-like GR-type FNRs Fig. 6. (A) Multiple alignment of the amino acid sequences of FNR from T. thermophilus HB27, TR from E. coli, and TR from A. pernix. Accession numbers (NCBI) are: FNR from T. thermophilus HB27, YP_004071; TR from E. coli, NP_415408; and TR from A. pernix, NP_147693. Asterisks indicate identical amino acid residues. Colons indicate conservative replacements, and single dots indicate less conservative replacements. Underlines 1, 2 and 3 indicate the FAD-binding site, the redox-active site, and the NADPH-binding site, respectively. (B) Phylogenetic tree of FNR from different sources. The phylogenetic tree was constructed using the program CLUSTALW (http://align.genome.jp/). The accession numbers are: FNR from Spinacia oleracea, AAA34029; FNR from Nostoc sp. PCC 7120, NP_488161; FNR from Zea mays, NP_001105568; FNR from E. coli, NP_418359); FNR from Azotobacter vinelandii, ZP_00417949; FNR from Rhodobact- er capsulatus, AAF35905; ADR from Homo sapiens, AAB59498; adrenodoxin reductase from Saccharomyces cerevisiae, AAB64812; FprA from Mycobacterium tuberculosis, O05783; BphA4 from Pseudomonas sp. KKS102, BAA04112; putidaredoxin reductase from P. putida, AAA25758; FNR from M. tuberculosis H37Rv, NP_215202; YumC from B. subtilis, CAB15201; and FNR from C. tepidum, NP_662397. T. Mandai et al. Thermostable electron transport system FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2425 [...].. .Thermostable electron transport system T Mandai et al by MALDI-TOF-MS (Ultraflex; Bruker Daltonics GmbH) Protein identification was carried out by a search of the database NCBInr using mascot (Matrix Science, Boston, MA, USA) and a Mono Q column under the conditions described above The purified FNR was dialyzed against buffer A, and stored at )80 °C The concentration of the purified FNR was determined... were separated using an UPLC system (ACQUITY UPLC system; Waters, Milford, MA, USA) equipped with an ACQUITY UPLC BEH C18 column (1.7 lm, 2.1 · 150 mm; Waters, Ireland), and the same mobile phase as described above was used at a flow rate of 0.2 mLÆmin)1 MS was carried out using a NanoFrontier LD mass spectrometer (Hitachi, Tokyo, Japan) The MS parameters were as follows: atmospheric pressure chemical ionization... by a Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science and a special Grant-in-Aid of the Advanced Program of High Profile Research for Academia-Industry Cooperation, sponsored by the Ministry of Education, Science, Culture, Sports and Technology of Japan References 1 Hannemann F, Bichet A, Ewen KM & Bernhardt R (2007) Cytochrome P450 systems – biological variations... carotenoid-glycoside-esters from thermophilic eubacterium Thermus thermophilus Tetrahedron Lett 36, 4901– 4904 Hara M, Yuan H, Yang Q, Hoshino T, Yokoyama A & Miyake J (1999) Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters Biochim Biophys Acta 1461, 147–154 Sato S, Nakazawa K, Hon-Nami K & Oshima T (1981) Purification, some properties and amino acid sequence of Thermus thermophilus. .. were harvested by centrifugation at 5000 g for 20 min, and the pellet was suspended in buffer B containing 1 mm phenylmethanesulfonyl fluoride and 0.1 mm EDTA The crude extract of E coli was prepared as described above The extract was incubated at 70 °C for 30 min, and then centrifuged at 20 000 g for 30 min at 4 °C The heat-treated supernatant was purified with a DE52 column, a 2¢,5¢-ADP–Sepharose column... 27 Aliverti A, Pandini V, Pennati A, De Rosa M & Zanetti G (2008) Structural and functional diversity of ferredoxin-NADP(+) reductases Arch Biochem Biophys 474, 283–291 28 Quinlan RF, Jaradat TT & Wurtzel ET (2007) Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases Arch Biochem Biophys 458, 146–157 29 Chun YJ, Shimada T, Sanchez-Ponce R, Martin MV, Lei L, Zhao... The tubes were placed on ice for 5 min, and then centrifuged at 13 000 g for 10 min The supernatant was directly analyzed by RP-HPLC The HPLC analysis was performed using an HPLC system (Prominence; Shimadzu, Kyoto, Japan) equipped with an ODS-100S column (150 · 4.6 mm; Tosoh, Tokyo, Japan), and acetonitrile ⁄ methanol ⁄ isopropanol (85 : 10 : 5) was used as the mobile phase, at a flow rate of 1 mLÆmin)1... modeling, molecular dynamics simulations, and analysis of CYP119, a P450 enzyme from extreme acidothermophilic archaeon Sulfolobus solfataricus Biochemistry 39, 2484–2498 9 Puchkaev AV, Koo LS & Ortiz de Montellano PR (2003) Aromatic stacking as a determinant of the FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS 2427 Thermostable electron transport system 10 11 12... desalted on a BioGel P6 DG column equilibrated with buffer A and stored at )80 °C The concentration of the purified Fdx was determined using a molar extinction coefficient of 29.0 mm)1Æcm)1 at 408 nm [13] Cloning, expression and purification of FNR FNR (TTC0096) was amplified by PCR using genomic DNA as a template and two oligonucleotide primers, 5¢-GGAATTCCATATGGCGGCGGACCACACGGA CGT-3¢ (forward primer) and... Mandai et al thermal stability of CYP119 from Sulfolobus solfataricus Arch Biochem Biophys 409, 52–58 Blasco F, Kauffmann I & Schmid RD (2004) CYP17 5A1 from Thermus thermophilus HB27, the first b-carotene hydroxylase of the P450 superfamily Appl Microbiol Biotechnol 64, 671–674 Momoi K, Hofmann U, Schmid RD & Urlacher VB (2006) Reconstitution of b-carotene hydroxylase activity of thermostable CYP17 5A1 . A novel electron transport system for thermostable CYP17 5A1 from Thermus thermophilus HB27 Takao Mandai, Shinsuke Fujiwara and Susumu Imaoka Nanobiotechnology. was a gift from Kao Chemical (Tokyo, Japan). NADPH, NADH and NADP + were purchased from Oriental Yeast (Tokyo, Japan). a- Cyano-4-hydroxycinnamic acid was