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

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

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

18.3 ± 0.6 nmol

and

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- locus in the genome, TTC1809) and ferredoxin–NAD(P)+ doxin (Fdx; 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 (Km for NADH = 2440 ± 546 lm; Km for NADPH = 4.1 ± 0.2 lm). Furthermore, the FNR reduced cytochrome c in the presence of NADPH and Fdx. The Tm value of the FNR was 99 (cid:2)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 (cid:2)C, and the Km and Vmax values for b-carotene hydroxylation were b-cryptoxanthinÆmin)1Ænmol)1 14.3 ± 1.6 lm CYP175A1, respectively. This is the ﬁrst report of a native electron trans- port system for CYP175A1.

reactions,

possess

stability,

extreme

chrome P450s for industrial applications. Thermophilic cytochrome P450s 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].

ligand-bound states

Cytochrome P450s are associated with a number of physiologically essential 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 cyto- the has

usefulness

generally

limited

of

CYP119 is well characterized, and its crystal struc- ture has been determined in the ligand-free state and [5,6]. As expected, in several CYP119 is highly resistant to both high temperatures (Tm = 91 (cid:2)C) and high pressures (up to 2 kbar) [7].

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.

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2416

T. Mandai et al.

Thermostable electron transport system

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.

signiﬁcantly to the

system. The

catalytic

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 electron transport system was composed of Fdx and Fdx– NAD(P)+ reductase (FNR), and these components were characterized at high temperature.

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 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 (Tm = 88 (cid:2)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].

Results

To

oxidative

reactions,

perform their

Isolation and identiﬁcation of the components of the CYP175A1 electron transport system

catalytic

[1,12]. The electron transport

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 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].

hydroxylation

b-Carotene

To ﬁnd the electron donor of the electron transport system for CYP175A1, we initially measured the b-carotene hydroxylation activity in the presence of the cytosol of T. thermophilus, puriﬁed CYP175A1, and the electron donors NADH, NADPH, and pyru- vate (+CoA), which are generally used in cyto- chrome P450 systems. The activities of CYP175A1 in the presence of NADH and NADPH were 0.03 and 0.43 nmol b-cryptoxanthinÆmin)1Ænmol)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- system for CYP175A1 was dependent on port 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. activity was detected in a major peak when it was reacted with

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-

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2417

T. Mandai et al.

Thermostable electron transport system

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 (A400 ⁄ A280 = 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

kDa

A

62

47.5

32.5 to that

25

16.5

1

2

3

4

5 B

1.0

0.5 e c n a b r o s b A

the electron transport

[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- calculated from the amino acid sponds 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 characteristic of ﬂavoproteins 473 nm, which is (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 system for CYP175A1 belongs to class I.

0.0

500

600

700

300

400 Characterization of recombinant FNR

Wavelength (nm)

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 mM 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 lM) was measured in buffer A (50 mM potassium phosphate buffer, pH 7.4, 10% glycerol).

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 (cid:2)C and at pH 7.4 in the presence of NADH as well as NADPH, the Km value of the FNR for NADPH was about 600-fold lower than that for NADH, and the Vmax value of the FNR with NADPH was about 55-fold higher

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2418

T. Mandai et al.

Thermostable electron transport system

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 lM), 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 (cid:2)C for 2 min.

Total protein (mg)

Puriﬁcation steps

Puriﬁcation (fold)

Yield (%)

Total activity (nmolÆmin)1)

Speciﬁc activity (nmolÆmin)1Æmg)1)

53.2 47.0 26.4 24.1

0.1 1.7 15.4 34.8

1 17 154 348

100 88 50 45

Crude extract DE52 Butyl–Sepharose Mono Q

474.5 28.1 1.7 0.7

transport system for CYP175A1 is composed of Fdx, FNR, and NADPH (Fig. 4B).

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 (cid:2)C with saturating concentrations of NADPH (1 mm) and ferricyanide (1 mm) than at 25 (cid:2)C (data not shown).

To determine the optimal pH of the FNR, we mea- sured ferricyanide reduction activity at 50 (cid:2)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 Tm values of the FNR at pH 7.4 and at pH 5.0 were 99 and 95 (cid:2)C, respectively. These results indicate that the FNR is an extremely thermostable protein at both pH 7.4 and pH 5.0.

by TTC0096,

electrons

transfers

The FNR reduced cytochrome c at 50 (cid:2)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 from NADPH to Fdx.

Characterization of the CYP175A1 system reconstituted from its recombinant components

the

excess

puriﬁed

All quantitative analyses were performed at 65 (cid:2)C for 2 min, to limit the production of a second metabo- the degradation of lite, zeaxanthin, and to inhibit 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 turnover required to obtain maximal 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)1Ænmol)1 CYP175A1. Furthermore, the Km and Vmax 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 Km and Vmax values were 14.3 ± 1.6 lm and 18.3 ± 0.6 nmol b-cryptoxanthinÆmin)1Ænmol)1 CYP175A1, respectively.

and

Discussion

In the present study, we isolated an electron transport system for CYP175A1 from T. thermophilus HB27 by

We attempted to reconstitute b-carotene hydroxylation recombinant activity with 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 zeaxanthin b-cryptoxanthin (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

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2419

T. Mandai et al.

Thermostable electron transport system

A

kDa 175

thermophilic

cytochrome P450

83

62

47.5

32.5

25

16.5

1

2

3

4

5

0.4 B

0.04

0.3

0.02 e c n a b r o s b A

from rial cytochrome P450s, and is very different another (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.

0.2

0.00 TTC0096, which actually encodes FNR,

450

350

550 e c n a b r o s b A

Wavelength (nm)

0.1

0.0

300

400

500

600 Wavelength (nm)

the

and

binding

protein

thioredoxin. Thus,

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 mM 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 lM) was measured in buffer A. The inset shows the absorption spectrum between 350 and 600 nm.

electrons, and was

multistep chromatography, and identiﬁed the electron transport proteins. The system utilized NADPH as a source of 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-

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 of NADPH GXFAAGD) by encoded the (GXGXXA), whereas TTC0096 lacks a redox-active site (CXXC), which par- ticipates in various redox reactions, such as the reduc- tion of 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 from various sequence identity with genuine TRs

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2420

T. Mandai et al.

Thermostable electron transport system

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 lM), 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 (cid:2)C for 2 min.

Total protein (mg)

Puriﬁcation steps

Puriﬁcation (fold)

Yield (%)

Total activity (nmolÆmin)1)

Speciﬁc activity (nmolÆmin)1Æmg)1)

118.3 85.4 69.6 29.9

0.2 1.7 46.2 77.8

1 7 185 312

100 72 59 25

Crude extract DE52 ADP–Sepharose Mono Q

474.5 51.0 1.5 0.4

Table 3. Kinetic parameters for the ferricyanide reduction activity of FNR. Ferricyanide reduction activities were measured in 50 mM potassium phosphate buffer (pH 7.4) containing potassium ferri- cyanide (1 mM). The Km value for NADH was determined in the pre- sence of FNR (200 nM) and NADH (0.5–7.0 mM), and the Km value for NADPH was determined in the presence of FNR (20 nM) and NADPH (2–100 lM).

NADH

NADPH

2440 ± 546 152 ± 15

4.1 ± 0.2 8318 ± 71

Km (lM) Vmax (nmolÆmin)1Ænmol)1 of FAD)

under the optimized conditions, with the exception of temperature. This was about 54-fold greater than the turnover rate (0.23 nmol b-cryptoxanthinÆmin)1Ænmol)1 CYP175A1) reported by Momoi et al. [11], who car- reconstitution using an artiﬁcial electron ried out system, putidaredoxin and putidaredoxin transport 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.

study,

In this

Table 4. Cytochrome c reduction activities. Cytochrome c reduc- tion activities were measured in 50 mM potassium phosphate buffer (pH 7.4) containing horse heart cytochrome c (0.1 mM), FNR (50 nM), Fdx (50–500 nM), and NADPH (0.5 mM) at 50 (cid:2)C.

Ratio (FNR : Fdx)

1 : 0

1 : 1

1 : 2

1 : 5

1 : 10

105 ± 2 150 ± 4 186 ± 1 346 ± 6 544 ± 10

(nmolÆmin)1Ænmol)1 of FAD)

rate of b-carotene the turnover 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].

encoded on a chromosome,

As noted above, the CYP175A1 system produces thermozeaxanthins and thermobiszeaxanthins for rein- forcement of the cell membrane at high temperature including CYP175A1, that are [16]. Most enzymes, related to the carotenoid biosynthetic pathway are encoded on a megaplasmid, pTT27 [24]. However, the electron transport system components, Fdx and FNR, are suggesting that the chromosome controls the carotenoid biosynthetic pathway.

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)1Ænmol)1 CYP175A1

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 Tm values of CYP175A1, Fdx and FNR are 88, 114, and 99 (cid:2)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

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2421

T. Mandai et al.

Thermostable electron transport system

industrial envi-

A

b-carotene, even in the context of ronments.

60

Experimental procedures

40 ) 1 – D A F f o l o m n

Materials

· 1 – n i m

20 · l o m µ (

y t i v i t c a n o i t c u d e r e d i n a y c i r r e F

0

4

5

7

8 6 pH

B 100

)

%

80

60

40

(Osaka,

( y t i v i t c a l a u d i s e R

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 Japan). Tween 20 was purchased from Bio-Rad Laboratories (Hercules, CA, USA).

20 Cloning, expression and puriﬁcation of CYP175A1

0

40

60

80

100 Temperature (°C)

Fig. 3. Characterization of FNR. (A) Effect of pH on the activity of FNR. The buffers used in this experiment were 50 mM potassium acetate buffer of pH range 4.0–6.0 (closed circles and solid line) and 50 mM 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 mM potassium ferricyanide, FNR (30 nM) and 1 mM NADPH at 50 (cid:2)C. The values represent the mean ± standard deviation (SD) of triplicate experiments. (B) Ther- mostability of FNR. FNR (60 nM) was incubated at various tempera- tures (40–110 (cid:2)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 mM potassium phos- phate buffer (pH 7.4) or 50 mM potassium acetate buffer (pH 5.0) containing 1 mM potassium ferricyanide, heat-treated FNR and 1 mM NADPH at 25 (cid:2)C. The values represent the mean ± SD of triplicate experiments.

20 min. The pellet was suspended for

T. thermophilus HB27 was cultured at 70 (cid:2)C in Thermus 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 (cid:2)C for 5 min, and this was followed by 30 cycles of 94 (cid:2)C for 30 s, 55 (cid:2)C for 30 s, and 68 (cid:2)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 (cid:2)C up to an D600 of 1.0, and CYP175A1 expression was induced by treatment with 0.5 mm IPTG for 24 h at 25 (cid:2)C. Cells were harvested by centrifugation at 5000 g in buffer A (50 mm potassium phosphate buffer, pH 7.4, and containing 1 mm phenylmethanesulfonyl 10% glycerol) ﬂuoride, 0.1 mm EDTA, and 0.1% Emulgen 911. Lysozyme was added to a ﬁnal concentration of 1 mgÆmL)1, and the

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 than towards

industrially useful compounds other

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2422

T. Mandai et al.

Thermostable electron transport system

100A

80 Zeaxanthin

β-carotene

60 4 5 4 A

40

β-cryptoxanthin

absorbance at 418 ⁄ 280 nm above 1.3 were pooled, dialyzed against buffer A, and stored at )80 (cid:2)C until use. The con- centration of puriﬁed CYP175A1 was determined with an extinction coefﬁcient of 104 mm)1Æcm)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.

20

0 Puriﬁcation of an electron transport system for CYP175A1 from T. thermophilus HB27

0

10

30 20 Retention time (min)

B

β-carotene

Fdx

NADPH

CYP175A1

FNR FAD

heme

e–

(99 °C)a

(114 °C)b

β-cryptoxanthin zeaxanthin

(88 °C)c

NADP+

1 mm phenylmethanesulfonyl ﬂuoride

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 lM), Fdx (0.8 lM), FNR (0.4 lM) and b-carotene (30 lM) in buffer A (total volume, 200 lL). The reactions were performed at 65 (cid:2)C for 5 min without (solid line) or with (dotted line) 1 mM 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 Tm value of each protein at neutral pH. aData from this study. bData from Grifﬁn et al. [32]. cData from Yano et al. [4].

30 mL) volume: (column

in buffer A. The

column

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2423

solution was stirred at 4 (cid:2)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 (cid:2)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) 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 T. thermophilus HB27 was cultured in Thermus medium (total volume: 6 L) at 70 (cid:2)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) and containing 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 (cid:2)C, and the cytosolic fraction was then loaded onto a DE52 (Whatman, Maidstone, UK) column 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 mLÆmin)1. The puriﬁed protein was desalted on a Bio-Gel P6 DG column equilibrated with buffer A, and stored at )80 (cid:2)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 (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 1 mm NADP+. The fraction eluted from the 2¢,5¢-ADP– Sepharose column was dialyzed against buffer B. The

T. Mandai et al.

Thermostable electron transport system

)

A

B

8

5

4

6 1 – 1 A 5 7 1 P Y C

3

4

2

· e t a r r e v o n r u T

2

1 f o l o m n 1 – n i m

0

8

4

5

7

0

300

900 1200

· l o m n (

6 pH

600 Fdx (nM)

)

C

D

15

20 1 – 1 A 5 7 1 P Y C

15

10 1 – 1 A 5 7 1 P Y C

f o

10

·

5

· e t a r r e v o n r u T

5 l o m n 1 – n i m

0 0 0.0

0.5

1.5

2.0 · l o m n (

0

20

40

60

80

100 · l o m n (

1.0 Tween 20 (%)

β-carotene (µM)

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 nM), Fdx (60 nM), FNR (30 nM), 20 lM b-carotene (containing 0.1% Tween-20) and NADPH (1 mM) at 65 (cid:2)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 mM 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 nM), Fdx (30–960 nM), FNR (30 nM), 20 lM b-carotene (containing 0.1% Tween-20) and NADPH (1 mM) in 50 mM potassium acetate buffer (pH 5.0) containing 10% glycerol (total volume, 200 lL). The reactions were performed at 65 (cid:2)C for 2 min. (C) Effect of Tween-20 on b-carotene hydroxylation activity. The reaction mixtures contained CYP175A1 (30 nM), 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 mM) in 50 mM potassium acetate buffer (pH 5.0) containing 10% glycerol (total volume, 200 lL). The reactions were performed at 65 (cid:2)C for 2 min. (D) Kinetic analysis of b-carotene hydroxylation by the reconstitution system. The reaction mixtures contained CYP175A1 (30 nM), Fdx (240 nM), FNR (30 nM), 0.8% Tween-20, b-carotene (1–80 lM) and NADPH (1 mM) in 50 mM potassium acetate buffer (pH 5.0) containing 10% glycerol (total volume, 200 lL). The reactions were performed at 65 (cid:2)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.

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 (cid:2)C.

Identiﬁcation of the puriﬁed electron transport proteins

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2424

ﬁed protein, which was analyzed by an automated amino (PPSQ-21A; Shimadzu, Kyoto, Japan), acid sequencer 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 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-

T. Mandai et al.

Thermostable electron transport system

1 -------------MAADHTDVLIVGAGPAGLFAGFYVGMRGLSFRFVDPLPEPGGQLTAL 47 1 ------------MGTTKHSKLLILGSGPAGYTAAVYAARANLQPVLITGM-EKGGQLTTT 47 1 MPLRLSAVRAPKIPRGEEYDTVIVGAGPAGLSAAIYTTRF-LMSTLIVSM-DVGGQLNLT 58

A T. thermophilus E. coli A. pernix

.. . :*:*:**** *..*. * :: : : ****.

48 YPEKYIYDVAG-FPKVYAKDLVKGLVEQVAPFNPVYSLGERAETLE-REGDLFKVTTSQG 105 48 T---EVENWPGDPNDLTGPLLMERMHEHATKFETEIIFD-HINKVD-LQNRPFRLNGDNG 102 59 N---WIDDYPG-MGGLEASKLVESFKSHAEMFGAKIVTGVQVKTVDRLDDGWFLVRGSRG 114

T. thermophilus E. coli A. pernix

: : .* : . *:: : .:. * . . : :.:: :. * : ..*

T. thermophilus E. coli A. pernix

106 NAYTAKAVIIAAGVGAFEPRRIGAPGEREFEGRGVYYAVKSKA-EFQGK-RVLIVGGGDS 163 103 -EYTCDALIIATGASA---RYLGLPSEEAFKGRGVSACATCDG-FFYRNQKVAVIGGGNT 157 115 LEVKARTVILAVGSRR---RKLGVPGEAELAGRGVSYCSVCDAPLFKGKDAVVVVGGGDS 171

.. ::*:*.* * :* *.* : **** . ... * : * ::***::

T. thermophilus E. coli A. pernix

164 AVDWALNLLDTARRITLIHRRPQFRAHEASVKELMKAHEEGRLEVLTPYELRRVEGDER- 222 158 AVEEALYLSNIASEVHLIHRRDGFRAEKILIKRLMDKVENGNIILHTNRTLEEVTGDQMG 217 172 ALEGALLLSGYVGKVYLVHRRQGFRAKPFYVEEARKK-PNIEFILDS--IVTEIRGRDR- 227 *:: ** * . . .: *:*** ***. ::. . : .: : : : .: * :

T. thermophilus E. coli A. pernix

223 VRWAVVFHNQTQEELA-LEVDAVLILAGYITKLGPLANWGLALEKNKIK-----VDTTMA 276 218 VTGVRLRDTQNSDNIESLDVAGLFVAIGHSPNT-AIFEGQLELENGYIKVQSGIHGNATQ 276 228 VESVVVKNKVTGEEKE-LRVDGIFIEIGSEPPK-ELFEA-IGLETDSMG--NVVVDEWMR 282 * . : .. . :: * * .::: * . : : : **.. : .

T. thermophilus E. coli A. pernix

277 TSIPGVYACGDIVTYPGKLPLIVLGFGEAAIAANHAAAYAN-PALKVNPGHSSEKAAPGT 335 277 TSIPGVFAAGDVMDHI--YRQAITSAGTGCMAALDAERYLD--GLADAK----------- 321 283 TSIPGIFAAGDCTSMWPGFRQVVTAAAMGAVAAYSAYTYLQEKGLYKPKPLTGLK----- 337

*****::*.**

: . . ..:** * * : .*

T. thermophilus FNR

B

New type

B. subtilis YumC

C. tepidum FNR

Pseudomonas sp. BphA4

GR-type FNRs

ONFR-like

P. putida PDR

M. tuberculosis FNR

H. sapiens ADR

M. tuberculosis FprA

ADR-like

S. cerevisiae ADR

S. oleracea FNR

Z. mays FNR

Plastidic-type

Nostoc sp. PCC 7120 FNR

Plant-type FNRs

A. vinelandii FNR

R. capsulatus FNR

Bacterial-type

E .coli FNR

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.

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2425

T. Mandai et al.

Thermostable electron transport system

by MALDI-TOF-MS (Ultraﬂex; Bruker Daltonics GmbH). Protein identiﬁcation was carried out by a search of the data- base 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 (cid:2)C. The concentration of the puriﬁed FNR was determined using a molar extinction coefﬁcient of 12.5 mm)1Æcm)1 at 473 nm.

Cloning, expression, and puriﬁcation of Fdx

Characterization of FNR

and

The molar extinction coefﬁcient of the puriﬁed FNR was determined by extracting the total enzyme-bound FAD. The puriﬁed FNR was incubated at 110 (cid:2)C for 30 min in an aluminum block in 50 mm potassium phosphate buffer (pH 7.4), and denatured proteins were then removed by centrifugation at 10 000 g for 10 min. The concentration of free ﬂavin was determined from its absorption coefﬁ- cient of 11.3 mm)1Æcm)1 at 450 nm [37]. The molecular mass of the puriﬁed FNR under nondenaturing condi- tions was determined by gel ﬁltration on a Superdex- 200HR column, which was calibrated using molecular mass standards and equilibrated with buffer A containing 150 mm KCl. Gel ﬁltration was carried out with buffer A containing 150 mm KCl at a ﬂow rate of 0.4 mLÆmin)1.

Measurement of b-carotene hydroxylation activity

in dissolved

2.3% Tween 20 and (v ⁄ v),

a molar coefﬁcient extinction using chloroform (4 mm). b-Carotene was The solution (5 lL) was diluted 10-fold with acetone containing then mixed vigorously and vacuum-dried. The resulting residue was dissolved in 99 lL of the reaction buffer (200 lm b-caro- in 2 mL tene solution). All reactions were carried out tubes with caps. Fdx (TTC1809) was ampliﬁed by PCR using genomic DNA as a template and two oligonucleotide primers, 5¢-GGA ATTCCATATGCCGCACGTGATCTGCGAG-3¢ (forward primer) 5¢-CGCGGATCCTTACTCTAGGCCCGC GAGCT-3¢ (reverse primer). The PCR product was inserted into the pET-21a vector, and the construct was designated pET–Fdx. E. coli BL21(DE3) Codon Plus cells were trans- formed with pET–Fdx. The transformant was grown in 2 · YT medium containing chloramphenicol and ampicillin at 37 (cid:2)C up to an D600 of 1.0, and Fdx expression was induced by treatment with 1.0 mm IPTG for 24 h at 25 (cid:2)C. Cells were harvested by centrifugation at 5000 g for 20 min, and the pellet was suspended in buffer A contain- ing 1 mm phenylmethanesulfonyl ﬂuoride. The crude extract of E. coli was prepared as described above. The extract was incubated at 80 (cid:2)C for 30 min, and then centri- fuged at 20 000 g for 30 min at 4 (cid:2)C to remove denatured proteins. The heat-treated supernatant was diluted two-fold with buffer A containing 3.0 m ammonium sulfate. The diluted solution was puriﬁed with a butyl-Sepharose 4 Fast Flow column and a Mono Q column under the conditions described above. The puriﬁed Fdx was desalted on a Bio- Gel P6 DG column equilibrated with buffer A and stored at )80 (cid:2)C. The concentration of the puriﬁed Fdx was deter- mined of 29.0 mm)1Æcm)1 at 408 nm [13].

Cloning, expression and puriﬁcation of FNR

ice-cold acetonitrile ⁄ chloroform [4 : 1

(85 : 10 : 5) was used as

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2426

To purify the electron transport system for CYP175A1, reactions with CYP175A1 b-carotene hydroxylation (0.5 lm) and the electron transport system were per- formed in buffer A containing 20 lm b-carotene (total volume, 200 lL). The reaction mixtures were incubated at 65 (cid:2)C for 3 min, and the reactions were initiated by the addition of 2 lL of 100 mm NADPH. After 2 min at (v ⁄ v), 65 (cid:2)C, 1.0 mL] was added to extract the reaction products. The tubes were placed on ice for 5 min, and then centrifuged at 13 000 g for 10 min. The supernatant was directly ana- lyzed 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 ⁄ metha- nol ⁄ isopropanol the mobile phase, at a ﬂow rate of 1 mLÆmin)1. To determine the optimal reaction conditions, we assessed the effects of pH (4.0–7.4), Fdx (30–960 nm) and Tween 20 (0.1–1.6%) on b-carotene hydroxylation activity. The b-carotene hydrox- ylation reactions were carried out under the conditions described above, and the products were extracted with ice-cold acetonitrile (1.0 mL). For the kinetic analysis, FNR (TTC0096) was ampliﬁed by PCR using geno- mic DNA as a template and two oligonucleotide prim- ers, 5¢-GGAATTCCATATGGCGGCGGACCACACGGA (forward primer) and 5¢-CGCGGATCCTAGG CGT-3¢ TCCCGGGGGCGGCCTTCTC-3¢ (reverse primer). The PCR product was inserted into the pET-21a vector, and the construct was designated pET–FNR. E. coli BL21(DE3) Codon Plus cells were transformed with pET–FNR. The transformant was grown in 2 · YT medium containing chloramphenicol and ampicillin at 37 (cid:2)C up to an D600 of 1.0, and FNR expression was induced by treatment with 1.0 mm IPTG for 5 h at 37 (cid:2)C. Cells were harvested by cen- trifugation at 5000 g for 20 min, and the pellet was sus- pended 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 incu- bated at 70 (cid:2)C for 30 min, and then centrifuged at 20 000 g for 30 min at 4 (cid:2)C. The heat-treated supernatant was puri- ﬁed with a DE52 column, a 2¢,5¢-ADP–Sepharose column

T. Mandai et al.

Thermostable electron transport system

Construction of a phylogenetic tree

A phylogenetic tree was constructed with FNRs from different sources, using the neighbor-joining method of clustalw (http://align.genome.jp/).

(200 lm b-carotene solution). Other

Acknowledgements

b-carotene was completely solubilized, with minor modiﬁ- cations. A 4 mm b-carotene solution (5 lL) was diluted 10-fold with acetone containing 4.5% Tween 20 (v ⁄ v). The solution was mixed vigorously, and then vacuum- dried. The resulting residue was dissolved in 98 lL of 50 mm potassium acetate buffer (pH 5.0) containing 10% glycerol reaction conditions were identical to those described above, and the reaction products were extracted with 25-fold volumes of ice-cold acetonitrile.

This study was partially supported 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 sponsored by the Academia-Industry Cooperation, Ministry of Education, Science, Culture, Sports and Technology of Japan.

column

References

1 Hannemann F, Bichet A, Ewen KM & Bernhardt R

(2007) Cytochrome P450 systems – biological variations of electron transport chains. Biochim Biophys Acta 1770, 330–344. atmospheric pressure chemical follows: 2 Serizawa N (1996) Biochemical and molecular

The reaction products were identiﬁed by UPLC-MS. The products were extracted with chloroform (400 lL), and dis- tilled water (1 mL) was then added to the organic phase to remove Tween 20. The organic phase was dried, dissolved in methanol, and analyzed by UPLC-MS. The reaction products were separated using an UPLC system (ACQUITY UPLC system; Waters, Milford, MA, USA) equipped with (1.7 lm, an ACQUITY UPLC BEH C18 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 spec- trometer (Hitachi, Tokyo, Japan). The MS parameters were ionization as (APCI) positive ion mode; spray potential, 3500 V; N2 gas ﬂow, 5 LÆmin)1; N2 gas temperature, 250 (cid:2)C.

Ferricyanide and cytochrome c reduction assay

stated, ferricyanide approaches for production of pravastatin, a potent cho- lesterol-lowering drug. Biotechnol Annu Rev 2, 373–389. 3 Wright RL, Harris K, Solow B, White RH & Kennelly PJ (1996) Cloning of a potential cytochrome P450 from the archaeon Sulfolobus solfataricus. FEBS Lett 384, 235–239.

4 Yano JK, Blasco F, Li H, Schmid RD, Henne A & Poulos TL (2003) Preliminary characterization and crystal structure of a thermostable cytochrome P450 from Thermus thermophilus. J Biol Chem 278, 608– 616. dehydrogenase 5 Yano JK, Koo LS, Schuller DJ, Li H, Ortiz de Montel-

(50–500 nm) Fdx 50 (cid:2)C (total

lano PR & Poulos TL (2000) Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfo- lobus solfataricus. J Biol Chem 275, 31086–31092. 6 Park SY, Yamane K, Adachi S, Shiro Y, Weiss KE, Maves SA & Sligar SG (2002) Thermophilic cyto- chrome P450 (CYP119) from Sulfolobus solfataricus: high resolution structure and functional properties. J Inorg Biochem 91, 491–501. Unless otherwise reduction assays were performed in 50 mm potassium phosphate buffer (1 mm) at containing potassium ferricyanide (pH 7.4) 25 (cid:2)C (total volume, 500 lL). For the kinetic analysis, the concentration of NADPH was kept constant by regeneration with glucose 6-phosphate and glucose-6- from yeast. Cytochrome c phosphate reduction assays were performed in 50 mm potassium phosphate buffer (pH 7.4) containing horse heart cyto- (0.1 mm), NADPH (0.5 mm), FNR (50 nm) chrome c and volume, at 500 lL). Ferricyanide reduction activity was calculated from the decrease in absorbance at 420 nm (e420 nm = 1.02 mm)1Æcm)1). Cytochrome c reduction activity was calculated from the increase in absorbance at 550 nm (e550 nm = 21.0 mm)1Æcm)1).

Thermostability of FNR

7 McLean MA, Maves SA, Weiss KE, Krepich S & Sligar SG (1998) Characterization of a cytochrome P450 from the acidothermophilic archaea Sulfolobus solfataricus. Biochem Biophys Res Commun 252, 166–172.

8 Chang YT & Loew G (2000) Homology modeling, molecular dynamics simulations, and analysis of CYP119, a P450 enzyme from extreme acidothermophil- ic archaeon Sulfolobus solfataricus. Biochemistry 39, 2484–2498.

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2427

9 Puchkaev AV, Koo LS & Ortiz de Montellano PR (2003) Aromatic stacking as a determinant of the Puriﬁed FNR (60 nm) was incubated for 30 min at various temperatures (40–110 (cid:2)C) in buffer A or 50 mm potassium acetate buffer (pH 5.0) containing 10% glycerol. The heat treatment was stopped by placing the sample on ice for 5 min, and the denatured proteins were then removed by centrifugation at 10 000 g for 10 min at 4 (cid:2)C. The residual ferricyanide reduction activity of each sample was then measured as described above.

T. Mandai et al.

Thermostable electron transport system

thermal stability of CYP119 from Sulfolobus solfatari- cus. Arch Biochem Biophys 409, 52–58. 10 Blasco F, Kauffmann I & Schmid RD (2004) 22 Liu B & Lee YK (2001) In vitro biosynthesis of xantho- phylls by cell extracts of a green alga Chlorococcum. Plant Physiol Biochem 39, 147–154. 23 Goodman DS, Huang HS, Kanai M & Shiratori T

(1967) The enzymatic conversion of all-trans-beta-caro- tene into retinal. J Biol Chem 242, 3543–3554. CYP175A1 from Thermus thermophilus HB27, the ﬁrst b-carotene hydroxylase of the P450 superfamily. Appl Microbiol Biotechnol 64, 671–674. 11 Momoi K, Hofmann U, Schmid RD & Urlacher VB

(2006) Reconstitution of b-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase. Biochem Biophys Res Commun 339, 331–336. 24 Henne A, Bru¨ ggemann H, Raasch C, Wiezer A, Hart- sch T, Liesegang H, Johann A, Lienard T, Gohl O, Martinez-Arias R et al. (2004) The genome sequence of the extreme thermophile Thermus thermophilus. Nat Bio- technol 22, 547–553. 25 Seo D & Sakurai H (2002) Puriﬁcation and character-

ization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum. Biochim Biophys Acta 1597, 123–132. 12 McLean KJ, Sabri M, Marshall KR, Lawson RJ, Lewis DG, Clift D, Balding PR, Dunford AJ, Warman AJ, McVey JP et al. (2005) Biodiversity of cytochrome P450 redox systems. Biochem Soc Trans 33, 796–801. 13 Puchkaev AV & Ortiz de Montellano PR (2005) The

26 Seo D, Kamino K, Inoue K & Sakurai H (2004) Puriﬁ- cation and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC. Arch Microbiol 182, 80–89. Sulfolobus solfataricus electron donor partners of ther- mophilic CYP119: an unusual non-NAD(P)H-depen- dent cytochrome P450 system. Arch Biochem Biophys 434, 169–177. 14 Puchkaev AV, Wakagi T & Ortiz de Montellano PR

27 Aliverti A, Pandini V, Pennati A, De Rosa M & Zanetti G (2008) Structural and functional diversity of ferre- doxin-NADP(+) reductases. Arch Biochem Biophys 474, 283–291.

(2002) CYP119 plus a Sulfolobus tokodaii strain 7 ferre- doxin and 2-oxoacid:ferredoxin oxidoreductase consti- tute a high-temperature cytochrome P450 catalytic system. J Am Chem Soc 124, 12682–12683. 15 Yokoyama A, Sandmann G, Hoshino T, Adachi K, 28 Quinlan RF, Jaradat TT & Wurtzel ET (2007) Escheri- chia coli as a platform for functional expression of plant P450 carotene hydroxylases. Arch Biochem Biophys 458, 146–157.

Sakai M & Shizuri Y (1995) Thermozeaxanthins, new carotenoid-glycoside-esters from thermophilic eubacteri- um Thermus thermophilus. Tetrahedron Lett 36, 4901– 4904.

29 Chun YJ, Shimada T, Sanchez-Ponce R, Martin MV, Lei L, Zhao B, Kelly SL, Waterman MR, Lamb DC & Guengerich FP (2007) Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Strepto- myces coelicolor A3(2). J Biol Chem 282, 17486– 17500. 16 Hara M, Yuan H, Yang Q, Hoshino T, Yokoyama A & Miyake J (1999) Stabilization of liposomal mem- branes by thermozeaxanthins: carotenoid-glucoside esters. Biochim Biophys Acta 1461, 147–154.

17 Sato S, Nakazawa K, Hon-Nami K & Oshima T (1981) Puriﬁcation, some properties and amino acid sequence of Thermus thermophilus HB8 ferredoxin. Biochim Biophys Acta 668, 277–289. 30 Sielaff B & Andreesen JR (2005) Kinetic and binding studies with puriﬁed recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5. FEBS J 272, 1148–1159. 31 Mitani F (1979) Cytochrome P450 in adrenocortical mitochondria. Mol Cell Biochem 24, 21–43. 18 Hille R, Yoshida T, Tarr GE, Williams CH Jr, Ludwig ML, Fee JA, Kent TA, Huynh BH & Mu¨ nck E (1983) Studies of the ferredoxin from Thermus thermophilus. J Biol Chem 258, 13008–13013.

32 Grifﬁn S, Higgins CL, Soulimane T & Wittung-Stafsh- ede P (2003) High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin. Linear clusters form at high pH on polypeptide unfolding. Eur J Biochem 270, 4736–4743.

33 Carmichael AB & Wong LL (2001) Protein engineering of Bacillus megaterium CYP102. The oxidation of poly- cyclic aromatic hydrocarbons. Eur J Biochem 268, 3117–3125. 19 Ohnishi T, Blum H, Sato S, Nakazawa K, Hon-nami K & Oshima T (1980) A stable Thermus thermophilus iron–sulfur protein containing only one binuclear and one tetranuclear cluster. J Biol Chem 255, 345–348. 20 Laptenko O, Kim SS, Lee J, Starodubtseva M, Cava F, Berenguer J, Kong XP & Borukhov S (2006) pH-depen- dent conformational switch activates the inhibitor of transcription elongation. EMBO J 25, 2131–2141. 34 Otey CR, Bandara G, Lalonde J, Takahashi K & 21 Marasco E, Vay K & Schmidt-Dannert C (2006) Identi-

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2428

Arnold FH (2006) Preparation of human metabolites of propranolol using laboratory-evolved bacterial cyto- chromes P450. Biotechnol Bioeng 93, 494–499. ﬁcation of carotenoid cleavage dioxygenases from Nostoc sp. PCC 7120 with different cleavage activities. J Biol Chem 281, 31583–31593.

T. Mandai et al.

Thermostable electron transport system

35 Sulistyaningdyah WT, Ogawa J, Li QS, Shinkyo R,

FEBS Journal 276 (2009) 2416–2429 ª 2009 The Authors Journal compilation ª 2009 FEBS

2429

electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences. Proteome Sci 1, 6, doi:10.1186/1477- 5956-1-6. Sakaki T, Inouye K, Schmid RD & Shimizu S (2004) Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 and its mutant. Biotechnol Lett 26, 1857–1860. 36 Wang W, Sun J, Nimtz M, Deckwer W & Zeng A (2003) Protein identiﬁcation from two-dimensional gel 37 Jeon S-J & Ishikawa K (2002) Identiﬁcation and charac- terization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1. Eur J Biochem 269, 5423–5430.