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 65C, and the Km and Vmax
Trang 1CYP175A1 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 (Tm= 91C) 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 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 ferreferre-doxin–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 thermophilusHB27 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 Tmvalue of the FNR was 99C 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
65C, and the Km and Vmax values for b-carotene hydroxylation were 14.3 ± 1.6 lm and 18.3 ± 0.6 nmol b-cryptoxanthinÆmin)1Ænmol)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.
Trang 2The 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 (Tm= 88C), and its
substrate-bind-ing region is highly similar to the substrate-bindsubstrate-bind-ing
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 thermobiszeaxthermozeax-anthins into the cell mem-brane reduces memmem-brane 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)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-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
Trang 3purified 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
[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)1subunit, 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 Km value of the FNR for NADPH was about 600-fold lower than that for NADH, and the Vmaxvalue
of the FNR with NADPH was about 55-fold higher
A
62
47.5
32.5
25
16.5
kDa
B
Wavelength (nm) 0.0
1.0
0.5
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).
Trang 4than 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 50C with saturating
concentrations of NADPH (1 mm) and ferricyanide
(1 mm) than at 25C (data not shown)
To determine the optimal pH of the FNR, we
mea-sured ferricyanide reduction activity at 50C 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 Tmvalues of the FNR at
pH 7.4 and at pH 5.0 were 99 and 95C, 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 50C 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 65C 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)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)1CYP175A1, 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 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 C for 2 min.
Purification
Total activity (nmolÆmin)1)
Specific activity (nmolÆmin)1Æmg)1) Purification (fold) Yield (%)
Trang 5multistep 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
Wavelength (nm) 0.0
0.1
0.2
0.3
0.4
Absorbance Wavelength (nm) 450
0.00 0.02 0.04
550 350
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.
Trang 6species, 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
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-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
Tm values of CYP175A1, Fdx and FNR are 88, 114, and 99C, 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 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 C for 2 min.
Purification
Total activity (nmolÆmin)1)
Specific activity (nmolÆmin)1Æmg)1) Purification (fold) Yield (%)
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 Kmvalue for NADH was determined in the
pre-sence of FNR (200 nM) and NADH (0.5–7.0 mM), and the K m value
for NADPH was determined in the presence of FNR (20 nM) and
NADPH (2–100 lM).
Vmax(nmolÆmin)1Ænmol)1of FAD) 152 ± 15 8318 ± 71
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 C.
Ratio (FNR : Fdx)
1 : 0 1 : 1 1 : 2 1 : 5 1 : 10 (nmolÆmin)1Ænmol)1
of FAD)
105 ± 2 150 ± 4 186 ± 1 346 ± 6 544 ± 10
Trang 7be 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
(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)
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
Bio-Rad Laboratories (Hercules, CA, USA)
Cloning, expression and purification of CYP175A1
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
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
expression was induced by treatment with 0.5 mm IPTG
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
Ferricyanide reduction activity (µmol·min
–1 ·nmol of FAD
–1 )
pH
4 5 6 7 8
20
40
60
A
B
0
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 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 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 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 C The values represent the mean ± SD of
triplicate experiments.
Trang 8solution was stirred at 4C for 30 min The cell suspension
was disrupted by sonication, and the cell debris was then
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,
CYP175A1 was dialyzed against 50 mm potassium
phos-phate buffer (pH 6.3) containing 10% glycerol The
(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
con-centration of purified CYP175A1 was determined with an
Approximately 5 mg of purified CYP175A1 was obtained per 1 L of culture, and a single band was observed on
Purification of an electron transport system for CYP175A1 from T thermophilus HB27
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)
0.1 mm EDTA, and the cell suspension was disrupted by sonication The cell debris was removed by centrifugation
was then loaded onto a DE52 (Whatman, Maidstone, UK)
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
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
Bio-Gel P6 DG column equilibrated with buffer A, and
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
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
Sepharose column was dialyzed against buffer B The
β-carotene
β-cryptoxanthin
Retention time (min)
0
A454
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 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 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 Tmvalue of each protein at neutral
pH.aData from this study.bData from Griffin et al [32].cData from
Yano et al [4].
Trang 9dialyzed 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
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
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
0 1 2 3 4 5 A
C
B
D
–1 ·nmol of CYP175A1
–1 )
Tween 20 (%) 0.0
0 5 10 15
–1 ·nmol of CYP175A1
–1 )
Fdx (n M )
0 2 4 6 8
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 nM), Fdx (60 nM), FNR (30 nM), 20 lM b-carotene (containing 0.1% Tween-20) and NADPH (1 mM) at 65 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 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 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 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.
Trang 10T thermophilus 1 -MAADHTDVLIVGAGPAGLFAGFYVGMRGLSFRFVDPLPEPGGQLTAL 47
A pernix 1 MPLRLSAVRAPKIPRGEEYDTVIVGAGPAGLSAAIYTTRF-LMSTLIVSM-DVGGQLNLT 58
1
T thermophilus 48 YPEKYIYDVAG-FPKVYAKDLVKGLVEQVAPFNPVYSLGERAETLE-REGDLFKVTTSQG 105
A pernix 59 N -WIDDYPG-MGGLEASKLVESFKSHAEMFGAKIVTGVQVKTVDRLDDGWFLVRGSRG 114
: : * : *:: : : * : :.:: : * : *
T thermophilus 106 NAYTAKAVIIAAGVGAFEPRRIGAPGEREFEGRGVYYAVKSKA-EFQGK-RVLIVGGGDS 163
A pernix 115 LEVKARTVILAVGSRR -RKLGVPGEAELAGRGVSYCSVCDAPLFKGKDAVVVVGGGDS 171
::*:*.* * :* *.* : **** * : * ::***::
T thermophilus 164 AVDWALNLLDTARRITLIHRRPQFRAHEASVKELMKAHEEGRLEVLTPYELRRVEGDER- 222
A pernix 172 ALEGALLLSGYVGKVYLVHRRQGFRAKPFYVEEARKK-PNIEFILDS IVTEIRGRDR- 227
*:: ** * : *:*** *** :: : : : : : : * :
T thermophilus 223 VRWAVVFHNQTQEELA-LEVDAVLILAGYITKLGPLANWGLALEKNKIK -VDTTMA 276
A pernix 228 VESVVVKNKVTGEEKE-LRVDGIFIEIGSEPPK-ELFEA-IGLETDSMG NVVVDEWMR 282
* : :: * * ::: * : : : ** :
T thermophilus 277 TSIPGVYACGDIVTYPGKLPLIVLGFGEAAIAANHAAAYAN-PALKVNPGHSSEKAAPGT 335
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
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 cRhodobact-erevisiae, 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.