 |
INTRODUCTION |
Thromboxane A2 synthase
(TXAS)1 converts
prostaglandin H2 (PGH2) to TXA2,
which is a vasoconstrictor and a potent stimulator of platelet
secretion and aggregation (1). TXA2 is rather labile, being
hydrolyzed with half-life of about 30 s in aqueous solution to the
biologically inactive thromboxane B2 (2). This lability indicates that TXA2 is primarily an autocrine or paracrine
mediator, acting in the vicinity of its biosynthesis.
TXAS was characterized spectrophotometrically as a cytochrome P450
enzyme and was found to be associated with the endoplasmic reticulum
(3, 4). Unlike other microsomal P450s that require a P450 reductase as
electron donor to catalyze a mono-oxygenation reaction, TXAS catalyzes
an isomerization reaction without need for a reductase or other
electron donor or for molecular oxygen. Interestingly, TXAS also
catalyzes formation of the scission products, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and
malondialdehyde (MDA) at a ratio of 1:1:1 with TXA2
(3).
Although TXAS was purified from platelets more than a decade ago,
limited information has been obtained about the reaction mechanism or
about structural/functional relationships. This is mainly due to the
low abundance of the enzyme (~0.1% of platelet microsomal protein)
and the difficulty in purifying sufficient enzyme for kinetic and
spectroscopic studies. Conventional chromatography of platelet TXAS was
found to have poor yields and reproducibility (3). Immunoaffinity
purification of TXAS was accompanied by loss of >90% of the activity
during elution (5, 6). TXAS purified on a high pressure liquid
chromatographic, anion exchange column lost its P450 characteristics
and became an inactive P420 form (7). The outlook has been improved by
isolation of the TXAS cDNA (8-12). Several groups have described
systems for heterologous expression of TXAS. In prokaryotic expression
systems, fusion of TXAS with glutathione S-transferase
produced a protein of the correct size but that was enzymatically
inactive (4); insertion of TXAS cDNA into a common P450 expression
vector, pCW, resulted in a very low yield of recombinant protein (13).
In eukaryotic expression systems, plasmid-directed transient expression
in COS-1 cells and baculovirus-driven expression in Sf9 cells
generated enzymatically active TXAS but in amounts insufficient for
spectroscopic studies (4, 10, 14). Here, we report development of a
high level bacterial expression system for human TXAS and a rapid and reproducible purification procedure that affords milligram levels of
active enzyme suitable for detailed biophysical and biochemical characterization.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Nickel-nitrilotriacetate agarose (Ni-NTA) was
obtained from Qiagen. DEAE-Sephacel and octyl-Sepharose were from
Amersham Pharmacia Biotech. PGH2 was purchased from Biomol,
and the stable PGH2 analog, U44069
(15-hydroxy-9,11-[epoxymethano]prosta-5,13-dienoic acid), was from
Cayman. Imidazole, 1-phenylimidazole, 1-butylimidazole, 2-phenylimidazole, 4-phenylimidazole, pyridine, 4-ethylpyridine, 3,5-lutidine, pyrimidine, 2-aminopyrimidine, and 4-methylpyrimidine were from Aldrich. Clotrimazole
(1-[o-chloro-
,
-diphenylbenzyl]imidazole) and
miconazole
(1-[2,4-dichloro-
-([2,4-dichlorobenzyl]-oxy)phenyl]imidazole) were obtained from Sigma. Emulgen 913 was a gift from Kao Chemicals, Tokyo, Japan. The pCW and pT-groE expression vectors were kindly provided by Dr. Amy Roth (University of Oregon) and Dr. Shunsuke Ishii
(Institute of Physical and Chemical Research, Ibaraki, Japan), respectively.
Construction of TXAS Expression Plasmids--
The polymerase
chain reaction was used to amplify the human TXAS cDNA, isolated
from a lung cDNA library (8). The sense primers were designed to
introduce the initiator codon ATG within an NdeI site and to
make desired truncations of the TXAS amino-terminal region,
e.g. by replacing amino acid residues 1-28 with an
8-residue segment (MALLLAVF) favoring expression in Escherichia
coli (15). The antisense primers were designed to add a
BglII site near the carboxyl terminus, with or without a
4-histidine terminal segment to facilitate protein purification.
Individual polymerase chain reaction products were digested with
NdeI and BglII and ligated into the corresponding
restriction enzyme sites of the pCW plasmid, which uses the
tac promoter to drive expression (16). The resultant amino-
and carboxyl-terminal amino acid sequences of recombinant TXAS are
listed in Table I. Plasmids were transformed into a protease-deficient
E. coli strain BL21(DE3) containing the
chloramphenicol-resistant plasmid pT-groE (17), which expresses
chaperonins groES and groEL from the T7 promoter. Colonies were
isolated from LB-agar plates containing both ampicillin and chloramphenicol.
Expression and Purification of Recombinant TXAS--
Typically,
an overnight culture (20 ml) was inoculated into 1 liter of LB medium
with 100 µg/ml ampicillin and 25 µg/ml chloramphenicol. Bacteria
were grown at 37 °C in a shaker at 200 rpm until the Klett reading
reached 100-140. Isopropyl-
-D-thiogalactopyranoside (1 mM) and
-aminolevulinic acid (0.5 mM) were
then added, and the culture was continued at 160 rpm for 20 h at
30 °C before harvesting. The pelleted cells were kept at
70 °C
until protein purification.
Harvested cells (17 g from 5 liters of culture medium) were resuspended
in 90 ml of Buffer A (0.1 M sodium phosphate, pH 7.5, 10%
glycerol, and 0.1 M NaCl) containing 50 µg/ml DNase, 2 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride and lysed
by two passes through a French pressure cell at 14,000 pounds/square
inch. All subsequent steps were performed at 4 °C. The cell
homogenates were stirred for 60 min with 1% Emulgen 913 to solubilize
the TXAS. The suspension was then centrifuged at 18,000 × g for 30 min, and the supernatant containing solubilized TXAS was diluted to 200 ml with Buffer A and applied to a Ni-NTA column
(1.5 × 5 cm) pre-equilibrated with Buffer A. The column was
washed with 5 column volumes of Buffer A, then with 10 column volumes
of Buffer B (0.02 M sodium phosphate, pH 7.5, 0.02% of Emulgen 913, and 10% glycerol) containing 5 mM histidine,
and then with 5 column volumes of Buffer B containing 10 mM
histidine to remove nonspecifically bound proteins. TXAS was eluted
with Buffer B containing 40 mM histidine. The brown-colored
fractions were pooled and loaded on a DEAE-Sephacel column (1.5 × 3 cm) pre-equilibrated with Buffer B. The flow-through fractions, which contained the majority of the TXAS activity, were collected and applied
to an octyl-Sepharose column (1.5 × 3 cm) pre-equilibrated with
Buffer C (0.02 M sodium phosphate, pH 7.5, and 10%
glycerol) containing 0.5 M NaCl. The column was washed with
10 volumes of the same buffer and then with 10 volumes of Buffer C. TXAS was eluted with Buffer C containing 0.2% Emulgen 913 and stored
at
70 °C until used.
The level of recombinant TXAS expression was assessed by Coomassie Blue
staining after separation of proteins by polyacrylamide gel
electrophoresis under denaturing conditions; the identity of TXAS was
confirmed by Western blot analysis using antiserum raised against a
glutathione transferase-TXAS fusion protein (4).
Heme and Protein Assays--
Heme content was determined by the
formation of pyridine hemochromogen (18). Briefly, the purified TXAS
(~150 µg) was incubated in 0.15 M NaOH and 1.8 M pyridine and then reduced by addition of a few grains of
dithionite. Absorbance differences at 556 nm and 540 nm were recorded
for the reduced and oxidized heme pyridine complexes. A difference
extinction coefficient of 24 mM
1
cm
1 (reduced minus oxidized forms) was used for
calculation of heme concentration. Protein concentrations were
determined by bicinchoninic acid assay using bovine serum albumin as
standard (19). TXAS was also quantified for total number of tryptophan
residues by magnetic circular dichroism (MCD) using
L-tryptophan as standard (A282 = 5500 M
1 cm
1) (20).
Enzyme Assay--
TXAS activity was routinely measured by a
coupled assay as follows. Purified sheep prostaglandin H synthase
(about 1000 units; Ref. 21) was mixed with TXAS in 190 µl of 30 mM Tris, pH 7.5, containing 1 µM hemin at
23 °C. The reaction was initiated by adding 10 µl of 1.2 mM arachidonic acid (in ethanol) and continued for 3 min
before stopping with 5 µl of 2 N HCl. The reaction
mixture was neutralized with 20 µl of 1 M Tris, pH 8.0, prior to the radioimmunoassay for thromboxane B2 (22), the
stable hydration product of TXA2. To obtain
Km and Vmax values,
PGH2 in a small volume of isopropyl alcohol was added
directly to TXAS in 30 mM Tris, pH 7.5.
UV-Vis, MCD, and EPR Spectroscopy--
UV-Vis absorbance spectra
were recorded with an HP8452 diode array spectrophotometer or a
Shimadzu UV-2401PC spectrophotometer. Reduced CO difference spectra
were obtained by treating the protein with dithionite and then purging
the sample cuvette with carbon monoxide. Heme ligand perturbation
spectra were obtained by titration of the protein with a concentrated
stock solution of ligand. Dissociation constants
(Kd) were calculated by fitting the peak-trough amplitudes from the perturbation difference spectra and the
corresponding ligand concentrations to a one-site hyperbolic binding
model (23).
MCD spectra were obtained at room temperature using a Jasco J-500C
spectrometer with a 1.3 Tesla electromagnet calibrated with camphor
sulfonic acid and potassium ferricyanide (24). EPR spectra were
recorded at liquid helium temperature on a Varian E-6 spectrometer with
an Air Products liquid helium transfer line (25). EPR parameters,
including g values, V and D, the
rhombic and axial ligand field terms for low spin heme complexes, and the ratio of rhombic (E) and axial (D) ligand
field components for high spin heme complexes, were determined as
described previously (25).
 |
RESULTS |
Optimization of Expression Plasmid--
Expression of human TXAS
in E. coli was assessed for various TXAS cDNA
constructs. Initially, full-length, wild-type TXAS cDNA was ligated
into the pCW vector, a system that has been widely used in the
heterologous bacterial expression of other P450s (26). Coomassie
staining and immunoblot analysis indicated that the full-length
construct yielded low levels of expression in E. coli (Table
I), similar to results with other
unmodified P450 cDNAs (27). In light of earlier results with
P450c17 expression in E. coli (28), we modified the TXAS
cDNA by progressive truncation of the amino-terminal sequence and
insertion of the amino-terminal sequence MALLLAVF. In some constructs,
4 histidine residues were added at the carboxyl terminus to facilitate
protein purification. Five constructs, with deletion of residues 1-3,
1-24, 1-28, 1-59 or 1-65, were each found to be expressed to some
degree in E. coli strain JM109 (Table I). The highest levels
were obtained with deletion of residues 1-28 (
1-28), and addition
of the histidine tag to the
1-28 construct did not affect the
expression level. Previous studies have shown that deletion of the
first 28 amino acid residues had little effect on TXAS activity (13).
Therefore, the
1-28 TXAS construct with a modified P450C17 amino
terminus sequence and a histidine tag at the carboxyl terminus, termed pCW-TXAS(
1-28)mod(His)4, was used for further
studies.
View this table:
[in this window]
[in a new window]
|
Table I
TXAS constructs and their expression levels in JM109 cells
The sequences of sense primers (in 5' to 3' direction) for the modified
amino termini are as follows: AACATATGGCTCTGTTATTAGCAGTTTTT
(NdeI site is underlined), immediately followed by TXAS
cDNA sequences of the desired truncations. The sequences of
antisense primers (in 5' to 3' direction) are as follows:
GAAGATCTCAGTGATGGTGATGGCGGGATACGATCTTGATAT (for constructs
with a four-histidine tag) or GAAGATCTAACATCCACACTTAGGGT
(for constructs with native carboxyl terminus; the sequence corresponds
to 49-66 bases downstream from the TGA stop codon) (BglII
sites are underlined). The resultant amino acid sequences are shown
below. The cDNAs were constructed into the pCW expression vector
and transformed in JM109 cells. After induction with
isopropyl-1-thio- -D-galactopyranoside, cells were
sonicated and the homogenate was centrifuged at 10,000 × g for 10 min. Proteins in the supernatant were separated by
SDS-polyacrylamide gel electrophoresis, and the TXAS expression levels
were estimated by Coomassie staining and immunoblot analyses.
|
|
Immunoblot analyses indicated that the majority of recombinant TXAS
protein expressed from pCW-TXAS(
1-28)mod(His)4 in
JM109 was insoluble even after treatment with detergents Emulgen 913, Nonidet P-40, Triton X-100, cholate, or Lubrol, presumably reflecting misfolded protein in inclusion bodies. To improve the production of
properly folded TXAS, we utilized a co-expression system in which TXAS
was expressed along with chaperonins groES and groEL in the
protease-deficient BL21 host strain. These changes increased the yield
of detergent-soluble recombinant TXAS by at least 2-fold (data not shown).
Purification of Recombinant TXAS--
The expression of
chaperonins is under the control of T7 polymerase, which is induced by
isopropyl-1-thio-
-D-galactopyranoside in the BL21 cells.
Upon induction, production of groES (10 kDa), groEL (60 kDa), and
recombinant TXAS (56 kDa) were greatly increased. About 80% of the
total TXAS activity in the homogenate was found in the membrane
fraction, indicating that enzymatically active recombinant TXAS was
properly integrated in membrane. To facilitate purification, TXAS was
routinely solubilized with 1% Emulgen 913 directly from the cell
extract, without isolation of the membrane fraction. A protocol that
includes chromatography on Ni-NTA, DEAE-Sephacel, and octyl-Sepharose
was developed for purification of recombinant TXAS, as described under
"Experimental Procedures." Typical results are presented in Table
II and Fig.
1. The Ni-NTA affinity column step
produced the most dramatic purification, increasing the TXAS specific
activity by ~30-fold. The pooled TXAS fractions from Ni-NTA column
did not bind to the DEAE-Sephacel column, whereas some major
contaminants were retained. The octyl-Sepharose chromatography had
little effect on the TXAS specific activity, but it served to
concentrate the samples and remove histidine carried over from Ni-NTA
chromatography. The final TXAS fraction was nearly homogeneous as
judged from SDS-polyacrylamide gel electrophoresis (Fig.
1A), and the identity of the recombinant protein was
confirmed as TXAS by immunoblot analysis (Fig. 1B). Overall,
the recombinant TXAS was purified about 100-fold, with a yield near
14%.
View this table:
[in this window]
[in a new window]
|
Table II
Purification of recombinant TXAS from E. coli
Values shown are for a typical run for materials from 5 liters of
culture medium with the pCW-TXAS( 1-28)mod(His)4 construct
co-expressed with chaperonins groES and
groEL.
|
|

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of human TXAS expressed in
E. coli. Aliquots were taken at each purification step
for analysis by SDS-polyacrylamide gel electrophoresis. A,
Coomassie Blue staining. The amounts of protein loaded were as follows:
cell homogenate (35 µg); detergent extract (35 µg); Ni-NTA column
eluate (1.5 µg); DEAE Sephacel flow-through (1 µg); and
octyl-Sepharose eluate (1 µg). B, immunoblot analysis. The
protein loading was about one-tenth that in A. The position
of TXAS is indicated. The 80-kDa band is a bacterial protein
cross-reacting with the antibodies against the glutathione
S-transferase-TXAS fusion protein (4).
|
|
Characterization of Recombinant TXAS Activity--
The purified
recombinant TXAS was found to be catalytically active, with a
Km of 20 µM PGH2 and a
Vmax of 12 µmol of TXA2/min/mg of
protein at 23 °C. To determine the stoichiometric ratio of
TXA2, HHT, and MDA formed by the recombinant TXAS,
PGH2 was added to the enzyme, and the absorption spectra
were followed as a function of incubation time (Fig.
2). Formation of HHT and MDA, as
indicated by the absorption maxima at 234 and 268 nm, respectively, was
observed in the presence of TXAS but not in the control (data not
shown). At the end of the reaction, an aliquot was withdrawn and
assayed quantitatively for the TXA2 degradation product,
thromboxane B2. On the basis of the known extinction coefficients of HHT (33 mM
1 cm
1
at 234 nm) and MDA (31.5 mM
1
cm
1 at 268 nm) (29), the molar ratio of
TXA2:HHT:MDA formed by the recombinant TXAS was calculated
to be 0.94:1.0:0.93, consistent with published results using purified
platelet TXAS (3).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Conversion of PGH2 to HHT and MDA
by TXAS. Both sample and reference cuvettes contained purified
TXAS (8 pmol) in 0.3 ml of 30 mM Tris, pH 7.5, at 23 °C.
The reaction was initiated by addition of 4 µl of 1.4 mM
PGH2 to the sample cuvette. Absorption spectra were
recorded at 1 ( ), 2 ( ), 3 ( ), and 5 min ( ) after the
initiation of reaction.
|
|
Characterization of Recombinant TXAS UV-Vis and MCD
Spectra--
The heme content of the purified recombinant TXAS
determined by pyridine-hemochromogen analysis was 0.72 ± 0.06 mol
of heme/mol of TXAS, indicating that some 70% of the purified
recombinant protein was present as the holoenzyme. This is not due to
the inaccuracy of protein quantitation since results from bicinchoninic acid assay and noninvasive MCD quantitation for tryptophan residues were consistent. The purified TXAS exhibited an electronic spectrum typical for low spin cytochrome P450, with a Soret maximum at 418 nm
and
and
bands at 567 and 535 nm, respectively (Fig. 3). The heme-carbon monoxide complex of
dithionite-reduced TXAS had a major peak at 450 nm, and minor peaks at
420 nm and 546 nm, similar to the reduced CO spectrum reported for
purified platelet TXAS (3). The small fraction of 420 nm species in the
reduced heme-CO spectrum in Fig. 3 is probably not an inactive P420
form of TXAS because complete conversion to the 450 nm species was observed in the presence of methyl viologen, a mediator dye (data not
shown). The difference spectrum between the reduced heme-CO complex and
resting TXAS highlights the 450 nm peak, characteristic of cytochrome
P450 (Fig. 3).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Electronic absorption spectra of recombinant
TXAS. Purified TXAS (3 µg) was incubated in 0.3 ml of 20 mM sodium phosphate, pH 7.5, containing 10% glycerol and
0.2% Emulgen 913 (- - -). A few crystals of dithionite were added to
the enzyme sample before it was purged with CO gas for 1 min
(···). The difference between resting and reduced-CO spectra is
also shown (-·-·-).
|
|
To evaluate the effects of axial ligands on the TXAS heme behavior,
UV-Vis and MCD spectroscopy were used to characterize resting TXAS and
TXAS complexed with U44069 (O-based ligand) or imidazole
(N-based ligand). As shown in Fig.
4A, resting TXAS had optical
absorbance peaks at 360 (46.2 mM
1
cm
1), 418 (100 mM
1
cm
1), 535 (13.6 mM
1
cm
1), 567 (12.3 mM
1
cm
1), and 655 (4.9 mM
1
cm
1) nm. The U44069 complex exhibited a blue-shifted
Soret peak at 414 nm (101 mM
1
cm
1) and bands in the visible region at 531.5 (12.7 mM
1 cm
1), 563 (12.5 mM
1 cm
1), and 652 (4.7 mM
1 cm
1) nm. In contrast,
imidazole ligation of the TXAS yielded a bathochromic shift in the
electronic spectrum, with peaks at 424 (97.4 mM
1 cm
1) and 540 (4.4 mM
1 cm
1) nm. These spectra are
typical for low spin P450 hemoproteins in both peak positions and
amplitude (30). The MCD spectra for TXAS shown in Fig. 4B
display standard features typical for a low spin P450 heme with the
symmetric Soret crossovers closely matching the electronic absorbance
peaks. The two marker MCD bands at 556.1 and 520.7 nm in the visible
region for resting TXAS and its complex with imidazole (560.7 and 526.5 nm) or U44069 (549.9 and 515.1 nm) are very similar to those seen with
low-spin P450cam, chloroperoxidase (31), and nitric oxide
synthase (32, 33), each of which has a proximal thiolate ligand. The
lack of significant features in the 600-700 nm region of the MCD
spectrum (Fig. 4B) indicated that the TXAS complexes were
primarily low spin in nature.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Electronic (A) and MCD
(B) spectra of resting, imidazole-, and U44069-bound
TXAS. Spectra were recorded for purified recombinant TXAS (5 µM) in Buffer B containing 0.2% Emulgen 913 (solid
line) and for TXAS in the presence of 12 mM imidazole
(dotted line) or 350 µM U44069 (dashed
line).
|
|
Characterization of Recombinant TXAS EPR Spectra--
X-band EPR
spectra of TXAS derivatives were measured at liquid helium temperature.
Except for some nonspecific signals at g = 4.3 (adventitious iron) and at g = 2 (organic radicals), we only observed signals for low spin heme. Detailed EPR spectra in the
low spin heme region for TXAS and its complexes with imidazole and
U44069 are shown in Fig. 5. The rhombic
heme species have g tensor values of 2.419, 2.252, and 1.918 for resting TXAS (spectrum B) and of 2.464, 2.255 and 1.894 for the imidazole derivative (spectrum A), indicating the
presence of a single low spin heme species in both resting TXAS and its
imidazole derivative. The g values observed for resting
enzyme are very similar to those reported for platelet TXAS (3),
although the platelet enzyme showed two EPR species with slightly
different rhombicity. At saturating levels of U44069, the recombinant
TXAS heme did show two EPR species (Fig. 5, spectrum C); one
exhibited the spectral features of resting enzyme, whereas the other
appeared to be a complex with the ligand. Subtracting 36% of the
amplitude of spectrum B from spectrum C reveals a
new EPR species having g values of 2.484, 2.252, and 1.900 (Fig. 5, spectrum D). The closeness in g values
between O-based ligand (U44069) in spectrum D and
N-based ligand (imidazole) in spectrum A
indicates that the TXAS EPR is not very sensitive to the nature of the
TXAS distal heme ligand. Moreover, the lack of complete conversion to
the rhombic heme species even with excess U44069 implies that a portion
of the enzyme (~36%) is inaccessible to this ligand. These results
parallel our finding that a small fraction of the TXAS is impaired for reduction and CO complex formation (Fig. 3). The resting TXAS heme EPR
exhibited a heterogenous saturation behavior with half-saturation power
at 75 microwatts, similar to values reported for other low spin
cytochrome P450s (Fig. 6A;
Ref. 34).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
EPR spectra of TXAS. Shown are the EPR
spectra in the low spin heme region of resting TXAS (15 µM heme) (spectrum B), TXAS in the presence of
12 mM imidazole (spectrum A), and TXAS with 350 µM U44069 (spectrum C). Spectrum D
is the difference spectrum obtained by subtraction of 0.36 of
spectrum B from spectrum C. Anisotropic
g values are shown. EPR conditions were as follows: power, 1 milliwatt; modulation amplitude, 10 G; and temperature, 11 K.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
EPR signal characteristics of resting TXAS
and its complexes with imidazole and U44069. A, power
saturation. The signal amplitudes (solid circles) are shown
as a function of the microwave power. The curve shows the result of
fitting to the inhomogeous saturation function (Equation 1):
|
(Eq. 1)
|
where P1/2 is the power to achieve
half-saturation of the signal, and K is a proportionality
factor; the value of b is set to 1 for inhomogeneous
broadening as found for most hemeproteins (25). B, truth
diagram analysis. The rhombicity values (V/ ) are plotted
as a function of the tetragonal field values ( / ) for TXAS and its
derivatives with imidazole and U44069 (open triangles). The
six zones indicate the assignment reported previously (25). Complexes
in C zone are methionine and histidine ligands; in
B zone are bis-histidine or bis-imidazole ligands; in
H zone are histidine/imidazole or histidine/azide ligands;
in O zone are histidine/hydroxide or histidine/tyrosinate
ligands; in P zone are proximal thiolate ligands and in
CN zone are cyanide derivatives with proximal
ligands other than thiolate. Data for P450cam (filled
squares), chloroperoxidase (filled circles), and
endothelial nitric oxide synthase (open diamonds) are
included for comparison.
|
|
The EPR characteristics of resting, imidazole-, and U44069-bound TXAS
fit nicely into the "P" zone of the "Truth diagram" correlating
the low spin heme rhombicity and tetragonal field strength, as
originally devised by Blumberg and Peisach (Fig. 6B; Ref.
35), indicating that TXAS has a typical P450 electronic structure with
a proximal thiolate ligand. In addition, TXAS and its complexes with
imidazole and U44069 fall in a region within P zone overlapping with
P450cam but not chloroperoxidase or nitric oxide synthase.
Comparison of TXAS Interactions with Various Heme Ligands--
The
difference spectrum obtained upon addition of U44069 to TXAS exhibited
a maximum at 409 nm and a trough at 426 nm (data not shown). A similar
result was obtained for platelet TXAS (36) and interpreted to mean that
U44069 displaced the original heme ligand (probably water), with the
C-9 oxygen atom of U44069 interacting with the heme iron to form a
six-coordinate complex. The dissociation constant of the complex of
recombinant TXAS with U44069 was estimated by a spectral perturbation
method (see "Experimental Procedures") to be 28 ± 4 µM at 23 °C. To characterize further the structure of
distal heme site in recombinant TXAS, we extended our spectral perturbation studies to other heme ligands that have a nitrogen atom as
the distal heme ligand but that differ in the size and position of
substituent groups. The ligands were divided into three groups as
follows: imidazole-based, pyridine-based, and pyrimidine-based ligands
(Table III). All the ligands produced a
type II spectral change, with Soret peaks around 420-430 nm, an
indication that the exogenous ligand replaced the native distal ligand
and formed a six-coordinate complex (37). The imidazole-based ligands
showed a wide range of affinities for TXAS, with Kd values ranging from 0.5 µM for clotrimazole to more than
25 mM for 2-phenylimidazole. Interestingly, bulky and
hydrophobic derivatives, such as clotrimazole and miconazole, bound
strongly to TXAS. Preliminary molecular modeling using Alchemy III
software (Tripos Associates, Inc., St. Louis, MO) was conducted to
assess the geometry of the rigid imidazole analog, clotrimazole. The
tetrahedral carbon at the imidazole N-1 position is bonded with three
phenyl rings making clotrimazole a very rigid molecule, with vertical
and horizontal dimensions of ~8.5 and 9.5 Å. This provides
approximate minimal dimensions of the TXAS distal heme pocket. The
results in Table III suggest that the TXAS distal heme pocket can
accommodate relatively bulky substituents at the 1-position of
imidazole. On the other hand, the lower affinity of 4-imidazole
suggests that the distal heme pocket has less space to accommodate
substituents at the 4-position. The very much lower affinity for
2-phenylimidazole is likely due to steric hindrance between the bulky
2-substituent and heme porphyrin ring. The three pyridine-based ligands
tested had moderate affinities for the TXAS heme, with
Kd values of 40-180 µM (Table III).
All three pyrimidine-based ligands tested had quite low affinity for
the TXAS heme with Kd values of 1.9-12.7
mM (Table III), consistent with pharmacological studies with platelet microsomes which found that pyrimidine was a poor TXAS
inhibitor (38).
 |
DISCUSSION |
A common strategy for prokaryotic expression of mammalian P450s
involves replacement of the natural P450 amino terminus with the
MALLLAVF sequence derived from bovine steroid 17
-hydroxylase, CYP17
(26). This approach has been used successfully for expression in
E. coli of several other P450s, including CYP1A2 (39),
CYP3A4 (40), CYP55 (41), and several members of the CYP2C subfamily (42, 43). A previous attempt to express TXAS in E. coli
without the modified CYP17 amino-terminal sequence (13) resulted in a
much lower expression level than the present system (Table I), indicating that an amino-terminal sequence such as MALLLAVF promotes high level expression of recombinant TXAS. Furthermore, variation of
the extent of truncation at the TXAS amino-terminal had profound effects on TXAS expression (Table I), indicating that amino-terminal sequences beyond the modified CYP17 segment are also important in
achieving a high level of TXAS expression. Although the expression level with the modified amino-terminal sequence was high, the majority
of recombinant TXAS synthesized in E. coli was still enzymatically inactive and remained in inclusion bodies. Co-expression of TXAS with bacterial chaperonins, a strategy used with neuronal nitric oxide synthase (44), did improve the yield of active TXAS,
indicating that the post-translational maturation process may be
limiting in the bacterial expression system.
The purified recombinant TXAS shows some interesting heterogeneity in
its interactions with reductant and substrate analog. Upon reduction in
the presence of CO, about 30% of the material retained an absorbance
peak at 420 nm instead of shifting to 450 nm (Fig. 3); a small fraction
of 420 nm species was also observed with purified platelet TXAS (3).
The 420 nm species in TXAS was readily converted to the 450 nm form by
addition of the electron transfer mediator, methyl viologen, implying
that there is a barrier to electron transfer from dithionite in about
one-third of the recombinant TXAS molecules. A similar fraction of
recombinant TXAS appeared unable to bind U44069 (Fig. 5). Because 70%
of the purified TXAS preparation contains heme and thus contributes to the Soret absorbance, we estimate that just about half (0.7 × 0.7 = 0.49) of the purified recombinant TXAS is involved in
catalysis. This leads to an estimated intrinsic specific activity of 24 µmol of TXA2/min/mg of protein. This is almost exactly
the specific activity reported for purified platelet enzyme (24.1 µmol of TXA2/min/mg of protein; Ref. 3). Compared with
most microsomal P450 monooxygenases, which have specific activities of
only nmol of product/min/mg of protein (45), TXAS can be seen as a
relatively efficient catalyst. It should also be noted that recombinant
TXAS, like the purified platelet enzyme, catalyzes formation of
TXA2 along with HHT and MDA in approximately a 1:1:1 ratio.
The physiological roles of HHT and MDA are not clear, but MDA has been
reported to be important in formation of cellular DNA adduct, which may contribute to the etiology of human genetic disease and cancer (46).
Despite the fact that TXAS is a non-classical P450 and catalyzes an
isomerization reaction rather than monooxygenation, a typical low spin
P450 heme state was observed for TXAS in both absorption and EPR
spectroscopies. MCD is more reliable than EPR in assigning the axial
heme ligand for P450-type hemeproteins (47). Due to the dominant effect
of the thiolate proximal ligand in comparison with N- or
O-based distal ligand, the P450 g values measured
by EPR are rather insensitive to the nature of the axial heme ligand,
leaving the values of EPR parameters for various P450s clustered in a
very small region (47). In contrast, MCD spectra display distinctive
signatures originating from differences in the axial heme ligands, with
the Soret crossover and the peak positions in the visible region quite
diagnostic for the nature of the axial ligation in TXAS and its
derivatives (Fig. 4B). From a comparison of the present data
with the extensive MCD studies on other P450s by Dawson et
al. (31), both resting TXAS and its complex with U44069 are
assigned to have an oxygen-based axial ligand. The slight spectral
differences between resting TXAS and the U44069 complex indicates only
subtle differences in the oxygen coordination. On the other hand, the
MCD spectrum of the TXAS-imidazole complex shows an MCD signature very
similar in both the Soret and visible regions to N-liganded
P450cam complexes.
The similarity between the heme EPR properties of TXAS and
P450cam derivatives and their differences with
chloroperoxidase and nitric oxide synthase derivatives in the P zone of
Fig. 6B indicate that the polarity of the axial heme ligand
pockets in TXAS and P450cam are less polar than in
chloroperoxidase or nitric oxide synthase. This fits with the relative
substrate polarities in these enzymes, as the substrates for
chloroperoxidase and nitric oxide synthase are polar in nature, whereas
the substrates for P450cam and TXAS are relatively hydrophobic.
Pharmacological TXAS inhibitors have been actively sought because of
the anticipated beneficial effects of reducing platelet activation at
the site of acute vascular lesions. Existing TXAS inhibitors can be
divided into three structural groups as follows: substrate analogs,
imidazole-based inhibitors, and pyridine-based inhibitors (48-50). The
dissociation constants of some of these inhibitors measured with
purified recombinant TXAS (Table III) correlate well with drug potency
assayed using platelet microsomes (38, 48-50), confirming that the
purified, detergent-solubilized TXAS is functionally similar to the
membrane-bound form. This indicates that recombinant TXAS will be
useful for detailed characterization of enzyme interactions with
potential inhibitors.
In summary, catalytically active human TXAS has been expressed in
E. coli, and a rapid and inexpensive purification protocol was developed to provide the quantities of enzyme required for physiochemical characterization, reaction mechanism studies, and inhibitor design. Initial spectroscopic characterization of recombinant TXAS indicates the distal heme pocket is hydrophobic and relatively large compared with "classical" P450 enzymes.