(Received for publication, October 1, 1996, and in revised form, November 13, 1996)
From the Vascular Biology Research Center and Division of Hematology, University of Texas Medical School, Houston, Texas 77030
Prostacyclin synthase (PGIS), a cytochrome P450
enzyme, catalyzes the biosynthesis of a physiologically important
molecule, prostacyclin. In this study we have used a molecular
modeling-guided site-directed mutagenesis to predict the active sites
in substrate binding pocket and heme environment of PGIS. A
three-dimensional model of PGIS was constructed using
P450BM-3 crystal structure as the template. Our results
indicate that residues Ile67, Val76,
Leu384, Pro355, Glu360, and
Asp364, which were suggested to be located at one side of
lining of the substrate binding pocket, are essential for catalytic
activity. This region containing 1-1,
1-2,
1-3, and
1-4
strands is predicted well by the model. At the heme region,
Cys441 was confirmed to be the proximal axial ligand of
heme iron. The conserved Phe and Arg in P450BM-3 were
substituted by Leu112 and Asp439, respectively
in PGIS. Alteration of Leu112 to Phe retained the activity,
indicating that Leu112 is a functional substitution for
Phe. In contrast, mutant Asp439
Ala exhibited a slight
increase in activity. This result implies a difference in the heme
region between P450BM-3 and PGIS. Our results also indicate
that stability of PGIS expression is not affected by heme site or
active site mutations.
Prostacyclin (PGI2)1 is a
potent inhibitor of platelet activation, vasoconstriction, and
leukocyte interaction with endothelium (1, 2). It is considered to be
an important vasoprotective molecule. Its biosynthesis in vascular
endothelial cells is catalyzed by a series of enzymes of which
PGI2 synthase (PGIS) catalyzes the final conversion of
prostaglandin H2 (PGH2) to PGI2.
PGIS was purified from bovine aortic tissues to homogeneity and the purified enzyme was characterized as a cytochrome P450 (3-5). Bovine
and human PGIS cDNA have been isolated recently (6-8). The bovine
vascular PGIS cDNA is highly homologous to the human vascular
enzyme. The cDNA from both species contains an open reading frame
of 1500 nucleotides coding for a 500-amino acid peptide with a
calculated molecular mass of about 57 kDa. Sequence comparison with
known P450s shows less than 30% sequence identity between PGIS and
other cytochrome P450s. Interestingly, despite similarities in
enzymatic reactions, PGIS has only about a 16% sequence identity to
thromboxane A2 synthase (TXAS). Thus, PGIS has been
classified as a member of a new family of P450, CYP8. The active site
structure of PGIS has not been delineated. On sequence comparison with
P450s, we observed the conservation of several P450 structural elements in PGIS, including a putative membrane-anchoring segment, a helix I
which forms an -helix backbone through the center of the enzyme, and
a heme binding pocket. This raised the possibility of mapping the
active site amino acid residues by molecular modeling-guided site-directed mutagenesis. Cytochrome P450BM-3 from
Bacillus megaterium catalyzes the monooxygenation of fatty
acids, including arachidonic acid (AA). Its catalytic function and
primary structure resemble mammalian microsomal cytochrome P450s. The
heme domain of P450BM-3 shows about 25-30% sequence
identity to several microsomal P450 enzymes (9). Since AA is a
functional substrate of P450BM-3, and the substrate is
closely related to the substrate of TXAS and PGIS, we reasoned that the
crystal structure of the hemoprotein domain could serve as a useful
template for constructing a three-dimensional model of these two
enzymes. The three-dimensional model of TXAS has been reported by our
group (10) and proves to be useful in identifying TXAS active site
residues (11). In the present study, we constructed a PGIS
three-dimensional model based on the crystallographic structure of
P450BM-3. From this model, we identified several amino
residues potentially involved in substrate access and binding and in
heme binding. To test the functional role of these residues in
catalysis, we have altered these residues by site-directed mutagenesis.
Results from these experiments have allowed for identifying amino acid
residues in the substrate channel that are important in substrate
access and binding and in interaction with heme.
COS-1 cells (ATCC CRL-1650) were obtained from
the American Type Culture Collection. pBluescript (SK+) plasmid DNA and
eukaryotic expression vector pSG5 were from Stratagene. Human lung
cDNA was from CloneTech. Cell culture media and antibiotics were
from either Life Technologies, Inc. or HyClone Laboratories.
DEAE-dextran and pGEX-2T plasmid DNA were from Pharmacia Biotech Inc.
Isopropyl--D-thiogalactopyranoside, dimethyl sulfoxide,
and chloroquine were from Sigma.
[14C]Arachidonic acid ([14C]AA) was from
Amersham Corp. PGH2 was from Cayman Chemical (Ann Arbor,
MI). Oligonucleotides were synthesized by Genosys (The Woodlands, TX).
Linear-K preadsorbent TLC plates with silica gel (60 Å) and 250-µm
thickness were from Whatman (LK6D).
The strategy used for constructing the
PGIS three-dimensional model followed that developed for TXAS (10) and
other mammalian P450s (12). The strategy took into consideration six
procedures: (i) sequence alignment, (ii) framework construction, (iii)
loop structural determination, (iv) site-chain placement, (v) molecular docking, and (vi) energy minimization, which are briefly described below. A sequence similarity alignment was made for PGIS and the hemoprotein domain of the P450BM-3 (Fig. 1),
and the main-chain conformation of PGIS was built with the
Quanta-Charmm protein modeling package by transferring the crystal
coordinates of P450BM-3 to the aligned components of PGIS.
Thus, the conserved helices and strand framework in
P450BM-3 provided the backbone section coordinates of PGIS.
The backbone segments were linked to each other using a fragment
searching approach (13-15) and a data base containing 58-protein
three-dimensional structures, developed by Ruan et al. (10).
Several three-dimensional structural candidates were obtained from the
data base and one was chosen based on the best similarity of primary
structure and the best fit of C distance, with a cutoff value of 0.5 Å root mean square deviation (10, 12, 16). The three-dimensional
structural coordinates of the heme in PGIS was adopted directly from
the x-ray structure of P450BM-3 and then fixed into the
backbone structure of PGIS. The substrate, PGH2, structure
was constructed, energy-minimized, and subjected to conformation
search. One of the 200 conformations of the PGH2
three-dimensional structure with the best score was docked into the
proposed PGIS substrate binding pocket of the constructed
three-dimensional model of PGIS, which corresponded to the
P450BM-3 substrate binding cavity. An energy minimization with 500 steps of steepest descent was performed for the PGIS three-dimensional structural model containing heme and PGH2
structures. After the energy minimization, some steric clashes between
atoms of the model were removed, and a reasonable protein folding and the substrate in coordinate position were obtained.
Expression Vector of PGIS cDNA
The full-length cDNA of PGIS was amplified by a two-step polymerase chain reaction (PCR) with two pairs of primers (I34 (CAGCCCCGCGATGGCTTG) and I37 (TGTGCACACAGAAAGCTG), outer primer set; and I35 (CATGGATCCGCGATGGCTT GGGCC) and I36 (CGAGCACGTGGATCCATC), inner primer set). Outer primer set was used for first PCR in a 60-µl reaction mixture under the cycle schedule of 95 °C for 35 s, 53 °C for 1 min, and 72 °C for 1 min 10 s with 2-s extension for each additional cycle for a total of 30 cycles on a Perkin-Elmer DNA thermal cycler. 0.4 µg of human lung cDNA, 2.5 units of Taq DNA polymerase (Promega or Perkin-Elmer), 0.1 unit of Pfu DNA polymerase (Stratagene), 100 nM of each primer, and buffer for Pfu DNA polymerase were used. 1 µl of the first PCR product (using outer primers) was used as template for second PCR amplification using inner primers in a 60-µl reaction mixture under the cycle schedule of 95 °C for 35 s, 55 °C for 1 min, and 72 °C for 1 min 10 s with 2-s extension for each additional cycle for a total of 30 cycles. The final PCR products, which contained the full-length PGIS cDNA, were gel-purified and subcloned into the BamHI site of pBluescript. The subcloned PGIS cDNA was verified by DNA sequencing.
Site-directed MutagenesisThe PGIS cDNA was subcloned
into pSG5 plasmid at either BamHI or
BamHI-BglII sites for transient expression in
COS-1 cells. Orientations of insert were verified by both
AccI and EcoRI restriction enzyme digestions. The
final construct containing the full-length PGIS cDNA (pSG5-PGIS,
wild-type) was verified by DNA sequencing. The following single mutants
of PGIS, Ile67 Lys, Arg72
Glu,
Arg72
Gln, Val76
Asp,
Thr206
Val, Leu210
Asp,
Leu213
Glu, Pro355
Val,
Glu360
Gly, Asp364
Val,
Leu384
Asp, Phe476
Val,
Cys231
Ser and Cys441
Ser,
Leu112
Asp, Leu112
Gly,
Leu112
Phe, Pro113
Ala, and
Asn439
Ala, were produced according to the method
described previously (17) with some modifications. In brief, a primer
bearing the mutated sequence and its paired primer were used in PCR to
amplify a 400-500-base pair product using pSG5-PGIS DNA as the
template. The PCR products were purified with Wizard PCR prep (Promega) to get rid of primers. Purified DNA of the first PCR product was used
as a mega-primer for the second PCR with a primer on the other side of
the mutation. The PCR product (about 5 ng), which contained the
full-length cDNA and a portion of the vector, was used as the
template for the second PCR. The final products were gel-purified,
restriction enzyme-digested, and subcloned into pSG5-PGIS to replace
the target segment. DNA sequencing was used to verify the sequence at
mutation and its surrounding region. The plasmids for transfection were
prepared using Qiagen plasmid purification kits (Qiagen).
To generate the expression vector for
glutathione S-transferase (GST) and PGIS (GST-PGIS) fusion
protein, PGIS cDNA (wild-type) was subcloned into pGEX-2T vector
(pGEX-PGIS). Transformed JM105 cells containing pGEX-PGIS DNA were
cultured at 37 °C in 2 × YT medium to
A600 = 0.8. Isopropyl--D-thiogalactopyranoside (0.2 mM)
was then added to induce protein expression, and cells were further
incubated at 37 °C for 3 h. Harvested cells were sonicated for
35 s and centrifuged at 1000 × g for 10 min. The
pellet which contained the fused GST-PGIS protein was washed with lysis
buffer (100 mM NaCl, 50 mM Tris-Cl, pH 8.0, 0.5% Triton X-100, 10 mM EDTA) and centrifuged before
being applied on SDS-polyacrylamide gel electrophoresis gels. The
~68-kDa GST-PGIS protein was dissected out from the
SDS-polyacrylamide gel electrophoresis gel and electroeluted. The
antibody against purified GST-PGIS was prepared in rabbits by H.T.I.
Bio-products (Ramona, CA).
COS-1 cells were grown to near-confluence in 100-mm tissue culture dishes in the presence of Dulbecco's modified Eagle's medium containing 2% fetal calf serum and 8% bovine calf serum at 37 °C in a humidified 5% CO2 atmosphere. Transfection procedure was performed as described by Wang et al. (11) with some modifications. In brief, cells were activated in fresh medium for 2 h before transfection. After washing with phosphate-buffered saline (PBS), COS-1 cells were incubated in 1 ml of PBS containing DEAE-dextran (0.5 mg/ml) and DNA (5 µg/ml) for 30 min in a lamina hood. 5 ml of Dulbecco's modified Eagle's medium containing 2% fetal calf serum and chloroquine (60 µg/ml) was then added, and the cells were incubated for additional 3 h at 37 °C in 5% CO2 atmosphere. After incubation, the cells were shocked with dimethyl sulfoxide (10%) for 2 min, and the medium was replaced with 12 ml of complete growth medium. The transfected cells were then incubated for 42-60 h. To harvest cells, COS-1 cells were scraped from the plate, pelleted by centrifugation, washed with PBS, and resuspended in 350 µl of PBS. The protein concentration of each sample was determined using BCA protein assay reagent kit (Pierce).
Determination of PGIS Protein Level by Western BlotImmunoblotting was performed by a procedure described previously (18). 15-20 µg of whole cells were boiled in an electrophoresis sample buffer (19) for 8-10 min before being applied to a 10% polyacrylamide minigel for electrophoresis. The resolved proteins were electrotransferred at 300-450 mA for 1 h to nitrocellulose membrane. Subsequently, the membrane was blocked with 3% non-fat milk in PBS and probed with a 1 to 1000 dilution of rabbit serum containing antibody against GST-PGIS fusion protein at 25 °C for 1 h or 4 °C overnight. A second antibody of goat anti-rabbit IgG conjugated with horseradish peroxidase was used as recommended. The protein bands were visualized by incubation with either the peroxidase substrate 4-chloro-1-naphthol (Bio-Rad) or Supersignal CL-HRP substrate system (Pierce). The levels of wild-type and mutant proteins on the Western blots were scanned by densitometer as described below.
Assay of PGIS ActivityThe assay was performed by mixing
whole cell homogenates (60-80 µg in PBS) with 5.5 µg of purified
sheep PGH2 synthase (20) provided by Dr. A.-L. Tsai, and
[14C]AA (10 µM) was added to the mixture in
a total volume of 100 µl. After addition of [14C]AA,
the tube was vortexed for the initial 30 s and incubated for 3 min. The reaction was terminated with 40 µl of methanol and 1 M citric acid (4:1, v/v). Organic products generated were extracted with 300 µl of diethyl ether twice. The organic extract which contained 92% ± 2% (n = 10) of the
radioactivity was concentrated under nitrogen to less than 60 µl and
applied to a TLC plate. The TLC plate was chilled on ice before placing
in a developing tank (on ice) in the organic phase of ethyl
acetate/2,2,4-trimethylpentane/acetic acid/water (110:50:20:100,
v/v/v/v; upper phase) (21). After development, the radioactive signals
on the TLC plate were detected by autoradiography. PGI2 was
hydrolyzed to 6-keto-PGF1 during extraction, therefore
the amount of [14C]6-keto-PGF1
produced
reflects the amount of PGI2 converted from
PGH2. The position of 6-keto-PGF1
on TLC
plate was determined by co-migration of enzymatic products of a
wild-type sample and nonlabeled 6-keto-PGF1
which was
visualized by iodine vapor. The radioactive signal of
[14C]6-keto-PGF1
on the autoradiography
was detected by using a densitometer (UMAX Vista-T630 or Epson ES-800C
scanners with the Adobe Photoshop program), and the intensity of each
signal was analyzed on a Macintosh computer using the public domain NIH Image program.
Radioimmunoassay was also used to determine the catalytic activity of
PGIS. Whole cell homogenate (60 µg of protein) was incubated with
PGH2 (5 µM) at room temperature for 3 min,
the reaction was terminated, and the organic products were extracted by
the procedure described above. The organic extract was dried under
nitrogen and resuspened in methanol. The extract was applied to
reverse-phase HPLC as described previously (22). The fractions
containing 6-keto-PGF1 were collected, dried, and
resuspended in methanol. 6-Keto-PGF1
contents in these
extracts were measured by radioimmunoassay as described previously
(23).
A three-dimensional model based on
the crystal structure of the hemoprotein domain of P450BM-3
was constructed. Details of the PGIS model will be published elsewhere.
The backbone structure of the PGIS model matched well with that of
P450BM-3, especially the helical regions (data not shown).
The crystal structure of P450BM-3 reveals a long binding
pocket lined with mostly nonaromatic hydrophobic residues (9). We
compared the substrate access channel of the PGIS model with that of
P450BM-3 by superimposing the substrate binding pocket
structure of PGIS model on that of P450BM-3 crystal
structure. Residues which were predicted from the three-dimensional
model to be important in substrate binding pocket and heme site are
shown in Fig. 2. Along the substrate access channel of
PGIS were hydrophobic residues, Ile67, Val76,
Thr206, Leu210, Leu213, and
Phe476, which correspond to Phe42,
Tyr51, Leu181, Met185,
Leu188, and Leu437 of P450BM-3
channel, respectively. To test the hypothesis that these hydrophobic
residues were important in PGIS catalytic activity, the cDNA
sequences at these six positions were altered individually to code for
recombinant PGIS mutants: Ile67 Lys (on
1-1),2 Val76
Asp (on
1-2), Thr206
Val, Leu210
Asp,
Leu213
Glu (on
F), and Phe476
Val
(between
4-1 and
4-2). Transient expression of each of these
mutants in COS-1 cells generated a level of PGIS protein comparable
with that of the wild-type (Fig. 3, A and
B). 6-Keto-PGF1
levels
produced by recombinant mutants Ile67
Lys,
Val76
Asp, and Leu210
Asp were markedly
reduced to the level of the mock-transfected control as analyzed by TLC
(Fig. 4). The TLC results were confirmed by
radioimmunoassay of 6-keto-PGF1
fractions separated by reverse-phase HPLC (data not shown). The catalytic activities of PGIS
mutants from multiple experiments expressed as percent of the wild-type
PGIS activity are summarized in Table I. The activities
of Ile67
Lys, Val76
Asp, and
Leu210
Asp were 5.1, 8.3, and 6.8% of the wild-type
enzyme, respectively. By contrast, catalytic activity of mutants
Thr206
Val, Leu213
Glu, or
Phe476
Val was not significantly different from that of
the wild-type (100, 97, and 83%, respectively; Table I and Fig. 4).
These results indicate that, as predicted by the three-dimensional
model, Ile67, Val76, and
Leu210 of PGIS are important in substrate access to
the active site, while contrary to prediction from the model,
Thr206, Leu213, and Phe476 are not
critically involved in substrate access.
|
Arg47, corresponding to Arg72 in PGIS model, is considered to be a "gate-keeper" for P450BM-3 enzyme. To determine whether this charged residue plays a similar role in PGIS, we altered it to Glu or Gln by site-directed mutagenesis. These two mutants expressed a similar quantity of proteins on Western blots as the wild-type (Fig. 3A) and had comparable catalytic activities as the wild-type enzyme (Table I and Fig. 4).
Ravichandran et al. (9) predicted from the crystal structure
of P450BM-3 that 1-4 strand was part of the substrate
binding sites and residues Ala328, Ala330, and
Met354 (on
1-3) were involved in the substrate binding.
In this region, according to sequence alignment and PGIS modeling,
three corresponding amino acid residues, Pro355,
Glu360, and Leu384, respectively, were
suggested to be involved in substrate binding (Fig. 2). A nearby
residue in TXAS, Arg413 (on
1-4) corresponding to
Asp364 in PGIS, was suggested to have interaction with
substrate (11). Each of these four amino acid residues in the
full-length PGIS cDNA was individually altered to
Pro355
Val, Glu360
Gly,
Asp364
Val, and Leu384
Asp. The level
of each mutant protein expressed in transient transfected COS-1 cells
was comparable with that of the wild-type (Fig. 3, A and B). PGIS
activity was significantly diminished in Asp364
Val and
Leu384
Asp mutants (6.8 and 6.9%, respectively; Table
I and Fig. 4), whereas Pro355
Val and
Glu360
Gly mutants retained a fraction of the wild-type
enzymatic activity (34 and 44%, respectively). These results indicate
that Pro355, Glu360, Leu384, and
Asp364 are important in substrate binding as predicted by
molecular modeling.
A cysteine residue which serves as the
proximal axial ligand for the heme iron through a thiolate bond is
conserved among all P450s. There are only two Cys residues in PGIS,
Cys231 (on G) and Cys441, of which
Cys441 corresponds to the consensus P450 cysteine (Figs. 1
and 2). Mutation of Cys441 to Ser by site-directed
mutagenesis resulted in a diminished enzyme activity (13%; Table I and
Fig. 4), without alteration in the expressed protein level (Fig.
3B). Mutation of Cys231 to Ser did not alter the
catalytic activity (98%) (Table I and Fig. 4).
In the heme binding region of cytochrome P450, an Arg or a His residue
is conserved which forms a hydrogen bond with the D-ring propionate group of the heme moiety. Alignment of amino acid sequence of the three-dimensional model of PGIS with that of
P450BM-3 did not reveal a corresponding Arg or His.
Instead, it suggested that Asn439 of PGIS was the
corresponding residue (Figs. 1 and 2). This raised the possibility that
the heme binding environment of PGIS may differ from that of other
P450s and Asn439 may be functionally important in heme
binding. To test this hypothesis, Asn439 was altered to
Ala. The protein level of mutant Asn439 Ala in
transient transfected COS-1 cells is similar to that of the wild-type.
Contrary to the hypothesis, mutation of Asn439 to Ala
resulted in a slight increase in the PGIS activity.
Crystallographic structure of P450BM-3 suggests that
Phe87 of P450BM-3 is involved in heme
interaction (9). However, neither sequence alignment nor molecular
modeling disclosed a corresponding Phe residue around this region in
PGIS. The corresponding residue derived from molecular modeling was
Leu112 (Fig. 2). To test if this Leu residue can provide
hydrophobic interactions with heme and substrate as the Phe residue in
other P450s does, Leu112 was mutated to Asp, Phe, or Gly by
site-directed mutagenesis. The catalytic activity of mutant
Leu112 Phe was only slightly lower (89%) than that of
the wild-type, while the activity of mutants Leu112
Asp
and Leu112
Gly was reduced to 11 and 15% of the
wild-type enzyme, respectively (Table I).
Pro113 was mutated as a control. Although situated next to
Leu112, this residue was not located at the vicinity of
heme or substrate pocket on the PGIS model. Pro113 Ala
mutant as predicted from the model retained most of the catalytic
activity (Table I).
Molecular modeling coupled with site-directed mutagenesis is a powerful tool in studying the structure-activity relationship of cytochrome P450 enzymes. PGIS is a new family of P450 which shares several enzymatic and spectral characteristics with TXAS. Based on our previous observations that the crystallographic structure of P450BM-3 serves as a suitable template for constructing the three-dimensional model of TXAS (10), we have taken a similar approach in constructing the three-dimensional model of PGIS, and the results indicate that the generated model is valuable for identifying the amino acid residues involved in substrate binding and heme environment.
Substrate Binding PocketThe PGIS model suggests that the substrate access channel of PGIS is similar to that of P450BM-3 in that it is very long and lined with hydrophobic residues. This result is not surprising since the substrate (PGH2) for PGIS is a 20-carbon metabolite of arachidonate, a substrate for P450BM-3. Our site-directed mutagenesis results indicate that Ile67, Val76, and Leu384, which are situated along the lower portion of the channel according to the PGIS model (Fig. 2), are critically important in PGIS catalytic activity. Mutations of these hydrophobic residues to charged residues reduced the catalytic activity to the background value. Hence, this portion of the binding pocket in PGIS is comparable with that of P450BM-3.
Two residues (Pro355 and Glu360) are located
inside the lower portion of the binding pocket adjacent to heme (Fig.
2). Mutations of these two residues, Glu360 Gly and
Pro355
Val retained 44 and 34% activity, respectively,
suggesting that these two charged residues are involved in catalytic
activity. We have shown previously that TXAS Arg413, which
is not predicted to be located at the binding pocket based on
P450BM-3 structure, is important in TXAS catalytic activity (11). This residue corresponds to PGIS Asp364. Mutation of
Asp364 to Val reduced the catalytic activity to the
background value, indicating that these two corresponding residues are
similarly important in PGIS and TXAS activities. According to the PGIS
model, this charged residue was located near the lining of the
substrate binding pocket but not in the vicinity of PGH2 or
heme (Fig. 2). Taken together, these results indicate that, with minor
exceptions, the lower part of the substrate binding pocket containing
1-1,
1-2,
1-3, and
1-4 strands in PGIS model (Fig. 2)
is comparable with that of P450BM-3.
By contrast, the upper portion of the substrate binding pocket of PGIS
was not as well predicted from the P450BM-3 structure. Only
one of the four predicted residues is important in substrate binding
and catalytic activity: Leu210 Asp lost the activity,
whereas Thr206
Val, Leu213
Glu, and
Phe476
Val retained the activity completely. It has
been indicated that Met185 and Leu437 in
P450BM-3 form strong van der Waals interactions (9). These two residues correspond to Leu210 and Phe476 in
PGIS by modeling. As mentioned above, the mutant Leu210
Asp completely lost the catalytic activity as predicted from the
P450BM-3 structure, whereas mutation of Phe476
to Val surprisingly had no effect on the activity. The discrepancy between the predicted and experimental data by site-directed
mutagenesis indicates that the molecular model around the upper portion
of the channel of PGIS (Fig. 2) containing most of
F helix
(Thr206, Leu210, and Leu213) is
different from that of P450BM-3. Since the polarity and
size of AA are different form PGH2, it is expected to find
disagreement between the structure of P450BM-3 (using AA as
substrate) and PGIS (using PGH2 as substrate) around the
substrate access channel.
Arg72 in PGIS model is corresponding to Arg47
in P450BM-3 structure. This P450BM-3 Arg
residue is located at the mouth of its access channel in strand 1-2
close to the molecular surface and is not well defined in the crystal
structure (9). Mutation of Arg to Glu in P450BM-3 blocks
the enzymatic reaction, and this residue was suggested to be important
in substrate recognition and binding (9, 24). Surprisingly, mutation of
the conserved Arg in PGIS to charged residue, Glu or Gln, of comparable
size did not significantly change the catalytic activity (101 and 80%, respectively). These results imply that the charge group of this Arg is
not important in substrate access and binding in PGIS. Unlike the
P450BM-3, which is a soluble enzyme, the PGIS has a trans-membrane domain at its N-terminal, which is close to the entrance
of the substrate binding pocket. We speculated that the entrance of
substrate channel of PGIS differs from that of P450BM-3 because of the influence of membrane topology of PGIS on substrate channel orientation.
The three-dimensional model of PGIS predicts Cys441 to be the proximal axial ligand for heme iron. This prediction was confirmed by site-directed mutagenesis. Hatae et al. (25) have recently obtained a similar result. This cysteine provides the thiol moiety to coordinate the heme. It has been shown that P450s exhibit a conserved sequence motif (F-G/S-X-G-X-R/H-X-C-hy-G, where hy denotes any hydrophobic residue) at the cysteine region near the C terminus of P450 proteins (26). This motif in PGIS, WGAGHNHCLG, differs from the conserved motif by two residues: substitutions of Trp (W) for Phe (F) and Asn (N) for Arg/His (R/H). Since Phe to Trp substitution also occurs in nitric oxide synthase, we altered the second substitution from Asn439 to Ala. This mutant surprisingly exhibited a slight increase in the catalytic activity. Asn439 is unlikely to be the corresponding conserved Arg or His. This result implies that structure around the heme environment of PGIS is different from that of other P450s.
The crystal structure of P450BM-3 predicted that Phe87 forms close van der Waals interactions with the heme on the distal side and the corresponding residue in P450cam provides hydrophobic interactions with the substrate camphor (27). The corresponding residue in PGIS is Leu112. It is interesting to note that alteration of Leu112 to Phe retained the catalytic activity whereas change of it to a charged residue (Asp) or a smaller residue (Gly) reduced the activity markedly. This result indicates that Leu can substitute Phe in this region to provide hydrophobic interaction with heme and probably also with PGH2.
Despite a marked loss of catalytic activity, mutants,
Cys441 Ser and Leu112
Asp or
Leu112
Gly, in which the heme environment is severely
perturbed still expressed intact proteins as detected by Western blots.
By contrast, TXAS in which the heme ligation or environment is
perturbed by site-directed mutation expresses a very low level of
proteins in cells (11). The stability of TXAS protein expression
requires heme in a correct orientation. On the other hand, stability of PGIS protein appears to be less dependent on heme. These results epitomize major structural differences between PGIS and TXAS despite a
similar substrate binding pocket.
Guided by the three-dimensional model, we have
identified nine amino acid residues which are near or line the
substrate binding pocket and are important in catalytic activity,
especially the residues in 1-1,
1-2,
1-3, and
1-4
strands region. We have also confirmed Cys441 as the
proximal axial ligand of heme iron. However, the model is imperfect and
fails to predict amino acid residues in other regions of the active
site pocket especially in the
F helix region, heme environment and
the substrate entrance gate-keeper. These inconsistencies between the
model and the experimental data offer an interesting opportunity for
further experiments to elucidate the different and potential new
structural characteristics of PGIS. One approach is to refine the model
based on the site-directed mutagenesis data from which additional
functionally important residues can be identified. It is hoped that,
through these experiments, the PGIS active site pocket and heme site
can be more precisely mapped. Eventually, these structures will have to
be confirmed by three-dimensional structure derived from x-ray
crystallography and/or NMR spectroscopy.
We thank Dr. Ah-Lim Tsai for providing sheep PGH2 synthase and Dr. Richard J. Kulmacz and Pei-Yung Hsu for valuable technical assistance.