(Received for publication, October 3, 1994; and in revised form, November 18, 1994)
From the
A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino
acids derived from cytochrome P450 2C2 sequence and 184 C-terminal
amino acids from cytochrome P450 2C1 acquires a novel progesterone
21-hydroxylase activity which is absent in the parent enzymes.
Extension of the cytochrome P450 2C2 sequence to residue 382 reduced
progesterone hydroxylase activity to 5% of that of C2MstC1, while
further extension to residue 411 or 462 increased activity back to
about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2
sequence to residue 382, substitution of cytochrome P450 2C1 amino
acids at positions 368, 369, and 374 increased progesterone hydroxylase
activity to a level equivalent to that of C2MstC1. In the chimera with
cytochrome P450 2C2 sequence extending to residue 411, substitutions of
P450 2C1 amino acids at positions 386 and 388, in addition those at
368, 369, and 374, were required to obtain activities equivalent to
that of C2MstC1, which suggests an interaction between these two
regions. The lauric acid hydroxylase activities of all chimeras and
mutant cytochromes P450 differed by 2-fold or less, demonstrating that
the changes in progesterone hydroxylase activity reflected altered
interactions with the substrate. Alignment of cytochrome P450 2C1
sequence with cytochromes P450, P450
, and
P450
predicts that residues 368/369 and 386/388 are in
adjacent antiparallel strands of the same
-sheet, in agreement
with the experimental data suggesting an interaction between these two
regions.
Cytochromes P450 (P450), ()constitute a superfamily
of proteins involved in oxidative metabolism of various endogenous
substances such as steroids, fatty acids, biogenic amines,
prostaglandins, and leukotrienes as well as exogenous substances
including drugs, plant metabolites, and a variety of
pollutants(1) . The P450 superfamily consists of at least 221
genes and 12 putative pseudogenes present in species from bacteria to
man with the 51 P450s described for the rat being the largest number
described for a single species(2) . The large number of P450s
accounts in part for the wide variety of substrates metabolized by
P450s, but individual P450s also may metabolize molecules with quite
different structures. Among the P450 subfamilies, subfamily C with 28
mammalian members has the greatest diversity.
In rabbit, eight
subfamily 2C genes have been reported(3) . These P450s
metabolize a variety of xenobiotics but most also metabolize endogenous
steroid molecules(3) . P450 2C3 is the predominant progesterone
16-hydroxylase in rabbit liver, but P450 2C14 and P450 2C16 also
are progesterone 16
-hydroxylases. A variant of P450 2C3, 2C3v, has
progesterone 6
in addition to 16
-hydroxylase activity. P450
2C5 is the major progesterone 21-hydroxylase in liver and the closely
related P450 2C4 has low progesterone 21-hydroxylase activity. In
contrast, hydroxylation of steroids by P450 2C1 and P450 2C2 either
cannot be detected or occurs at a very low
rate(3, 4) . Substrates of these two P450s include the
fatty acids, lauric acid and arachidonic acid. Both catalyze the
(
-1)-hydroxylation of lauric acid, and 11,12- and
14,15-epoxygenation of arachidonic
acid(4, 5, 6) . P450 2C2, but not P450 2C1,
also catalyzes the 19-hydroxylation of arachidonic acid. Interestingly,
a chimeric P450 consisting of the N-terminal 1-306 amino acids of
P450 2C2 and C-terminal 307-490 amino acids of P450 2C1 acquired
progesterone C21-hydroxylase activity, while the (
-1)-lauric acid
hydroxylase activity of P450 2C2 was retained(7) .
We have
shown that the amino acids in the N-terminal portion of P450 2C2 are
important for the progesterone hydroxylase activity of the chimera and
for the lauric acid hydroxylase activity of both the chimera and P450
2C2(5, 7, 8) . These amino acids are within
SRS-1, one of six such sites predicted from the alignment of P450 2C
sequences with P450, for which the three-dimensional
structure is known(9) . The remaining SRSs are scattered along
the entire P450 molecule. In the absence of a three-dimensional
structure for the mammalian P450 2C proteins, the validity of such
alignments with P450
can be tested by analysis of
chimeric and mutated P450s. The acquisition of progesterone hydroxylase
activity when the C-terminal 184 amino acids of P450 2C1 were
substituted for those of P450 2C2 indicates that regions in the
C-terminal portion of the P450 also are critical for recognition of
steroid substrates. In order to define the C-terminal regions
responsible for recognition of the progesterone substrate, several
chimeric genes encoding hybrid proteins containing N-terminal P450 2C2
sequence and successively shorter substitutions of P450 2C1 C-terminal
sequence have been constructed. Specific critical amino acids have been
identified by site-directed mutagenesis. The results indicate that
three C-terminal regions are important for progesterone hydroxylase
activity and that two of these regions interact with each other. These
experimental data are consistent with the relative positions of these
regions predicted by alignment with the amino acid sequences of
bacterial P450s for which three-dimensional structures have been
determined.
For mutations at residues 386 and 388,
pCMV5-C2tmBalC1 was used as a template in a PCR reaction. The primer at
one end hybridized to the pCMV5 sequence just 3` of the C2tmBalC1
insert and had the sequence 5`-CCACCCGGGGATCC-3`. The other mutating
primer, O386/388, extended from the StyI site to the region to
be mutated. The PCR fragment was digested with StyI and BamHI and ligated with MstII/BamHI-digested
pCMV5-C2tm DNA and the MstII-StyI fragment of
pCMV5-C2tm containing the mutations at 368, 369, and 374, to introduce
the additional mutations at 386 and 388 into C2tmBalC1. This
penta-mutant is referred to as C2pmBalC1. The mutations at 386 and 388
were then introduced into C2tm and C2tmHincC1 by substituting the StyI-BamHI fragments containing the mutations and are
referred to as C2pm and C2pmHincC1, respectively. Mutated sequences
were confirmed by sequencing using the Sanger's dideoxynucleotide
chain termination method with Sequenase version 2 (U. S.
Biochemical Corp.) according to the manufacturer's protocol.
Mutants in pCMV-C2StyC1 and pCMV5-C2tmBalC1 were sequenced using the
primers 5`-GTACAGCATAGAAGTCAG-3` and 5`-AAGTGGCCAGGGTC-3`,
respectively.
Figure 1: Schematic representation of the cDNAs of the parental, chimeric, and the mutant P450s that were analyzed. The P450 2C1 cDNA is represented by solid black, and the P450 2C2 cDNA is represented by cross-striped rectangles. The numbering at the top, represents the amino acid positions of the coding region of the protein. The six SRSs(9) , are represented by a horizontal line at the appropriate amino acid positions and are labeled as SRS1-SRS6. Splice sites represent the restriction enzyme sites that were used to make the chimeric P450s. The protein encoded by each of the cDNAs is labeled on the right-hand side of the diagram. The chimeras are designated by the N-terminal sequence, the restriction site at which the switch was made, and the C-terminal sequence in that order. In the case of C2StyC1 mutants, the sequence between amino acid residues 368 and 378 is shown, and, in the case of other mutants, the sequence between the amino acid positions 368 and 388 is shown. The P450 2C2 residues were mutated to P450 2C1 residues, by PCR, as described under ``Experimental Procedures.'' The P450 2C2 sequences are shown at the top, and the mutant amino acids at the corresponding positions are shown below.
Figure 2: Analysis of progesterone metabolites produced by the cell lysates of COS-1 cells. A mock-transfected culture, used as a negative control, is labeled as MOCK. The cell lysates were obtained and assayed for progesterone hydroxylase activity as described under ``Experimental Procedures.'' The positions of 21-hydroxyprogesterone and progesterone are indicated. Individual lanes are labeled by the corresponding proteins which catalyzed the reaction. A, a representative autoradiogram of a TLC of progesterone metabolites produced after transfection with the P450 2C1, P450 2C2, and their chimeric P450s. B, a representative autoradiogram of the progesterone metabolites, produced by the cell lysates of the COS-1 cells, transfected with the C2StyC1 mutants. As a control, activities of C2MstC1 and C2StyC1 are also included. The single and the double mutants are designated as wild type amino acid, the position, and the mutant amino acid in that order. A triple mutant (tm), of C2StyC1 with H368R, T369A, and L374V, is referred to as C2tmStyC1.
Figure 3:
Progesterone and lauric acid hydroxylase
activity in COS-1 cells transfected with the P450s 2C1, 2C2, their
chimeras, and the C2StyC1 mutants. Values plotted represent means and
standard errors for seven independent transfections. Top
panel, progesterone hydroxylase activity was determined as
described in Fig. 2. The position of the 21-hydroxylated product
was determined by co-migration with the unlabeled
21-hydroxyprogesterone. The bands containing the product were scraped,
and radioactivity was assayed by scintillation counting. Values for the
individual hydroxylase activities were normalized against that of
C2MstC1. Middle panel, (-1)-lauric acid hydroxylation. A
second aliquot of the cell lysates from COS-1 cells, which had been
used for the progesterone hydroxylase assay, was assayed for lauric
acid hydroxylase activity and analyzed by HPLC as described under
``Experimental Procedures.'' The means of seven independent
transfections for each of the proteins are plotted with the standard
error indicated. Values for the individual hydroxylase activities were
normalized against activity of the wild type P450 2C2. Bottom
panel, ratio of the means of the progesterone 21-hydroxylase
activity and the lauric acid hydroxylase
activity.
These changes in progesterone hydroxylase
activity potentially could be due to changes in the levels of
expression of functional enzymes for each chimera in the COS-1 cells.
To assess the effect of mutations on the stability of the proteins,
transfected COS-1 cells were labeled for 4 h with
TranS-label, and the expressed proteins were
immunoprecipitated(5) . A weak band co-migrated with P450 for
the mock-transfected cells, and a second band, of unknown origin,
migrated slightly faster than P450 2C2 and served as an useful internal
control for labeling and precipitation (Fig. 4). Since the
half-life of the newly expressed P450 in COS-1 cells has been shown to
be less than 1 h, labeling the cells for 4 h provides a reasonable
measure of the steady state levels of the protein(5) . No
differences greater than 2-fold in the amount of radioactive proteins
immunoprecipitated for P450 2C1, P450 2C2, the chimeric proteins, and
all the other mutants analyzed in this study were detected in five
independent experiments. The results demonstrate that the differences
in progesterone activity are not the result of altered stability or
expression of the P450 proteins.
Figure 4:
Immunoprecipitation of the P450s expressed
in COS-1 cells. Expression of immunoreactive P450 2C1, 2C2, their
chimeras, and mutants in COS-1 cells was analyzed. Forty-eight h after
transfection, COS-1 cells were labeled for 4 h with 60 µCi/ml
Tran[S]-label. After lysis, P450s were
immunoprecipitated using a polyclonal antiserum raised against P450 2C3 (12) . Immunoprecipitated proteins were analyzed by SDS-PAGE
and fluorography. P450 protein bands are marked by an arrow at
the left. Individual lanes are labeled by the corresponding
parental, chimeric, or the mutant P450 expressed, and the sample from
mock-transfected cells is labeled MOCK.
It is possible that either
differences in the fraction of total immunoprecipitable protein that
folds into a functional P450 or general effects of the mutations on the
catalytic activity rather than specific effects on interactions of the
substrate with P450 might cause the changes in progesterone activity.
The lauric acid hydroxylase activity of each of the chimeric proteins,
therefore, was analyzed in duplicate aliquots of the whole cell
extract. Results from seven experiments are summarized in Fig. 3, middle panel. Mock-transfected cells do not
show any detectable lauric acid hydroxylase activity. P450 2C1 is a
weak (-1)-lauric acid hydroxylase with activity about 10% of that
of P450 2C2 under these conditions. The activities of the chimeric
P450s ranged from 75% to 150% relative to that of P450 2C2 indicating
that the mutations did not have substantial effects on the expression
of functional enzyme or general catalytic activity. These changes are
similar to those reported for a chimera in which the last 28 amino
acids of P450 2C2 were replaced with those of P450 2C14(11) .
In Fig. 3, bottom panel, the progesterone hydroxylase activities of the chimeric proteins are normalized to those of the lauric acid hydroxylase activity by calculating the ratio of the two activities. The relative activities for the chimeras were similar for either the progesterone activity (Fig. 3, top panel) or normalized activity (Fig. 3, bottom panel). These results indicate that the P450 2C1 coding sequence between the MstII and StyI sites is important for interaction of progesterone with the P450. Further, P450 2C1 coding sequence between the StyI site and the BalI site interfered with these interactions since the C2BalC1 and C2HincC1 chimeras had similar activities severalfold greater than C2StyC1. Finally, the P450 2C1 sequence C-terminal of the HincII site, encoding 28 amino acids, was critical for progesterone interactions since the C2HincC1 chimera was a progesterone 21-hydroxylase, while P450 2C2 was not.
These three substitutions, which increase progesterone hydroxylase activity of C2StyC1 by 20-fold, were also introduced into P450 2C2, C2BalC1, and C2HincC1 to test whether they could similarly increase activity in these enzymes. No progesterone hydroxylase activity was detected when a single substitution was made in P450 2C2 at position 369, and barely detectable levels were observed for substitutions at 368 and 369 or at 368, 369, and 374 ( Fig. 5and Fig. 6). This result shows that in addition to these three P450 2C1 amino acids, there is still a requirement for the C-terminal P450 2C1 sequence for progesterone recognition. To identify the C-terminal P450 2C1 sequence required for the progesterone 21-hydroxylase activity, the three substitutions were introduced into C2BalC1 and C2HincC1 to produce C2tmBalC1 and C2tmHincC1, respectively. In these mutants, the three mutations are combined with the P450 2C2 sequence between the StyI and BalI sites and the P450 2C1 sequence from the HincII site to the termination codon, both of which increase progesterone hydroxylase activity based on the studies with the chimeras. Activities equivalent to C2MstC1 or greater were, therefore, anticipated. The results, however, showed that introduction of the three mutations into C2BalC1 and C2HincC1 had little or no effect on the activity of these two chimeras in contrast to the effects of these substitutions in C2StyC1 ( Fig. 5and Fig. 6). These results indicate that there is a requirement for the P450 2C1 sequence from the StyI to BalI sites encoding amino acids 382-411 for the increase in progesterone hydroxylation mediated by the three mutations introduced between the MstII and StyI sites encoding amino acids 306-382. Similar progesterone 21-hydroxylase activities of C2tmBalC1 and C2tmHincC1 eliminate a specific role for the P450 2C1 sequence between BalI and HincII for progesterone hydroxylation. These results suggest that amino acids at 368, 369, or 374 might interact with amino acids between residues 382 and 411.
Figure 5: A typical autoradiogram of the TLC showing the separated products of progesterone metabolism, catalyzed by the mutants of P450 2C2, C2StyC1, C2BalC1, and C2HincC1, expressed in COS-1 cells. The assays were done as described in the legend for Fig. 2. The ``tm'' refers to the triple mutation, involving H368R, T369A, and L374V. Activities of C2MstC1 and P450 2C2 are shown as controls. The positions of C21-hydroxyprogesterone and progesterone are indicated.
Figure 6: Summary of progesterone and lauric acid hydroxylase activities of mutations of P450 2C2, C2StyC1, C2BalC1, and C2HincC1. Values plotted represent means and standard errors for seven independent transfections. Both the progesterone and lauric acid hydroxylase activity assays were done on the same cell lysates. Analysis of progesterone hydroxylation (top panel), lauric acid hydroxylation (middle panel), and the ratio of the means of progesterone hydroxylation to lauric hydroxylation (bottom panel) were carried out as described in the legend to Fig. 3.
Figure 7: A typical autoradiogram showing the separated products of progesterone metabolism of COS-1 cells expressing P450 mutants at positions 386 and 388. The activity assays were done on the cell lysates as described in the legend for Fig. 2A. ``tm'' refers to the triple mutant indicated in the legend for Fig. 2B, and ``pm'' refers to the penta mutant, involving H368R, T369A, L374V, D386A, and L388I. When these five mutations are in P450 2C2, C2BalC1, and C2HincC1, the constructs are referred to as C2pm, C2pmBalC1, and C2pmHincC1, respectively. The mobilities of 21-hydroxyprogesterone and progesterone are indicated.
Figure 8: Summary of progesterone and lauric acid hydroxylase activities of P450s with mutations at 386 and 388. The values plotted here represent the means and standard errors of four independent transfections. Both the progesterone and lauric acid hydroxylase activities were performed on the same cell lysates of COS-1 cells transfected with the indicated constructs. Analysis of progesterone hydroxylation (top panel), lauric acid hydroxylation (middle panel), and the ratio of the means of progesterone hydroxylation to lauric hydroxylation (bottom panel) were carried out as described in the legend to Fig. 3.
These five substitutions were introduced into P450 2C2 and C2HincC1, producing C2pm and C2pmHincC1, respectively, to determine if they were sufficient to explain the progesterone 21-hydroxylase activity of C2MstC1. The introduction of the five mutations conferred detectable progesterone hydroxylase activity to P450 2C2 but only about 10% that of C2MstC1 ( Fig. 7and Fig. 8). This indicates that there is still a requirement for additional C-terminal amino acids of P450 2C1 in addition to the 5 residues identified, for maximum progesterone 21-hydroxylase activity. The introduction of the five mutations into C2HincC1 increased activity to levels comparable with those of C2MstC1 and C2pmStyC1 and about 10-fold higher than that of C2pm. This result indicates that the additional C-terminal P450 2C1 sequence required is in the C-terminal 28 residues encoded by sequence from the HincII site to the termination codon.
We have exploited the novel progesterone hydroxylase activity
in a chimera with N-terminal P450 2C2 sequence and C-terminal P450 2C1
sequence to identify regions and amino acids of P450 2C1 that confer
this activity. Previously, position 113 of P450 2C1 had been shown to
be important for progesterone 21-hydroxylase activity(12) . In
addition, amino acids near the C terminus were implicated in steroid
substrate recognition by the observation of a novel testosterone
16-hydroxylase activity if the 28 C-terminal amino acids of P450
2C2 were replaced with those of P450 2C14(11) , but not with
those of P450 2E1 or P450 2B5(13) . In this study, the
C-terminal 28 amino acids and two additional regions in the C-terminal
half of P450 2C1 were shown to contribute to progesterone
21-hydroxylase activity. Remarkably, the activity of lauric acid
hydroxylation in all of the chimeras and mutations tested differed by
only 2-fold while progesterone hydroxylation varied by 20-fold or more.
The relatively constant activity for lauric hydroxylation indicates
that the variations in progesterone hydroxylase activity are not due to
changes in either the expression levels of functional P450 in the COS-1
cells or the inherent activity of the enzyme and can be ascribed to
altered interactions of the substrate with the enzyme.
The experimental data indicate that the regions containing amino acids 368-374 and amino acids 386-388 may interact with each other. If P450 2C1 sequence was present in both regions as in C2MstC1 or C2tmStyC1, maximal activity was observed, but if P450 2C2 sequence replaced the P450 2C1 sequence in either the first region, C2StyC1, or the second region, C2tmBalC1, activity was reduced. Further, substitution of P450 2C1 amino acids at 368, 369, and 374 into C2StyC1 increased progesterone activity 20-fold. However, the same substitutions in C2BalC1 or C2HincC1 resulted in no increase in activity. Therefore, in order for the P450 2C1 substitutions at 368, 369, and 374 to increase progesterone 21-hydroxylase activity, P450 2C1 sequence had to be present in the region encoded by sequence between the StyI and BalI sites which indicates that amino acids in these two regions interact with each other. In the StyI/BalI region, substitutions of P450 2C1 amino acids at 386 and 388 in combination with those at 368, 369, and 374 resulted in maximal progesterone hydroxylase activity, suggesting that these 2 residues were near 368, 369, or 374 in P450 2C1.
The
experimental results indicating an interaction between the
368-374 and 386-388 regions are reinforced by predicted
positions of these amino acids in P450 2C1 based on alignments with
bacterial P450s for which the three-dimensional structure is known.
Alignment of rabbit P450 2C proteins with P450 as
proposed by Gotoh (9) and the alignment of P450
and P450
with P450
based on the
three-dimensional structure(14, 15) are shown in Fig. 9. In this alignment, residues 368 and 369 of P450 2C1
correspond to P450
residues Arg-299 and Ile-300 which are
in one strand of
-sheet 3 and line the substrate binding pocket.
P450 2C1 residues, 386 and 388, correspond to Glu-317 and Leu-319,
respectively, which are in the second strand of the same
3 sheet.
The side chains of Glu-317 and Leu-319 also protrude into the substrate
binding pocket. This alignment suggests, therefore, that these 4
residues are near the substrate binding pocket and that Ala-386 and
Ile-388 can interact with Arg-368 and Ala-369 or nearby residues
through hydrogen bonding, influencing the conformation of the
3
sheet and the shape of the substrate binding pocket. The effect of
changes at 374 which aligns at the end of the
3 sheet strand are
not as easily rationalized. Presumably amino acid changes at this
position might indirectly affect the
-strand lining the substrate
binding pocket. Based on the structure of P450
, Poulos
and his co-workers (16) have suggested that in order to
accommodate larger substrates yet maintain the required reaction
trajectory and interactions with the iron-bound oxygen atom, the
3
sheet is one of the segments that may undergo topological adjustments.
Our results suggesting an interaction between amino acids on separate
strands of the
3 sheet which affects interactions with a steroid
more than with a fatty acid substrate are consistent with this
proposal.
Figure 9:
Comparison of the sequences of rabbit P450
2C sequences with those of the bacterial P450s, P450,
P450
, and P450
. The P450 2C sequences are
aligned with P450
as described by (9) , and the
P450
and P450
are aligned with P
based on the three-dimensional structure as
described(14, 15) . Residues corresponding to
positions 368, 369, 374, 386, and 388 of P450 2C1, which contribute to
progesterone hydroxylase activity, are shaded. Brackets at the bottom indicate which of the bacterial amino acids
are present in two antiparallel strands of the same
-sheet.
Alignment of P450 2C1 and P450 2C2 amino acid sequences
with P450 is also consistent with interactions between
the amino acids at 368/369 and 386/388. Residues 363 to 370 of P450 2C1
and P450 2C2 correspond to residues 329 to 335 of P450
which forms the
-strand 1-4, and that between 384 and
390 corresponds to residues 350 to 356 in the adjacent antiparallel
-strand 1-3. In P450
, these two
strands
line the substrate binding pocket. Similar results were obtained when
the amino acid sequences of P450 2C1 and P450 2C2 were aligned with
that of P450
. The region between amino acids 366 and 371
of P450 2C1 and P450 2C2 corresponds to the
-strand 1-4 of
P450
(from residues 317 to 322), and that between
residues 384 and 390 corresponds to the adjacent antiparallel
-strand 1-3 (from residues 335 to 341). These two strands
also line the substrate binding pocket although the substrate-bound
models of P450
have implicated only one amino acid,
Phe-317 (corresponding to Val-366 of P450 2C1), in substrate binding.
The predicted positions of the residues 368/369 and 386/388 in P450 2C1
on the basis of alignment with all the P450s for which the
three-dimensional structure is known is consistent with the
experimental results that residues in these two regions interact and
affect interactions of the substrate with the enzyme.
Gotoh (9) proposed 6 SRSs scattered along the P450 molecule which
might be involved in the P450 substrate recognition. Of the five amino
acids we have identified in the C-terminal portion of P450 2C1 that
contribute to progesterone hydroxylase activity, only 368 and 369 are
within an SRS (SRS-5, residues 359 to 369) and would have been
predicted to affect substrate interactions. Mutations within SRS-5 have
been shown to affect the activity of other P450s. A S364(365)T change
is responsible for the difference in progesterone 6-hydroxylase
activity in two variants of P450 2C3(17) . The equivalent
residue in P450 2C1 as aligned by Gotoh and Fujii-Kuriyama (18) is shown in parentheses. An Ala substitution for
Val-363(362) conferred androgen 15
-hydroxylase activity to P450
2B1, and an Ala substitution for Val-367(366) conferred androgen
6
-hydroxylase activity to this enzyme(19, 20) .
An I380(369)F mutation in P450 2D1 affected bufuralol but not
debrisoquine metabolism (21) and a M366(361)L change was one of
three changes required for the alteration in substrate specificity of
P450 2A4 to that of 2A5(22) . The effects of mutations at 368
and 369 are, therefore, consistent with observations in other P450s and
provide additional support for the assignment of this region as an SRS.
In contrast, the other mutations contributing to progesterone
hydroxylase activity at positions 374, 386, and 388 are not within
SRSs. It seems most likely that these residues exert their effects by
interactions indirectly altering the geometry of the substrate pocket.
This possibility is consistent with the proposed interactions of
residues 386 and 388 with 368 and 369.
Since all the rabbit P450 2C enzymes, except P450 2C1 and P450 2C2, are steroid hydroxylases, it is possible that mutations of ``steroid-determining'' amino acids in the N- and C-terminal regions of P450 2C1 and P450 2C2, respectively, may explain the lack of progesterone 21-hydroxylase activity in the parent proteins, which is recovered in the C2MstC1 hybrid. Comparison of P450 2C1 and P450 2C2 sequences with P450 2C5 should be most instructive since P450 2C5, like C2MstC1, is a progesterone 21-hydroxylase. There is, however, no consistent similarity between the amino acids in P450 2C5 or the other P450 2C steroid hydroxylases and the amino acids at five positions in P450 2C1 that contribute to the steroid hydroxylase activity (Fig. 9). At position 368, all the steroid hydroxylases have His as in P450 2C2 rather than Arg as in P450 2C1. Most of the steroid hydroxylases have the P450 2C1 amino acid at 369 and the P450 2C2 amino acid at 386. The contribution of specific amino acids at these positions to steroid hydroxylase activity, therefore, is not simple but is context-dependent.
Previous studies in which C-terminal 28 amino acid substitutions conferred steroid hydroxylase activity led to the suggestion that the 28 C-terminal amino acids prevented or were not permissive for steroid substrates in P450 2C2(11) . The observation that 5 substitutions outside of the 28 C-terminal positions can also confer progesterone hydroxylase activity demonstrates the this C-terminal sequence does not completely block steroid substrate interactions. The substitutions in both the residue 368 to 388 region and the C-terminal region independently confer steroid hydroxylase activity and are additive in combination.