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INTRODUCTION |
Until the recent elucidation of the rabbit cytochrome
P450 2C5 x-ray crystal structure (1), chimeragenesis, site-directed mutagenesis, and homology modeling based on bacterial structures have
been the primary tools available to identify key residues responsible
for the substrate specificities of mammalian P450 enzymes (2). Most of
these residues belong to the substrate recognition sites
(SRSs)1 proposed by Gotoh (3)
based on analogy with the crystal structure of bacterial P450 101 and
have direct counterparts in the active site of P450 2C5. However, most
P450 structures reveal that the heme group is buried deep within the
protein matrix, indicating that residues outside of the active site may
also be required to guide the substrate into the heme pocket by
recognizing substrates at the protein surface and/or comprising
part of a substrate access channel (4-6).
The role of SRS and non-SRS residues in differential substrate
specificity and stereo- and regioselectivity within P450 subfamilies has been studied thoroughly, especially in the case of P450 2A, 2B, and
2C enzymes. In most cases, the functions of the enzymes could be
interconverted by making multiple reciprocal substitutions at SRS
residues (7-10). However, in a number of other studies, non-SRS
residues were shown to play a crucial role in determining substrate
specificities (11-17). These non-SRS residues were predicted to be
part of a substrate access channel near a region between the F and G
helices, as seen in bacterial P450 101, or between the B-C loop and N
terminus of the I helix, as seen in bacterial P450 51 (18-20). The
mammalian P450 2C5 structure supports the existence of both substrate
access channels (1).
Structure-function studies across P450 subfamilies have been largely
neglected until very recently (21, 22). Renewed interest in this area
was sparked by the considerable effort in a number of laboratories to
use the P450 2C5 structure to model other mammalian P450 enzymes and to
predict their substrate specificities and stereo- and regioselectivity
(2, 23-25). An implicit assumption in all such models based on a
single template is that the backbones of the enzymes are essentially
invariant and that active-site differences alone are responsible for
specificity differences. In this study, we sought to confer the
progesterone hydroxylation specificity of 2C5 on 2B1. 2B1 is a high
Km progesterone 16
-hydroxylase, whereas 2C5 is a
low Km progesterone 21-hydroxylase (26). 2B1 was
used as a model enzyme because of extensive previous site-directed
mutagenesis studies that have verified experimentally all 13 active-site residues inferred from the 2C5 structure (2, 21, 27-32).
Through a systematic approach of site-directed mutagenesis, a 2B1
enzyme (V103I/I114A/F206V/S294D/F297G/V363L/I477F) was constructed that
showed a 3-fold higher kcat compared with 2C5
and 80% regioselectivity for progesterone 21-hydroxylation. The
results suggest a dominant role of active-site side chains in
determining regioselectivity differences across these two P450 subfamilies and extend previous evidence for the reliability of 2B
models based on the 2C5 structure (24).
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EXPERIMENTAL PROCEDURES |
Materials--
Oligonucleotide primers were obtained from the
University of Texas Medical Branch Molecular Biology Core Laboratory.
[4-14C]Progesterone and [4-14C]testosterone
were obtained from PerkinElmer Life Sciences and Amersham Biosciences),
respectively. All other chemicals were purchased from sources
previously described (33) or from standard suppliers. Rat
NADPH-cytochrome P450 reductase and cytochrome b5 were prepared as described (34).
Site-directed Mutagenesis--
The truncated version of 2B1 that
served as the background for all mutations described in this study,
2B1dH, was generated using overlap extension PCR as described (33).
Mutants were constructed either by overlap extension PCR or by
subcloning using pKK2B1dH as a template. The primers and templates used
are shown in Fig. 1. Primary forward and
reverse primers were as previously described (33). To confirm the
desired mutation and to verify the absence of unintended mutations, all
constructs were sequenced at the University of Texas Medical Branch
Protein Chemistry Laboratory.

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Fig. 1.
Primers for the construction of 2B1dH
mutants. The codons changed to make the desired mutation are shown
in boldface, and nucleotide(s) changed to remove a
restriction site are underlined. Multiple mutants
constructed by subcloning are also shown with the restriction sites
used.
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Expression and Purification of P450 2B1dH Mutants--
2B1dH and
its mutants were expressed in Escherichia coli TOPP3
(Stratagene) and purified as described (33). In brief, bacteria were
grown in Terrific Broth salts for ~2.5 h at 37 °C before induction using isopropyl-
-D-thiogalactopyranoside and
supplementation with the heme precursor
-aminolevulinic acid. After
72 h at 30 °C, the cells were harvested by centrifugation, and
protein was extracted from lysed membranes using potassium phosphate
buffer (pH 7.4). Protein extract was loaded onto a Ni2+
affinity column, which was washed with 20 volumes of buffer, and then
eluted with 200 mM imidazole. Cytochrome P450 was
quantitated using the reduced CO difference spectrum (35). Specific
content was determined using the Pierce BCA kit with bovine serum
albumin as a standard. The specific content for 2B1dH was 18 nmol of
P450/mg of protein, whereas the specific content for most of the
mutants was between 8 and 16. However, the
I114A/F206V/S294D/F297G/V363L, I114A/F206V/S294D/F297G/V363L/I477F, and
V103I/I114A/F206V/S294D/F297G/V363L/I477F multiple mutants had specific
contents of 4, 5, and 2 nmol, respectively. Low specific content in
these mutants is accounted for by the presence of significant amounts
of P420.
Enzymatic Assays--
Progesterone and testosterone
hydroxylation assays were carried out essentially as described (33, 36)
using a 1:4:2 molar ratio of P450/cytochrome P450 reductase/cytochrome
b5 in the absence of lipid. 16
- and
15
-hydroxyprogesterone were not resolved. However, the progesterone
16
-hydroxylase activity of 2B1 has been measured previously using
two-dimensional chromatography (21). Km and
kcat values were determined by regression analysis using Sigma Plot (Jandel Scientific, San Rafael, CA).
Computer Modeling--
A molecular model of P450 2B1 was
constructed using the InsightII software package (Homology, Discover_3,
Biopolymer, Builder, and Docking from Molecular Simulations Inc., San
Diego, CA) and P450 2C5 as the template as described previously (24).
For the 2B1 mutant (V103I/I114A/F206V/S294D/F297G/V363L/I477F), the
coordinates of the corresponding residues were changed in the 2B1
three-dimensional model by Biopolymer, and the resulting 2B1 mutant was minimized.
The structure of progesterone was constructed using the Builder module.
The parameters for heme and ferryl oxygen were those described by
Paulsen and Ornstein (37, 38). During the docking calculations, the
system energy minimization and molecular dynamics simulations were
carried out with the Discover_3 program using the consistent valence
force field with a non-bond cutoff of 10 Å to a maximum gradient of 5 kcal mol
1 Å
1. Progesterone was
automatically docked into the three-dimensional models of 2B1 and
V103I/I114A/F206V/S294D/F297G/V363L/I477F in a reactive binding
orientation with the Docking module of InsightII, leading to 16
- or
21-hydroxylation. Because the initial oxidation step involves hydrogen
abstraction, the C-16 or C-21 atom was placed 3.7 Å from ferryl
oxygen, with the 16
-hydrogen or one of the hydrogen atoms bonded to
C-21 directed toward ferryl oxygen (C-H-ferryl oxygen angle of 180°)
to promote hydrogen bond formation. During the subsequent energy
minimization process, the substrate molecule, along with the side
chains of protein residues within 5 Å of the substrate, was allowed to
move. The non-bond interaction energies were evaluated with the Docking
module of InsightII, and the lowest energy orientation obtained after
molecular mechanics minimization of 2B1 and its mutant is shown in Fig.
6.
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RESULTS |
2B1dH Single Mutants I114A, F297G, and V363L Show Significant
Progesterone 21-Hydroxylase Activity--
2B1dH single mutants were
constructed at the nine active-site positions where 2B1 and 2C5 differ
and tested for oxidation of progesterone. Consistent with previous
findings, 2B1dH had negligible progesterone 21-hydroxylase activity,
but significant progesterone 16
-hydroxylase activity (>2
min
1) at 150 µM progesterone (Fig.
2), which was the highest substrate concentration obtainable under our conditions. However, the 2B1dH single mutants I114A, F297G, and V363L had 5-15% of 2C5dH
progesterone 21-hydroxylase activity. With regard to progesterone
16
- and 15
-hydroxylation, the I114A mutant showed enhanced
activity, whereas F297G and V363L showed decreased activity (Fig. 2).
Although V363L demonstrated lower activity for progesterone
21-hydroxylation compared with I114A, it showed high regioselectivity
for this reaction (>70%). The F206V substitution yielded relatively
high activity (6 min
1) and >95% regioselectivity for an
unknown product (Fig. 2). The progesterone hydroxylation profile of
V103I was similar to that of 2B1dH, whereas S294D, V367L, I477F, and
G478V showed negligible progesterone hydroxylase activity.

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Fig. 2.
Progesterone hydroxylation profiles of 2C5dH,
2B1dH, and 2B1dH single mutants. The progesterone hydroxylase
activities determined are indicated. The activity was measured at 150 µM substrate as described under "Experimental
Procedures." The bars represent the means obtained from
two independent determinations. 16 - and
15 -hydroxyprogesterone do not separate under the conditions
used. The structure of progesterone is shown in the inset,
with the 16 - and 21-positions indicated.
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The Km for Progesterone Is Decreased by ~13-Fold upon
F206V Substitution--
As shown in Table
I, the Km for
progesterone of 2B1dH was determined to be 200 µM.
Mutants I114A and F297G showed a 3-fold decrease in
Km, whereas V363L showed a 2-fold decrease.
Interestingly, a F206V substitution decreased the Km by ~13-fold. The kcat values for progesterone
21-hydroxylation by I114A, F297G, and V363L were in the range of 1-2
min
1 versus 16 min
1 for 2C5dH
(Table I).
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Table I
Steady-state kinetics of 2B1dH single mutants for progesterone
16 +15 - and 21-hydroxylase and unknown activities
Results are the means ± S.D. of three independent experiments.
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I114A/F206V/F297G/V363L Shows
Enhanced kcat and Regioselectivity for Progesterone
21-Hydroxylation--
A number of multiple mutants were constructed by
combining I114A, F297G, and V363L to test the additive effect on
progesterone 21-hydroxylase activity. In addition, F206V was also added
to assess whether this mutation decreases the Km.
The progesterone hydroxylation profiles of the multiple mutants are
presented in Fig. 3. As expected,
I114A/F297G (Fig. 3, A bars) showed enhanced progesterone
21-hydroxylase activity compared with either single mutant. Addition of
V363L to I114A/F297G increased the regioselectivity for progesterone
21-hydroxylation, although activity was suppressed (Fig. 3,
B bars versus A bars). Addition of
F206V to I114A and I114A/V363L caused high activity for unknown
products while suppressing progesterone 21- and 16
/15
-hydroxylase
activities (Fig. 3, C and E bars). However,
addition of F206V to I114/F297G did not suppress progesterone 21- and
16
/15
-hydroxylase activities (Fig. 3, D bars).
Interestingly, addition of F206V to I114A/F297G/V363L enhanced activity
for progesterone 21-hydroxylation and suppressed progesterone 16
-
and 15
-hydroxylase activity, but still allowed significant
production of unknown products (Fig. 3, F bars). Unlike 2B1dH, which produced only 16
-hydroxyprogesterone, and 2C5dH, which
produced only 21-hydroxyprogesterone, the quadruple mutant I114A/F206V/F297G/V363L (Q) produced two major and two minor unknown products (Fig. 4). 21-Hydroxyprogesterone
was determined to compose 57% of all the products in the
quadruple mutant. I114A/F206V, I114A/F206V/F297F, and I114A/F206V/V363L
had a decreased Km for progesterone
versus those mutants without Val206. The
Km for progesterone was unaltered in I114A/F297G and
I114A/F297G/V363L compared with the individual single mutants (Table
II), further suggesting the crucial role
of F206V in enhancing the affinity for the substrate. The
kcat for the quadruple mutant was 60% of that
for 2C5dH, and the Km was decreased by 3-4-fold
compared with 2B1dH (Tables I and II). The 10-fold increase in
kcat for progesterone 21-hydroxylation with no
change in Km upon addition of F206V to
I114A/F297G/V363L is striking and is different from other multiple
mutants that include F206V.

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Fig. 3.
Progesterone hydroxylation profiles of 2B1dH
multiple mutants. A bars, I114A/F297G; B
bars, I114A/F297G/V363L; C bars, I114A/F206V; D
bars, I114A/F206V/F297G; E bars, I114A/F206V/V363L;
F bars, I114A/F206V/F297G/V363L (quadruple mutant). The
progesterone hydroxylase activities determined are indicated. The
activity was measured at 150 µM substrate as described
under "Experimental Procedures." The bars represent the
means obtained from two independent determinations. 16 - and
15 -hydroxyprogesterone were isolated together.
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Fig. 4.
Thin-layer chromatography of progesterone
metabolites produced by 2B1dH, 2C5dH, and the 2B1dH quadruple
mutant. The quadruple mutant is 2B1dH I114A/F206V/F297G/V363L.
Unknown 3 is identical to the unknown obtained with F206V in
Fig. 2. 16 - and 15 -hydroxyprogesterone co-migrate and were
isolated together.
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Table II
Steady-state kinetics of 2B1dH multiple mutants for progesterone
21-hydroxylase and unknown activities
Results are the means ± S.D. of three independent experiments.
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The Testosterone Hydroxylation Profiles of S294D and I477F Are
Similar to That of 2C5dH--
Testosterone hydroxylation profiles were
determined for 2B1dH single mutants and compared with those of 2B1dH
and 2C5dH. As reported above, 2B1dH produced equal amounts of
16
- and 16
-hydroxytestosterone, whereas 2C5dH demonstrated mainly
testosterone 16
-hydroxylase activity (Fig.
5). Of the nine single mutants tested,
S294D, V363L, and I477F had decreased testosterone 16
-hydroxylase
activity and enhanced 16
-hydroxylase activity (similar to 2C5dH).
The Km of 2C5dH for testosterone (Table
III) was similar to the reported
Km of 2B1dH (39). However, the S294D and I477F
single mutants demonstrated a 2-fold lower Km for testosterone. Interestingly, F206V showed decreased 16
- and
16
-testosterone hydroxylase activities and a new testosterone
6
-hydroxylase activity. This profile of testosterone hydroxylation
is similar to the progesterone hydroxylation profile in that F206V
primarily showed activity for a new product (Figs. 2 and 5). On the
other hand, V103I showed unaltered testosterone hydroxylation, and
F297G, V367L, and G478V showed decreased testosterone 16
- and
16
-hydroxylase activities. The similar testosterone hydroxylation
profiles and affinities for substrate of 2C5dH, S294D, and I477F
suggested that addition of S294D and/or I477F to the 2B1dH quadruple
mutant might enhance progesterone 21-hydroxylase activity and/or
regioselectivity.

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Fig. 5.
Testosterone hydroxylation profiles of 2B1dH,
2C5dH, and 2B1dH single mutants. The testosterone hydroxylase
activities determined are indicated. The activity was measured at 200 µM substrate as described under "Experimental
Procedures." The bars represent the means obtained from
two independent determinations.
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Table III
Steady-state kinetics and regioselectivity for testosterone
16 -hydroxylase activity of 2B1dH, 2C5dH, and 2B1dH single mutants
Results are the means ± S.D. of two independent experiments.
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Q/I477F and Q/S294D/I477F Show
Enhanced Progesterone 21-Hydroxylase Activity and
Regioselectivity--
Q/S294D, Q/I477F, and Q/S294D/I477F were
examined for progesterone hydroxylation using 150 µM
substrate (Table IV). Progesterone 21-hydroxylase activity was largely unaffected, whereas
regioselectivity for Q/S294D, Q/S294D/I477F, and Q/I477F was increased
to 73, 69, and 67%, respectively, compared with 57% for the quadruple
mutant. Steady-state kinetic parameters were also measured for these
multiple mutants (Table IV). The kcat for
Q/S294D and Q/S294D/I477F was similar to that for the quadruple mutant,
but was increased for Q/I477F (similar to 2C5dH). However, the
Km was either unaffected in the case of Q/S294D and
Q/S294D/I477F or increased in the case of Q/I477F. These observations
suggested that addition of S294D to the quadruple mutant increased
regioselectivity for progesterone 21-hydroxylation, whereas I477F
enhanced the activity as well as the regioselectivity. The
kcat and regioselectivity for progesterone
21-hydroxylation were much improved over those for the quadruple mutant
and close to those for 2C5dH. To test further improvement of
progesterone 21-hydroxylase activity and regioselectivity, the
remaining three mutants (V103I, V367L, and G478V) were added to Q/I477F
and Q/S294D/ I477F.
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Table IV
Progesterone hydroxylation at 150 µM substrate and
steady-state kinetics and regioselectivity for progesterone
21-hydroxylase activity
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Addition of V103I to Q/I477F and
Q/S294D/I477F Further Enhances kcat
and Regioselectivity for Progesterone
21-Hydroxylation--
Q/V103I/I477F and Q/V103I/S294D/I477F
showed ~80% regioselectivity for progesterone 21-hydroxylation
(Table V). The
kcat for progesterone 21-hydroxylation by
Q/V103I/I477F and Q/V103I/S294D/I477F was 1.5- and 3-fold higher than
that for 2C5dH, respectively. The Km for
progesterone was largely unchanged from that of the mutants lacking
V103I (Table V versus Table IV). Addition of S294D to
Q/I477F and Q/V103I/I477F yielded high P420, as described under
"Experimental Procedures," whereas addition of S294D/V367L to
Q/V103I/I477F and Q/V103I/I477F/G478V yielded only P420 (data not shown). As expected based on the V367L and G478V single mutants, Q/V103I/V367L/I477F, Q/V103I/I477F/G478V, and Q/V103I/S294D/I477F/G478V showed much lower activity and regioselectivity for progesterone 21-hydroxylation compared with Q/V103I/I477F and Q/V103I/S294D/I477F (Table V). The Km for substrate was not
substantially affected compared with Q/V103I/I477F and
Q/V103I/S294D/I477F.
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Table V
Progesterone hydroxylation at 150 µM substrate and
steady-state kinetics and regioselectivity for progesterone
21-hydroxylase activity
Q/V103I/S294D/V367L/I477F and Q/V103I/S294D/V367L/I477F/G478V do not
express.
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Docking of Progesterone into the Active Site of 2B1dH and
Q/V103I/S294D/I477F Models--
To
explain the changes in regioselectivity observed for progesterone in
2B1 Q/V103I/S294D/I477F, a molecular model was constructed. Fig.
6 (A and B) shows
progesterone docked into the active site of the wild-type 2B1 and 2B1
Q/V103I/S294D/I477F models, respectively. The substrate fit well in the
wild-type 2B1 active site, with no van der Waals overlaps when docked
in an orientation that leads to formation of 16
-hydroxyprogesterone
(Fig. 6A). However, the substrate did not fit in a 21-OH
orientation. The estimated angle (C-H-ferryl oxygen) and distance
(between C-16 and ferryl oxygen) in 2B1 are 153.0° and 4.43 Å,
respectively. Active-site residues Ile114,
Phe206, Phe297, Val363, and
Ile477 are within 5 Å of the substrate. In contrast,
progesterone fit well in the 2B1 Q/V103I/S294D/I477F active site when
docked in a 21-OH orientation, but not in a 16
-OH orientation (Fig.
6B). The estimated angle (C-H-ferryl oxygen) and distance
(between C-21 and ferryl oxygen) are 160.6° and 3.69 Å,
respectively. These are close to the angle and distance required for
hydrogen bond formation (180° and 3.7 Å, respectively) and are
similar to those for 2C5 (Ref. 1 and data not shown). Active-site
residues Ile103, Ala114, Val206,
Asp294, Gly297, Leu363, and
Phe477 are within 5 Å of the substrate, with
Ala114 and Phe477 lying closest at 3 Å. Our
modeling results are consistent with the biochemical data that 2B1
favors 16
-hydroxylation, whereas 2B1 Q/V103I/S294D/I477F favors the
formation of 21-hydroxyprogesterone. Consistent with the experimental
data, modeling of progesterone in 2B1 mutants that included V367L
and/or G478V along with Q/V103I/S294D/I477F showed poor fits (data not
shown). This suggests that V367L and G478V mutations, either
individually or in combination with others, change the active-site
structure in a way that is unfavorable for progesterone
21-hydroxylation.

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Fig. 6.
Docking of progesterone into the active site
of P450 2B1 models. A, substrate was docked in
2B1dH in an orientation leading to formation of
16 -hydroxyprogesterone; B, substrate was docked into
Q/V103I/S294D/I477F in an orientation leading to
21-hydroxyprogesterone. The heme (red sticks), progesterone
(brown space-filling representation), and active-site
residues (purple sticks) are shown. The carbon atoms at
positions 16 and 21 of progesterone are shown in blue and
green, respectively.
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DISCUSSION |
The recent elucidation of the x-ray crystal structure of the first
mammalian P450 (rabbit 2C5) has sparked intense interest in
understanding the structural basis of P450 function to facilitate drug
discovery/design and engineering of novel biocatalysts (1). This
breakthrough has led to advanced homology models of drug-metabolizing cytochromes P450, especially the enzymes from the P450 2 subfamily, relative to those constructed based on bacterial enzymes (25). The P450
2C5 crystal structure has also provided an enhanced framework for
identifying active-site residues and investigating their role in
differential substrate specificities and stereo- and regioselectivity across subfamilies (2, 22). By extensive site-directed mutagenesis studies and more recently by analogy with 2C5, 13 2B1 active-site residues have been identified, nine of which differ from those of 2C5
(2). In the present study, 2B1 residues have been replaced systematically by the corresponding active-site residues of 2C5 to
confer a novel progesterone hydroxylase activity (progesterone 21-hydroxylation).
The major finding is that simultaneous substitution of seven 2B1
active-site residues (positions 103, 114, 206, 294, 297, 363, and 477)
with the corresponding 2C5 residues resulted in a 3-fold higher
kcat for progesterone 21-hydroxylation compared with 2C5 with 80% regioselectivity. However, the Km
for the substrate remained an order of magnitude higher than that for
2C5. Consistent with the experimental data, substrate docking in the
active site of a model of the multiple mutant showed that the formation
of 21-hydroxyprogesterone is favored, unlike wild-type 2B1, which
favors 16
-hydroxyprogesterone. To produce a 2B1 multiple mutant with
the desired phenotype, a three-tiered approach was used. First, 2B1
single mutants were made at all nine non-identical active-site
positions, and three mutants that showed progesterone 21-hydroxylation
(I114A, F297G, and V363L) and a fourth with a decreased
Km (F206V) were combined to yield a quadruple mutant. Second, additional substitutions (S294D and I477F) were added
based on a 2C5-like testosterone hydroxylation profile. Finally, V103I,
which had no effect on progesterone or testosterone hydroxylation
profiles on its own, was added to Q/S294D/ I477F.
New insights can be gained by comparing and contrasting the present
study with our recent investigation of active-site determinants of
specificity differences between P450 2B6 and 2E1 (22). In that case, a
single point mutation at the alignment position corresponding to
residue 477 in 2B enzymes conferred on 2E1 significant activity for the
2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin and
abolished activity for the 2E1-selective substrate
p-nitrophenol. However, none of six 2B6 single mutants
gained activity for p-nitrophenol. The two major advances in
this investigation were the focus on regioselectivity differences
for a common substrate, progesterone, and the generation of multiple 2B
mutants, many of which included substitutions that, on their own, did
not enhance the activity of interest. Based on the results, it appears
that, in addition to direct interactions of active-site residues with
substrate, residue-residue interactions and/or an influence of
active-site backbone residues on orientation of the substrate may also
be important (4, 24). Residue-residue interactions have been implicated
in determining stereo- and regioselectivity for androstenedione hydroxylation and differential inhibition by 4-phenylimidazole in 2B4
and 2B5 (7, 24). For example, mutagenesis experiments and molecular
modeling suggest that the side chains of residues 114 and 294 in 2B4
and 2B5 move in concert to influence the 4-phenylimidazole binding orientation (24). In the present case, there were increases in
progesterone 21-hydroxylase activity of 10-, 2-, and 3-fold upon
addition of F206V to I114A/F297G/V363L, I477F to
I114A/F206V/F297G/V363L, and V103I to Q/I477F, respectively,
even though F206V, I477F, and V103I alone showed negligible
progesterone 21-hydroxylase activity. These observations may reflect an
additional interaction between Phe477 and/or
Phe206 and progesterone that leads to tighter packing in
the active site and an increased frequency of productive collisions
(Fig. 6). Ile103 is also closer to progesterone in
Q/V103I/S294D/I477F compared with 2B1.
The almost complete conversion of stereoselectivity for testosterone
16-hydroxylation by 2B1 S294D, V363L, and I477F to that of 2C5 aided in
making multiple mutants with higher progesterone 21-hydroxylase
activity and regioselectivity. However, it should be recognized that
the 2C5-like testosterone hydroxylation profiles of S294D, V367L, and
I477F resulted from abolished testosterone 16
-hydroxylase activity
and enhanced 16
-hydroxylase activity, rather than acquisition of a
novel activity as with progesterone 21-hydroxylation by I114A, F297G,
and V363L. There are numerous prior examples of stereoselective loss of
activity upon single amino acid substitutions in bacterial and
mammalian P450 enzymes (28, 40, 41). On the other hand, F206V exhibited
a novel activity with both progesterone and testosterone along with
greatly suppressed original activities. Residue 206 in 2B1 has
previously been shown to be critical in converting steroid 16- to
15
-hydroxylation (21), and the analogous residue in 2a4
and 2a5 is a major determinant of differences in substrate specificity
(40). The F205V substitution in 2C5 results in almost a complete loss
of progesterone 21-hydroxylase activity and gain of new activity,
suggesting that this residue is critical in determining P450
regioselectivity (26). However, the structural basis for these
observations remains unclear.
In the absence of deleterious steric interactions with 2B1, the more
hydrophobic progesterone should be characterized by a lower
Km compared with testosterone, as observed with 2C5.
However, wild-type 2B1 exhibited a 7-fold higher Km for progesterone than for testosterone. This suggests that the larger
17
-acetyl group in progesterone, as opposed to the hydroxyl group in
testosterone, clashes with one or more residues in the 2B1 active site.
Furthermore, a F206V substitution in 2B1, either individually or in
combination with I114A, I114A/F297G, or I114A/V363L, decreased the
Km for progesterone by 6-13-fold, suggesting a
major role of Val206 in determining affinity. Other
multiple mutants that included F206V exhibited an increased
Km, which may be due to the occurrence of additional
unfavorable interactions. The Km is generally
affected by parameters such as size, shape, and hydrophobicity of the
substrate and of active-site residues (42). A similar Phe-to-Val
substitution at residue 226 in P450 1A2 (analogous to residue 206 in
2B1) has also been shown to have a strong bearing on substrate affinity
(43). An excellent correlation has been observed between
Km values and side chain size at residue 209 in P450
2a5 (analogous to residue 206 in 2B1), in which the Km values decrease as the side chains become larger
regardless of the hydrophobicity (44). The larger side chain of
Phe206 in 2B1 may interact sterically with the additional
acetyl group in progesterone compared with testosterone (Fig. 6) (1).
The F206V substitution may provide more room for entry in a particular orientation leading to increased affinity for substrate.
In summary, our results demonstrate that active-site
residues are mainly responsible for determining differences in
regioselectivity for progesterone hydroxylation between 2B1 and 2C5. A
synergistic effect on progesterone 21-hydroxylation activity and
regioselectivity by certain multiple substitutions suggests a role of
residue-residue interactions in determining active-site topology and
substrate orientation. This report suggests the feasibility of rational redesign of mammalian P450 specificity based on analogy with P450 2C5,
as previously performed for bacterial P450 enzymes of known three-dimensional structure (45). This approach provides an excellent
complement to directed evolution by random mutagenesis, which tends to
mainly pinpoint non-active-site residues (46, 47).