Molecular Modeling of Human P450c17 (17
-Hydroxylase/ 17,20-Lyase): Insights into Reaction Mechanisms and Effects of Mutations
Richard J. Auchus and
Walter L. Miller
Departments of Pediatrics (R.J.A., W.L.M.) and Internal Medicine
(R.J.A.) and The Metabolic Research Unit (W.L.M.) University of
California San Francisco, California 94143-0978
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ABSTRACT
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P450c17 (17
-hydroxylase/17,20-lyase) catalyzes
steroid 17
-hydroxylase and 17,20-lyase activities in the
biosynthesis of androgens and estrogens. These two activities are
differentially regulated in a tissue-specific and developmentally
programmed manner. To visualize the active site topology of human
P450c17 and to study the structural basis of its substrate specificity
and catalytic selectivity, we constructed a second-generation
computer-graphic model of human P450c17. The energetics of the model
are comparable to those of the principal template of the model,
P450BMP, as determined from its crystallographic coordinates. The
protein structure analysis programs PROCHECK, WHATIF, and SurVol
indicate that the predicted P450c17 structure is reasonable. The
hydrophobic active site accommodates both
4
and
5 steroid substrates in a catalytically
favorable orientation. The predicted contributions of positively
charged residues to the redox-partner binding site were confirmed by
site-directed mutagenesis. Molecular dynamic simulations with
pregnenolone, 17-OH-pregnenolone, progesterone, and 17-OH-progesterone
docked into the substrate-binding pocket demonstrated that
regioselectivity of the hydroxylation reactions is determined both by
proximity of hydrogens to the iron-oxo complex and by the stability of
the carbon radicals generated after hydrogen abstraction. The model
explains the activities of all known naturally occurring and synthetic
human P450c17 mutants. The model predicted that mutation of lysine 89
would disrupt 17,20-lyase activity to a greater extent than
17
-hydroxylase activity; expression of a test mutant, K89N, in yeast
confirmed this prediction. Hydrogen peroxide did not support catalysis
of the 17,20-lyase reaction, as would be predicited by mechanisms
involving a ferryl peroxide. Our present model and biochemical data
suggest that both the hydroxylase and lyase activities proceed from a
common steroid-binding geometry by an iron oxene mechanism. This model
will facilitate studies of sex steroid synthesis and its disorders and
the design of specific inhibitors useful in chemotherapy of sex
steroid-dependent cancers.
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INTRODUCTION
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Most steroid biosynthetic reactions are catalyzed by cytochrome
P450 enzymes (1). P450c17 (17
-hydroxylase/17,20-lyase), a microsomal
enzyme, catalyzes both steroid 17
-hydroxylation (needed for
glucocorticoid production), in which an oxygen is inserted into a C-H
bond, and 17,20-lyase activity (needed for sex steroid production), in
which 21-carbon 17
-hydroxysteroids are cleaved to 19-carbon,
17-ketosteroids, and acetic acid (2, 3, 4, 5). P450c17 can also catalyze a
modest degree of 16
-hydroxylase activity (6, 7, 8, 9) but does not
catalyze hepatic 16
-hydroxylase activity. Thus, P450c17 catalyzes
two fundamentally different reactions on a single active site, both of
which are crucial for human physiology.
In human steroidogenesis, P450c17 is the key branchpoint: in the
adrenal zona glomerulosa, where P450c17 is absent, steroidogenesis is
directed toward the mineralocorticoid aldosterone; in the adrenal zona
fasciculata, where the 17
-hydroxylase activity of P450c17
predominates, the glucocorticoid cortisol is produced; in the adrenal
zona reticularis and in the gonads, the presence of both
17
-hydroxylase and 17,20-lyase activities results in the synthesis
of sex steroids. For human P450c17, both
5-pregnenolone
and
4-progesterone are good substrates for the
17
-hydroxylase reaction, but
5 17-OH-pregnenolone is
preferred 100-fold over
4-17-OH-progesterone for the
17,20-lyase reaction (9). Furthermore, the 17
-hydroxylase and
17,20-lyase activities of human P450c17 can be regulated independently
(10). The 17,20-lyase activity of P450c17 is preferentially increased
by increased abundance of the redox partner P450-oxidoreductase (OR)
(7, 11, 12, 13), by serine/threonine phosphorylation of P450c17 (14), and
by the presence of cytochrome b5 (6, 7, 15, 16, 17, 18), which acts
as an allosteric facilitator but not as an electron donor (9). These
factors may contribute to human adrenarche, when adrenal synthesis of
C-19 steroids increases without a change in C-21 steroid synthesis (19, 20), and to the pathogenesis of the polycystic ovary syndrome (10, 14, 21).
A detailed understanding of the mechanisms that control the substrate
and reaction selectivity of P450c17 requires a detailed understanding
of the enzymes structure, active site topology, and substrate
reactivity. Crystals of membrane-bound mammalian P450 enzymes suitable
for crystallography have not been reported; hence all available
knowledge of the fine structure of P450 enzymes derives from the
crystal structures of soluble bacterial P450s. Most of these bacterial
enzymes are class I P450s, receiving electrons from a ferredoxin
intermediate, analogously to mammalian mitochondrial P450 enzymes.
Models of microsomal (class II) P450s based on bacterial class I P450s
have been suboptimal (22). However, the P450 moiety (termed P450BMP) of
the unique bacterial class II enzyme P450BM-3 (23) has been
crystallized (24) and its structure solved to 2.0 Å resolution (25).
Using this crystal structure as our principal template, we have now
constructed a second-generation model of human P450c17 that exhibits
highly favorable energetics, predicts the basis for its enzymatic
activities and redox partner interactions, explains the activities of
all published P450c17 mutants, and accurately predicts the activities
of novel mutants.
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RESULTS
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Model Construction
The modeling approach relies on the identification of predicted
core elements first and allows thermodynamics and molecular dynamics
(MD) to drive the final topology. The amino acid alignment used is
shown in Fig. 1
(see Materials and
Methods). Although conceptually straightforward, the process is
tedious and complicated by discrepancies in the lengths of the P450c17
and P450BMP sequences. Initial energy minimization attempts failed due
to prohibitively unfavorable residue overlaps in the initial model.
These regions were identified and repositioned by manual bond rotations
in Midas Plus. The model was then subjected to energy minimization,
first by applying 1000 cycles in the SANDER module of Amber 4.1 to each
of 13 isolated 28- to 33-amino acid segments in vacuo using
the BELLY option of Amber 4.1, and then by applying 1100 cycles of
energy minimization on the entire model (Fig. 2
). Each step in the modeling process
reduced the free energy of the model (Table 1
).

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Figure 1. Alignment of Core Elements of Human P450c17 and
P450BMP
The P450c17 model begins with the A-helix of P450BMP (residue 24 in
P450BMP; residue 48 in P450c17) and ends with the last residue traced
in the electron density map of P450BMP. Residues that comprise the core
helices and sheets are in boldface type, and the most
conserved residues (the C-helix tryptophan, catalytic threonine in the
I-helix, the ExxR sequence in the K-helix, and the heme-liganding
cysteine) are underlined.
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Figure 2. Tracing of P450c17 Model Intermediates Showing the
Carbon Backbone without the Amino Acid Side Chains
The heme is seen edge-on as a nearly straight line in the
center. A, Assembled core elements and loops after manual
adjustments without energy minimization (total energy 3.3 x
1022 kcal/mol). Note three areas that refold significantly
in subsequent panels: the F-G loop (arrow 1), the H-I loop (arrow 2),
and the loop between the meander and the heme-binding domain (arrow 3).
B, After initial energy minimization, the gaps in the backbone have
been eliminated, and regions 1, 2, and 3 have refolded significantly
but remain protruding from the main protein core (total energy
-4.3 x 103 kcal/mol). C, After sequential dynamics,
the total energy is unchanged but the 1, 2, and 3 regions have now been
folded against the main protein core. D, Final model, obtained as the
energy-minimized average structure during the final 40 psec of MD
simulation (total energy -4.6 x 104 kcal/mol).
Energetics have refolded numerous regions of the protein, in addition
to repositioning the side chains, which are not shown in the figure.
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Once the initial energy-minimized model was completed (Fig. 2B
),
inspection revealed that the three regions in which P450c17 contains
more amino acids than P450BMP settled into local energy minima rather
than sampling conformations that exploit hydrophobic packing
interactions with adjacent parts of the protein. To overcome energy
barriers preventing access to a greater range of conformations, these
three regions were sequentially isolated using the BELLY option but
this time subjected to MD simulations for 1545 psec at 300° K until
realistically packed stable conformations were achieved. The structure
obtained after these preliminary dynamics runs yielded the model in
Fig. 2C
with an energy of -4.0 x 103 kcal/mol.
Finally, to optimize and verify the stability and robustness of the
model, the structure was solvated via the EDIT module of Amber 4.1
using a 33.5-Å sphere of water molecules, centered asymmetrically to
exclude the hydrophobic patch near the amino terminus, which is the
presumed membrane attachment site. The water molecules were
pre-equilibrated using the BELLY option during 1000 cycles of energy
minimization, followed by 15 psec of MD at 300° K before a 100 psec
MD simulation of the entire solvated model using the SANDER module of
Amber 5.0 on the Cray T3E at the San Diego Supercomputing Center. An
average structure, encompassing the last 40 psec, was captured using
the CARNAL module of Amber and was energy minimized for another 1000
cycles using the steepest-descent method with SANDER (Table 1
),
yielding the final model (Fig. 3
). The
final energy of the solvated model is virtually identical to the free
energy of the comparably solvated P450BMP crystal structure (Table 1
).

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Figure 3. Final Model of P450c17
The protein is shown as a ribbon diagram in yellow with
the heme in red and the 33.5 Å sphere of hydration,
comprising 2508 water molecules, in cyan. The I-helix
can be seen above and to the left of the heme; the
ß-sheet domain and presumed membrane attachment site (which is not
hydrated) are to the right. The coordinates for all
atoms in the model are available from the Research Collaboratory for
Structural Bioinformatics, PDB ID code 2c17, RCSB ID RCSB001146 at
http://www.rcsb.org
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There are small but significant differences between the P450BMP
structure and the final P450c17 model. These include reduced bowing of
the I-helix, a slight kink in the F-helix, and the more spherical shape
in the P450c17 model. In some regions, there are significant
discrepancies in length: the F-G loop, which is longer and is
disordered in P450terp (26), adopts a gradual twisted turn, while the
H-I loop folds along the face of the protein in a twisted ß-sheet.
The loop before the heme-binding region puckers at residues G422 and
P428 to form a flap that tucks up into the body of the protein during
MD.
The model was evaluated with PROCHECK, WHATIF, and SurVol to
identify residues that deviated from expected values. The model scored
very well for side chain parameters, bad contacts, and distortion of
atom geometries, but less well for some main chain parameters (Table 2
). We improved the scores for main chain
parameters by isolating the regions of the protein containing residues
in "disallowed" areas of the Ramachandran plot (Table 2
) and
manipulating them manually. By changing dihedral angles of relevant
residues and returning the polypeptide to its original orientation, the
plot was "corrected." However, energy minimization, hydration, and
100 psec of MD with the corrected model returned some of the subject
residues to the "disallowed" regions of the Ramachandran plot.
Thus, although more favorable phi/psi angles are accessible during MD
simulations, the Amber force field consistently returns these residues
to other conformations.
Because the omega (amide) bond angles fell above the acceptable limit,
these were constrained by increasing the rotational barrier from 10
kcal/mol to 30 kcal/mol. Energy minimization and MD then produced
acceptable omega angle parameters, had minimal effects on the
Ramachandran plot or on other parameters, and improved the overall
G-factor from -0.41 to -0.31 in PROCHECK. However, these manipulated
structures had root-mean-square deviations of only <0.01 Å to 0.02 Å
from the original model. These attempts to refine the structure show
that 1) the default parameters in the parm94 or parm96 database of
Amber 4.1 tend to underconstrain amide bonds; 2) Amber 4.1 effectively
sets side-chain conformation and limits van der Waals overlaps, but may
not optimize phi/psi angles of a dynamic structure; and 3) because all
the residues in disallowed regions of the Ramachandran plot lie in
peripheral loops, MD returns the corrected structures nearly to their
original positions. Thus subsequent studies were done with the original
model.
The Heme-Binding Site
The highly conserved heme-binding site consists of residues P434
to I443, with C442 serving as the axial ligand of the heme iron. R440
forms a hydrogen bond with the heme proprionate moiety, a theme seen in
all P450 crystal structures (25). The mutation R440C causes complete
17
-hydroxylase deficiency (27), and the corresponding mutation R435C
in P450arom causes complete aromatase deficiency (28). The related
mutant H373L fails to bind heme and causes complete 17
-hydroxylase
deficiency (29). H373 does not interact with the heme, but forms a
hydrogen bond with the carboxylate of E391 in the adjacent strand of a
ß-sheet near the membrane attachment site. Thus, H373L appears to
create a global alteration of P450c17 structure that secondarily
prohibits heme binding.
The Substrate-Binding Pocket
The substrate-binding pocket is a space defined by the heme group,
which forms the floor of the pocket; the I-helix, which runs along one
edge of the heme ring; strands 4 and 5 of ß-sheet 1, opposite the
I-helix; I112 in the B'-C loop to one side; the loop after the K-helix
on the other side; and finally, V482 and V483, which form a turn in
ß-sheet 3, form the top of the pocket (Fig. 4A
). The side chains of
V482 and V483 lie above the heme, 3.3 Å closer to the oxene oxygen
than the corresponding side chain of I395 in the P450cam structure
(Fig. 4B
). The presence of three additional residues in P450c17, Q472,
L473, and P474 (Fig. 1
) forces the turn of ß-sheet 3 further into the
protein, limiting the extent of the pocket above the heme (Fig. 4B
).
Thus, our current model does not possess a bilobed substrate-binding
pocket as was predicted by preliminary models of P450c17 based on
P450cam (22, 30) or P450BMP (31). The smaller substrate-binding pocket
in the current model prohibits binding of steroids perpendicular to the
heme ring as proposed previously (22) and limits substrates to a
position in which the plane of the steroid is roughly parallel to the
plane of the heme ring (Fig. 4C
). This geometry constrains steroid
substrates to a single orientation with C17 above the heme
iron. In this orientation the C18 and C19
methyl groups participate in hydrophobic interactions with the side
chains of P368, V482, V483, and A302 (Fig. 4D
). The oxygen atom at
steroid carbon C3 lies adjacent to G95 in strand 5 of
ß-sheet 1. In interacting with G95,
4 (3-keto)
steroids form a hydrogen bond with the amide hydrogen while
5 (3ß-hydroxy) steroids form a hydrogen bond with the
carbonyl oxygen, during MD simulations.

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Figure 4. Active Site Region of P450c17 Containing
Pregnenolone Substrate
A, Stick diagram generated using the neon option of
Midas Plus. The heme group is shown in red; the ferryl
oxygen is green; the hydroxyl of T306 is
orange; hydrophobic residues of P450c17 are shown in
gray while hydrophilic residues are shown in
cyan. The steroid is yellow with carbon
17 and its attached hydrogen in blue. Note that the
entire substrate-binding pocket is defined by hydrophobic residues; the
two hydrophilic residues do not contribute to the surface boundary of
the pocket. B, Comparison of the crystal structures of P450cam
(cyan), P450BMP (yellow), and our model
of P450c17 (gray) showing the heme group and ß-sheet
3. This sheet extends closer to the heme and occupies more space in
P450BMP and P450c17 than in P450cam, restricting the size of the
substrate-binding pocket. Although the -carbons of P450c17 were
initially positioned in the same locations as the corresponding
-carbons of P450BMP, the MD procedures moved the P450c17 backbone
considerably, further restricting the size of the P450c17
substrate-binding pocket. C, The same perspective, scale, and color
scheme used in panel A are used with the conic
space-filling option of Midas Plus. This view shows that there is room
for water molecules above the steroid, but that the
steroid fills virtually all of the remainder of the substrate-binding
pocket. D, Cutaway view (saggital section using the
conic option) through T306 (orange), the
ferryl oxygen (green), and C17 of the steroid
(blue), showing the proximity of these three components
of the 17 -hydroxylase reaction.
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Although our modeling procedure did not presuppose an active site
topology, it is remarkable that the energy minimization and MD
coalesced distant residues to form an entirely hydrophobic
substrate-binding pocket whose geometry precisely fulfills the criteria
needed to catalyze the activities of P450c17. Valine residues 482 and
483 descend from their initial positions on the P450BMP template during
the energy minimization and dynamics procedures (Fig. 4B
) to reduce the
size of the substrate-binding pocket so that it can only accommodate
planar substrates such as steroids. It is of interest that the residues
corresponding to V482 and V483 in the P450c17 sequences of other
species are also both valines in mouse, rat, chicken, and trout,
Leu-Val in cattle and pigs, and Ile-Val in the guinea pig (32). The
critical position of the conserved V483 residue, which lies at the apex
of one side of the contiguous ß-sheets 3 and 4 (Fig. 1
) explains why
carboxyl-terminal mutations such as a frame shift after residue 480
(33), a premature stop codon at 461 (34), or deletion of the three
residues, D487-S488-F489 (35), completely eliminate all P450c17
enzymatic activity, while the far-carboxyl mutation R496C eliminates
about 90% of activity (34). All these mutations should disrupt this
extended ß-sheet structure, malpositioning the valines that form the
top of the substrate-binding pocket. Similarly, duplication of I112, a
highly conserved residue that forms one edge of the substrate-binding
pocket, also ablates all enzymatic activity (36). The positioning of
I112 is altered by the mutation S106P, which destroys all P450c17
activity (37) by disrupting the last turn of the B'-helix (Fig. 1
),
thus moving the B'-C loop that contains I112.
Aromatic hydrophobic residues, principally F53 and F54, line the cleft
between the ß-sheet domain and the
-helical domain inside the F-G
loop and may form an access pathway for entry of steroid substrates.
Deletion of either F53 or F54 reduces both the 17
-hydroxylase and
17,20-lyase activities of P450c17 equally but does not eliminate all
activity (38). A highly hydrophobic domain on the periphery of the
ß-sheet domain, including F93, F384, Y60, and Y64, and the
hydrophobic loop between F417 and F433 appear to contribute to membrane
attachment. The mutation Y64S destroys P450c17 activity (36), but it is
not clear whether this is associated with altered membrane binding.
Threonine 306 of P450c17 is the proton-donating threonine that is
generally required for catalysis and is conserved in almost all
cytochrome P450 enzymes (39). The hydroxyl group of T306 occupies the
corner of the substrate-binding pocket across the heme ring from the
D-ring of the steroid (Fig. 4D
). This location corresponds closely to
the location of the homologous threonine in three of the four available
cytochrome P450 crystal structures, permitting the hydroxyl proton to
participate in the dioxygen protonation and O-O bond cleavage needed
for the 17
-hydroxylase reaction. The precise mechanism for scission
of the dioxygen is uncertain; however, the P450c17 model suggests that
the T307 and acidic E305 that are adjacent to T306 may participate in a
charge-relay mechanism (40), consistent with the complete loss of
activity by the E305A/T306A double mutant (41). The
substrate-containing crystal structures of P450cam (39) and P450BMP
(25) contain several water molecules near this threonine hydroxyl;
however, the model of P450c17 containing pregnenolone accommodates only
one molecule of water near the heme proprionate and none closer
to T306. A lack of solvent in the active site when the steroid
substrate is bound may simply be a function of the parameters used for
hydrating the model; however, this result also suggests that
catalysis by wild-type P450c17 involves water molecules only after
formation of iron-oxygen complexes.
The Redox-Partner Binding Site
In P450BM3, the flavoprotein reductase domain donates
electrons through the redox-partner binding site on the
"underbelly" of the P450, on the opposite side of the heme moiety
from the substrate-binding pocket (25). This region has a large number
of basic residues, mostly lysines in P450BMP and arginines in P450c17,
yielding a surface charge distribution that is predominately positive.
Previous site-directed mutants in this region of rat P450c17, then
thought to contribute to the substrate-binding site (42), selectively
altered 17,20-lyase activity (43, 44). Using an intermediate version of
our current model, we recently showed that neutralization of two of
these surface charges with the naturally occurring mutations R347H or
R358Q resulted in selective loss of 17,20-lyase activity (12); the
final model remains completely consistent with this earlier
interpretation.
The recently determined x-ray crystal structure of the FMN and P450
domains of P450BM-3 (45), which is analogous to the interaction of the
FMN domain of OR with P450c17, illustrates how these mutations of the
P450c17 redox-partner binding site may preferentially impair
17,20-lyase activity. The surface of the FMN domain that interacts with
the P450 contains a cluster of acidic residues that interact with the
basic residues in the P450s redox-partner binding site. However,
these interactions are weak and indirect, as the protein surfaces
remain separated by several angstroms; furthermore, electron transfer
from the FMN to the heme does not occur across these charged surfaces,
but occurs at an adjacent site. We suggest that when one of these basic
residues is neutralized in P450c17, electron transfer still occurs, but
there is a subtle disruption in the P450c17OR(b5)
complex. Thus, the R347H and R358Q mutants have nearly the same
Michaelis-Menten constant (Km) as the wild-type P450c17,
but substantially slower Vmax for the 17
-hydroxylase
reaction (13). The preferred reaction is 17
-hydroxylation; it occurs
at the most reactive C-H bond in this part of the steroid and it is
relatively insensitive to perturbations of the P450OR geometry. By
contrast, the 17,20-lyase reaction is a complex cleavage of a C-C bond
that few P450 enzymes catalyze; this C-C bond scission can occur in a
very narrow range of P450OR geometries and is markedly stimulated by
the allosteric action of b5, presumably by optimizing the
topology of this complex (9, 13).
It has been suggested that F417 also lies in the redox-partner binding
site, as the mutation F417C also selectively affects 17,20-lyase
activity (46). Our model indicates that F417 is positioned just
C-terminal to the "meander" decapeptide but is not accessible to
solvent and hence cannot form part of the redox-partner binding site.
However, F417 appears to contribute to hydrophobic interactions that
help to stabilize the flap between the heme-binding domain and the
meander; this flap lacks basic residues but is adjacent to R358. Thus
F417 may help to form an edge of the redox-partner binding site, but is
not itself a component of that surface. P342 is located at the start of
the J'-helix, which contains the R347 residue that is crucial for
redox-partner binding (12). The mutation P342T, which causes a partial
loss of both 17
-hydroxylase and 17,20-lyase activities (47), creates
a Thr-Thr-Thr motif that would extend and misdirect the J'-helix,
introducing substantial structural changes in the redox partner binding
site. Thus, not all mutations in the redox-partner binding site cause a
selective loss of 17,20-lyase activity; rather, this behavior appears
to be confined to mutations affecting a cluster of basic residues. To
test this, we built a novel mutant by site-directed mutagenesis.
Site-Directed Mutagenesis
Arginine residues 347 and 358 contribute two large positive
charges to the proposed redox-partner binding site, but the model
predicts that other positive charges also lie on this surface beneath
the heme. Among these, K89 is closest to both the heme and to R347 and
R358 and might also contribute to interactions with OR. Therefore we
hypothesized that mutation of K89 to a neutral, polar residue would
preferentially disrupt 17,20-lyase activity while preserving substrate
binding and most 17
-hydroxylase activity, similarly to the R347H and
R358Q mutations. When the K89N mutant was coexpressed in yeast with
human OR, about 84% of 17
-hydroxylase activity was retained but
only 22% of 17,20-lyase activity was retained (Fig. 5
). As predicted, the binding of
17-OH-pregnenolone was not affected appreciably, with a Km
of 0.8 µM as substrate and an inhibitory constant
(Ki) of 0.5 µM as a competitive inhibitor of
the 17
-hydroxylase reaction, as compared with a Km of
0.4 µM and a Ki of 0.7 µM for
the wild-type enzyme and Ki values of 0.3 µM
and 0.8 µM for the R347H and R358Q mutants, respectively
(13). Thus, the K89N mutation does not alter substrate binding yet
reduces the ratio of 17,20-lyase activity to 17
-hydroxylase activity
(in the absence of b5) from 1:30 to 1:100. These data
suggest that K89, which is quite distant from R347 and R358 in the
linear amino acid sequence, is part of the redox-partner binding site,
folded into close proximity with R347 and R358, as the model
predicts.

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Figure 5. Kinetics of the K89N Mutant and Wild-Type Human
P450c17
The graphs show Lineweaver-Burk plots of enzymatic activity measured in
microsomes from yeast coexpressing the P450c17 enzyme and human OR. A,
Measurement of 17 -hydroxylase activity for K89N mutant using
[3H] pregnenolone as substrate in the absence
(squares) or presence (circles) of 5
µM 17-OH-pregnenolone as inhibitor. B, Measurement of
17,20-lyase activity for K89N mutant using
[3H]17-OH-pregnenolone as substrate. C, 17 -Hydroxylase
activity of wild-type P450c17 using [3H]pregnenolone as
substrate (conditions identical to those used to obtain data
represented by squares in panel A, which is a range of
substrate concentrations that are below saturation). D, Measurement of
17,20-lyase activity for wild-type P450c17 using
[3H]17-OH-pregnenolone as substrate (conditions identical
to those used to obtain data in panel B). Km,
Ki, and Vmax values were determined from
lines fitted by least-squares (13 ).
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Our model also explains the results of mutagenesis experiments that
were based on our previous model but could not be explained by that
model (22). The substitutions D298V, D298S, and G301I lie in the
I-helix, where partially disrupted hydrogen bonding near the
catalytically important T306 appears to be critical for P450 activity
(25). These nonconservative substitutions probably perturb the ability
of this long helix to participate in catalysis. The mutation G111D,
which is adjacent to the I112 that borders the active site, eliminates
all activity, probably by disrupting substrate binding. Conversely, the
model predicts that the conservative mutants M369I and I371L, which lie
in an adjacent strand of the ß-sheet that interacts with the C3
hydroxyl or keto group, and the conservative mutant L102T, which is
midway through the B'-helix, should not alter the locations of
functionally important residues, and indeed all of these mutants retain
more than 50% of activity (22).
MD: 17
-Hydroxylase Reaction
During unconstrained MD simulations with pregnenolone or
progesterone bound to the model, these steroid substrates remain
tightly bound to the active site without major reorientation. This
behavior is consistent with the slow dissociation rates (0.1
sec-1) of steroids from guinea pig P450c17 (48) and
provides strong evidence that the active site was modeled accurately.
Five hydrogen atoms (H16
, H17
, and the three H21 atoms) approach
sufficiently close to the heme reaction center to be susceptible to
oxygen insertion reactions (Table 3
).
Curiously, it is the three C21 hydrogens that have the shortest average
and minimum distances from the oxene oxygen, but these hydrogens would
be the least amenable to hydrogen atom abstraction due to the
instability of the resulting primary carbon radicals (49). H17
is
the next closest hydrogen atom to the activated oxygen. Abstraction of
this atom would yield a tertiary carbon radical; H16
is only
slightly more distant from the reaction center (Table 3
). Thus, our
model predicts that, during the hydroxylation reaction, carbons 16, 17,
and 21 straddle the heme iron oxene, presenting five hydrogen atoms as
potential sites for oxygenation. The actual reactions chosen are
determined by both reactivity and geometry, so that the H17
methine
and H16
methylene hydrogens become the principal and secondary sites
of reaction, respectively.
MD: 17,20-Lyase Reaction
Because the chemical mechanism of the 17,20-lyase reaction has
been controversial, it was not obvious how to model the heme moiety
during this reaction. A ferryl peroxide has been suggested (50, 51),
but mechanisms using the same iron oxene believed to mediate the
hydroxylase reaction appear equally likely on theoretical grounds. An
iron oxene mechanism is wholly consistent with the isotopic labeling
data that led to the ferryl peroxide proposal (50). A mechanism that
involves ferryl peroxides has been shown for the deformylation of
cyclohexane carboxaldehyde by P450-LM2 (52) and implies
that H2O2 alone should support the 17,20-lyase
reaction. Therefore, we tested the ability of
H2O2 and other oxidizing agents to support
17,20-lyase activity in our yeast microsome system (9) containing human
P450c17 with or without human OR. We measured the 17,20-lyase activity
supported by iodosobenzene or cumene hydroperoxide, which would suggest
an oxene mechanism, or lyase activity supported by
H2O2, which would suggest a peroxide mechanism.
However none of these reagents alone supported either the
17
-hydroxylase or 17,20-lyase reactions (Fig. 6
). It is quite possible that bulky
organic reagents such as iodosobenzene and cumene hydroperoxide
cannot reach the active site; hence these experiments may not be
informative. However, H2O2 is about the same
size as H2O and O2, both of which reach the
active site. We believe that the inability of
H2O2 to support catalysis makes a peroxide
mechanism more problematic. While these data do not exclude a ferryl
peroxide mechanism during the conversion of pregnenolone to alternate
products (50) or the metabolism of synthetic substrates (41), the lack
of catalysis in the presence of H2O2 prompted
us to model the heme as the iron oxene species for the lyase reaction.
The orientations of the steroid substrates were not changed from those
of the hydroxylase substrates, since the 17-OH-derivatives are products
of the hydroxylase reaction that do not need to dissociate from the
enzyme before 17,20-lyase conversion to 19-carbon 17-ketosteroids (48, 53). Throughout our dynamics simulations, the hydrogen of the
17-OH-group remained much closer to the oxene than any other steroid
atoms. Only subtle differences were calculated between the
distances of the potentially reactive steroid atoms in
17-OH-pregnenolone and 17-OH-progesterone from the oxene oxygen
(Table 3
).

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Figure 6. Evidence against a Ferryl Peroxide Mechanism for
the 17,20-Lyase Reaction
Yeast microsomes containing 1 pmol human P450c17 (measured by
difference spectra) and an excess of human OR were incubated with 2
pmol of [3H]pregnenolone or 2 pmol of
[3H]17OH-pregnenolone with 1 mM of the
indicated oxidizing agent at 37° for 1 h, and the steroidal
products were analyzed by TLC and autoradiography. PhIO, Iodosobenzene;
CumOOH, cumene hydroperoxide; H2O2, hydrogen
peroxide. In the experiments with the pregnenolone substrate, the trace
of radioactivity comigrating with 17-OH-pregnenolone may represent
trace catalysis with NADPH found in the microsomes or may represent the
formation of 20 -hydroxy-pregnanediol by endogenous yeast
20 -hydroxysteroid dehydrogenase. Note that no DHEA is
formed in any reaction.
|
|
Formation of the iron oxene with 17-OH steroids in the active site
predicts two consequences. First, formation of a hydroxyl radical by
abstraction of the hydrogen from C17-OH is the overwhelmingly favored
route of oxene catabolism, due both to proximity of this hydrogen atom
with the iron oxene and to the greater stability of hydroxyl radicals
over most carbon radicals (49), suggesting the radical fragmentation
mechanism in Fig. 7
. Second, crowding of
the active site, with 17-OH-progesterone closer to the heme iron
than 17-OH-pregnenolone, suggests that the enzyme may be
relatively unable to accommodate both 17-OH-progesterone and oxygen to
generate an active oxidizing species, thus accounting for the 50- to
100-fold preference for 17-OH-pregnenolone as the substrate for the
17,20-lyase reaction (9). In fact, the close apposition of
17-hydroxysteroids to the heme might preclude the reaction of bound
steroid with a ferryl peroxide.

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Figure 7. Proposed Mechanism of the 17,20-Lyase Reaction
The ferryl oxene permits hydrogen abstraction at the C17 hydroxyl of
17-OH-pregnenolone (I) to generate a hydroxyl radical (II) that
fragments into DHEA (III) and an acetyl radical (IV).
Recombination with the heme hydroxyl yields acetic acid and regenerates
the resting-state heme.
|
|
 |
DISCUSSION
|
---|
Human adrenal 17,20-lyase activity, as indicated by serum
concentrations of dehydroepiandrosterone (DHEA) and
DHEA-sulfate, increases sharply at adrenarche at age
610, peaks at age 2535, and then falls slowly with advancing age
(54). The basis of these changes is unclear, although several factors
influence the ratio of the 17,20-lyase to 17
-hydroxylase activities
of human P450c17 (10). 17,20-Lyase activity is preferentially increased
by 1) an increased abundance of OR (7, 9, 11); 2) by the presence of
cytochrome b5 (7, 9, 15, 17, 18); 3) by serine/threonine
phosphorylation of P450c17 (14); and 4) by proper electrostatic charge
distribution in the redox-partner binding site of P450c17 (12, 13, 55).
Furthermore, 17-OH-pregnenolone is by far the preferred substrate for
the 17,20-lyase activity of human P450c17 (7, 9, 17, 37). These
observations suggest that subtle changes in active site topology
profoundly alter the ability of P450c17 to catalyze the 17,20-lyase
reaction. Therefore, we sought to define the three-dimensional
structure for human P450c17 with and without binding of its four
principal substrates.
Our present model is a significant advance over previous models of
human P450c17 based on the structure of P450cam (22, 30) or P450BMP
(31). Those previous models indicated that P450c17 had a bi-lobed
substrate-binding pocket, with each lobe binding steroid substrates
differently to catalyze either the 17
-hydroxylase or the 17,20-lyase
reactions. By contrast, our current model is based on the hypothesis
that evolution tends to conserve secondary and tertiary structures to a
greater extent than amino acid sequences (56) and depends heavily on
both energetics and MD to adjust the folding of the final structure. As
a result, our model has at least six characteristics required of an
accurate P450c17 model. First, the model scores well in a variety of
programs that evaluate protein structures. Second, the energetics of
the model are as good as those of the x-ray crystal structure of
P450BMP. Third, the model accommodates the expected substrates with
sufficient flexibility near the A-ring to accept both
5-
and
4-steroids. Fourth, the model correctly predicts
that P450c17 will have 16
-hydroxylase activity. Fifth, the model is
consistent with all available data on the activity of human P450c17
mutants. Sixth, the model correctly predicts that the K89N mutant will
exhibit selective reduction in 17,20-lyase activity without affecting
the active site. While this manuscript was in preparation, others
reported that the mutations K83A, K88A, K91A, R126A, R347A, and R358A
in an altered soluble form of P450c17 also had a greater effect on
lyase activity than hydroxylase activity, so that the hydroxylase/lyase
ratio increased 2- to 4.5-fold (55). Our model predicts that all of
these basic residues lie in or near the redox-partner binding site.
While it is likely that our model differs in some respects from the
true structure of P450c17, the goodness of fit described above
indicates that this model can be used to test hypotheses about P450c17
structure and enzymology.
Our model prompts us to propose that the enzyme uses the same active
oxygenating intermediate for both the 17
-hydroxylase and 17,20-lyase
reactions. The proposed iron oxene mechanism requires that the steroid
occupy only a single position in the substrate-binding pocket, thus
avoiding the awkward and energetically improbable shifting of the
steroid A-ring from one pocket to another, as required by prior
bi-lobed models but which is not supported by kinetic data (48, 53).
Our recent kinetic data with the wild-type enzyme (9), with the R347H
and R358Q mutants (13), and with the novel K89N mutant (Fig. 5
), plus
the inability of hydrogen peroxide to support catalysis (Fig. 6
) are
all consistent with our current structure and the proposed iron-oxene
reaction mechanism. Thus, our model and complementary experimental
results challenge the prevailing biochemical concepts about how P450c17
binds substrates and catalyzes the 17,20-lyase reaction, thus opening
potential new pharmacological approaches to the selective inhibition of
17,20-lyase activity.
P450c17 and P450c21 are closely related enzymes in amino acid
sequence, gene structure, and ability to use the same steroid,
progesterone, as substrate (57). Our P450c17 model offers some insights
into the apparent requirements of the active site of P450c21. Our model
indicates that when the
-hydrogens at steroid positions C16 and C17
are sufficiently close to the oxene oxygen to allow 16
- and
17
-hydroxylation, the C21 hydrogens also becomes susceptible to the
radical abstraction reaction. Therefore, P450c21 must suppress
reactivity at C16 and C17 by presenting only the less reactive C21
hydrogens to the oxene oxygen by preventing the steroid D-ring from
approaching the center of the heme.
Our heuristic model of P450c17 permits us to examine the structural
factors that govern the interactions of P450c17 with the OR and of this
complex with b5, which strongly influence 17,20-lyase
activity. Previous studies (9, 11, 12, 13, 17, 18) clearly show that a
description of the 17,20-lyase reaction is incomplete without detailed
understanding of how OR facilitates the generation of the oxygenating
species from which catalysis occurs. We have proposed a model of how
b5 allosterically promotes the interaction of OR with
P450c17 to facilitate electron donation and catalysis (9) and have
shown how the R347H and R358Q mutants support the model (12, 13);
others have initiated efforts to dock b5 with models of
other eukaryotic P450 enzymes (58). The determination of the x-ray
crystal structure of rat OR (59) will facilitate modeling of the
reductase-P450c17 catalytic complex. Understanding the structure of
this complex and its mechanism of 17,20-lyase catalysis may permit the
design of orally active agents that selectively inhibit this reaction.
Such compounds may be useful as drugs to inhibit both estrogen and
androgen synthesis in sex steroid-dependent malignancies such as breast
and prostate cancer. Furthermore, understanding 17,20-lyase catalysis
may prove useful in understanding the basis of hyperandrogenic states
such as the polycystic ovary syndrome.
 |
MATERIALS AND METHODS
|
---|
Alignment and Graphics
The amino acid sequence of human P450c17 was aligned with the
sequence of P450BMP to map known core structural elements (
-helices
and ß-sheets) from P450BMP onto predicted core elements of human
P450c17 rather than aligning the amino acid sequences by sequence
homology programs alone. The similarities in core structures of the
crystals of P450BMP, -cam, and -terp have been described (26), and the
rationale for constructing models of eukaryotic P450s based on the
structure of P450BMP has been presented elsewhere (60). The amino acid
alignments were done essentially as described by Graham-Lorence and
Peterson (61). The sequences of P450s -cam, -terp, and -BMP were
aligned with a series of P450s from different families using the
University of Wisconsin GCG Pileup program (62). The results were then
manually inspected for the presence of helix-breaking residues,
C-capping residues that balance helix dipoles, and helix
amphipathicity. The initial alignment was adjusted to accommodate the
changes in secondary structure dictated by the positions of specific
residues located by this detailed analysis, while preserving the
location of the core elements and most conserved residues. This
sequence alignment was then imposed upon the P450BMP structure (25),
which was chosen as principal template because it is the only available
structure of a type II P450 that accepts electrons directly from a
flavoprotein reductase as do microsomal P450s such as P450c17.
The side chains of P450c17 were substituted for those of the P450BMP
core elements using the Midas Plus graphics program (63, 64) on a
Silicon Graphics Indigo-2 workstation with a Maximum Impact graphics
card. The lengths of the core elements were adjusted as determined by
the alignment, and connecting loops were added as an
-carbon
replacement from the P450BMP structure when the lengths for the P450c17
and P450BMP loops were approximately equal. In three cases (the F-G and
H-I loops and the loop before the heme-binding region), the loops were
extended to accommodate the additional residues in the P450c17
sequence, relying on energetics and dynamics to dictate the final
conformation (see below). The amino terminus with its membrane-spanning
region and the C-terminal residues that correspond to disordered
P450BMP residues that do not yield a clear diffraction pattern were
deleted from the analysis.
Energy Minimization
Energy minimization and MD calculations were performed with the
Amber 4.1 suite of programs (65) running on a Digital Equipment Corp.
(Marlboro, MA) AlphaServer 2100 computer (A21064A
microprocessor, clock speed, 275 MHz). Prolonged (100 psec) MD
simulations used Amber version 5.0, upgraded to enable parallel
processing, running on a Cray T3E using 16 Digital Equipment Alpha
21164 microprocessors at the San Diego Supercomputing Center. Amber
parameters for the heme thiolate were obtained from literature values
(66, 67) and from the Amber data subdirectory. Amber files for P450BMP
were generated using the same cysteinyl-heme residue file as for the
P450c17 model to permit calculation of P450BMP energies for comparison.
Protein database (PDB) files were generated with the programs
"ambpdb" and "fixatname" and viewed with MidasPlus.
Docking Studies and Dynamics
Amber files for
5-pregnenolone,
4-progesterone, and the corresponding 17-hydroxylated
steroids were constructed using PDB files in the Cambridge Database and
steroid parameters derived from ab initio quantum mechanical
charge density calculations using the program Gaussian 94 (68) followed
by RESP point charge fitting (69). The steroids were manually placed in
the substrate-binding pocket such that a nearly linear alignment of the
C17-(O)H-Fe was achieved without superposition of steroid atoms and
amino acid side chains. Using the BELLY option of Amber, the energy of
each of the four enzyme-substrate complexes was minimized for 200
cycles to accommodate the manually docked steroids, and the water
molecules (a 25-Å sphere centered about the catalytic
O-H of threonine 306) were preequilibrated for 10
psec. Each substrate-protein complex was subjected to 100 psec of MD as
the iron oxene species, added in the EDIT module of Amber using
literature parameters (66). Each complex was equilibrated during the
first 60 psec, and the final 40 psec were analyzed using the CARNAL
module of Amber to obtain the data shown in Table 3
.
Model Evaluation
PDB files for P450BMP and for the final P450c17 model were
submitted to the server for the Biotech Validation Suite for Protein
Structures via their Internet Website
(http://biotech.pdb.bnl.gov:8400) for evaluation by the programs
PROCHECK Version 3.0 (70), SurVol Version 1.0 (71), and WHATIF Version
4.99 (72).
Site-Directed Mutagenesis
The K89N mutation was constructed by primer mismatch extension
using human P450c17 cDNA (73) in the vector V10-c17 as template (9) and
the sense oligonucleotide
5'-GGTGCTTATTAAGAACGGCAAGGACTTCTCTGG-3' and the
antisense oligonucleotide
5'-CCAGAGAAGTCCTTGCCGTTCTTAATAAGCACC-3', where the mutated
codon is underlined. The sequence was
amplified in 200 µM deoxynucleoside triphosphates
and 5 U of pfu polymerase (Stratagene,
La Jolla, CA) using 14 cycles of 95 C for 30 sec, 55 C for 1 min, and
68 C for 15 min. The unpurified amplified product was digested with 10
U of DpnI at 37 C for 1 h to cleave the methylated
template plasmid, and then used directly to transform Escherichia
coli DH5
. After confirming the accuracy of the mutagenesis by
sequencing the entire cDNA, the plasmid was used to transform
Saccharomyces cerevisiae strain W303B. Yeast microsomes were
prepared and used for kinetic analyses as described previously (9, 13).
Note Added in Proof
B. J. Brock and M. R. Waterman have recently presented
additional evidence against a ferryl peroxide mechanism for 17,20-lyase
activity (Biochemistry 38:15981606, 1999).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Sandra Graham and Julian A. Peterson (University
of Texas Southwestern Medical Center, Dallas, TX) for abundant advice,
assistance, and encouragement and Dr. Elaine C. Ming, Dr. Richard
Dixon, and Mr. David E. Konerding for assistance with the Amber
programs at University of California, San Francisco, and at the San
Diego Supercomputer Center.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Professor Walter L. Miller, Department of Pediatrics, Building MR-IV, Room 209, University of California, San Francisco, San Francisco, California 94143-0978.
This work was supported by the National Cooperative Program for
Infertility Research Grant U54-HD-34449 (to W.L.M.), NIH Grants
DK-37922 and DK-42154 (to W.L.M.), a fellowship from the Howard Hughes
Medical Institute, and NIH Clinical Investigator Award DK-02387 (to
R.J.A.). Molecular graphics images from the UCSF Computer Graphics
Laboratory were supported by NIH Grant P41 RP-01081.
Received for publication March 4, 1999.
Revision received April 15, 1999.
Accepted for publication May 10, 1999.
 |
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