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-Hydroxylase/ 17,20-Lyase): Insights into Reaction Mechanisms and Effects of Mutations
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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 enzyme’s 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(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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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 15–45 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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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.
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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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) 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 P450’s 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 P450c17•OR•(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 P450•OR 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 P450•OR 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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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.
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-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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. 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 38:1598–1606, 1999).
|
ACKNOWLEDGMENTS |
|---|
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FOOTNOTES |
|---|
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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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-hydroxylase and C17, 20-lyase) associated with one protein.
Biochemistry 20:4037–4042[CrossRef][Medline]
-hydroxylase-C17, 20
lyase). J Biol Chem 256:3871–3876-hydroxylase/C17,20 lyase cytochrome P450. J Biol
Chem 259:3971–397616-C19 steroids by homogeneous cytochrome
P-450 C21 side-chain cleavage from pig testis. Biochem Biophys Res
Commun 132:708–713[CrossRef][Medline]
-hydroxylase and 17,20 lyase activities of P450c17: contributions
of serine106 and P450 reductase. Endocrinology 132:2498–2506-hydroxylase activity is catalyzed by human
cytochrome P450 17-hydroxylase. J Clin Endocrinol Metab 77:98–102[Abstract]
-hydroxylase-17,20-lyase
(CYP17) by cytochrome b5: endocrinological and mechanistic
implications. Biochem J 308:901–908
-hydroxylase
deficiency. J Clin Endocrinol Metab 79:160–164[Abstract]
-hydroxylase deficiency. J Biol Chem 268:25811–25817, a major target
for the chemotherapy of prostatic cancer. Biochem Biophys Res Commun 171:1160–1167[CrossRef][Medline]
-hydroxylase/17,20-lyase–a target for prostate cancer
chemotherapy–from the crystal structure of P450BM-3. Anti-Cancer Drug
Design 12:113–123[Medline]
-hydroxylase/17, 20 lyase):
tissue distribution, regulation, and substrate specificity of the
expressed enzyme. DNA Cell Biol 13:1199–1212[Medline]
-hydroxylase/17,20 lyase deficiency in two Mennonite families. Hum
Genet 82:285–286[CrossRef][Medline]
Cys, Gln(461)Stop) actually cause combined
17-hydroxylase/17,20-lyase deficiency. Biochim Biophys Acta 1139:275–279[Medline]
-hydroxylase deficiency. J
Clin Endocrinol Metab 77:489–493[Abstract]
-hydroxylase/17,20
lyase (P450c17) in Escherichia coli. J Biol Chem 268:19681–19689 Pro causes 17-hydroxylase
deficiency. J Biol Chem 266:15992–15998 results in the partial combined
17-hydroxylase/17,20-lyase deficiency. J Biol Chem 264:18076–18082-hydroxylase-17,20-lyase
(P45017 also CYP17). Biochem J 330:967–974
. Mol Endocrinol 5:1373–13804-lyase
versus 5-lyase activity of rat CYP17. J Biol Chem 268:18267–18271 Stop,
Pro342Thr) in the CYP17 (P450–17) gene
lead to ambiguous external genitalia in a male patient with partial
combined 17-hydroxylase/17,20 lyase deficiency. J Clin
Endocrinol Metab 74:667–672[Abstract]
, lyase dependent androstenedione formation from
progesterone. Biochemistry 34:10939–10945[CrossRef][Medline]
). Biochemistry 33:4410–4418[CrossRef][Medline]
; 17-hydroxylase-17,20-lyase).
Biochemistry 34:14104–14113[CrossRef][Medline]
-hydroxylase/17, 20 lyase):
similarity to the gene for P450c21. DNA 6:439–448[Medline]
-hydroxylase/17,20 lyase): cloning of human adrenal and testis
cDNAs indicates the same gene is expressed in both tissues. Proc Natl
Acad Sci USA 84:407–411This article has been cited by other articles:
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A. Biason-Lauber, B. Kempken, E. Werder, M. G. Forest, S. Einaudi, M. B. Ranke, N. Matsuo, V. Brunelli, E. J. Schönle, and M. Zachmann 17{alpha}-Hydroxylase/17,20-Lyase Deficiency as a Model to Study Enzymatic Activity Regulation: Role of Phosphorylation J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1226 - 1231. [Abstract] [Full Text]
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W. L. Miller and R. J. Auchus Role of Cytochrome b5 in the 17,20-Lyase Activity of P450c17 J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1346 - 1346. [Full Text]
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G. I. Lepesheva, L. M. Podust, A. Bellamine, and M. R. Waterman Folding Requirements Are Different between Sterol 14alpha -Demethylase (CYP51) from Mycobacterium tuberculosis and Human or Fungal Orthologs J. Biol. Chem., July 20, 2001; 276(30): 28413 - 28420. [Abstract] [Full Text] [PDF]
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