Molecular Modeling of Human P450c17 (17{alpha}-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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
P450c17 (17{alpha}-hydroxylase/17,20-lyase) catalyzes steroid 17{alpha}-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 {Delta}4 and {Delta}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{alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Most steroid biosynthetic reactions are catalyzed by cytochrome P450 enzymes (1). P450c17 (17{alpha}-hydroxylase/17,20-lyase), a microsomal enzyme, catalyzes both steroid 17{alpha}-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{alpha}-hydroxysteroids are cleaved to 19-carbon, 17-ketosteroids, and acetic acid (2, 3, 4, 5). P450c17 can also catalyze a modest degree of 16{alpha}-hydroxylase activity (6, 7, 8, 9) but does not catalyze hepatic 16{alpha}-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{alpha}-hydroxylase activity of P450c17 predominates, the glucocorticoid cortisol is produced; in the adrenal zona reticularis and in the gonads, the presence of both 17{alpha}-hydroxylase and 17,20-lyase activities results in the synthesis of sex steroids. For human P450c17, both {Delta}5-pregnenolone and {Delta}4-progesterone are good substrates for the 17{alpha}-hydroxylase reaction, but {Delta}5 17-OH-pregnenolone is preferred 100-fold over {Delta}4-17-OH-progesterone for the 17,20-lyase reaction (9). Furthermore, the 17{alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go (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. 2Go). Each step in the modeling process reduced the free energy of the model (Table 1Go).



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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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Table 1. Free Energies Achieved during P450c17 Modeling

 
Once the initial energy-minimized model was completed (Fig. 2BGo), 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. 2CGo 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 1Go), yielding the final model (Fig. 3Go). The final energy of the solvated model is virtually identical to the free energy of the comparably solvated P450BMP crystal structure (Table 1Go).



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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

 
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 2Go). 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 2Go) 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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Table 2. Model Evaluation Parameters

 
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{alpha}-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{alpha}-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. 4AGo). 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. 4BGo). The presence of three additional residues in P450c17, Q472, L473, and P474 (Fig. 1Go) forces the turn of ß-sheet 3 further into the protein, limiting the extent of the pocket above the heme (Fig. 4BGo). 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. 4CGo). 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. 4DGo). The oxygen atom at steroid carbon C3 lies adjacent to G95 in strand 5 of ß-sheet 1. In interacting with G95, {Delta}4 (3-keto) steroids form a hydrogen bond with the amide hydrogen while {Delta}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 {alpha}-carbons of P450c17 were initially positioned in the same locations as the corresponding {alpha}-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{alpha}-hydroxylase reaction.

 
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. 4BGo) 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. 1Go) 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. 1Go), 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 {alpha}-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{alpha}-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. 4DGo). 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{alpha}-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{alpha}-hydroxylase reaction (13). The preferred reaction is 17{alpha}-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{alpha}-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{alpha}-hydroxylase activity, similarly to the R347H and R358Q mutations. When the K89N mutant was coexpressed in yeast with human OR, about 84% of 17{alpha}-hydroxylase activity was retained but only 22% of 17,20-lyase activity was retained (Fig. 5Go). 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{alpha}-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{alpha}-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{alpha}-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{alpha}-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 ).

 
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{alpha}-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{alpha}, H17{alpha}, and the three H21 atoms) approach sufficiently close to the heme reaction center to be susceptible to oxygen insertion reactions (Table 3Go). 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{alpha} is the next closest hydrogen atom to the activated oxygen. Abstraction of this atom would yield a tertiary carbon radical; H16{alpha} is only slightly more distant from the reaction center (Table 3Go). 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{alpha} methine and H16{alpha} methylene hydrogens become the principal and secondary sites of reaction, respectively.


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Table 3. Distances From Heme Oxene Oxygen to Steroid Atoms

 
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{alpha}-hydroxylase or 17,20-lyase reactions (Fig. 6Go). 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 3Go).



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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{alpha}-hydroxy-pregnanediol by endogenous yeast 20{alpha}-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. 7Go. 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human adrenal 17,20-lyase activity, as indicated by serum concentrations of dehydroepiandrosterone (DHEA) and DHEA-sulfate, increases sharply at adrenarche at age 6–10, peaks at age 25–35, 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{alpha}-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{alpha}-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 {Delta}5- and {Delta}4-steroids. Fourth, the model correctly predicts that P450c17 will have 16{alpha}-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{alpha}-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. 5Go), plus the inability of hydrogen peroxide to support catalysis (Fig. 6Go) 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 {alpha}-hydrogens at steroid positions C16 and C17 are sufficiently close to the oxene oxygen to allow 16{alpha}- and 17{alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alignment and Graphics
The amino acid sequence of human P450c17 was aligned with the sequence of P450BMP to map known core structural elements ({alpha}-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 {alpha}-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 {Delta}5-pregnenolone, {Delta}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 3Go.

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{alpha}. 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
 
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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