©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stereoselective Hydroxylation of Norcamphor by Cytochrome P450
EXPERIMENTAL VERIFICATION OF MOLECULAR DYNAMICS SIMULATIONS (*)

(Received for publication, August 18, 1994; and in revised form, October 18, 1994)

Paul J. Loida Stephen G. Sligar (§) Mark D. Paulsen Gregory E. Arnold Rick L. Ornstein (§)

From the  (1)School of Chemical Sciences and The Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois 61801 (2)Environmental Molecular Sciences Laboratory, Pacific Northwest Laboratory, Richland, Washington 99352

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The stereoselectivity of cytochrome P450 hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor. (1R)- and (1S)-norcamphor (>92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450 with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M. B., and Ornstein, R. L.(1993) J. Comput. Chem. 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products. With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450.


INTRODUCTION

The superfamily of monooxygenase enzymes referred to as cytochrome P450 plays a critical role in a variety of biological processes, including clearance of xenobiotics, steroid synthesis, and lipid peroxygenation(1) . Many of the exogenous small molecules metabolized by this class of enzymes are of great clinical and commercial importance in both animals and plants(2) . While different enzymes, or ``forms,'' of the cytochrome P450 system are responsible for oxidizing various substrates, the chemical substituent (heme-thiolate ligation) and mechanism of oxidation appear to be largely invariant(3, 4) . Thus, the device by which the protein matrix determines the substrate specificity of a given enzyme within the expansive superfamily of cytochrome P450s has been an area of active investigation(5, 6, 7, 8, 9, 10, 11) .

The mammalian enzymes are membrane-bound and as such have been resistant to crystallization and detailed structural characterization. In the absence of atomic coordinates, topology mapping of the active site by means of suicide substrates and amino acid scanning via site-directed mutagenesis are being used to determine the general characteristics of cytochrome P450 active sites(9, 10, 11, 12, 13, 14, 15) . Still, the analysis of specific electrostatic, hydrophobic, and steric contributions to the reaction efficiency and oxidative specificity cannot be accomplished without detailed structural information.

The x-ray structure of the bacterial cytochrome, P450, was the first to become available nearly a decade ago(16) . This enzyme catalyzes the 5-exo hydroxylation of d-camphor as the first in a series of oxidative steps which allow its host, Pseudomonas putida, to live with camphor as its sole source of carbon. As it has been available in large quantities for many years, P450 has served as the prototypical cytochrome P450 in providing detailed chemical, physical, and structural information for the superfamily(7) . The x-ray structure of cytochrome P450 reveals that the active site is a buried cleft, essentially isolated from bulk solvent(17) . There is a single hydrogen bond formed between Tyr-96 and the ketone of camphor that enhances substrate affinity and influences substrate positioning in the active site(30, 32) . Extensive hydrophobic contacts are also made with camphor, and the shape of the active site is highly complementary to the three-dimensional structure of this molecule. Recently, cytochrome P450 has been the subject of several theoretical studies of protein-small molecule recognition(18, 19, 20, 21, 22, 23, 24, 25) . Our efforts to characterize structure-function relationships in the enzyme-substrate complex of P450 have focused on utilizing camphor analogs to probe specific features of the active site(26, 27, 28, 29, 30, 31, 32, 33, 34) . In previous reports, racemic norcamphor was used to characterize the contribution of hydrophobic contacts to the regiospecificity of the hydroxylation reaction(33) . However, the interpretation of these experiments is complicated by the fact that racemic substrate was used; therefore, the observed changes in the reaction specificity cannot be unequivocally attributed to specific structural features of the alternate substrate(35, 36) . Herein we describe investigations which utilize the pure stereoisomers of norcamphor to unambiguously evaluate the role of steric contacts and hydrogen bonding interactions in the P450-substrate complex. The experimental results verify predictions made previously by Bass and Ornstein (37) that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon.


MATERIALS AND METHODS

The preparation of (1R)- and (1S)-norcamphor has been reported previously. (^1)Other chemicals were purchased from Aldrich and Sigma. Cytochrome P450, putidaredoxin, and putidaredoxin reductase were overexpressed in E. coli and purified according to previously established procedures(38, 39) .

The cytochrome P450 reaction system was reconstituted under the following conditions: 1 µM P450, 5 µM putidaredoxin, 1 µM putidaredoxin reductase, 50 mM Tris-HCl, pH 7.0, 200 mM KCl, 500 µM substrate, 250 µM NADH in a final volume of 1 ml. Steady state reaction kinetics were monitored spectrophotometrically by the oxidation of NADH ( = 6.23 mM cm). Each reaction mixture was extracted three times with an equal volume of methylene chloride, and the combined organic portion was dried over anhydrous magnesium sulfate and concentrated under a slow stream of dry nitrogen. The product profile was determined by GC analysis of the reaction extract (J & W DB-5, inner diameter: 0.32 mm, 30 m; 130 °C for 5 min, 15 °C/min). Retention times are 5.2 min, norcamphor; 6.3 min, 3-hydroxynorcamphor; 7.6 min, 5-hydroxynorcamphor; 8.1 min, 6-hydroxynorcamphor. The total hydroxylated product was quantitated by adding a known amount of internal standard (norborneol, retention time: 5.6 min) to the reaction mixture immediately after the complete oxidation of NADH. The ferric spin equilibrium and equilibrium dissociation constants (K) were determined as reported previously(34) .

Simulations were performed on substrate-bound P450 using the cvff force field (40, 41) and version 2.9 of the Discover simulation package. The x-ray structure of (1R)-norcamphor-bound P450(42) , obtained from the Protein Data Bank(43) , served as the starting point for the simulations. An all hydrogen representation for the protein was used, and all acidic and basic groups were modeled in their ionized forms. A 3-Å layer of solvent was also included in the simulations, and a constant dielectric of 1.0 (40, 41) was used. Group-based twin cutoffs of 12.0 and 15.0 Å were employed with no switching function. Prior to the dynamics trajectories, minimization was performed first on the added hydrogens, then on the solvent, and finally on the entire protein. For the trajectories, velocities were initialized at 50 K and the system slowly warmed to 300 K. For each enantiomer, four separate trajectories of 200 ps each were calculated. In the simulations of the (1S)-enantiomer, the substrate was superimposed on (1R)-norcamphor in the crystal structure of substrate-bound cytochrome P450. The current simulations differ from our earlier simulations (37) in that a ferryl oxygen intermediate has now been included and a more widely used Coulombic -hydration model has now been used. Previously the iron was unligated, a distance-dependent dielectric constant was used, physiologically charged residues were made net neutral, and only waters of crystallization were explicitly treated.


RESULTS

The steady state rate of NADH oxidation (nmol min NADH/nmol/min P450), stoichiometry of NADH consumption to hydroxynorcamphor production (expressed as percent coupling), and product profile are summarized in Table 1. Cytochrome P450 hydroxylates (1R)-norcamphor and (1S)-norcamphor to form three regioisomers, 5-hydroxynorcamphor, 6-hydroxynorcamphor, and 3-hydroxynorcamphor. The (1R)-enantiomer is structurally analogous to the physiologic substrate d-camphor and is preferentially hydroxylated at the 5-exo position: 65:30:5 for the 5-, 6-, and 3-carbons. This is in contrast to the racemic mixture which results in a ratio of 47:45:8(31) . As expected, the product profile of the (1S)-enantiomer compensates for the difference with a ratio of 28:62:10 for 5-, 6-, and 3-hydroxylation. The steady state NADH oxidation rate, 90 nmol min NADH/nmol/min P450, and the overall efficiency of NADH consumption, 15%, is nearly the same for each enantiomer, and all values are similar to those reported previously for the racemic mixture(33, 44) . Under saturating conditions, both (1R)- and (1S)-norcamphor result in an equilibrium spin conversion of about 45%. The free energy of binding is also similar with DeltaG of approximately -5.6 kcal mol and -5.3 kcal mol, respectively. With the exception of the regiospecificity, all of the reaction and physical parameters are very similar for each enantiomer of norcamphor.



For comparison with the experimental results, product profiles for (1R)- and (1S)-norcamphor were predicted from an analysis of molecular dynamics simulations. Structures saved at 0.5-ps intervals from the final 125 ps of 200-ps molecular dynamics simulations of (1R)- and (1S)-norcamphor bound P450 were analyzed to determine the frequency of reactive substrate conformations. The geometric criteria used to define a reactive geometry were the same as had been used previously (23) and included the substrate hydrogen-ferryl oxygen distance and the corresponding carbon-hydrogen-ferryl oxygen angle. A substrate orientation was considered reactive if the oxygen distance was less than 3.5 Å and the C-H-O bond angle was within 45 °C of linear. For both (1R)- and (1S)-norcamphor, in a large majority of structures examined, neither the 3-, 5-, nor 6-carbon was in a reactive orientation relative to the ferryl oxygen. Thus, a predicted product profile was based on a very small number of structures. To improve the regiospecificity statistics, four separate trajectories were calculated for each substrate. The number of reactive conformations found for the 3-, 5-, and 6-carbon of norcamphor in each of the trajectories is shown in Table 2along with the total number of reactive conformations. The predicted product profiles calculated from these trajectories are reported in Table 1. The (1R)-enantiomer is predicted to be preferentially hydroxylated at the 5-carbon, whereas the (1S)-enantiomer is predicted to form excess 6-hydroxynorcamphor. The values reported in Table 1were obtained by averaging the profiles from each set of trajectories. Also shown in Table 1is the fraction of conformations classified as reactive (shown as ``Percent Coupling''). Previous calculations suggested that this quantity is correlated with the experimentally observed uncoupling (23) .




DISCUSSION

In the case of the physiological substrate, d-camphor, the high resolution x-ray structure and experimental measures of the reaction efficiency provide convincing evidence that the three-dimensional features of the P450 active site are complementary to those of this small molecule(17, 32, 33, 42, 44, 45) . The active site is completely buried, and multiple contacts are made between each d-camphor carbon and the heme and amino acid residues of the protein. (1R)-d-camphor essentially fills the negative image of the active site without atomic overlap and without leaving open space which might be occupied by solvent. (1R)-norcamphor is an exceptionally good camphor analog for probing the steric contacts in the binary complex because it retains the (2.2.1) bicyclic skeleton and the ketone of camphor, yet lacks the 8, 9, and 10 methyl groups which protrude from the center of the molecule. These methyl groups account for approximately 36% of the total surface area of camphor. Even without an x-ray structure of P450 with norcamphor bound(42) , one could imagine this substrate oriented in the active site with a hydrogen bond formed between Tyr-96 and the substrate ketone and the carbons 1 through 7 in approximately the same positions as the corresponding atoms of d-camphor. In this orientation the 5-carbon of norcamphor is adjacent to the iron as is the case with d-camphor, and the active site contacts between Val-295, Ile-395, Val-396, Thr-185, and Val-247 of P450 and the 8-, 9-, and 10-carbon of norcamphor are absent(42) .

Previous investigations using the racemic mixture of norcamphor have shown that the loss of the specific contacts results in a less efficient catalytic system(31, 33) . Not only is the steady state rate of NADH oxidation slowed by half, but the overall conversion of reducing equivalents to hydroxylated product is decreased by a factor of 10(31) . The spectrally determined K is 100-fold larger, and the spin state equilibrium is reduced by 50%(33) . These experimentally measured parameters are known to be sensitive markers for detailed features of the substrate bound complex, such as active site hydration, substrate positioning and orientation in the active site, substrate-active site complementarity, and relative free energy of noncovalent interactions(30, 34, 46) . Thus, the loss of steric contacts, or hydrophobic interactions, apparently results in a binary complex in which norcamphor is not bound in a ``hand-in-glove'' fashion. Additional experimental evidence in this regard is the change in the product profile from 100% 5-hydroxycamphor with d-camphor to multiple hydroxylation products with norcamphor: 5-hydroxynorcamphor, 6-hydroxynorcamphor, and 3-hydroxynorcamphor in a ratio of 47:45:8, respectively(31) . Theoretical calculations of radical stability show that the norcamphor 3-carbon is the most reactive position and suggest that the 3-carbon hydroxylation is favored in solution phase(18) . The flattening of the product profile and corresponding increase in 3-hydroxynorcamphor is due to a loss of stereochemical constraints on the substrate by the surrounding amino acid residues in the P450 active site. Norcamphor is either mobile in the active site or is bound in multiple orientations such that not only the 5-carbon, but also the 6- and 3-carbon can approach the putative oxidizing intermediate ([FeO]) and be positioned for hydroxylation. While the ratio of products is not taken as an absolute measure of the relative residence time of each substrate carbon near the [FeO] (due to complex geometric and chemical constraints), the values do reflect a loss of substrate recognition by P450 and serve as a rough measure of the structural complementarity between substrate and active site. The fact that 1-methylnorcamphor exhibits an intermediate decrease in 5-hydroxy product provides additional support for this line of reasoning(33) . The results are consistent with a conceptual model of enzyme-small molecule recognition in which substrate mobility and/or accessible binding orientations increase as the structure of the substrate diverges from that of d-camphor and protein-substrate contacts are lost.

Although preliminary investigations utilizing the racemic mixture of norcamphor have provided many important insights into the mechanism of substrate recognition by P450, extending this line of investigation requires the purified enantiomers of norcamphor. For example, in previous reports, active site mutations were shown to perturb the regiospecificity of the reaction in a predictable manner (33) . The prediction was based on molecular modeling of (1R)-norcamphor, the structural analog of d-camphor. Any rationalization of the results cannot be accurate without acknowledging the possibility that the experimentally observed change in product profile was a result of the (1S)-enantiomer. The percent 5-hydroxy product formed from the (1R)-enantiomer may be altered in a counter intuitive fashion and this fact masked by corresponding changes in the product profile of (1S)-norcamphor. Therefore, the pure stereoisomers are needed to accomplish the detailed mapping of specific interactions in the binary complex.

This problem was first addressed theoretically by Bass and Ornstein (37) who foresaw the stereoselective oxidation of (1R)- and (1S)-norcamphor based on molecular dynamics simulations. These calculations predicted (1R)-norcamphor to be hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon. Additional calculations using a more sophisticated molecular model are reported here and are in agreement with the results of the initial simulations (Table 1). The predicted regiospecificity of (1R)- and (1S)-norcamphor hydroxylation is confirmed by the experimental determination of the product profile as summarized in Table 1. This is one of the few instances that a theoretical prediction of stereoselective and regiospecific oxidation by P450 has actually preceded the experimental account. The initial reports were published almost 2 years before the preparation of the pure enantiomers of norcamphor^1; therefore, the validity of the original predictions is undisputed(37) . The results suggest that the dynamics of a substrate-active site complex can be accurately represented over a limited time scale by a molecular mechanics level calculation. These interpretations are consistent with a series of similar investigations which, collectively, establish computational analysis as a valid approach to the characterization and prediction of small molecule binding and metabolism with P450(18, 19, 20, 21, 22, 23, 24, 25, 48, 49, 50) .

Theoretically determined ratios for the 5-, 6-, and 3-hydroxy products presented herein are consistent with experiment in a qualitative sense and are also in good agreement quantitatively. The calculations reported differ most significantly from those described previously by the inclusion of a single oxygen atom as a distal ligand to the iron, intended to explicitly model the putative [FeO] intermediate. In addition, the previous study used a distance-dependent dielectric and included only crystallographic waters, whereas the present set of trajectories was calculated with a constant dielectric and a layer of added solvent. These differences in the model do result in some quantitative differences between the two sets of predicted product profiles, but do not affect the qualitative trends. To obtain an estimate of the precision of our predicted product profiles, multiple trajectories were analyzed for each enantiomer. For both of the enantiomers, one of the four trajectories predicted a product profile in qualitative disagreement with the other three trajectories (see Table 2). For (1R)-norcamphor this resulted in a particularly large estimate of the uncertainty in the predicted product profile. Although norcamphor is significantly more mobile than d-camphor, the simulation time for a single trajectory may be insufficient to properly sample substrate orientations. For highly mobile substrates such as norcamphor(21) , the vast majority of a trajectory is spent sampling unreactive conformations, making a precise prediction for the product profile difficult. A substantially large investment of computer resources is required to attain the same number of reactive conformations with norcamphor as a highly coupled substrate such as d-camphor.

(1R)- and (1S)-norcamphor might be expected to behave similarly in P450-catalyzed hydroxylation due to the high degree of symmetry between the pair of stereoisomers. In this light it is not surprising that the efficiency of the P450-catalyzed hydroxylation of (1R)- and (1S)-norcamphor is nearly identical as measured by: 1) the steady state rate of NADH oxidation, 2) the coupling of NADH to hydroxylated product, 3) the equilibrium spin conversion, and 4) the spectrally determined dissociation constant. The unique difference is in the product profile. The ratio of 5-hydroxynorcamphor to 6-hydroxynorcamphor produced is inverted for the pair of stereoisomers with (1R)-norcamphor favoring the 5-carbon and (1S)-norcamphor the 6-carbon (Table 1). The reversal in the regiospecificity is especially interesting in light of the observation that l-camphor, as well as d-camphor, is oxidized to form only 5-hydroxycamphor(51, 52) . This may be due to the smaller size of norcamphor, a property which could allow it to undergo more extensive rearrangements relative to the iron(53) . Inasmuch as hydroxylation regiospecificity reports on individual orientations of the substrate in the active site, the ratio of 5-carbon to 6-carbon hydroxylation can be expressed as a relative free energy. The value of 0.8 kcal mol reflects a quantitative measure of the difference in the recognition of (1R)- versus (1S)-norcamphor by P450 and is comparable with recent reports of the stereospecific hydroxylation of ethylbenzene and epoxidation of styrene and stereoselective hydroxylation of (1R)- and (1S)-nicotine(19, 20, 47) . Given the fact that 3-hydroxynorcamphor is the favored product free in solution, the predominance of 5-carbon hydroxylation suggests that extensive constraints are placed on (1R)-norcamphor despite the loss of several protein-substrate contacts. The increased 3-carbon oxidation observed with the (1S)-enantiomer and the larger K may be an indication of increased mobility in the active site. Nevertheless, the 34% excess of 6-hydroxynorcamphor production is evidence that (1S)-norcamphor makes a significant number of specific interactions with P450 and is highly oriented in the active site, regardless of its stereochemistry.

When the hydrocarbon portions of (1R)- and (1S)-norcamphor are superimposed, the structural difference between the stereoisomers is the position of the ketone on the hydrocarbon ring. In consideration of the experimentally determined reversal in the product profile, this observation indicates that the position of the carbonyl on the hydrocarbon skeleton of norcamphor contributes the most to the average orientation of these substrates in the active site. Molecular docking of (1S)-norcamphor in the P450 active site shows that when the ketone oxygen is positioned on the same atom of d-camphor the 6-carbon ends up adjacent to the iron (Fig. 1). Moving the carbonyl by one carbon appears to rotate the hydrocarbon skeleton of (1S)-norcamphor in the active site. These observations suggest that hydrogen bonding interactions can be used to direct the regiospecificity of the hydroxylation reaction. To test this hypothesis, genetically engineered variants of cytochrome P450 which contain novel hydrogen bonding groups are being constructed and characterized as an approach to the rational redesign of the enzyme active site.


Figure 1: The cytochrome P450 active site with (1R)- and (1S)-norcamphor bound (A and B, respectively). A, side-on view of the cytochrome P450 active site with (1R)-norcamphor bound(24) . The hydrogen bond between the substrate ketone and Tyr-96 is intact and the 5-carbon is adjacent to the iron. B, same view of the active site with (1S)-norcamphor modeled in. The substrate is positioned to form the hydrogen bond with Tyr-96. Note that the 6-carbon of the substrate is oriented near the iron.



In summary, the reaction of (1R)- and (1S)-norcamphor with cytochrome P450 is stereoselective with respect to the regiospecificity of hydroxylation. The experimental characterization confirms the previously reported prediction that the product profile resulting from the (1R)-enantiomer would reflect a preference for 5-hydroxynorcamphor and the major product with the (1S)-enantiomer would be 6-hydroxynorcamphor. The results provide evidence that hydrogen bonding interactions in the P450-substrate complex make a significant contribution to the specificity of the oxygenation reaction and play an important role in the mechanism of substrate recognition.


FOOTNOTES

*
This work was supported by Grant GM 33775 from the National Institutes of Health and Grant KP0402 from the Health Effects and Life Science Research Division of the Office of Health and Environmental Research of the Department of Energy (to R. L. O.). Pacific Northwest Laboratory is operated for the United States Department of Energy by Battelle Memorial Institute under Contract DE-AC06-76RLO-1830. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Authors to whom correspondence should be addressed.

(^1)
P. J. Loida and S. G. Sligar, to be submitted for publication.


ACKNOWLEDGEMENTS

We thank E. J. Mueller and H. Yeom for critical reading of the manuscript.


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