Protein engineering of cytochrome P450cam (CYP101) for the oxidation of polycyclic aromatic hydrocarbons

Charles F. Harford-Cross, Angus B. Carmichael, Fiona K. Allan, Paul A. England, Duncan A. Rouch and Luet-Lok Wong1

Department of Chemistry, Inorganic Chemistry Laboratory,South Parks Road, Oxford OX1 3QR, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations of the active site residues F87 and Y96 greatly enhanced the activity of cytochrome P450cam (CYP101) from Pseudomonas putida for the oxidation of the polycyclic aromatic hydrocarbons phenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-type P450cam had low (<0.01 min–1) activity with these substrates. Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, while fluoranthene gave mainly 3-fluoranthol. Pyrene was oxidized to 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with small amounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrene gave 3-hydroxybenzo[a]pyrene as the major product. The NADH oxidation rate of the mutants with phenanthrene was as high as 374 min–1, which was 31% of the camphor oxidation rate by wild-type P450cam, and with fluoranthene the fastest rate was 144 min–1. The oxidation of phenanthrene and fluoranthene were highly uncoupled, with highest couplings of 1.3 and 3.1%, respectively. The highest coupling efficiency for pyrene oxidation was a reasonable 23%, but the NADH turnover rate was slow. The product distributions varied significantly between mutants, suggesting that substrate binding orientations can be manipulated by protein engineering, and that genetic variants of P450cam may be useful for studying the oxidation of polycyclic aromatic hydrocarbons by P450 enzymes.

Keywords: monooxygenase/mutagenesis/P450cam/PAH/protein engineering


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are hazardous and persistent environmental contaminants (Dipple, 1985Go; Jacob et al., 1986Go). They are procarcinogens and their carcinogenicity is expressed via oxidation by human cytochrome P450 enzymes such as CYP1A1 (Guengerich, 1995Go). The persistence of large PAHs such as pyrene and benzo[a]pyrene in the environment is due to their low solubility in water (bioavailability) and the lack of naturally occurring microbial enzyme systems for their efficient degradation (Heitkamp and Cerniglia, 1988Go; Cerniglia, 1992Go; Schneider et al., 1996Go). It has been reported that, once such PAHs are sequestered inside microorganisms, the rate limiting step for biodegradation may be the initial oxidation by P450 monooxygenases and non-haem iron dioxygenases (Herbes and Schwall, 1978Go; Heitkamp et al., 1988Go; Cerniglia, 1992Go). The involvement of P450 enzymes in both PAH carcinogenesis in mammals and PAH bioremediation by microorganisms is of interest because all P450 enzymes appear to follow the same mechanism of generating a highly reactive ferryl intermediate species which then rapidly attacks diverse substrates (Mueller et al., 1995Go). The product formed depends primarily on the orientation of substrate binding. Therefore, increased understanding of PAH oxidation by P450 enzymes could have significant implications in many areas.

The camphor-hydroxylating enzyme cytochrome P450cam (CYP101; Nelson et al., 1993) from the soil bacterium Pseudomonas putida (Gunsalus and Wagner, 1978Go) has been studied in detail (Mueller et al., 1995Go). P450cam was the first P450 enzyme to be structurally characterized (Poulos et al., 1987Go), and the role of active site residues in substrate recognition has been examined by mutagenesis studies (Atkins and Sligar, 1988Go; Atkins and Sligar, 1989Go; Mueller et al., 1995Go). We have investigated the active site engineering of P450cam for the oxidation of a wide range of organic molecules (Wong et al., 1997Go). We showed that mutations of an active site residue Y96 to more hydrophobic residues dramatically increased the activity of P450cam towards the oxidation of hydrophobic molecules smaller than camphor, e.g. simple alkanes and styrene (Stevenson et al., 1996Go; Nickerson et al., 1997Go), as well as larger molecules including naphthalene and pyrene (Fowler et al., 1994Go; Jones et al., 1996Go; England et al., 1998Go). The oxidation of naphthalene by wild-type P450cam and the Y96A and Y96F mutants gives 1-naphthol as the dominant product, with small amounts of 2-naphthol, which parallels the activity of mammalian P450 enzymes. We reported that pyrene is oxidized to 1,6- and 1,8-pyrenequinone, which is more akin to the P450 enzyme of the detoxification pathway in the fungus Cunninghamella elegans (Cerniglia et al., 1986Go) than mammalian enzymes for which K-region oxidation products dominate (Jacob et al., 1982Go).

We have suggested that hydrophobic substitutions at Y96 may broaden the P450cam substrate range through two effects. First, the increased active site hydrophobicity should promote the binding of organic molecules and also reduce uncoupling by disfavouring the entry of water molecules into the active site during the catalytic cycle (Stevenson et al., 1996Go; England et al., 1998Go). Second, the Y96 side chain has been proposed to be part of the substrate access channel (Poulos et al., 1985Go), and hydrophobic substitutions may also promote the entry of hydrophobic molecules such as pyrene.

We report here the effect of mutations at F87 on the PAH oxidation activity of P450cam. F87 is involved in camphor binding and also proposed to play a key role in substrate access on the basis of structural data (Poulos et al., 1985Go) and molecular dynamics calculations (Paulsen et al., 1991Go). We prepared the double mutants F87A–Y96F and F87L–Y96F and compared their activity for the oxidation of phenanthrene, fluoranthene, pyrene and benzo[a]pyrene with wild-type P450cam and the Y96A and Y96F mutants. Our rationale was that the smaller side chain of alanine and the more flexible side chain of leucine compared with the rigid phenylalanine at the 87 position might promote the entry and binding of PAHs, and also alter the PAH binding orientation and hence the products formed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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General

Buffer components were from Anachem Ltd. HPLC grade acetonitrile and chloroform were from Aldrich. Phenanthrene, 9-phenanthrol, fluoranthene, pyrene, 1-pyrenol, benzo[a]pyrene and 4-phenylphenol were from Aldrich. NADH was from Boehringer Mannheim. PD10 gel filtration columns were from Pharmacia. UV/Vis spectra were recorded on a Varian CARY 1E double-beam spectrophotometer. The cell holders were equipped with magnetic stirrers and the cell temperature was maintained (±0.1°C) by a Peltier temperature controller unit. NMR spectra were recorded at 500 MHz on a Bruker WH500 or a Varian UnityPlus spectrometer. HPLC experiments were performed on a Gilson Gradient system using analytical (5mm i.d.x250mm) or semi-prep (10mm i.d.x250mm) C18 modified reverse phase columns and monitoring the eluent at 254 nm. Preparative scale incubation and product purification by HPLC were carried out as described previously (England et al., 1998Go).

DNA manipulations and recombinant protein preparation

General DNA manipulations followed standard literature methods (Sambrook et al., 1989Go). Site-directed mutagenesis was performed by the method of Kunkel (1985) using the Bio-Rad Mutagene kit. All possible mutations at the 96 position were generated using a degenerate oligonucleotide and the Y96A and Y96F mutants identified by DNA sequencing. The oligonucleotide 5'-GCGAGTGCCCGNNNATCCCTCGTGAA-3' was used for generating the F87A (NNN = GCC) and F87L (NNN = TTG) mutations. The nucleotide sequence of the entire gene for each mutant was confirmed by automated DNA sequencing at the DNA sequencing facility of the Department of Biochemistry, University of Oxford.

Expression and purification of P450cam and the associated electron transfer proteins putidaredoxin and putidaredoxin reductase were carried out following standard literature methods (Unger et al., 1986Go; Peterson et al., 1990Go; Yasukochi et al., 1994Go), except that the final purification step for all three proteins was anion exchange chromatography on a 50 ml Source Q column (Pharmacia). For wild-type P450cam, only samples with purity ratio A392/A280 > 1.60 were used (Gunsalus and Wagner, 1978Go). The mutants did not show complete conversion of the haem to the high-spin state even in the presence of a large excess (1 mM) of camphor and therefore their purities were assessed by the ratio of the absorbances at 404 nm (the isosbestic point for the low-spin/high-spin conversion) and 280 nm. The minimum A404/A280 ratio acceptable was 1.20, equivalent to A392/A280 = 1.60 for the wild-type enzyme. The purity criterion for putidaredoxin was A325/A280 > 0.65, and for putidaredoxin reductase A280/A415 < 8.0. The three proteins were stored in 50% glycerol buffered solutions at –20°C, and a further 0.5 mM camphor was added to the storage solution for the P450cam enzymes. Glycerol and camphor were removed by gel filtration on a 5 ml PD10 column, eluting with 50 mM Tris–HCl, pH 7.4, immediately before experiments.

All the P450cam enzymes studied in this work contain the base mutation C334A. This substitution of Cys334 on the surface of P450cam had been shown to remove completely the tendency of wild-type P450cam to dimerize via disulphide bond formation. The C334A mutant is monomeric at all concentrations, and has identical activity to the monomeric form of wild-type P450cam but, unlike the wild-type enzyme, it does not give insoluble aggregates during purification (Nickerson and Wong, 1997Go). However, for simplicity, the C334A base mutant is referred to as `wild-type' P450cam, and the Y96F–C334A double mutant as `Y96F', etc.

NADH turnover rate measurements

Incubation mixtures (3.75 ml) contained 50 mM Tris–HCl buffer, pH 7.4, 1 µM P450cam, 10 µM putidaredoxin, 2 µM putidaredoxin reductase and 200 mM KCl. Substrates were added as 100 mM stocks in ethanol (5 mM for benzo[a]pyrene) to a nominal final concentration of 100 µM (50 µM for benzo[a]pyrene), but the low solubility of PAHs in aqueous buffers meant that precipitation occurred for all the target substrates. The incubation mixture was thoroughly oxygenated and, after equilibrating at 30°C for 2 min, NADH was added to 500 µM and the absorbance at 340 nm monitored. The incubation mixtures were stirred at a fixed rate throughout the pre-equilibration and NADH consumption measurements. We found that only a small proportion of the PAH substrate was oxidized and absorbance changes due to substrate and products did not affect the time-course measurements.

The fully reconstituted P450cam system has a background rate of NADH oxidation even in the absence of substrates. This arises primarily from air oxidation of the reduced forms of the electron transfer proteins putidaredoxin reductase and putidaredoxin. Whilst the rate of this background process is slow compared with the rate with camphor as substrate, it is significant or even comparable to the rate with many unnatural substrates such as PAHs. To take this background rate into account, incubations were carried out with all the components of a substrate oxidation reaction except the P450cam enzyme and substrate (Nickerson et al., 1997Go). The average rate of background NADH consumption was determined to be 0.037 ± 0.003 (O.D. units)(min)–1 under the experimental conditions. For each PAH oxidation incubation, the amount of NADH oxidized by the background process was calculated from the background rate and the incubation time. This was then subtracted from the total amount of NADH added, and the PAH-dependent NADH consumption rate was calculated using {varepsilon}340nm = 6.22 mM–1cm–1.

HPLC analysis and the activity of the P450cam enzymes

After the incubations were completed, the internal standard 4-phenylphenol was added as a 100 mM stock in ethanol to a final concentration of 25 µM. The mixtures were extracted by thorough vortexing with 2 ml pre-chilled (4°C) chloroform. After centrifugation at 4000 g at 4°C for 20 min, 1 ml chloroform was removed with a calibrated syringe and evaporated under a stream of nitrogen. For phenanthrene, fluoranthene and pyrene oxidation reactions, the residues were redissolved in 1 ml 40% v/v acetonitrile in water. A 400 µl aliquot was injected onto an analytical scale C18 reverse phase HPLC column (4 mm i.d.x250 mm). For the phenanthrene and fluoranthene extracts, a gradient of 40–50% v/v acetonitrile in water was developed over 30 min at a flow rate of 1 ml(min)–1. The retention times were 2-phenanthrol, 23.5 min; 3-phenanthrol, 24.5 min; 1-phenanthrol, 26.7 min; 4-phenanthrol, 29.1 min; 3-fluoranthol, 27.6 min and the internal standard 4-phenylphenol, 22.0 min. For extracts of pyrene oxidation reactions, the products were eluted with 40% v/v acetonitrile water for 30 min, and then a gradient of 40–100% acetonitrile was developed over 30 min. The retention times were 1,6-pyrenequinone, 13.5 min; 1,8-pyrenequinone, 14.3 min; 2-pyrenol, 43.5 min; 1-pyrenol, 46.0 min and the internal standard 4-phenylphenol, 29.0 min. For benzo[a] pyrene, the residues were redissolved in 1 ml 80% v/v methanol/water and the extracts analysed by reverse phase HPLC by eluting with 80% v/v methanol/water (Mushtaq et al., 1987Go).

The rates of phenanthrene oxidation were determined using 9-phenanthrol as the product standard. The 4-phenylphenol internal standard was added to mixtures containing different concentrations of 9-phenanthrol and all the incubation components except phenanthrene and NADH, and the mixtures were extracted and analysed as described above. A calibration plot of the 9-phenanthrol peak area to that of the internal standard against the 9-phenanthrol concentration was linear and passed through the origin. The amount of 1-, 2-, 3- and 4-phenanthrol formed by P450cam-catalysed oxidation was calculated from the plot by scaling to the extinction coefficients at 254 nm: 57.0 mM–1cm–1 for 1-phenanthrol; 63.1 mM–1cm–1 for 2-phenanthrol; 66.4 mM–1cm–1 for 3-phenanthrol; 36.9 mM–1cm–1 for 4-phenanthrol; 41.1 mM–1cm–1 for 9-phenanthrol (Djerassi et al., 1955Go).

The fluoranthol formation rates were also determined from the 9-phenanthrol calibration plot by scaling with the extinction coefficients. The potential error here was that the extraction efficiency of fluoranthols may be different from that of 9-phenanthrol. Control experiments in which the 3-fluoranthol product peak was collected from the HPLC analysis and quantitated using the literature extinction coefficient ({varepsilon}254 nm = 16.6 mM–1cm–1 in ethanol; Rice et al., 1983Go) showed that this error was acceptably small (<10%).

The rates of formation of pyrene oxidation products were determined by calibration with authentic samples. The 1,6- and 1,8-pyrenequinones were synthesized by hydrogen peroxide oxidation of pyrene in refluxing acetic acid (Arnold and Larson, 1940Go), and were purified by HPLC on a semi-prep C18 reverse phase column (10 mm x 250 mm) eluting with 40% v/v acetonitrile/water at 2.5 ml (min)–1. Stock solutions of the purified compounds in ethanol were prepared and quantitated using {varepsilon}254 nm = 12.0 mM–1cm–1 for 1,6-pyrenequinone and 27.5 mM–1cm–1 for 1,8-pyrenequinone (Fatiadi, 1965Go). Mixtures containing different concentrations of these compounds and all the components of an incubation except NADH and pyrene were extracted and analysed by HPLC as described above. Calibration plots of the ratio of the two pyrenequinone product peak areas to the internal standard passed through the origin and were linear within the range 0–50 µM. An authentic commercial sample of 1-pyrenol was used for calibration by the method described above for the pyrenquinones. Samples of 2-pyrenol from preparative scale turnover reactions were pooled, quantitated as an ethanol solution using {varepsilon}337 nm = 44.7 mM–1cm–1 (Jones, 1945Go), and calibrated as above.

The area of the internal standard peak was identical to within 5% for HPLC analysis of all the reactions, indicating good reproducibility for the extraction and analysis protocols. Similarly, the ratio of the total product peak areas to the internal standard were also reproducible to within 5% for all the reactions. The total product concentrations and hence their rates and coupling efficiencies of formation were readily calculated.


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PAH binding and NADH turnover activities of the P450cam enzymes

Binding of phenanthrene, fluoranthene, pyrene and benzo[a]pyrene by the P450cam enzymes was examined by the haem spin-state shift assay. The spin-state shift gives an indication of the likelihood of catalytic turnover because it is accompanied by an increase in the haem reduction potential from –300 mV (low-spin form) to –170 mV (high-spin form), which promotes electron-transfer from putidaredoxin (E' = –240 mV) to P450cam to initiate the catalytic cycle (Sligar, 1976Go; Sligar and Gunsalus, 1976Go). The target PAHs induced only small spin-state shifts (<10%) with all of the P450cam enzymes. Therefore, either the PAHs did not enter the active site of the P450cam enzymes or, if they did, the binding orientations are such that the haem iron sixth ligand water was not displaced. The minimal spin-state shifts also precluded the determination of PAH binding constants by spectroscopic titrations.

The absence of any significant haem spin-state shift suggested that the P450cam enzymes should have very low NADH oxidation activity with the PAHs. The data in Table IGo show that indeed many of the P450cam/PAH combinations had NADH oxidation rates that were two orders of magnitude slower than camphor oxidation by the wild-type. The observed rates for wild-type P450cam with PAHs were so close to the background rate that it was difficult to determine the PAH-dependent rates with confidence. The mutants were more active than the wild-type with phenanthrene and fluoranthene as substrate. In fact, the rate for phenanthrene with the F87A–Y96F double mutant was 31% of the rate of camphor oxidation by wild-type P450cam. The rates were much lower for pyrene, and the NADH oxidation activity of wild-type P450cam and the Y96F mutant with benzo[a]pyrene as substrate were not readily detectable above background. The two double mutants showed low but detectable activity, while theY96A mutant had the highest activity.


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Table I. NADH turnover activities of wild-type (WT) cytochrome P450cam and mutants with camphor and polycyclic aromatic hydrocarbons as substrates
 
Products and rates of PAH oxidation

Phenanthrene Phenanthrene was oxidized by all the P450cam enzymes to four products, which were isolated and purified from a preparative scale reaction catalysed by the Y96A mutant. The mass spectra showed all four to be monooxygenated phenanthrenes, and detailed analysis by one- and two-dimensional 1H NMR spectroscopy identified them as 1-phenanthrol, 2-phenanthrol, 3-phenanthrol and 4-phenanthrol (Figure 1Go). The UV/Vis spectra were also identical to those in the literature (Djerassi et al., 1955Go; Bao and Yang, 1991Go). The other hydroxylation product, 9-phenanthrol, was not formed. The distribution of the four phenanthrol products are shown in Table IIGo. The major product shifted from 4-phenanthrol for the Y96 single-site mutants to 1-phenanthrol for the F87–Y96 double mutants, indicating that mutations at F87 resulted in altered orientations of phenanthrene binding.



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Fig. 1. The products from the oxidation of polycyclic aromatic hydrocarbons (PAHs) catalysed by wild-type cytochrome P450cam and the active site mutants Y96A, Y96F, F87A–Y96F and F87L–Y96F. The distribution of products varied between mutants. The wild-type enzyme did not give detectable amounts of product with fluoranthene, and only the F87A–F96F double mutant gave detectable products from benzo[a]pyrene.

 

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Table II. Activity and selectivity of phenanthrene oxidation by wild-type (WT) cytochrome P450cam and mutants
 
The monooxygenase activity of P450 enzymes is well-known to undergo uncoupling side reactions, i.e. not all of the NADH consumed by the system were necessarily utilized for substrate oxidation (Kuthan and Ullrich, 1982Go; Zhukov and Arachov, 1982Go; Gorsky et al., 1984Go). The data in Table IIGo show that, despite the very reasonable NADH turnover rates of the mutants, the actual rates of phenanthrol product formation were low. Therefore, all the enzymes showed high degrees of uncoupling, which was consistent with the small haem spin-state shifts and incomplete exclusion of water when phenanthrene is bound within the active site of the enzymes. However, all the mutants showed higher coupling than the wild-type which channelled only 0.2% of the NADH consumed towards phenanthrol formation.

Fluoranthene Incubations of wild-type P450cam with fluoranthene did not give detectable quantities of product by HPLC. The mutants gave three products, with one compound constituting >70% of the products for all the mutants. The mass spectra indicated that all were monooxygenated fluoranthrenes (M+ = 217). The major product was purified from a preparative scale incubation with the F87L–Y96F double mutant and characterized by 1H NMR spectroscopy as 3-fluoranthol. The UV/Vis spectrum was also identical to that in the literature (Rice et al., 1983Go). The two minor products were not formed in sufficient quantities for detailed characterization.

The rate and coupling of fluoranthene oxidation by the mutants are given in Table IIIGo. The F87L–Y96F double mutant was the most active, but for all the enzymes the coupling efficiencies were low and comparable to those for phenanthrene oxidation. Unlike for phenanthrene where mutations at F87 increased the substrate oxidation rate by increasing the NADH turnover rate, the higher activity of the F87L–Y96F double mutant compared with the Y96F single site mutant was due to increases in both the NADH turnover rate and the coupling.


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Table III. Product distribution, rate and coupling of fluoranthene oxidation by mutants of cytochrome P450cam
 
Pyrene We had previously reported that pyrene oxidation by wild-type P450cam and the Y96A and Y96F mutants gave 1,6- and 1,8-pyrenequinone (England et al., 1998Go). It was proposed that the quinone products were formed by initial oxidation of pyrene to 1-pyrenol, followed by a second oxidation to give 1,6- and 1,8-dihydroxypyrene which were then oxidized by air, and 1-pyrenol was not observed by HPLC.

In the present work, when the peak previously assigned to 1-pyrenol in the HPLC analysis of what was thought to be pure 1-pyrenol was collected and analysed by NMR and UV/Vis spectroscopy, it was found to be another compound. The sample of 1-pyrenol had presumably decomposed significantly, leading to the incorrect assignment. Authentic 1-pyrenol had a much longer retention time with the previous analysis program which was also used for the phenanthrols and fluoranthols, and it was actually in the 100% acetonitrile column wash at the end of the program. The HPLC analysis protocol was altered to incorporate a 40–100% v/v acetonitrile gradient, and 1-pyrenol was actually found to be present in all the reactions, including that with the wild-type enzyme. In addition, small amounts of 2-pyrenol were also identified by 1H NMR and UV/Vis spectroscopy, particularly in the reactions catalysed by the Y96A mutant.

Wild-type P450cam gave 1-pyrenol as the predominant product (Table IVGo). The 1,6- and 1,8-pyrenequinones were just detectable but reliable peak area integration was not possible. The Y96A mutant gave the highest proportion of 2-pyrenol, and there was little further oxidation of 1-pyrenol. The extent of further oxidation was increased for the Y96F mutant, and the regioselectivity of pyrene oxidation was significantly shifted so that almost no 2-pyrenol was formed. The F87 mutations maintained the selectivity for the 1-position, but dramatically shifted the selectivity of further oxidation of 1-pyrenol to the 6-position. The different selectivities indicated that the mutations altered the binding orientation of pyrene as well as 1-pyrenol.


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Table IV. Product distribution, rates and couplings for the formation of pyrenequinones by wild-type (WT) cytochrome P450cam and mutants
 
The rate and overall coupling efficiency for product formation are given in Table IVGo. The pyrenequinone concentrations were counted twice in the coupling calculations because the P450cam enzymes catalysed two oxidation steps to give these products. The Y96 single-site mutants were much more active than the wild-type, and the F87 mutations further increased the activity. The major factor for the higher activity of all the mutants was their much higher coupling efficiencies. The approximately 20% couplings of the double mutants are reasonably high considering the very different structures of camphor and pyrene, and certainly remarkable compared with the coupling for phenanthrene and fluoranthene oxidation. Finally, although the absolute product formation rates of the double mutants were not high (~1 min–1), they nevertheless represent a striking acceleration compared with the wild-type.

Benzo[a]pyrene

HPLC analysis of the reactions showed that, although the Y96A mutant had the highest NADH turnover rate, only the F87A–Y96F double mutant gave detectable amounts of one major and one minor product. These results strongly suggested that the F87A–Y96F double mutant had much higher coupling efficiency, which was consistent with the trend for the other PAHs studied here. The major product was isolated by HPLC from a preparative scale incubation and comparison of the 1H NMR and UV/Vis spectra with literature data (Yagi et al., 1976Go) showed that it was 3-hydroxybenzo[a]pyrene. The UV/Vis spectrum of the minor product did not match with any hydroxybenzo[a]pyrene, and it was not formed in sufficient quantities for further characterization. Consequently we did not attempt to measure the product formation rate and coupling efficiency.


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
We have shown in the present work that the bacterial cytochrome P450cam can be genetically engineered to have high NADH turnover rates with polycyclic aromatic hydrocarbons such as phenanthrene and fluoranthene while the wild-type enzyme has very low activity. The mutants carry substitutions at F87 and Y96—both are active site residues which contact the camphor substrate but they are also proposed to form part of the putative substrate access channel. The coupling efficiency, and consequently the absolute rate of PAH oxidation are low, but the increased activities compared with the wild-type are sufficient for the major oxidation product of benzo[a]pyrene by the F87A–Y96F double mutant to be characterized by NMR spectroscopy. Benzo[a]pyrene oxidation is particularly interesting because the crystal structures of P450cam show that there is no substrate access channel from the external medium into the enzyme active site, and yet dynamic fluctuations of the structure are sufficient to allow the entry of such a large and rigid molecule. Since the distribution of PAH oxidation products varied between mutants, the mutations at F87 altered substrate binding but the data did not rule out significant effects on substrate access.

PAHs induce only small (<10%) haem spin-state shifts, and the NADH turnover rates are less than 20 min–1 in many cases. These slow rates are consistent with previous reports for substrates which induce such small shifts (Fruetel et al., 1992Go; Loida and Sligar, 1993Go; Stevenson et al., 1996Go). However, it should be noted that the small spin-state shifts might arise from weak binding of the PAH substrates by the P450cam enzymes and the low solubility of the PAHs. This problem is particularly acute for pyrene and benzo[a]pyrene. Therefore detailed comparison between the NADH turnover rates for different PAHs is difficult while direct comparison between mutants for the same PAH is more valid because the same concentration of substrate is present in solution. From all the data, the NADH turnover rates for phenanthrene oxidation by the Y96A mutant, and for phenanthrene and fluoranthene oxidation by the two F87–Y96 double mutants are quite remarkable because the values are up to 31% of the rate of camphor oxidation by wild-type P450cam. Under the conditions employed in the experiments the first electron transfer from reduced putidaredoxin to the P450cam enzymes is the rate limiting step. Evidently the activation energy of electron transfer, which is a combination of the thermodynamic driving force (the haem reduction potential) and reorganization energy (primarily the need or otherwise of water ligand dissociation), is not controlled solely by the haem spin-state equilibrium in P450cam.

Although PAH oxidation by the P450cam enzymes is often highly uncoupled, the absolute rates of approximately 1 min–1 for the mutants are all 2–3 orders of magnitudes higher than for the wild-type, and are comparable to the activities of PAH oxidizing mammalian P450 enzymes. Mutations at both the 87 and 96 positions of P450cam can increase the NADH turnover activity as well as coupling, and in most cases the effects at the two sites are mutually reinforcing. However, there is a clear difference between phenanthrene and fluoranthene on the one hand, and pyrene and benzo[a]pyrene on the other. The major effect of the F87 mutations for phenanthrene and fluoranthene is the increased NADH turnover rate, with only small effects on the coupling (Tables II and IIIGoGo). Since the product distribution was also significantly perturbed, the substrate binding orientation has been altered to promote electron transfer but the uncoupling branch points in the catalytic cycle are not greatly affected. The converse is observed for pyrene where the large rate enhancements over the single-site Y96 mutants are mainly due to increased coupling efficiencies. For benzo[a]pyrene, no product is observed with the Y96A mutant, which has a higher NADH turnover rate than the F87A–Y96F double mutant, and so the F87A mutation must also significantly increase the coupling efficiency. Taken together, the results indicate that the two F87 mutations altered PAH binding either to promote electron transfer or to increase the coupling efficiency, but not both. We have therefore achieved different halves of the twin protein engineering objectives of fast NADH turnover activity and high coupling for these unnatural substrates, which suggests that suitable combinations of mutations for both objectives should exist. We have shown, for example, that the rate and coupling efficiency can be engineered for the oxidation of linear and branched alkanes (Stevenson et al., 1998Go).

The oxidation products of fluoranthene and benzo[a]pyrene do not give any information on the mechanism of PAH oxidation by the P450cam enzymes. However, the predominance of 1- and 4-phenanthrol from phenanthrene oxidation is of interest. The NIH shift mechanism of aromatic oxidation, which involves the spontaneous rearrangement of an arene oxide intermediate, was first proposed for PAH oxidation by mammalian P450 enzymes (Guroff et al., 1967Go; Jerina and Daly, 1974Go). The rearrangement of synthetic phenanthrene 1,2- and 3,4-epoxide has been reported to give 1- and 2-phenanthrol in a ratio of 94:6, and 19:81 for 3- and 4-phenanthrol (Bruice et al., 1976Go). Although the predominance of 1- and 4-phenanthrol is also observed with the P450cam enzymes, many of the ratios are significantly different from the synthetic oxides, and there was no evidence of any arene oxides in the products. In the case of pyrene, the Y96A mutant gave a significantly different ratio of attack at the 1- and 2-positions from the other P450cam enzymes. Therefore, although the phenanthrene oxidation products indicate that the NIH shift may be occurring, alternative mechanisms such as direct hydroxylation or electron transfer oxidation cannot be ruled out.

Mammalian P450 enzymes oxidize phenanthrene at all positions, with attack at the activated K-region (9,10-positions) predominating. Pyrene oxidation is more complex, with the proportions of K-region oxidation products, 1-hydroxypyrene, and 1,6- and 1,8-products varying between enzymes (Boyland and Wolf, 1950Go; Jacob et al., 1982Go; Cerniglia et al., 1986Go). Also, most sites of benzo[a]pyrene are attacked (Dipple, 1985Go). These data suggest that the PAHs have significant mobility and multiple binding orientations in the active site of mammalian enzymes. The preferential attack of non K-region positions by the P450cam enzymes suggests more specific binding. The aromatic side chains of F87 and Y96 are in the upper part of the P450cam active site, and the mutations described here could enable specific binding of one aromatic ring of the PAH in the vicinity of the side chains at these positions, thus presenting only non-K region positions to the ferryl intermediate. Molecular dynamics calculation of PAH binding by the P450cam mutants would be of interest.

In conclusion, we have shown that P450cam can be engineered to have high NADH turnover activity with a four-ring PAH such as fluoranthene, and even benzo[a]pyrene can be oxidized, albeit slowly. Mutations at F87 and Y96 altered the turnover rate, coupling efficiency and product distribution, indicating that all three parameters may be further manipulated by protein engineering.


    Acknowledgments
 
This work was supported by the Higher Education Funding Council for England (HEFCE), and in part by a British Gas–Biotechnology Directorate–Clean Technology Unit–Department of Trade and Industry LINK programme. P.A.E. and C.F.H.C. thank the EPSRC for Quota Studentships.


    Notes
 
1 To whom correspondence should be addressed Email: luet.wong{at}chem.ox.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 17, 1999; revised November 22, 1999; accepted December 8, 1999.