Finding of a New Fluorescent Substrate Probe - American Chemical

301 University Boulevard, Galveston, Texas 77555-1031. Received January 3, 2002. The main objective of the present study was to find a fluorescent sub...
0 downloads 0 Views 136KB Size
Download PDF
806

Chem. Res. Toxicol. 2002, 15, 806-814

7-Benzyloxyquinoline Oxidation by P450eryF A245T: Finding of a New Fluorescent Substrate Probe Kishore K. Khan* and James R. Halpert Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1031 Received January 3, 2002

The main objective of the present study was to find a fluorescent substrate probe for cytochrome P450eryF (P450eryF). P450eryF is a bacterial P450 that catalyzes the hydroxylation of 6-deoxyerythronolide B at the 6S position, a necessary step in the biosynthesis of erythromycin. The lack of a conserved threonine residue in the I-helix, in contrast to other P450s, makes P450eryF unable to oxidize other substrates. A recent study [Xiang et al. (2000) J. Biol. Chem. 275, 35999-36006] has shown that the substitution of Ala-245 by threonine confers on P450eryF significant testosterone hydroxylase activity. Therefore, we investigated various known fluorescent P450 substrates with P450eryF wild-type as well as two mutants, A245S and A245T. Among the various fluorescent compounds tested, 7-benzyloxyquinoline (7-BQ) was found to be the most suitable probe for P450eryF A245T, with rates of oxidation being lower for A245S and wild-type enzyme. The steady-state kinetics of 7-BQ oxidation by A245T are sigmoidal (Vmax ) 0.71 nmol/min/nmol, n ) 2.18, and S50 ) 132 µM). R-Naphthoflavone (R-NF), a well-known activator of CYP3A4, did not stimulate 7-BQ oxidation by A245T, although the S50 value for R-NF binding to wild-type P450eryF was similar to P450 3A4. Interestingly, spectral binding studies of wild-type P450eryF and A245T with ketoconazole and miconazole showed differential binding behaviors. Titration of wild-type with ketoconazole and miconazole and of A245T with miconazole showed the expected type-II binding. However, titration of A245T with ketoconazole produced a spectrum similar to type-I. Inhibition studies showed that both ketoconazole and miconazole are able to inhibit 7-BQ oxidation by A245T, although miconazole showed a slightly higher potency. In brief, the present study reports the discovery of 7-BQ as the first fluorescent and only the second unnatural substrate, and of miconazole as an effective P450eryF inhibitor. Cytochrome P450eryF (CYP107A1) is a bacterial P4501 that catalyzes the hydroxylation of 6-deoxyerythronolide B (6-DEB) at the 6S position, a necessary step in the synthesis of erythromycin (1, 2). P450eryF is a unique P450 that lacks the conserved threonine residue in the I-helix of other P450s. This residue is thought to participate in the important step of delivering a proton required for conversion of iron-linked dioxygen to ferryl oxygen (3). Examination of the crystal structure of 6-DEB-bound P450eryF showed that the conserved threonine is replaced with alanine and a complex hydrogen bonding network involving two amino acid side chains, three water molecules, and the substrate (3). Although this arrangement amply satisfies the requirement for substrate-assisted catalysis in the 6-hydroxylation of 6-DEB, the resulting enzyme is ineffective in oxidizing other substrates (4). An earlier study reported that the substitution of Ala-245 by a serine or threonine decreases the hydroxylation rate of 6-DEB by approximately 6- and 1000-fold, respectively (5). However, a more recent study * To whom correspondence should be addressed. E-mail address: [email protected]. Phone: (409) 772-9677. Fax: (409) 772-9642. 1 Abbreviations: P450, cytochrome P450; 6-DEB, 6-deoxyerthronolide B; DHEA, dehydroepiandrosterone; 7-BR, 7-benzyloxyresorufin; 7-PR, 7-pentoxyresorufin; R-NF, R-naphthoflavone; β-NF, β-naphthoflavone; 7-BFC, 7-benzyloxy-4-(trifluoromethyl)coumarin; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; 7-BQ, 7-benzyloxyquinoline; 7-HQ, 7-hydroxyquinoline; IPTG, isopropyl-β-D-thiogalactopyranoside; δ-ALA, δ-aminolevulinic acid.

has shown that the substitution of Ala-245 by threonine leads to a significant gain-of-function that enables the enzyme to oxidize testosterone (6). P450eryF is the only bacterial P450 to show cooperative behavior (7). A recent X-ray crystallographic study has demonstrated that P450eryF could simultaneously bind two molecules of androstenedione or 9-aminophenanthrene (7). Cooperativity is a well-known phenomenon among some of the mammalian P450s, and the existence of multiple substrate-binding sites has been proposed in CYP3A4 on the basis of substrate oxidation kinetics, sitedirected mutagenesis, and ligand binding studies (8-16). However, thus far no structural information is available with respect to how effectors bind to the enzyme or influence the binding of substrate. The recent study of P450eryF provided the first structural proof of the simultaneous presence of two ligand molecules in a P450 active site. It is noteworthy that several of the activesite residues that were found to be in contact with the first or second molecule of the substrate in the P450eryF crystal structure have direct counterparts in human CYP3A4 residues that were suggested to play a crucial role in cooperativity based on site-directed mutagenesis studies (11-15). Unlike several other bacterial P450s, not much is known about structure-function relationships of P450eryF. However, its known crystal structure, high

10.1021/tx0200010 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/21/2002

7-BQ as a New Substrate Probe of P450eryF

water solubility, and cooperative behavior make P450eryF an attractive model system to facilitate studies of human P450 cooperativity. Furthermore, the observation that substitution of Ala-245 with threonine confers significant testosterone hydroxylase activity on P450eryF also suggests that, with a few appropriate alterations in activesite architecture, this enzyme could be rendered more versatile. However, these studies are very difficult to carry out without a better probe of P450eryF. The HPLCbased testosterone hydroxylase assay used by Xiang et al. is cumbersome and time consuming (6). In the past, fluorescence-based assays have been used very successfully to provide a quick and easy way to characterize different P450 enzymes. In the present study, we have investigated some of the well-known fluorescent P450 substrates as alternates for P450eryF using wild-type and mutants A245S and A245T. The His-tagged proteins generated for easier purification were initially characterized by spectral binding with androstenedione, testosterone, and 9-aminophenanthrene. The investigation of various fluorescent substrates showed the highest rate of oxidation by P450eryF A245T with 7-benzyloxyquinoline (7-BQ), and the steady-state kinetics of the reaction were found to be sigmoidal. Interestingly, spectral titrations of P450eryF A245T with ketoconazole and miconazole showed differential binding behaviors, although both compounds are able to inhibit 7-BQ oxidation by A245T. The finding of a rapid and simple assay and an effective inhibitor should significantly add to structure-function studies of P450eryF.

Experimental Procedures Materials. Testosterone, androstenedione, 9-aminophenanthrene, imidazole, 1-phenylimidazole, 2-methylimidazole, 4-phenylimidazole, 7-benzyloxyresorufin (7-BR), 7-pentoxyresorufin (7PR), resorufin, flavone, R-naphthoflavone (R-NF), β-naphthoflavone (β-NF), isopropyl-β-D-thiogalactopyranoside (IPTG), and δ-aminolevulinic acid (δ-ALA) were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC), and 7-hydroxy-4-(trifluoromethyl)coumarin were purchased from Molecular Probes Inc. (Eugene, OR). 7-Benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), 7-BQ, and 7-hydroxyquinoline (7-HQ) were purchased from Gentest Corp. (Woburn, MA). 7-Alkoxyquinoline derivatives were synthesized by Dr. Eugene Mash (Department of Chemistry, The University of Arizona, Tucson, AZ). Miconazole and ketoconazole were obtained from Spectrum Quality Products, Inc. (Gardena, CA). Oligonucleotide primers for PCR were either obtained from The University of Texas Medical Branch Molecular Biology Core Laboratory (Galveston, TX) or from Sigma Genosys (Woodlands, TX). The Expand PCR Kit and Rapid Ligation kits were obtained from Roche (Indianapolis, IN). Restriction enzymes were purchased from GIBCO-BRL (Grand Island, NY). The GeneClean kit and TA Cloning kit were from BIO 101 (Carlsbad, CA) and Invitrogen (San Diego, CA), respectively. Ni2+-NTA affinity resin was purchased from Qiagen (Valencia, CA). All other chemicals were of the highest grade available and were obtained from standard commercial sources. His-Tagging of P450eryF, A245S, and A245T. The original P450eryF clone (pWHM808) was created using the pTrc99c vector and genomic DNA (2). To generate the His-tagged construct, a small segment of the C-terminus of this plasmid was PCR amplified using the forward primer (5′-GCTGGCCAAGCTG-3′) and reverse primer (5′-AAGCTTTCAATGGTGATGGTGTCCGTCGAGCCGCACCGGTAGG-3′). The reverse primer had codons for four histidines, a new HindIII restriction site, and a sequence complementary to the cDNA of the P450eryF C-terminus. Reaction conditions were as follows: one

Chem. Res. Toxicol., Vol. 15, No. 6, 2002 807 cycle of 94 °C for 2 min, followed by 30 cycles of 94 °C for 1 min, 37 °C for 1.5 min, and 68 °C for 1 min (the temperature was achieved over a period of 2 min), and finally one cycle of 68 °C for 15 min for TA extension. The PCR product obtained was gel-purified and cloned into the pCRII vector using the TA Cloning Kit (Invitrogen, San Diego, CA). Subsequently, the subcloned PCR product and the P450eryF expression plasmid (pWHM808) were digested with MscI and HindIII, and the appropriate bands were gel-purified and ligated. The sequence of the final His-tagged P450eryF was confirmed to verify the absence of any undesirable mutations and the presence of the His-tag. This modification also deleted a large segment of cDNA (of about 900 bp) present after the stop codon in the original plasmid. The construction of the mutants, A245T and A245S (without His-tag) has been described earlier (5). For His-tagging of A245T and A245S, both the cDNA of the mutants and His-tagged wildtype plasmid were cut with MscI and HindIII, and the smaller fragment containing the His-tagged segment of wild-type cDNA was ligated with the larger fragment of the mutant cDNA after gel purification. The presence of the His-tag as well as the desired mutation was confirmed by sequencing. Expression and Purification of His-Tagged P450eryF. The plasmid containing the cDNA of His-tagged P450eryF was transformed into Escherichia coli DH5R host cells. The transformed colonies were grown in 3 mL of LB media containing 50 mg/L ampicillin at 37 °C overnight. This culture was subsequently used for inoculation of 250 mL of TB media supplemented with ampicillin. The culture was grown at 37 °C with shaking at 240 rpm until OD (600 nm) reached 0.8-1.2, and 1 mM IPTG and 80 mg/mL δ-ALA were added. The culture was allowed to grow for an additional 48-72 h at 30 °C with shaking at 190 rpm. The cells were harvested by centrifugation (3840g, 10 min) and resuspended in 20 mM Tris-HCl, pH 8.0, and 500 mM KCl (buffer A). The resuspended cells were lysed by sonication (5 × 5 pulses for 5 s each, 60% capacity, flat tip, output 50%). The lysed cells were centrifuged (113000g, 35 min), and P450 in the supernatant was determined using the reducedCO difference spectrum. For purification, a nickel affinity column (Qiagen, Valencia, CA) was packed (approximately 100 nmol P450/mL of resin) and preequilibrated using buffer containing 20 mM Tris-HCl, pH 8.0, and 500 mM KCl (buffer A). The loaded column was washed with 10-column volumes each of buffer A containing 0, 1, and 10 mM imidazole, and finally with a 2-column volume of buffer A containing 20 mM imidazole. The protein was eluted with buffer A containing 200 mM imidazole. The eluted protein was dialyzed against two changes of buffer containing 2 L of 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM DTT. The concentration of P450 was determined using the reduced-CO difference spectrum. Spectral Binding Studies. Binding spectra were recorded on a Shimadzu-2600 spectrophotometer fitted with a temperature controller (TCC-240A). A solution (2.0 mL) containing 2 µM protein in 50 mM Tris-HCl, pH 7.5, and 10 mM EDTA was divided into two quartz cuvettes (10 mm path length) and a baseline was recorded between 350 and 500 nm. An aliquot of substrate in a suitable solvent (water/methanol) was then added to the sample cuvette, and the same amount of the solvent was added to the reference cuvette. The difference spectra were obtained after the system reached equilibrium (3 min). All spectra were recorded at 25 °C. Fluorescence Assays. In initial reactions, the buffer and assay conditions used were similar to those described for the testosterone hydroxylation assay (6). A fixed concentration of fluorescent substrate (100 µM for 7-BR, 7-PR, 7-EFC, and 7-BFC, and 200 µM for the quinoline derivatives) dissolved in methanol was added to 10 µM of the enzyme in 100 mM TrisHCl, pH 7.5, and 10 mM EDTA such that the final concentration of methanol was 2% in all assays. The mixture was preincubated for 5 min at 37°C before initiation of the reaction by adding hydrogen peroxide (10 mM final). The total reaction volume of

808

Chem. Res. Toxicol., Vol. 15, No. 6, 2002

Khan and Halpert

Figure 1. Binding spectra of wild-type P450eryF with androstenedione (A), testosterone (B), and 9-aminophenanthrene (C). The concentration of enzyme was 2 µM in all cases. The lines through the experimental points in panels D, E, and F show hyperbolic (- - -) and sigmoidal (-) fits to the change in absorbance against substrate concentrations for androstenedione, testosterone, and 9-aminophenanthrene, respectively. the assay was 100 µL. After 5 min of incubation, the reactions were stopped by adding 340 units of catalase. The determination of the amount of the various products formed were carried out as described (14, 17-18). In all cases, the final activity was calculated by comparison to a standard curve. The final 7-BQ assay conditions as optimized after a detailed study of various experimental conditions were as follows (also see Results). A fixed concentration of 7-BQ was added to 4 µM enzyme in a buffer containing 50 mM Tris-HCl, pH 7.5, and 200 mM KCl. The rest of the assay procedure was the same as described above except that the reactions were carried out at room temperature and stopped after 5 min by adding 50 µL of 6.8 unit/µL catalase. Finally, 2 mL of a buffer containing 50 mM Tris-HCl, pH 7.5, and 200 mM KCl was added, and the reaction mixture was vortexed. The total amount of product was deter-

mined by fluorescence, using excitation and emission wavelengths of 410 and 510 nm, respectively, and a bandwidth of 20 nm in each case. Activity was calculated by comparison to a standard curve of 7-HQ. Data Analysis. Nonlinear regression (Sigma Plot Windows, Jandel, San Rafel, CA) was used to determine Vmax and S50 by using the equation v ) (VmaxSn)/(S50n + Sn) (12). In the case of spectral titrations, ∆Amax and KS were determined by nonlinear regression of the plot of ∆A vs S using the hyperbolic equation [∆A ) ∆AmaxS/(KS + S)], the Hill equation (∆A ) ∆AmaxSn/ (S50n + Sn)], or the tight binding equation [∆A ) ∆Amax/2E(E + S + KS - [{E + S + KS}2 - 4ES]1/2)], where E and S represent the concentrations of P450eryF and substrate, respectively, ∆A and ∆Amax are the changes in the absorption at the substrate concentration S and a saturating substrate concentration,

7-BQ as a New Substrate Probe of P450eryF

Chem. Res. Toxicol., Vol. 15, No. 6, 2002 809

Table 1. S50 Values, Hill Coefficients (n Values) and Maximal Absorbance Changes from Spectral Titrations of P450eryF Wild-Type and A245T with Various Ligands testosterone ∆Amax (µM-1 cm-1) WT A245T

0.030 (0.003)a b

androstenedione

n

S50 (µM)

∆Amax (µM-1 cm-1)

1.64 (0.13)

278 (35)

0.070 (0.005) 0.044 (0.003)

DHEA

n

S50 (µM)

∆Amax (µM-1 cm-1)

1.20 (0.07) 1.09 (0.04)

261 (32) 277 (29)

0.018 (0.004) ND

9-aminophenanthrene

n

S50 (µM)

∆Amax (µM-1 cm-1)

n

S50 (µM)

1.55 (0.02)

326 (93)

0.065 (0.01) 0.039 (0.005)

1.9 (0.10) 1.7 (0.01)

10.3 (0.2) 31.2 (2.7)

a The values in parentheses show the deviation obtained from fitting to the Hill equation. parameters could not be determined. ND, Not determined.

b

The change was very small, and the various

respectively, and KS is the equilibrium dissociation constant of the enzyme-substrate complex.

Results Expression and Purification of P450eryF WildType, A245S, and A245T. The original vectors containing cDNA of the wild-type and mutants (2, 5) were modified to put a 4-His tag at the C-terminus for easier purification of the expressed proteins. The expression level was also maximized by adding 80 mg/L of δ-ALA and 1 mM of IPTG during culture growth. Purification of the protein was achieved using a single metal-affinity column with the final yields between 800 and 1200 nmol/L and an A418/A280 ratio of 1.5-1.7. The yield for the wild-type P450eryF was originally reported to be 3 mg/L (i.e., approximately 60 nmol/L) with an A418/A280 ratio of 1.76 (19). Thus, the modification of the cDNA of wild-type and mutants, as well as changes in expression and purification procedures, led to much higher yields of protein with significant purity, avoiding the need for a fermentor or the complexity of multi-column protein purification procedures. The expression level of similarly expressed 6-His tag wild-type P450eryF was reported to be ∼80 mg (1600 nmol) of protein/L of the culture (6). Substrate Binding Studies. The wild-type P450eryF and A245T were initially characterized by spectral titration with androstenedione, testosterone, and 9-aminophenanthrene. Androstenedione and 9-aminophenanthrene binding to the wild-type P450eryF is known to be cooperative (7). The crystal structures have also revealed the simultaneous presence of two molecules of androstenedione or 9-aminophenanthrene in the wildtype P450eryF active site (7). Testosterone binding and formation of one of the four metabolites by A245T have also been reported to be sigmoidal (6). The binding of androstenedione or testosterone to the enzyme induces a type-I spectral change, whereas 9-aminophenanthrene induces a type-II spectral change. As expected, binding of androstenedione and testosterone to the recombinant wild-type P450 also showed sigmoidal behavior, although the n value for androstenedione binding was smaller than testosterone (see Figure 1 and Table 1). Furthermore, although the binding of androstenedione showed a much higher ∆Amax compared with testosterone, the enzyme showed similar S50 values for both steroids. Binding of another steroid molecule dehydroepiandrosterone (DHEA), which has a 3-OH instead of 3-keto group, to the wildtype enzyme was also found to be sigmoidal. The binding affinity of this ligand was similar to that of the other two steroids, although the ∆Amax was the smallest among the three compounds. A245T exhibited a very small spectral change on testosterone binding, such that an exact determination

Figure 2. The chemical structures of various 7-substituted (A) coumarin, (B) resorufin, and (C) quinoline derivatives examined in this study.

of binding affinity was not possible. Titration of A245T with androstenedione and 9-aminophenanthrene also revealed a smaller spectral change compared with the wild-type (Table 1). These results are consistent with studies of 6-DEB binding to wild-type, A245S, and A245T, where the physiological substrate produced the smallest spectral change with the A245T mutant (5). Fluorescent Substrates of P450eryF. Fluorescence assays provide one of the easiest and quickest ways of screening a large number of compounds. In this study, we tested five different fluorescent substrates often used with other P450 enzymes: 7-BR, 7-PR, 7-EFC, 7-BFC, and 7-BQ (Figure 2). All of these substrates were assayed using wild-type P450eryF, as well as mutants A245S and A245T, with H2O2 as an oxidizing agent. No oxidation activity was observed with 7-BR, 7-PR, or 7-EFC (Figure 3A). Low activity was observed using 7-BFC. However, 7-BQ showed the best activity among all the substrates tested, with A245T being the most active among the wildtype P450eryF, A245S, and A245T. The relative rates of 7-BFC oxidation by wild-type P450eryF, A245S, and A245T were 1, 5, and 9%, respectively, of the rate of 7-BQ oxidation by A245T. The relative rates of 7-BQ oxidation by A245S and P450eryF were 48% and 7%, respectively, compared with A245T. This observed order of activity was the same as for testosterone hydroxylation (6). A survey of different 7-alkoxyquinolines showed the best activity with 7-butyloxyquinoline. However, this activity was approximately 5-6-fold less than that with 7-BQ (Figure 3B). To find the most suitable conditions for 7-BQ oxidation, various parameters were optimized using P450eryF A245T. Among the three different buffers tested (HEPES, phosphate, and Tris-HCl), the rate of oxidation was found to be maximal using Tris-HCl buffer. While the addition of MgCl2 and EDTA showed negligible effects on the rate of oxidation of 7-BQ by A245T, the addition of glycerol caused a decrease in activity. However, the addition of KCl (50-500 mM) showed a 30-50% increase in 7-BQ

810

Chem. Res. Toxicol., Vol. 15, No. 6, 2002

Figure 3. (A) A plot of the percent activity for oxidation of various fluorescent substrates by wild-type P450eryF, A245S, and A245T compared with 7-BQ oxidation by P450eryF A245T. (B) A plot of the percent activity for oxidation of various 7-alkoxyquinoline derivatives by P450eryF A245T compared with 7-BQ. C1, C2, C3, C4, C5, C6, and C7 represent 7-methoxy-, 7-ethoxy-, 7-propoxy-, 7-butoxy-, 7-pentoxy-, 7-hexoxy-, and 7-heptoxyquinoline, respectively. The actual rate of 7-BQ oxidation was 0.3 nmol/min/nmol of P450eryF A245T under initial assay conditions as described in Experimental Procedures.

oxidation by A245T. The use of cumene hydroperoxide (0.1-2.0 mM) as an alternate oxidizing agent yielded much lower activity. The highest activity observed was at least four to 5-fold less than the oxidation rate at 10 mM H2O2. A detailed analysis of time, protein concentration, and H2O2 concentration was carried out at 37 °C and room temperature (22 °C). On the basis of the above experiments, subsequent assays were performed at 4 µM enzyme and 10 mM H2O2 in 50 mM Tris-HCl, pH 7.5, containing 200 mM KCl. The reaction was linear for more than 5 min under these conditions (Figure 4A), and a 5-min reaction time was used for future reactions. The steady-state kinetics of 7-BQ oxidation by A245T showed sigmoidal behavior (Vmax ) 0.71 ( 0.02 nmol/min/ nmol, n ) 2.18 ( 0.13, and S50 ) 132 ( 5 µM) (Figure 4B), indicating the cooperative nature of 7-BQ oxidation by P450eryF. The kinetics of testosterone consumption by A245T has also been reported to be sigmoidal (6). The Vmax value for 7-BQ oxidation by A245T is comparable to the rate of consumption of testosterone (0.48 min-1), but at least four times higher than that of the rate of formation of any single metabolite (6). In comparison, the 6-DEB hydroxylation rates have been reported to be approximately 15-times higher with the wild-type, but

Khan and Halpert

Figure 4. (A) Time dependence of 7-BQ oxidation by P450eryF A245T at 200 µM substrate concentration under final assay conditions (see Experimental Procedures for details). (B) The steady-state kinetics of 7-BQ oxidation by A245T. The solid line through the experimental points shows the fit to the Hill equation.

100-fold lower with A245T2 (5). However, the steady-state kinetics of 6-DEB hydroxylation by wild-type P450eryF showed hyperbolic behavior (5). Flavones are known to be some of the best activators of CYP3A4 (9). A spectral binding study of wild-type P450eryF with R-NF showed positive cooperativity (n ) 2.0 ( 0.2) and an S50 value (5.0 µM) similar to the wildtype P450 3A4 (14, 16). However, the addition of various flavones (flavone, R-NF, and β-NF) at two different concentrations (10 and 25 µM) showed no significant effect on the rate of 7-BQ oxidation by A245T at 200 µM substrate concentration. Interestingly, at least for R-NF, these concentrations have previously been reported to be optimal for activation of substrate oxidation by P450 3A4 (10-12). A detailed kinetic analysis in the presence of 25 µM R-NF showed a small decrease in the rate of 7-BQ oxidation by A245T, with no significant effect on any of the three kinetic parameters. Interestingly, 7-BQ is a well-known substrate of human CYP3A4 (26, 27), and has been reported to exhibit heterotropic but not homotropic cooperativity (27). Spectral Binding and Inhibition Studies Using Imidazole Derivatives. Imidazole derivatives constitute some of the most effective inhibitors of cytochrome P450 enzymes due to their ability to form a coordination bond between the lone electron pair of nitrogen and the heme without being oxidized (18, 28, 29). However, a 2 The rate of 6-DEB oxidation by wild-type and A245T were measured using ferredoxin, spinach ferredoxin reductase, and NADPH, and not H2O2 as in case of 7-BQ and testosterone.

7-BQ as a New Substrate Probe of P450eryF

Chem. Res. Toxicol., Vol. 15, No. 6, 2002 811

Figure 5. Spectral titration of A245T with ketoconazole (A) and miconazole (B). The concentration of A245T was 2 µM in the case of ketoconazole titration and 1 µM with miconazole. (C) The lines through ∆ absorbance against ketoconazole concentration show fits to the tight-binding (- - -) and Hill equation (-), respectively. The ∆Amax, n, and S50 values were 0.083 ( 0.004 cm-1, 1.49 ( 0.12, and 22.1 ( 1.9 µM, respectively. (D) The change in absorbance against miconazole concentrations was fit to the tight-binding equation. Table 2. Dissociation Constants and Maximal Absorbance Changes from Spectral Titrations of Various Imidazole Derivatives with P450eryF Wild-Type, A245T and A245S WT KS (µM) ketoconazole imidazole 1′-phenylimidazole 4-phenylimidazole 2-methylimidazole miconazole

5.1 (2.0)a 1190 (30)b 231 (6)b 432 (14)b d 0.180 (0.087)a

A245T ∆Amax (µM-1 cm-1) 0.071 (0.008)a 0.075 (0.001)b 0.057 (0.001)b 0.078 (0.001)b 0.067 (0.004)a

KS (µM)

A245S

∆Amax (µM-1 cm-1)

c

0.212 (0.062)a

KS (µM)

∆Amax (µM-1 cm-1)

23 (1.1)a

0.058 (0.002)a

0.059 (0.003)a

a The values in parentheses show the deviation obtained from tight-binding equation. b The values in parentheses show the deviation obtained from Michaelis-Menton fitting. c Showed no significant change at lower concentrations of ketoconazole. At higher concentrations type-I like spectra were observed (see Figure 3, panels A and C, for details). d Did not show any spectral changes.

recent study reported that P450eryF A245T failed to give a typical type-II spectrum upon titration with ketoconazole, and the attempt to crystallize the A245T-ketoconazole complex was also unsuccessful (30). Renewed attempts to reproduce this spectral titration of A245T with ketoconazole showed rather interesting results. At lower concentrations of ketoconazole spectral changes were almost negligible, whereas at higher concentrations the peak and trough appeared at 384 and 417 nm (Figure 5A), respectively, unlike those in any type-II binding spectra (Figure 5B) but similar to type-I binding. A plot of ∆A vs [ketoconazole] could not be fit using either a

tight-binding or Michaelis-Menten equation but fit well to the Hill equation (Figure 5C). In contrast, wild-type and A245S showed typical type-II binding with ketoconazole, although the ∆Amax for A245S was smaller than for the wild-type enzyme (Table 2). The dramatic shift in binding pattern observed in the case of A245T may be due to a collapse of helices forming the P450eryF active site resulting from the introduction of a bulkier group in the I-helix and/or the disruption of a local hydrogen bonding network that was found essential for the substrate-assisted catalysis of 6-DEB by the enzyme (5, 30).

812

Chem. Res. Toxicol., Vol. 15, No. 6, 2002

Khan and Halpert

(Figure 6B). Automated fitting using the Enzyme Module Kinetics Program of Sigma Plot showed that the data only satisfy the conditions for noncompetitive inhibition, although the different parameters calculated showed large standard errors.

Discussion

Figure 6. (A) Ketoconazole (b) and miconazole (9) inhibition of 7-BQ oxidation by A245T ([7-BQ] ) 200 µM). The 7-BQ oxidation activity of 0.6 nmol/min/nmol of P450eryF A245T was considered 100% in both cases. (B) The steady-state kinetics of 7-BQ oxidation by A245T at various miconazole concentrations 0 (b), 2 (O), 5 (9), and 10 (0) µM miconazole. The lines through the experimental points show the fit to the Hill equation.

In light of the unusual binding of ketoconazole to A245T, we wanted to find an alternate imidazole derivative that could bind to A245T in a typical type-II mode. To find the strongest P450eryF binding imidazole derivative, the wild-type was initially tested (Table 2). As expected, all imidazole derivatives, except 2-phenylimidazole, showed the usual type-II binding spectra. This confirms the earlier observation of the requirement for an unhindered lone electron pair on a nitrogen, which is perturbed by the substitution at the 2-position (28). Furthermore, among various imidazoles, miconazole showed the highest binding affinity for the wild-type enzyme (even higher than ketoconazole). The titration of A245T also showed type-II binding with miconazole, and the mutant also had the same affinity as the wildtype (see Figure 5, panels B and D, and Table 2). This indicates that the much larger size of ketoconazole could be the reason for the drastic difference observed between the binding of ketoconazole, miconazole, and 9-aminophenathrene with A245T. Inhibition studies revealed that both miconazole and ketoconazole are able to inhibit 7-BQ oxidation by A245T (Figure 6A). An approximate estimation of IC50 values indicated that miconazole (IC50 ≈ 3-6 µM) may have slightly greater inhibition potency than ketoconazole (IC50 ≈ 8-10 µM). However, miconazole inhibition potency seems to be almost 15-30 times lower than its binding affinity. A detailed inhibition study with miconazole showed rather complex behavior, and the data were very difficult to fit to a particular inhibition mechanism

P450eryF is the only bacterial P450 that demonstrates cooperative ligand binding and has been crystallized with two ligand molecules bound simultaneously in the active site (7). Thus, the system is a useful mimic of mammalian P450 enzymes, such as P450 3A4, which is involved in the metabolism of a wide variety of drugs, steroids, and carcinogens and also shows homotropic and heterotropic cooperativity (8-16). The main objective of the present study was to find a fluorescent substrate probe of P450eryF amenable for high throughput assays using peroxides as the oxidizing support system. Although the identities of the natural electron donors to P450eryF have yet to be discovered, 6-DEB hydroxylation has been successfully reconstituted using spinach ferrodoxin and ferrodoxin NADP+ reductase (4). However, as recently reported by Xiang et al., the Ala-245 f Thr substitution confers testosterone hydroxylation upon P450eryF only in the presence of H2O2 and not in the presence of these artificial electron transport proteins (6). Although the addition of H2O2 could lead to slow decomposition of the enzyme, optimization of various parameters for 7-BQ oxidation yielded a linear reaction over a reasonable amount of time (Figure 4A). However, it is important to realize that the kinetic parameters obtained using the two different oxidizing support systems may be significantly different. Nevertheless, a better understanding of P450 catalyzed reaction kinetics performed using peroxide(s) has the potential to provide an inexpensive alternative to costly NADPH and other reconstitution enzymes for industrial use of these enzymes. Among the various coumarins, resorufins, and quinolines screened in this study, all quinoline derivatives yielded some activity with P450eryF A245T. Earlier, it has been suggested that the nitrogen atom in the ring system could confer special properties to quinolines as P450 substrates (17). However, both quinoline and resorufin have a N-atom in the ring system. Therefore, if the above suggestion is true, the strategic placement of the N-atom or steric constrains caused by the much larger size of resorufin could be the reason for the lack of activity with 7-benzyloxyresorufin. The benzyloxy side chain is also important, as both 7-BFC and 7-BQ showed much higher activity than 7-EFC and 7-alkoxyquinoline derivatives, respectively. Thus, a quinoline moiety in combination with a benzyloxy group as a side chain combine to make 7-BQ the best substrate tested. It is pertinent to point out here that the various substrates examined in this study (see Figure 2), as well as the previously discovered substrate testosterone (6), are much smaller than the physiological substrate of P450eryF, 6-DEB. Therefore, the number and identity of active site residues involved in protein-substrate interactions could be significantly different, as revealed by comparisons of various P450eryF-ligand crystal structures (3, 5, 7, 19, 20, 30). However, the availability of a such model system provides a good opportunity to better understand how an enzyme such as P450 3A4 is able to metabolize compounds as small as acetaminophen (mol. wt. 151) and as large as cyclosporins (mol. wt. 1201).

7-BQ as a New Substrate Probe of P450eryF

The cooperative nature of 7-BQ and testosterone oxidation kinetics by P450eryF A245T is among only a few examples of any P450 showing cooperativity in the presence of oxygen surrogates. With the exception of one report (21), no positive cooperativity had previously been detected in P450 systems supported with oxygen surrogates instead of NADPH/O2 (10, 22, 23). This observation had led to the hypothesis that the observed cooperativity of P450 may be due to effector stimulating better binding between P450 and reductase leading to a more efficient electron transfer (24, 25). However, the sigmoidal nature of testosterone and 7-BQ oxidation kinetics in the presence of H2O2 shows that this explanation may not be valid in the case of P450eryF. The discovery of a convenient fluorescent substrate probe of P450eryF also enabled the identification of miconazole and ketoconazole as inhibitors of the enzyme, although the precise mechanism of inhibition remains elusive. A recent study showed that P450eryF-ketoconazole crystals soaked in 6-DEB to exchange ligands exhibited a structure identical to that of the original P450eryF-6-DEB complex (30). Thus, at least ketoconazole inhibition of 6-DEB oxidation by the wild-type enzyme is expected to be reversible. However, inhibition kinetics is known to be dependent on the substrateinhibitor pair. Therefore, it will be difficult to extrapolate a specific mechanism of miconazole inhibition of 7-BQ oxidation by A245T. In any event, the present spectral and inhibition results clearly indicate that miconazole is an effective inhibitor of P450eryF. In conclusion, the present study reports the discovery of only the second xenobiotic substrate oxidized by P450eryF A245T. The rapid and simple 7-BQ assay should provide a more dynamic tool for better understanding of P450eryF cooperativity and its structurefunction relationships. The focus of future studies will involve further alterations in the active site based on docking of 7-BQ into P450eryF with the dual goals of obtaining higher activity and enhanced versatility.

Acknowledgment. The authors would like to thank Dr. Jill Cupp-Vickery for providing the original plasmids of wild-type P450eryF, A245S, and A245T, and Ms. YouAi He and You-Qun He for their help and suggestions provided during the modification of cDNA of mutants as well as of wild-type P450eryF. This work was supported by the Robert A. Welch Foundation, Houston, TX (H-1458), and Center Grant ES06676 from the National Institutes of Health.

References (1) Shafiee, A., and Hutchinson, C. R. (1988) Purification and reconstitution of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis. J. Bacteriol. 170, 1548-1553. (2) Andersen, J. F., and Hutchinson, C. R. (1992) Characterization of Saccharopolyspora erythraea cytochrome P-450 genes and enzymes, including 6-deoxyerythronolide B hydroxylase. J. Bacteriol. 174, 725-735. (3) Cupp-Vickery J. R., and Poulos, T. L. (1995) Structure of cytochrome P450eryF involved in erythromycin biosynthesis. Nat. Struct. Biol. 2, 144-153. (4) Andersen, J. F., Tatsuta, K., Gunji, H., Ishiyama. T., and Hutchinson, C. R. (1993) Substrate specificity of 6-deoxyerythronolide B hydroxylase, a bacterial cytochrome P450 of erythromycin A biosynthesis. Biochemistry 32, 1905-1913. (5) Cupp-Vickery, J. R., Han, O., Hutchinson, C. R., and Poulos, T. L. (1996) Substrate-assisted catalysis in cytochrome P450eryF. Nat. Struct. Biol. 3, 632-637.

Chem. Res. Toxicol., Vol. 15, No. 6, 2002 813 (6) Xiang, H., Tschirret-Guth, R. A., and Ortiz De Montellano, P. R. (2000) An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates. J. Biol. Chem. 275, 35999-36006. (7) Cupp-Vickery, J., Anderson, R., and Hatziris, Z. (2000) Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity. Proc. Natl. Acad. Sci. U.S.A. 97, 3050-3055. (8) Shou, M., Grogan, J., Mancewicz, J. A., Krausz, K. W., Gonzalez, F. J., Gelboin, H. V., and Korzekwa, K. R. (1994) Activation of CYP3A4: evidence for the simultaneous binding of two substrates in a cytochrome P450 active site. Biochemistry 33, 6450-6455. (9) Shou, M., Dai, R., Cui, D., Korzekwa, K. R., Baillie, T. A., and Rushmore, T. H. (2001) A kinetic model for the metabolic interaction of two substrates at the active site of cytochrome P450 3A4. J. Biol. Chem. 276, 2256-2262. (10) Ueng, Y. F., Kuwabara, T., Chun, Y. J., and Guengerich, F. P. (1997) Cooperativity in oxidations catalyzed by cytochrome P450 3A4. Biochemistry 36, 370-381. (11) Harlow, G. R., and Halpert, J. R. (1997) Alanine-scanning mutagenesis of a putative substrate recognition site in human cytochrome P450 3A4. Role of residues 210 and 211 in flavonoid activation and substrate specificity. J. Biol. Chem. 272, 53965402. (12) Harlow, G. R., and Halpert, J. R. (1998) Analysis of human cytochrome P450 3A4 cooperativity: construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics. Proc. Natl. Acad. Sci. U.S.A. 95, 66366641. (13) Domanski, T. L., He, Y. A., Harlow, G. R., and Halpert, J. R. (2000) Dual role of human cytochrome P450 3A4 residue Phe304 in substrate specificity and cooperativity. J. Pharmacol. Exp. Ther. 293, 585-591. (14) Domanski, T. L., He, Y. A., Khan, K. K., Roussel, F., Wang, Q., and Halpert, J. R. (2001) Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and the effect on substrate oxidation and cooperativity. Biochemistry 40, 10150-10160. (15) Khan, K. K., He, Y. Q., Domanski, T. L., and Halpert, J. R. (2002) Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation. Mol. Pharmacol. 61, 495-506. (16) Hosea, N. A., Miller, G. P., and Guengerich, F. P. (2000) Elucidation of distinct ligand binding sites for cytochrome P450 3A4. Biochemistry 39, 5929-5939. (17) Mayer, R. T., Netter, K. J., Heubel, F., Hahnemann, B., Buchheister, A., Mayer, G. K., and Burke, M. D. (1990) 7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases. Biochem. Pharmacol. 40, 1645-1655. (18) Spatzenegger, M., Wang, Q., He, Y. Q., Wester, M. R., Johnson, E. F., and Halpert, J. R. (2001) Amino acid residues critical for differential inhibition of CYP2B4, CYP2B5, and CYP2B1 by phenylimidazoles. Mol. Pharmacol. 59, 475-484. (19) Cupp-Vickery, J. R., Li, H., and Poulos, T. L. (1994) Preliminary crystallographic analysis of an enzyme involved in erythromycin biosynthesis: cytochrome P450eryF. Proteins 20, 197-201. (20) Cupp-Vickery, J. R., and Poulos, T. L. (1997) Structure of cytochrome P450eryF: substrate, inhibitors, and model compounds bound in the active site. Steroids 62, 112-116. (21) Ingelman-Sundberg, M., and Johansson, I. (1980) Catalytic properties of purified forms of rabbit liver microsomal cytochrome P-450 in reconstituted phospholipid vesicles. Biochemistry 19, 4004-4011. (22) Maenpa¨a¨, J., Hall, S. D., Ring, B. J., Strom, S. C., and Wrighton, S. A. (1998) Human cytochrome P450 3A (CYP3A) mediated midazolam metabolism: the effect of assay conditions and regioselective stimulation by alpha-naphthoflavone, terfenadine and testosterone. Pharmacogenetics 8, 137-155. (23) Lee, C. A., Manyike, P. T., Thummel, K. E., Nelson, S. D., and Slattery, J. T. (1997) Mechanism of cytochrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes. Drug Metab. Dispos. 25, 1150-1156. (24) Huang, M. T., Chang, R. L., Fortner, J. G., and Conney, A. H. (1981) Studies on the mechanism of activation of microsomal benzo[a]pyrene hydroxylation by flavonoids. J. Biol. Chem. 256, 6829-6836. (25) Miller, M. S., Huang, M. T., Jeffrey, A. M., and Conney, A. H. (1983) Betamethasone-mediated activation of biphenyl 2-hydroxylation in rat liver microsomes. Studies on possible mechanisms. Mol. Pharmacol. 24, 137-145. (26) Stresser, D. M., Blanchard, A. P., Turner, S. D., Erve, J. C. Dandeneau, A. A. Miller, V. P., and Crespi, C. L. (2000) Substratedependent modulation of CYP3A4 catalytic activity: analysis of

814

Chem. Res. Toxicol., Vol. 15, No. 6, 2002

27 test compounds with four fluorometric substrates. Drug Metab. Dispos. 28, 1440-1448. (27) Lu, P., Lin, Y., Rodrigues, A. D., Rushmore, T. H., Baillie, T. A., and Shou, M. (2001) Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin bind to different domains within the active site of cytochrome P450 3A4. Drug Metab. Dispos. 29, 1473-1479. (28) Murray, M., and Wilkinson, C. F. (1984) Interactions of nitrogen heterocycles with cytochrome P-450 and monooxygenase activity.

Khan and Halpert Chem.-Biol. Interact. 50, 267-275. (29) Poulos, T. L., and Howard, A. J. (1987) Crystal structures of metyrapone- and phenylimidazole-inhibited complexes of cytochrome P-450cam. Biochemistry 26, 8165-8174. (30) Cupp-Vickery, J. R., Garcia, C., Hofacre, A., and McGee-Estrada, K. (2001) Ketoconazole-induced conformational changes in the active site of cytochrome P450eryF. J. Mol. Biol. 311, 101-110.

TX0200010