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Research ArticleArticle

Structure-Function Analysis of Human Cytochrome P-450 2B6 Using a Novel Substrate, Site-Directed Mutagenesis, and Molecular Modeling

Tammy L. Domanski, Kathleen M. Schultz, Fabienne Roussel, Jeffrey C. Stevens and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics September 1999, 290 (3) 1141-1147;

Abstract

The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarinO-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.

The cytochrome P-450 (P-450) superfamily catalyzes the monooxygenation of a large number of endogenous and exogenous compounds. Some of the first P-450s purified and cloned included members of the 2B subfamily, due to their high activity and level of expression (Waxman, 1988). Early P-450 2B cDNAs cloned include rat 2B1 and 2B2 (Fujii Kuriyama et al., 1982;Aoyama et al., 1989), rabbit 2B4 and 2B5 (Gasser et al., 1988), mouse 2B9 and 2B10 (Noshiro et al., 1988), and dog 2B11 (Graves et al., 1990). Studies of the purified or heterologously expressed enzymes revealed a specificity for steroid hydroxylation (Waxman, 1988), and subsequent work focused on elucidation of the structural determinants of this selectivity. Initial studies examined allelic forms of 2B1 and illustrated the importance of residues L58, I114 (Aoyama et al., 1989), and G478 (Kedzie et al., 1991).

The group-to-group alignments of CYP2 sequences by Gotoh (1992)proposed the presence of six substrate recognition sites (SRSs) within the P-450 2 family. Significant progress has been made toward defining the molecular basis of rat 2B1, rabbit 2B4 and 2B5, and dog 2B11 specificity by testing predictions made by similar alignments (reviewed in Von Wachenfeldt and Johnson, 1995). However, the human member of the 2B subfamily has defied thorough characterization due to its highly variable expression in the human liver (Ekins et al., 1998) and to the lack of a useful isoform-selective substrate or inhibitor (Mimura et al., 1993). Initial characterizations found that 2B6 activates several carcinogens, including dibenzo[a,h]anthracene (Shou et al., 1996) and 6-aminochrysene (Mimura et al., 1993), and hydroxylates the anticancer drug cyclophosphamide (Chang et al., 1993), activities shared by other 2B and non-2B enzymes. In addition, P-450 2B6 hydroxylates testosterone at a much lower rate than other members of the 2B subfamily (Imaoka et al., 1996; Yang et al., 1998). Recently, new 2B6 substrates have been identified. Evidence has shown that 2B6 is responsible for the N-demethylation ofS-mephenytoin (Heyn et al., 1996) and the hydroxylation of RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxyl-benzamide;Stevens et al., 1997]. These new substrates and the ability to heterologously express 2B6 (Yang et al., 1998) have provided the necessary tools to perform detailed characterization of P-450 2B6.

This report is the first to describe specific residues that affect 2B6 specificity. The study took advantage of the differences in steroid and RP 73401 hydroxylation that exist between 2B1 and 2B6 to identify residues that help to determine 2B6 substrate specificity. Ten unique 2B6 SRS residues were mutated to the corresponding residues in 2B1, and two additional mutants, L363V and V477I, were produced to test sites known to be important in 2B1. In an attempt to confer androstenedione hydroxylation on P-450 2B6, SRS exchanges were performed between 2B6 and 2B1. A model of P-450 2B6 was also developed and used to dock a molecule of RP 73401 within the predicted active site.

Experimental Procedures

Materials.

All oligonucleotides were obtained from the University of Arizona Macromolecular Structure Facility (Tucson, AZ). Restriction endonucleases were purchased from Life Technologies, Inc. (Grand Island, NY), and the Expand PCR (polymerase chain reaction) kit was purchased from Boehringer Mannheim (Indianapolis, IN). 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), NADPH, dilauroyl-l-3-phosphatidylcholine (DLPC), androstenedione, and testosterone were obtained from Sigma Chemical Co. (St. Louis, MO). [14C]Androstenedione and [14C]testosterone were purchased from DuPont-New England Nuclear (Boston, MA). 7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) and 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) were purchased from Molecular Probes (Eugene, OR). HEPES was obtained from Calbiochem (La Jolla, CA), and silica gel thin-layer chromatography plates and acetonitrile were purchased from J.T. Baker (Phillipsburg, NJ). RP 73401 and RPR 113406 were obtained from RPR Research Center (Dagenham, UK). All other reagents and supplies not listed were obtained from standard sources. Purified P-450 2B1 was kindly provided by You-Ai He (University of Texas Medical Branch, Galveston, TX).

Subcloning and Mutagenesis of P-450 2B6.

The 2B6 cDNA was kindly provided by Dr. Frank Gonzalez (National Cancer Institute, National Institutes of Health, Bethesda, MD). Subcloning of the 2B6 cDNA into the plasmid pFastBac1 was accomplished using the PCR and primers designed to incorporate BamHI and SpeI sites upstream of the start codon and downstream of the termination codon, respectively (Fig. 1A). The 3′ primer also included six histidine codons, which allowed the potential for protein purification using a metal affinity column. After amplification of the cDNA and subcloning of the desired fragment into pFastBac1 to create pFast2B6H, the insert was verified by sequencing (University of Arizona Sequencing Facility, Tucson, AZ).

Figure 1
Figure 1

Primers used for amplification of 2B6 cDNA (A) single mutants and (B) multiple mutants. The mutated nucleotides are underlined, and silent mutations that altered restriction sites are in bold.

Plasmid pFast2B6H was used as the template for construction of all of the mutants described in the text and presented in tables. Mutants M103V, F107I, and R109K were produced using a one-step PCR method followed by subcloning into pF2B6H to create the desired mutation. A unique restriction site, SalI, was located close to the codons for residues 103, 107, and 109; therefore, this site was incorporated into the mutagenic primers (Fig. 1A). After amplification of the desired regions with the mutagenic primers and 2B6,5′B, in the case of M103V, and 2B6,3′S, for F107I and R109K, the products were digested with SalI and a second restriction enzyme (BamHI for M103V and SpeI for F107I and R109K). The appropriate fragments were purified with GeneClean II (Bio 101, Vista, CA), ligated into pF2B6H, and transformed into DH5α cells. The DNA from resulting colonies was isolated, and the subcloned region was sequenced to check for the presence of the desired mutation and to ensure that no PCR errors had occurred. This method was also used to create S284H, N289M, N291S, T292L, and T292A, except that the mutagenic primers incorporated an EcoRI site at the 5′ terminus (Fig.1A).

For the remaining single mutants, L363V, M365I, I370R, C475S, and V477I, the Expand PCR kit was used according to the manufacturer’s directions. As above, plasmid pF2B6H was the template for these reactions. Each mutagenic primer contained a silent mutation that changed one restriction recognition site for later selection. For L363V, M365I, and I370R, each mutagenic primer was used with the S5ANTI primer (Fig. 1A). In the case of C475S and V477I, the S6ANTI primer was included in the reaction. In this procedure, two overlapping primers were used, which resulted in amplification away from the overlap. The length of the amplification step (5 min) allowed for amplification of the entire template plasmid. After amplification, most of the reaction was incubated with DpnI, a four-base cutting enzyme that only digests methylated DNA. In this way, all wild-type template DNA was digested into small fragments. The uncut and cut portions of the reaction were then transformed into DH5α. The DNA was then isolated from several of these possible mutant-containing colonies and digested with the appropriate restriction enzyme: NcoI for the SRS-5 mutants and BstXI for the SRS-6 mutants. Those clones missing either the NcoI or the BstXI site were sequenced through the entire length of the coding sequence to ensure that the desired mutations were the only ones present.

The multiple mutants ΔS1, ΔS2, ΔS5, and ΔS6 (mutants containing conversion of 2B6 SRS amino acid sequence to 2B1) were produced with the Expand PCR kit as described above, except that a silent mutation was not introduced into the mutagenic primer (Fig. 1B). The mutations made in S1 and S5 each caused the loss of anNcoI site, the changes in S2 resulted in the loss of anXmnI site, and the mutations in S6 removed aBstXI site. The sequence of each construct was then verified.

To ensure that the Expand amplification did not introduce extraneous mutations into regions of the pFastBAc plasmid that were required for proper infection and protein expression, subcloning was performed. For each mutant, a BamHI-to-SpeI fragment encompassing the entire 2B6 coding sequence was subcloned from the amplified mutant-containing plasmid into digested pFastBac.

Expression of P-450 2B6 and Mutants in Spodoptera frugiperda Insect Cells.

The Bac-to-Bac System from Life Technologies was chosen for the heterologous expression of P-450 2B6. After strains were generated containing the 2B6 or mutant cDNA in pFastBac, plasmid δ-aminolevulenic acid was isolated and transformed into DH10Bac cells according to the manufacturer’s instructions. Transfection of the recombinant bacmid DNA into S. frugiperda (Sf9) cells was performed as described (Felgner et al., 1987). A large-scale amplification of the viral stock was done, and the viral titer was determined by serial dilution and plaque assay.

Sf9 cells were grown in SF-900 M serum-free media (Life Technologies) at 27°C and shaken at 120 rpm. For protein expression, infection of Sf9 cells was performed as described (Roussel et al., 1998) with several modifications. A multiplicity of infection of 3 pfu/cell was chosen for the expression of 2B6 and its mutants. At 24 h postinfection, ferric citrate and ALA were added at a final concentration of 100 μM. The cells were harvested 48 h later and microsomal membranes were prepared as described previously (Roussel et al., 1998). P-450 content was measured according to the method of Omura and Sato (1964), and total protein was determined by the Bradford method (1976).

Enzyme Assays.

Androstenedione and testosterone hydroxylase assays were performed as described previously (Domanski et al., 1998) with the following modifications. Microsomal samples containing 2.5 pmol of 2B1, 2B6, or a mutant were reconstituted with rat NADPH:P-450 reductase and cytochrome b5 in a 1:2:1 ratio, followed by the addition of 0.03 mg/ml DLPC and CHAPS at a final concentration of 0.05%. The reactions contained HEPES (50 mM HEPES at pH 7.6, 15 mM MgCl2, 0.1 mM EDTA) and [14C]androstenedione or [14C]testosterone (25 μM) with a final methanol concentration of 1%. The reactions were initiated by the addition of NADPH (1 mM) and run for 20 min at 37°C. The reactions were stopped with tetrahydrofuran. The analytes were separated by thin-layer chromatography and detected by autoradiography (Halpert and He, 1993).

Samples for 7-EFC O-deethylase assays were prepared as described above with reductase, cytochromeb5, DLPC, and CHAPS. Reactions were carried out in HEPES (50 mM HEPES at pH 7.6, 15 mM MgCl2, 0.1 mM EDTA) at a final 7-EFC concentration of 0.12 mM, started by the addition of NADPH (1 mM) and incubated for 20 min at 37°C. Aqueous trichloroacetic acid (20%) was added to stop the reaction, and the tubes were centrifuged (1 min at 14,000g). An aliquot of the reaction was then transferred to a glass tube containing 0.1 M Tris (pH 9.0), and fluorescence was determined with excitation at 410 nm and emission at 510 nm. A blank was run for each sample, and the final activity was calculated by comparison to a standard curve for 7-HFC.

RP 73401 hydroxylase assays were performed as described previously (Stevens et al., 1997) using 25 pmol of P-450 and 100 μM RP 73401 for 30 min in an NADPH-regenerating system. The metabolites were separated on an LC Module 1 (Waters Corp., Milford, MA) using a Spherisorb ODS-2, 250 × 4.6 mm, 5-μm analytical column (Phase Separations, Norwalk, CT) with an ODS-2 5-cm guard column and a flow rate of 1 ml/min. Solvents A (80:20, 10 mM ammonium acetate, pH 4.0/acetotnitrile) and B (55:45, 10 mM ammonium acetate, pH 4.0/acetonitrile) were mixed by the following gradient: 0 to 12 min, 100% A; 12 to 35 min, 100% A to 100% B; and 35 to 40 min, 100% B to 100% A. The major hydroxylation product, RPR 113406, was detected at 254 nm and eluted at 22.4 min (Stevens et al., 1997).

Kinetic parameters were determined by incubating P-450 2B6 or mutant F107I with RP 73401 at concentrations ranging from 4 to 150 μM.Km andVmax values were calculated using the Michaelis-Menten nonlinear regression parameters within SigmaPlot (Jandel Scientific, San Rafael, CA).

Molecular Modeling of P-450 2B6 and Docking of RP 73401.

The P-450 2B1 model developed previously (Szklarz et al., 1995) was used as a template from which the 2B6 model was derived. Insight II software (Molecular Simulations Inc., Burlington, MA) was used for modeling, and the structures were displayed on a Silicon Graphics workstation (Silicon Graphics Inc., Mountain View, CA). Model building was performed as described earlier (Szklarz et al., 1996). Amino acid residue replacements (118) were made with the Biopolymer module of Insight II. The potential energy of the regions of amino acid replacement were then minimized using the Discover program with the consistent valence force field. The steepest descent method and harmonic potential were used as described earlier (Szklarz et al., 1995, 1996). Bond angles and bond lengths of the 2B6 structure were checked with the Prostat utility within the Homology program as described (Szklarz and Halpert, 1997).

The three-dimensional structure of RP 73401 (Stevens et al., 1997) was minimized within the Builder module to the lowest potential energy. The Discover module was used to dock RP 73401 into the active site of P-450 2B6 in an orientation that allows for trans-hydroxylation of the cyclopentyl group, and the oxidation site was fixed to allow for abstraction of the hydrogen atom (Szklarz et al., 1995). The final distance between the site of oxidation and the heme iron was fixed at 5.6 to 6.0 Å. After docking, conformational analysis of RP 73401 was performed with the Search Compare module of Insight II, and the enzyme-substrate interactions were optimized as described previously (Szklarz et al., 1995, 1996). The Docking module of Insight II was used to evaluate and minimize nonbonding interaction energy, both electrostatic and van der Waals, between RP 73401 and P-450 2B6 (Szklarz et al., 1995).

Results

Alignment and Comparison of 2B Amino Acid Sequences.

The amino acid sequences of 2B1, 2B4, 2B11, and 2B6 were aligned using the Pileup function in GCG (Wisconsin Package). P-450 2B6 is approximately 75% identical with the other members of the 2B subfamily and was found to contain 10 unique amino acids within its proposed SRSs (Table1). These 10 amino acids were altered to the corresponding 2B1 residues. Two additional 2B6 mutants were made, L363V and V477I, due to the known role of V363 and I477 in 2B1 substrate specificity (He et al., 1995; Szklarz et al., 1995).

View this table:
Table 1

Unique 2B6 SRS residues

Expression of P-450 2B6 and Its Mutants in Sf9 Insect Cells.

Initial attempts were made to express P-450 2B6 in Escherichia coli using methods that have been used for other members of the 2B subfamily. Approaches included modification of the N terminus (John et al., 1994), expression of a 2B11/2B6 chimera (Szklarz et al., 1996), and truncation of the 3′-untranslated region (Richardson et al., 1995). In the case of the 2B11/2B6 chimera, no expression was detected. Expression of P-450 2B6 with a modified N terminus resulted in protein levels of <1 nmol 2B6/liter cells. Western blotting revealed a protein recognized by polyclonal anti-2B1 that migrated slightly faster than purified 2B1, although no 7-EFC O-deethylase activity was detected from this sample (data not shown). Due to this lack of 2B6 significant expression and activity in E. coli, the Bac-to-Bac baculovirus expression system from Life Technologies was chosen and used according to the manufacturer’s instructions. This system proved successful for the expression and isolation of active P-450 2B6, the 13 single mutants, and the 4 multiple mutants studied. The specific content of P-450 in the microsomal membranes varied from 150 of 800 pmol P-450/mg total protein, within the typical range that is reported for 2B6 expression in Supersomes (170 pmol/mg; Genetest Corp., Woburn, MA) and in Sf9 cells (120 pmol/mg; Yang et al., 1998).

Enzyme Activities of P-450 2B1, 2B6, and Single SRS Mutants.

Initial characterizations of P-450 2B6 and 2B1 were performed. Purified P-450 2B1 exhibited no significant RP 73401 hydroxylase activity, consistent with results obtained from incubation with liver microsomes from phenobarbital-treated rats (data not shown). Androstenedione hydroxylase assays showed the P-450 2B6 16β-hydroxylase activity to be 400-fold lower than 2B1 (Table 2). P-450 2B1 and 2B6 exhibited 7-EFC O-deethylase activities of 7.12 and 3.74 nmol 7-HFC/min/nmol P-450, respectively (Table 2).

View this table:
Table 2

Comparison of activities of mutants with an entire SRS converted from the 2B6 sequence to the 2B1 sequence

Each 2B6 construct was tested for three enzyme activities: androstenedione hydroxylation, 7-EFC O-deethylation, and RP 73401 hydroxylation. Control samples transfected with bacmid lacking the 2B6 insert were also tested for these activities. These samples did not produce an absorbance peak at 450 nm, and background activities for the three assays performed were insignificant (data not shown). BecauseE. coli-expressed rat reductase was added exogenously, reconstitution conditions were optimized and performed as described inExperimental Procedures.

Results from androstenedione hydroxylase assays revealed that of the 13 P-450 2B6 single SRS mutants, only L363V exhibited a significant increase in 16β-hydroxylase activity (0.23 nmol/min/nmol) compared with 2B6 (0.04 nmol/min/nmol) (data not shown).

Figure 2 illustrates the results of the 7-EFC O-deethylase and RP 73401 hydroxylase assays. In most instances, with the notable exception of T292L, all of the mutants were active and metabolized both substrates to some extent. Mutant T292A, made to test the possibility that the hydroxyl group of T292 was necessary for some type of residue-residue or residue-substrate interaction, retained 74% of the 7-EFC O-deethylase activity and 79% of the RP 73401 hydroxylase activity. N289M displayed higher than wild-type levels of both activities (137% 7-EFC and 122% RP 73401). The 7-EFC O-deethylation and RP 73401 hydroxylation activities of S284H, N291S, C475S, and V477I were affected in parallel (Fig. 2), suggesting that these sites are not involved in P-450 2B6-specific functions. M103V and R109K exhibited a greater loss of 7-EFC O-deethylation (51 and 43%, respectively) than of RP 73401 hydroxylase activity (33 and 0%, respectively). M365I retained 72% of the 7-EFCO-deethylase activity and 107% of the wild-type RP 73401 hydroxylase activity. F107I, L363V, and I370R showed a greater loss of RP 73401 hydroxylation than 7-EFC O-deethylation, with I370R showing 104% of 7-HFC production and 68% of RP 73401 hydroxylation. Mutants F107I and L363V, while retaining 62 and 110% of 7-EFCO-deethylase activity, respectively, retained only 26 and 58% of RP 73401 hydroxylase activity.

Figure 2
Figure 2

Comparison of 7-EFC O-deethylase (filled bars) and RP 73401 hydroxylase (hatched bars) activities. The activities of the mutants are expressed as percentages of 2B6 wild-type activities. The results represent the average of two assays. Error bars are included. **Mutants F107I and L363V, while retaining 62 and 110% of 7-EFC O-deethylase activity, respectively, retained only 26 and 58% of RP 73401 hydroxylase activity.

Kinetic Analysis of P-450 2B6, F107I, and L363V RP 73401 Metabolism.

The Km andVmax values for 2B6 were found to be 29.9 μM and 11.2 nmol RPR 113406/min/nmol P-450, respectively (Fig.3). For F107I, these values were 31.7 μM and 4.0 nmol RPR 13406/min/nmol P-450, respectively (Fig. 3). Similar results were found for L363V, which showed aKm value of 49.1 μM and aVmax value of 6.5 nmol RPR 113406/min/nmol P-450 (data not shown), indicating that the changes in RP 73401 hydroxylase activities for F107I and L363V were primarily due to changes in Vmax and, to a lesser extent, Km for L363V.

Figure 3
Figure 3

Kinetic analysis of 2B6 (●) and F107I (▪). RP 73401 hydroxylase assays were performed and analyzed with HPLC. The data represent the average of assays performed in duplicate. RP 73401 concentrations included in the assays were 4, 10, 20, 50, 75, 100, and 150 μM.

SRS Exchanges between 2B1 and 2B6.

One of the goals of this study was to confer androstenedione hydroxylase activity on P-450 2B6; this would provide insight into the basis of the species differences between rat 2B1 and human 2B6. As stated above, single residue changes did not accomplish this goal. Because many of the residues necessary to substrate specificity have been mapped to SRS residues, we chose to focus on these regions and convert several of the 2B6 SRSs to 2B1 SRSs. Consequently, overlapping primers containing multiple mutations were used to produce 2B6 cDNAs containing one SRS from 2B1. Four 2B6 SRSs (1, 2, 5, and 6) were individually changed, and the mutants were expressed and tested for 7-EFC O-deethylase and androstenedione hydroxylase activities (Table 2). Each of these proteins expressed at levels similar to 2B6 and retained some level of activity. ΔS6 exhibited a 3.5-fold increase in androstenedione hydroxylation, although this activity still remained less than 1% of the 2B1 activity.

Modeling P-450 2B6 and Docking of RP 73401 into Active Site.

Previously, Szklarz et al. (1995) developed a molecular model of P-450 2B1 that was used as a template for the production of a P-450 2B6 three-dimensional model. Figure 4illustrates a ribbon representation of the 2B6 model with the 12 side chains chosen for study displayed.

Figure 4
Figure 4

Model of P-450 2B6. The Discover module within the Insight II software (Molecular Simulations) was used to make the 2B6 model, shown as a ribbon structure. The heme and the side chains that were studied and mutated are shown as single black lines.

Using the 2B6 model, a molecule of RP 73401 was docked into the active site (Fig. 5). Although the side chains of residues M103, R109, M365, and V477 were predicted to lie within 5 Å of RP 73401, none of the mutants at these sites exhibited alterations in RP 73401 hydroxylase activity, except V477I, which had a similar decrease in 7-HFC production (Fig. 2). The methyl groups on the L363 side chain are located less than 5 Å from the substrate and may potentially be involved in direct substrate contact. F107, which lies more than 6 Å away from the docked molecule of RP 73401, had the most striking affect on substrate-specific activity. The side chains of these residues are illustrated in Fig. 5.

Figure 5
Figure 5

RP 73401 docked into the active site of P-450 2B6. Molecular Simulations Insight II was used to dock RP 73401 into 2B6 in an orientation for trans-hydroxylation of the cyclopentyl ring.

Discussion

This report is the first to study sites within P-450 2B6 that are involved in substrate specificity. Workers in our laboratory have used multiple amino acid sequence alignments in the past to develop homology models of several P-450s (Szklarz et al., 1995; Szklarz and Halpert, 1997), and a similar alignment was used here to choose 12 sites within the predicted 2B6 SRSs for study. The panel of marker substrates, androstenedione, 7-EFC, and RP 73401 was useful for separating mutations that caused nonspecific functional alterations from those that produced a change in an enzyme-specific function. We were most interested in mutants that would display only decreased 2B6-specific RP 73401 hydroxylase activity or demonstrate a significant increase in 2B1-specific androstenedione hydroxylase activity.

Each of the mutants was tested for the three marker activities, and the results were compared with 2B6 (Fig. 2). Only T292L exhibited an almost complete lack of any of the three activities tested. T292A retained near wild-type levels of all three activities, indicating that the hydroxyl group on T292 is not required. Of the remaining 11 single-residue mutants, none showed a significant increase in androstenedione hydroxylase activity. This suggests that a number of residue changes in 2B6 will be required to gain steroid hydroxylase activity similar to 2B1 or that there is an undiscovered residue within 2B6 that, when mutated, would result in P-450 2B6 steroid hydroxylase activity similar to P-450 2B1. Two mutants, F107I and L363V, exhibited a significant loss of RP 73401 hydroxylase with a small or no change in 7-EFC O-deethylase activity. A kinetic analysis was performed on these mutants that indicated the decrease in RP 73401 hydroxylase activity was predominantly caused by a loweredVmax value compared with 2B6. The finding that L363 plays a role in 2B6 substrate specificity is consistent with several previous studies on other 2B enzymes. A 2B1 mutant, V363L, exhibited a 2-fold decrease in total androstenedione hydroxylase activity (He et al., 1995), whereas P-450 2B11 mutant L363V exhibited a 5-fold increase in 16α-OH androstenedione activity (Hasler et al., 1994). Studies by Hanna et al. (1998a) demonstrated that 2B2 mutant A363V exhibited lidocaine N-deethylase activity similar to that of 2B1. In a second study, they reported that this mutant exhibited 7-EFC O-deethylase activity more like that of 2B1 than like that of 2B2 (Hanna et al., 1998b). Mutant 2B4 I363V also displayed an increase in androstenedione hydroxylase activity, and the reciprocal mutant in 2B5, V363I, demonstrated a decrease in activity (Szklarz et al., 1996). Although 2B6 mutant L363V displayed a 6-fold increase in androstenedione hydroxylase activity, the remaining gap between L363V and 2B1 activities, 0.23 and 16.0 nmol/min/nmol, respectively, suggests that 2B1 androstenedione hydroxylation involves a network of residues.

This is the first report to identify residue 107 as a site necessary for the activity of a member of the 2B subfamily. P-450 2B11 mutant V107I did not exhibit altered androstenedione hydroxylase activity (Hasler et al., 1994). The phenylalanine side chain at residue 107 in 2B6 is very different in structure than the isoleucine in 2B1 and 2B4 or the valine in 2B11. Docking of RP 73401 into the active site of the 2B6 model did not place F107 within 5 Å of the substrate (Fig. 5), suggesting that there is no direct interaction present. The possibility exists that this residue may reside in the RP 73401 channel of entry, although there is no direct evidence for this hypothesis.

Figure 4 represents the predicted structure of P-450 2B6 and shows the location of the residues mutated in this study. Of these, only M103, R109, L363V, M365I, and V477 lie within 5 Å of the docked molecule of RP 73401. M103V and R109K exhibited a greater loss of 7-EFCO-deethylation than of RP 73401 hydroxylase activity. V477I displayed a similar decrease in 7-EFC O-deethylation and RP 73401 hydroxylation. When 2B1 I477V was analyzed (Szklarz et al., 1995), it exhibited a 46% loss of androstenedione hydroxylase activity and an 83% loss of progesterone hydroxylase activity. This site appears important for androstenedione hydroxylase activity by 2B1, but this study provided no evidence of a role for this site in 2B6 RP 73401 hydroxylase activity. Mutants S284H, N289M, M365I, I370R, and C475S did not demonstrate changes in activity that warranted further study.

Because multiple sites, usually found within the SRSs, are responsible for conferring a specific function, it is typically easier to use site-directed mutagenesis to destroy P-450 function than to create it. In the above studies, single-site mutation studies were successful for identifying 2B6 residues that are involved in RP 73401 hydroxylation because this activity was selectively diminished. However, this approach did not reveal sites that are responsible for the species difference in steroid metabolism between human 2B6 and rat 2B1. To approach this problem, we chose to make multiple mutations within the 2B6 cDNA that would convert the amino acid sequence of an SRS to the amino acid sequence of 2B1. These SRS mutants, ΔS1, ΔS2, ΔS5, and ΔS6, contained between two and seven substitutions (Table 2). Unfortunately, none of these mutants exhibited a greater than 3.5-fold increase in steroid hydroxylase activity. However, all expressed at levels similar to wild-type 2B6 and retained some level of each of the three analyzed activities. These data show that 1) 2B6 will tolerate multiple mutations without destabilizing the protein and 2) substrate interconversion requires more complex substitutions than altering one SRS.

The 2B subfamily exhibits dramatic substrate specificity differences across species that have provided a basis for numerous structure-function analyses among rat P-450 2B1, rabbit 2B4 and 2B5, and dog 2B11. The lack of a marker function for P-450 2B6 has hindered similar studies of the human member of the subfamily. Recent data have identified new roles for P-450 2B6 and have shown that this enzyme, although a minor constituent of the liver, is involved in drug metabolism in vivo (Heyn et al., 1996; Stevens et al., 1997). Although this study identifies 2B6 residues F107 and L363 that are involved in RP 73401 hydroxylation, there is still relatively little known about this member of the 2B subfamily. The mutants produced in this study will be valuable for future studies of 2B6 inhibitors and other substrates. Hopefully, as new substrates for 2B6 are identified and additional sites are found that are key to these functions, the significance of this isoform in metabolizing xenobiotics will be learned.

Acknowledgments

We thank You-Ai He for providing purified P-450 2B1, Dr. Frank Gonzalez for providing the 2B6 cDNA, and Dr. Grazyna Szklarz for expert advice and assistance during the making of the 2B6 molecular model.

Footnotes

  • Send reprint requests to: Dr. Tammy L. Domanski, Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Route 1031, Galveston, TX 77555. E-mail:tadomans{at}utmb.edu

  • ↵1 This work was supported by National Research Service Award GM19058 and by National Institutes of Health Grant ES03619.

  • ↵2 Present address: Department of Drug Metabolism, Merck Research Laboratories, WP26 354, West Point, PA 19486.

  • Abbreviations:
    P-450
    cytochrome P-450
    CHAPS
    3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
    DLPC
    dilauroyl-l-3-phosphatidylcholine
    Sf9
    Spodoptera frugiperda
    7-EFC
    7-ethoxy-4-trifluoromethylcoumarin
    7-HFC
    7-hydroxy-4-trifluoromethyl-coumarin
    RP 73401
    3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide
    PCR
    polymerase chain reaction
    SRS
    substrate recognition site
    • Received December 18, 1999.
    • Accepted April 27, 1999.

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Structure-Function Analysis of Human Cytochrome P-450 2B6 Using a Novel Substrate, Site-Directed Mutagenesis, and Molecular Modeling

Tammy L. Domanski, Kathleen M. Schultz, Fabienne Roussel, Jeffrey C. Stevens and James R. Halpert
Journal of Pharmacology and Experimental Therapeutics September 1, 1999, 290 (3) 1141-1147;
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