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Vol. 290, Issue 3, 1141-1147, September 1999
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas (T.L.D., F.R., J.R.H.); and Department of Drug Metabolism and Pharmacokinetics, Rhône-Poulenc Rorer, Collegeville, Pennsylvania (K.M.S., J.C.S.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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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-trifluoromethylcoumarin O-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.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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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 of
S-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.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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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).
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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 DH5. 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 
S1, S2, S5, and S6 (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 an
NcoI site, the changes in S2 resulted in the loss of an
XmnI site, and the mutations in S6 removed a
BstXI 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 
-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.
) 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).
) 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 and
Vmax 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).
) 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).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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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 (Table
1). 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).
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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 16
-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).
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-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-EFC
O-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-EFC
O-deethylase activity, respectively, retained only 26 and
58% of RP 73401 hydroxylase activity.
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Kinetic Analysis of P-450 2B6, F107I, and L363V RP 73401 Metabolism.
The Km and
Vmax 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 a
Km value of 49.1 µM and a
Vmax 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.
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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. S6 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 4
illustrates a ribbon representation of the 2B6 model with the 12 side
chains chosen for study displayed.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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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 lowered
Vmax 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 16-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-EFC
O-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, S1, S2, S5, and
S6, 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.
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Acknowledgments |
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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.
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Footnotes |
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Accepted for publication April 27, 1999.
Received for publication December 18, 1999.
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.
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
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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.
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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-napthoflavone stimulation.
Arch Biochem Biophys
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) 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.
