roles of residues f206 and v367 in human cytochrome p450...

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Roles of Residues F206 and V367 in Human Cytochrome P450 2B6: Effects of

Mutations on Androgen Hydroxylation, Mechanism-based Inactivation and Reversible

Inhibition

Hsia-lien Lin, Haoming Zhang, Cesar Kenaan, and Paul F. Hollenberg Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071662

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Running title: Roles of F206 and V367 in the Catalytic Activity of CYP2B6

*To whom correspondence should be addressed: Paul F. Hollenberg, Department of

Pharmacology, 2220C MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI

48109-5632.

Phone: (734) 764-647-3121. Fax: (734) 763-5378.

E-mail: [email protected]

Number of text pages: 41

Number of Tables: 3

Number of Figures: 9

Number of References: 37

Number of words in the Abstract: 250

Number of words In the Introduction: 797

Number of words in the Discussion: 1408

Abbreviations: Andro, androstenedione; BG, bergamottin; BPA, 4-tert-

butylphenylacetylene; CYP2B1, rat cytochrome P450 2B1; CYP2B4, rabbit cytochrome

P450 2B4; CYP2B6, human cytochrome P450 2B6; EFC, 7-ethoxy-4-

(trifluoromethyl)coumarin; ESI, electrospray ionization; F206A, Phe206 to Ala mutation

of 2B6; GSH, glutathione; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; HPLC, high

pressure liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; LC-


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MS/MS, liquid chromatography-tandem mass spectrometry; reductase, NADPH-

cytochrome P450 reductase; SRS, substrate recognition site; Testo, testosterone; TFA,

trifluoroacetic acid; T302V, Thr302 to Val mutation of 2B6; V367A, V367 to Ala

mutation of 2B6; WT, wild type 2B6.


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Abstract

The crystal structures of human P450 2B6 (CYP2B6) indicate that Phe206 and Val367

are in close proximity to the substrate binding site and suggest that both residues may

play important roles in substrate metabolism and inhibitor binding. To test this

hypothesis, we investigated the effects of mutating these residues to Ala on the

regiospecificity of CYP2B6 for the metabolism of testosterone (Testo) and

androstenedione (Andro). For Testo metabolism, 16β-OH-Testo formation by F206A

was <5% of wild type (WT), whereas the V367A mutant exhibited a doubling of 16α-

OH-Testo formation with a 50% decrease in 16β-OH-Testo formation compared to WT.

Significant alterations in the regiospecificity for Andro metabolism were also observed.

To investigate the roles of these two residues in the metabolic activation of mechanism-

based inactivators, tert-butylphenylacetylene (BPA) and bergamottin (BG) were used to

test the susceptibility to inactivation. Although the rates of inactivation of both mutants

by BG were not significantly decreased compared to WT, the efficiency of inactivation

by BPA of both mutants was more than an order of magnitude lower. Our results

demonstrate that Phe206 plays a crucial role in determining the specificity of 2B6 for the

16β-hydroxylation of Testo and Andro and that it also plays an important role in BG

binding and mechanism-based inactivation by BPA. In addition, Val367 dramatically

enhances the catalytic activity of CYP2B6 toward Andro and plays an important role in

mechanism-based inactivation by BPA. The results presented here show the important

roles of Phe206 and Val367 in interactions of CYP2B6 with substrates and

inactivators/inhibitors and are consistent with the crystal structures.


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Introduction

The CYP2B enzymes are an outstanding model system for investigating structure-

function relationships of the P450s (Gotoh 1992; Zhao and Halpert, 2007). The 16β-

hydroxylations of testosterone (Testo) and androstenedione (Andro), are specific markers

for rat CYP2B1 activity (Waxman et al., 1983; Wood et al., 1983). From 1990 to 2000,

our understanding of the functional and structural importance of residues in the P450 2B

subfamily was based primarily on studies of the rat isoform. Site-directed mutagenesis

studies of CYP2B1 have shown that Phe115, Phe206, Leu209, Ser294, Ala298, Thr302,

and Gly478 are required for the 16β-hydroxylation of Testo and Andro (Kedzie et al.,

1991; He et al., 1994; Szklarz et al., 1995). A combination of approaches involving

mechanism-based inactivators and site-directed mutagenesis further identified Ile114,

Thr302, Leu363, Val367 and Gly478 as functionally important active site residues

(Kedzie et al., 1991; He et al., 1994, 1995; Kent et al., 1997). All of these residues are

located in the putative substrate recognition sites (SRSs) for the P450 2B subfamily as

proposed by Gotoh (1992). Prior to the determination of the first X-ray crystal structure

of a mammalian P450, the combined approaches of site-directed mutagenesis,

mechanism-based inactivators, and molecular modeling based on structures of bacterial

P450s proved to be invaluable for gaining insights into the structural and functional

relationships of CYP2B1 (Domanski and Halpert, 2001; Kent et al., 2001). A significant

breakthrough occurred when the first X-ray crystal structure of rabbit CYP2C5 was

solved in 2000 (Williams et al., 2000). Using the CYP2C5 crystal structure as a template,

a CYP2B6 homology model was constructed and used for analysis of the structure-


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activity relationships of human CYP2B6 (Wang and Halpert, 2002; Spatzengger et al.,

2003).

Because of its potential pharmacological/clinical significance, interest in CYP2B6

has increased significantly over the years (Turpeinen et al., 2006). CYP2B6 appears to be

the only functional member of the human 2B family and the average relative contribution

of CYP2B6 to the total hepatic CYP content is reported to range from 2% to 10% (Wang

and Tompkins, 2008). CYP2B6 is one of the most polymorphic CYP genes in humans

and it metabolizes a number of clinically relevant drugs (Zanger et al., 2007). However,

direct extrapolation of animal data based on CYP2B1 to human CYP2B6 has limitations

(Wang and Tompkins, 2008). For example, CYP2B6 metabolizes Testo and 7-ethoxy-4-

(trifluoromethyl)coumarin (EFC) at a much slower rate than CYP2B1, but readily

hydroxylates RP 73401 (3-cyclopentyloxy-N-(3,5-dichlor-4-methoxybenzamide), which

is not a substrate of CYP2B1 (Stevens et al., 1997; Domanski et al., 1999). So far, the

SRS residues believed to be involved in the structure-function relationship of CYP2B6

have not been investigated in detail.

Recently, the crystal structures of CYP2B6 complexed with 4-(4-

chlorophenyl)imidazole, 4-benzylpyridine, and 4-(4-nitrobenzyl)pyridine were solved

and revealed that Phe206 and Val367 are in the active site of CYP2B6 within 4 Å of the

bound inhibitors (Gay et al., 2010; Shah et al., 2011). Comparisons of the three crystal

structures showed that rearrangements of Phe206 and Phe297 were required to

accommodate the various ligands within the active site (Shah et al., 2011). Moreover,

crystal structures of the open and closed forms of CYP2B4 adducted with tert-


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butylphenylacetylene (BPA) were solved and revealed that Phe206 points into, while

Phe297 points out of the active site to avoid clashing with the tert-butyl and phenyl

groups of BPA (Gay et al., 2011). Docking of BPA into the CYP2B6 crystal structure

showed that Val367 is in close proximity to both the heme iron and the acetylene triple

bond of BPA (Lin et al., 2011). Our previous studies on Thr205, which is more than 6Å

away from the heme in CYP2B1, have demonstrated the importance of Thr205 in

determining the substrate specificity and product formation as well as influencing the

susceptibility of CYP2B1 to mechanism-based inactivation (Lin et al., 2003 and 2004).

Taken together, these observations suggest that Phe206 and Val367 may play a critical

role in substrate binding. Both Phe206 and Val367 are conserved in the active sites of

CYP2B1, CYP2B4 and CYP2B6. BPA and bergamottin (BG) have previously been

characterized as potent mechanism-based inactivators of CYP2B6. It was of great interest

to compare the effect of substituting Phe206 and Val367 of CYP2B6 on androgen

metabolism and mechanism-based inactivation by two structurally unrelated compounds

BPA and BG.

Our studies show that the product profiles for the metabolism of Testo and Andro

metabolism were dramatically altered by substitution of either of the two amino acid

residues in CYP2B6. These substitutions also altered the efficiency of inactivation by

BPA and the reversible inhibition of the enzyme by BG. These results are discussed in

the context of the functional and structural roles of Phe206 and Val367 in CYP2B6 and

clearly demonstrate that inferences from crystal structures of CYP2B6 have functional


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bearing with respect to product distribution and susceptibility to inhibition and

inactivation .

Materials and Methods

Chemicals. NADPH, GSH, catalase, and BPA were purchased from Sigma-Aldrich (St.

Louis, MO). BG was from Indofine Chemical Co., Inc. (Hillsborough, NJ). Testo, Andro,

16α-OH-Testo, 16β-OH-Testo, 16α-OH-Andro, and 16β-OH-Andro were all purchased

from Steraloids Inc. (Newport, RI). EFC was from Invitrogen Corp. (Carlsbad, CA). 7-

Hydroxy-4-(trifluoromethyl)coumarin (HFC) was obtained from Oakwood Products, Inc.

(West Columbia, SC). All other chemicals and solvents were of the highest purity

available from commercial sources.

Site-directed Mutagenesis. The human wild type CYP2B6 (WT) plasmid was used as a

template to construct two mutants at positions Phe206 and Val367. The mutations were

carried out using an in vitro QuickChange site-directed mutagenesis kit (Stratagene

product, Invitrogen, Santa Clara, CA). The primer 5’-CTTGTTCTACCAGACT

GCTTCACTCATCAGCTC-3’ was used for the Phe206 to Ala conversion (F206A

mutant). The primer 5’-CTCCCCATGGGTGCGCCCCACATTGTC-3’ was used for the

Val367 to Ala conversion (V367A mutant). The nucleotides that were substituted to yield

the altered amino acid residues are underlined and in bold. The mutations were confirmed

by DNA sequencing carried out at the University of Michigan Biomedical Core Facility

(Ann Arbor, MI).


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Assay for the Mechanism-Based Inactivation of the P450s. WT CYP2B6, the two

CYP2B6 mutants and NADPH-cytochrome P450 reductase (reductase) were expressed in

Escherichia coli and purified according to previously published procedures (Lin et al.,

2005). The purified P450s and reductase were reconstituted at 22ºC for 30 min as

previously described (Lin et al., 2003). The primary reaction mixtures contained 500

pmol P450, 1000 pmol reductase, 2 mM GSH, 100 units of catalase, and BPA or BG in a

total volume of 250μl in 100 mM potassium phosphate buffer (pH 7.7). After incubation

of the primary reaction mixture with 1 mM NADPH at 22ºC with various concentrations

of BPA (1 μM to 300 μM) or BG (1 μM to 20 μM), 3μl aliquots were removed at

different time points (0 to 6 min) and added to 300 μl of a secondary reaction mixture

containing 50 μM EFC and 200 μM NADPH for 10 min. The reactions were terminated

by adding 125 μl of acetonotrile and the formation of the HFC product was determined

using a fluorescence plate reader (excitation 410 nm; emission, 510 nm) on a Wallace

Victor II 1420-042 multilabel counter (PerkinElmer, Shelton, CT). The calculations to

obtain the kinetic values describing the mechanism-based inactivation were performed as

previously described (Lin et al., 2005; Zhang et al., 2009).

Metabolism of Testo and Andro. Aliquots (250 pmol) of the WT and F206A and

V367A mutants of 2B6 in the reconstituted system were prepared as described above and

were incubated with 0.2 mM Testo and 0.5 mM NADPH in 100 mM potassium

phosphate buffer (pH 7.7) at 37°C for 15 min. 6β-OH-Testo (internal standard) was

added to the reaction mixture and the metabolites were extracted with ethyl acetate, dried

down under nitrogen gas and analyzed by high pressure liquid chromatography (HPLC)


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as previously described (Lin et al., 2003). The dried products were dissolved in 65%

methanol and resolved using a Microsorb-MV C18 reverse-phase column (5 μm, 4.6 x

250 mm; Varian, Walnut Creek, CA). Testo and its metabolites were separated under

isocratic conditions with 65% methanol at a flow rate of 0.7 ml/min. The eluates

containing 16α-OH-Testo, 16β-OH-Testo, and Andro were monitored at 254 nm. For

Andro metabolism, aliquots (125 pmol) of the WT, F206A mutant and V367A mutant in

the reconstituted systems were prepared as described above for Testo except that the

internal standard was 16β-OH-Testo. The major metabolites are 16α-OH-Andro and 16β-

OH-Andro.

Metabolism of BG. Aliquots (500 pmol) of the WT, F206A and V367A 2B6 in the

reconstituted system were prepared as described above and incubated with 50 μM BG

and 1 mM NADPH in 100 mM potassium phosphate buffer (pH 7.7) at 37°C for 15 min.

5-Methoxypsoralen (internal standard) was added to the reaction mixture and the

metabolites were extracted with ethyl acetate for HPLC analysis and monitored at 310

nm according to our previous method (Lin et al., 2005; Kent et al., 2006).

Reversible Inhibition of the P450s. For the reversible inhibition studies, WT and F206A

were reconstituted with reductase as described above. Different concentrations of EFC (1

μM to 100 μM) were prepared and incubated with the 2B6 reconstituted systems together

with various concentrations of BG (0 to 4 μM) for 5 min and then the reactions were

initiated by adding 200 μM NADPH to measure the HFC product. The kinetic

parameters, Km and Kcat, were fitted to the Michaelis-Menten equation and the Ki was


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obtained by global fitting to a mixed model inhibition equation using Prism software

(GraphPad software Inc., San Diego, CA).

LC-MS/MS Analysis of GSH Conjugates of BPA. The samples (250 pmol in 250 μl

reaction mixture) of WT 2B6, F206A, and V367A were prepared in a reconstituted

system as described above and incubated with 1 mM NADPH and 100 μM BPA for 30

min. The reactions were terminated by adding 1 ml of acetonitrile. The mixtures were

centrifuged at 13,200 g at room temperature for 10 min. The supernatants were dried

under N2 gas and re-suspended in 100 µl of 50% methanol. The samples were analyzed

on a C18 reverse-phase column (Luna, 3 µm, 4.6 x 100 mm, Phenomenex, Torrance, CA)

with solvent A (0.1% acetic acid/water) and solvent B (0.1% acetic acid/acetonitrile).

Elution with 30% B for 5 min followed by a gradient to 40% B over 15 min and then

increasing linearly to 90% B over 15 min at a flow rate of 0.3 ml/min was used. The

column effluent was directed into the ESI source of a LCQ mass spectrometer (Thermo

Fisher Scientific). The ESI conditions were: sheath gas flow rate, 90 arbitrary units;

auxiliary gas, 30 arbitrary units; spray voltage, 4.5 kV; capillary temperature, 170°C;

capillary voltage, 30 V; and tube lens offset, 25 V. The data were acquired in positive

ion mode using the Xcalibur software (Thermo Fisher Scientific) with one full scan

followed by two data-dependent scans of the most intense and the second most intense

ions.


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Results

Location of the Residues and Structures of the Substrates. The locations of Phe206,

Thr302 and Val367 in the CYP2B6 crystal structure (Gay et al., 2010) are displayed in

Fig. 1A. Phe206 is located in the F-helix in the SRS-2 region (199LNLFYQTFSLIS209)

shown in magenta. Thr302 is located in the I-helix in the SRS-4 region

(290LNTLSLFFAGTETFS304) shown in green. Thr302 is included because it is the site of

covalent binding for the reactive intermediate of BPA (Lin et al., 2011). V367 is located

in the β1-4 sheet in the SRS-5 region (360SDLLPMGVPH369) shown in blue. The

chemical structures of the Testo, Andro, EFC, BPA and BG used in this study are shown

in Fig. 1B. The circled areas on the structures of BPA and BG indicate the sites of

metabolism resulting in the formation of reactive intermediates.

Catalytic Activities of the WT and F206A and V367A Mutants of CYP2B6. To

investigate whether or not mutations of residue Phe206 or Val367 affect the protein

function, the EFC deethylation activity was used to determine the specific activity and Km

values of the WT and mutant P450s. The Km values obtained for all three P450s were

very similar (~4 μM). The specific activity of WT was 2.8 nmol/min/nmol P450, whereas

the specific activities of the F206A and V367A mutants were 1.68 and 1.01

nmol/min/nmol P450 for EFC deethylation activities, respectively.

In order to investigate the effect of the mutation of F206 and V367 on the regio-

and stereo-specificity of 2B6, the metabolism of Testo by the WT and mutant proteins


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was investigated. Fig. 2A illustrates representative HPLC chromatographic profiles for

the products generated from the metabolism of Testo catalyzed by the WT, F206A

mutant and V367A mutant. 6β-OH-Testo (eluting at 8.1 min) was used as an internal

standard and three major metabolites: 16α-OH-Testo (8.9 min), 16β-OH-Testo (10.8

min), and Andro (18.4) were produced. In general, the conversion of the 17β-hydroxyl to

the 17-keto group to form Andro was catalyzed to a similar extent by all three P450s. For

F206A, the amount of 16α-OH-Testo produced was similar to that of WT, but the

formation of 16β-OH-Testo was almost completely suppressed as compared to the WT.

In contrast, the V367A mutant exhibited a greater than 2-fold increase in the 16α-

hydroxylase activity and more than a 50% decrease in the 16β-hydroxylase activity as

compared to WT. The unidentified product that eluted at 12.5 min was observed for all

P450s. Although the hydroxylation activity of F206A was low overall, a new metabolite

eluting at ~12 min was observed.

The stereo-selectivity of the enzymes was further studied by using Andro as the

substrate and 16β-OH-Testo (eluting at 10.0 min) as the internal standard. The HPLC

chromatographic metabolite profiles are shown in Fig. 3A. For WT, 16β-OH-Andro

(eluting at 7.6 min) was the predominant product and 16α-OH-Andro (eluting at 8.3 min)

was formed at very low levels. For the F206A mutant, the formation of 16β-OH-Andro

was almost completely suppressed whereas 16α-OH-Andro formation became more

prominent. For the V367A mutant, the generation of both the 16α-OH-Andro and 16β-

OH-Andro was dramatically enhanced as compared to the WT 2B6. To be able to better

visualize the regio- and stereo-selectivity for the metabolism of the two androgens by the

three P450s, 16β-OH-Testo was set as the 100% control for Testo metabolism and 16β-


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OH-Andro was set as the 100% control for Andro metabolism. The means from two

separate experiments are displayed in Fig. 2B for Testo metabolism and in Fig. 3B for

Andro metabolism. The total Testo metabolites formed by F206A and V367A are 30 and

80% of WT, respectively. The total Andro metabolites formed by F206A and V367A are

34 and 320% of WT, respectively.

To visualize the relative stereo-selectivity of the variants, the total activities for

the formation of the 16α-OH and 16β-OH products of Testo or Andro for each P450

were normalized as 100% and the relative amounts of 16α-OH and 16β-OH products

were calculated and displayed in Fig. 4. It is clear that WT selectively metabolizes both

androgens at the 16β-position whereas the F206A mutant selectively metabolizes both

androgens at the 16α-position. In contrast, V367A metabolizes Testo to the 16α-OH and

16β-OH products in almost equal amounts whereas its stereo-selectivity for Andro is

essentially similar to that of the WT.

Mechanism-based inactivation by BPA and BG. In addition to investigating the effects

of the mutation of the two residues on the catalytic activity of CYP2B6 for a single

substrate giving a single product (EFC) and for substrates giving multiple products

(Testso and Andro), we thought it would be important to investigate the effects of these

mutations on mechanism-based inactivation. To investigate whether the mutants were

still capable of being inactivated by BPA and BG, the WT and mutant P450s were

incubated with 10 μM BPA or 10 μM BG for varying lengths of time (0, 3, 6 and 9 min)

in the primary reaction mixture. The Ln % of activity remaining was calculated at each

time point and plotted against incubation time. As seen in Fig. 5, BPA and BG are both


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relatively efficient mechanism-based inactivators of the WT, as expected (Lin et al.,

2005; Lin et al., 2011). BPA inactivated both mutants much more slowly as compared

with WT, but inactivation by BG was not significantly altered by the mutation.

Kinetic Constants for the Mechanism-Based Inactivation. Mechanism-based

inactivation of CYP2B6 by BPA or BG was further characterized by incubating CYP2B6

with various concentrations of BPA or BG for time points varying from 0 to 9 min at

each concentration. For inactivation by BPA, 1 to 10 μM, 10 to 300 μM and 5 to 100 μM

BPA were used for WT, F206A, and V367A, respectively. For inactivation by BG, 1 to

20 μM BG was used for all three P450s. Similar to our previous studies, the present

results showed that the inactivation is time-and concentration-dependent (Lin et al., 2005;

Lin et al., 2011). The kinetic constants were calculated as described under Material and

Methods and are summarized in Table 1. For inactivation by BPA, the mutation of

Phe206 and Val367 to Ala did not significantly alter the kinact, but drastically increased

the KI. As a consequence, the efficiency of inactivation by BPA (as measured by the

kinact/KI) decreased by more than one order of magnitude, suggesting that Phe206 and

Val367 may contribute significantly to the mechanism of BPA-mediated inactivation. In

contrast to what was seen with BPA, the efficiency of the inactivation of both mutants by

BG did not decrease. Interestingly, although no significant change was observed with

V367A, a significant decrease in the KI resulted in a more than 4-fold increase in the

efficiency for inactivation of the F206A variant compared with WT.


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Reversible Inhibition of WT and the F206A Mutant by BG. The data presented in Fig.

5 clearly showed that, at time zero, BG inhibits the EFC deethylation activity of F206A

by ~80% of the WT. The observation that mutation of Phe206 to Ala potentiated

inhibition by BG was further investigated. The IC50 values for BG inhibition of WT and

F206A were characterized using 100 μM EFC as the substrate (Fig. 6). The IC50 values

for inhibition by the WT and F206A mutant by BG are 0.4 μM and 0.02 μM,

respectively. Thus, F206A is inhibited 50% by BG at a concentration that is 20-fold

lower as compared to that for the WT. Furthermore, the type of reversible inhibition was

assessed by using varying concentrations of EFC (1 μM to 100 μM EFC) as substrate

with various concentrations of BG as the inhibitor. For WT, the range of BG

concentrations used ranged from 0 to 1 μM. For F206A, the range of BG concentrations

was from 0 to 0.02 μM. The results are presented in Fig. 7A for WT and Fig. 8A for

F206A. The Km and kobs for the P450s are shown in Table 2. As can be seen in Table 2,

the Km increased and the kobs decreased with increasing concentrations of BG for both

P450s. Therefore, we determined the Ki values by nonlinear regression global fitting to

the mixed type inhibition equation. The apparent Ki values for WT and F206A are 0.12

and 0.004 μM respectively. Previously, Paine and coworkers used linear regression

Dixon plots to characterize a mixed type, reversible inhibition of human intestinal

CYP3A4 by BG (Paine et al., 2004). We have analyzed the inhibition of WT and F206A

in a similar manner. Dixon plots for both WT and F206A are also presented in Figs. 7B

and 8B, respectively. The Ki values were estimated to be ~0.2 μM for WT and ~0.005

μM for F206A. In short, the Ki values calculated using both methods are in good


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agreement and thus it is clear that the residue at position 206 has a significant effect (~30

fold) on the inhibitory potency of BG.

Metabolism of BG by WT, F206A and V367A. The metabolism of BG by the three

P450s was determined by HPLC analysis (Fig. 9). As reported previously, WT 2B6

generates two major metabolite peaks, M3 is 5’-OH-BG and M4 is a mixture of 6’-OH-

BG and 7’-OH-BG (Kent et al., 2006). The F206A mutant exhibited very low level

activity for BG metabolism compared to WT2B6, whereas the V367A mutant showed a

significant change in the regio-selectivity for the formation of the primary mono-

hydroxylated products.

Discussion

Although extensive studies have been performed to elucidate the functional

and structural importance of some SRS residues in rat CYP2B, very little is known about

the role of SRS residues in its human CYP2B counterpart (Domanski and Halpert, 2001;

Kent et al., 2001; Zhao and Halpert, 2007; Halpert, 2011; Zhang et al., 2011). The

studies described here were based on an analysis of the crystal structure of CYP2B6 that

suggested that Phe206 and Val367, two highly conserved SRS residues in human

CYP2B6, were in close proximity to the substrate binding site and thus might have

significant effects on substrate metabolism and susceptibility to inhibition/inactivation.


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CYP2B1 hydroxylates Testo to form equal amounts of 16β-OH and 16α-OH

(16β:16α = 1), whereas it hydroxylates Andro primarily at the 16β-position (16β:16α =

~8) (Waxman et al., 1983; He 1994; Lin et al., 2003). As shown here, CYP2B6

metabolizes both Testo (16β:16α = 5) and Andro (16β:16α = 17) primarily at the 16β-

position and is the only human P450 capable of hydroxylating Testo at the 16β-position

(Imaoka et al., 1996). Our data also demonstrate the highly stereo-selective

hydroxylation activity of CYP2B6 for the 16β position of both Testo and Andro, that is

much greater than that of CYP2B1. The total 16α-OH and 16β-OH metabolites formed

as well as the 16β-OH:16α-OH ratio for Testo and Andro observed with CYP2B6 were

compared to results published for CYP2B1 and CYP2B4 (Table 3). These data suggest

that when Phe206 was changed to Ala, the 16β-OH activities of CYP2B1 and CYP2B6

were markedly suppressed for both androgens. Substitution of Val367 with Ala resulted

in the following: (1) a decrease in the 16β:16α ratio for Testo with CYP2B6, but an

increase with CYP2B4; and (2) an increase in the 16β:16α ratio of Andro with CYP2B1

and 2B4, but not with CYP2B6. The total activities for Testo and Andro metabolism by

CYP2B6 and its V367A mutant show a similar pattern when compared to CYP2B4 and

its V367A mutant, but are quite different from what was seen with CYP2B1 and its

mutant. WT CYP2B4 exhibited a very low 16β:16α ratio with Testo as compared to

CYP2B1 and CYP2B6. In spite of some structural and catalytic similarities between

these proteins, our results clearly show the limitations of directly extrapolating data

obtained from animal CYPs to human CYPs.


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Although the total hydroxylation activities for both androgens by the F206A

mutant are much lower than that for WT (Fig. 2 and Fig. 3), the WT selectively produces

the 16β-OH metabolite, whereas the F206A variant selectively produces the 16α-

ΟΗ metabolite (Fig. 4). This dramatic conversion in stereo-selectivity was not observed

following mutation of Thr205 on the F-helix of CYP2B1 (Lin et al., 2003). Phe206 is

also located in the F-helix, which plays a role in substrate access to the active site. Based

on the results presented here, it appears that during the entrance into and binding of Testo

and Andro to the active site that the presence of Ala at position 206 may lead to a

distortion or twisting of the D-ring of both Testo and Andro that reorients the 16α-face

toward the heme iron. This finding agrees with a previous study using the A82W P450

mutant of BM3 as a tool for the regioselective hydroxylatiom of Testo at the 16β-position

(Rea et al., 2012). It is interesting to note that aromatic residues at this position favor

selectivity for steroid hydroxylation at the 16β-position for both CYP2B6 and P450

BM3. In contrast, the impact of Ala substitution at Val367 on the stereo-selectivity is not

as profound as substitution at Phe206. V367 is very close to the heme and the

replacement by Ala creates a more open active site leading to a 3-fold increase in the

formation of the products of Andro metabolism with no significant effect on the stereo-

selectivity.

When Phe206 or Val367 were replaced by Ala, the efficiency of this

inactivation reaction (kinact/KI), was drastically diminished, which can be primarily

attributed to an increase in the KI. Docking BPA into the active site of CYP2B6 in the

crystal structure and the crystal structure of BPA-adducted CYP2B4 revealed that Phe206


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and Val367 are in close contact with BPA (Gay et al., 2011; Lin et al., 2011). Thr302 has

previously been identified as the site for covalent modification of CYP2B6 by a reactive

intermediate of BPA resulting in mechanism-based inactivation (Lin et al., 2011). The

possible reasons contributing to the decrease in the susceptibility of F206A mutant to

inactivation would include the following: (1) the three dimensional structure of the active

site that facilitates covalent binding of the BPA reactive intermediate to the hydroxyl

moiety of Thr302 is distorted in the mutants and is less effective at steering the

intermediate to the Thr; (2) the reactive intermediate of BPA formed by the mutants

reacts more readily with water molecules in the active site than it reacts with the OH

moiety of Thr302; or (3) the mutations increase the mobility of BPA in the active site of

mutants, thereby facilitating its egress from the active site over its reaction with Thr302.

The substitution of Val367 with Ala creates more space in the active site that may either

increase the mobility of BPA in the active site or may allow BPA to move further away

from the heme iron. Therefore, the orientation of BPA’s triple bond relative to the side

chain of Thr302 in the V367A mutant may be very different from that in the WT. Any

one or a combination of these possibilities may be likely to reduce the interaction of BPA

with the mutant proteins and thus, higher concentrations of BPA would be required for

the inactivation.

Unlike the results observed with BPA, the efficiency for the mechanism-based

inactivation of the two mutants by BG actually improved. The KI for F206A is

approximately 5-fold lower than that for WT, indicating that BG binds with higher

affinity to F206A. This mutation also leads to increased sensitivity to reversible


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inhibition by BG with a Ki value that is ~30-fold lower than WT, indicating the

importance of residue 206 in determining the binding affinity and inhibitor sensitivity.

We investigated to see if the increase in the inhibitory potency observed for F206A was

seen with other furanocoumarin containing compounds and found that both the efficiency

of mechanism-based inactivation and the potency of reversible inhibition for F206A by

the furanocoumarins 5-methoxypsoralen and 8-methoxypsoralen were similar to WT. The

primary difference in the structures of these furanocoumarins is that the BG contains a

geranyl side chain at the methoxy position (+137 Da), whereas both 5-methoxypsoralen

and 8-methoxypsoralen have a methyl group (+15 Da). When BG enters the substrate

access channel, it is possible that the bulky geranyl side chain may impede access to the

active site due to steric hindrance by the phenyl moiety of Phe206 in the F-helix.

Removal of the Phe side chain then may open up the path for BG to enter the active site

for metabolic activation and to better expose the Thr302 for reaction with the activated

intermediate of BG. Thermodynamic measurements of the Phe206 to Ala substitution of

CYP2B4 support rearrangement of the active site observed in the crystal structure of the

1-(4-chlorophenyl)imidazole complex and may help to explain our results for BG

inactivation of the F206A mutant of CYP2B6 (Zhao et al., 2007). We have previously

demonstrated that CYP2B6 readily hydroxylates BG on the geranyl side chain to give the

5’-OH-BG, 6’-OH-BG and 7’-OH-BG (Kent et al., 2006). The very low level of these

hydroxylated metabolites formed by F206A suggests that for BG binding the

furanocoumarin moiety may be reoriented such that it is bound more tightly in the

vicinity of the heme iron than the geranyl side chain when compared to its binding to

WT. Because oxidation of the furan ring to generate a furanoepoxy intermediate is


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responsible for mechanism-based inactivation by BG, the preferential binding of the

furanocoumarin moiety near the heme iron may explain the 5-fold lower KI for

mechanism-based inactivation by BG of F206A when compared to the WT.

In conclusion, these studies demonstrate for the first time that Phe206 and

Val367, the two highly conserved SRS residues in close proximity to the substrate

binding site of CYP2B6, have important functional roles in the interaction of this P450

with substrates, inhibitors, and inactivators. Substitution of Phe206 or Val367 with

another amino acid residue such as alanine can have profound effects on substrate

metabolism, regiospecificity, mechanism-based inactivation, and inhibition.


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Acknowledgments

We thank Dr. Richard Neubig, Department of Pharmacology, University of Michigan

(Ann Arbor, MI) for the use of the Wallace Victor II plate reader. We are grateful to Dr.

Ute M. Kent for critical reading of the manuscript.


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Authorship Contributions

Participated in research design: Lin, Zhang, Kenaan

Conducted experiments: Lin and Kenaan

Contributed new reagents or analytic tool: Lin, Kenaan, Zhang

Performed data analysis: Lin, Kenaan, Zhang

Wrote or contributed to the writing of manuscript: Lin, Zhang, Hollenberg


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Footnotes

This work was supported in part by a National Institutes of Health Grant [CA-16954] to

PFH.


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Legends for Figures

Fig. 1. Locations of mutated residues in the CYP2B6 crystal structure and the chemical

structures of substrates utilized in the study. A, the locations of Phe206, Thr302, and

Val367 in the CYP2B6 crystal structure are shown (PDB 3IBD). The ribbon model

shows the spatial relationships between the heme (red) and these three residues. The

SRS-2 (magenta), SRS-4 (green) and SRS-5 (blue) residues are colored as indicated. B,

structures of BPA, BG, EFC, Testo and Andro. The carbon atoms of the geranyl side

chain of BG are numbered and the sites of oxidation of BPA and BG to form the reactive

intermediates are circled.

Fig. 2. Metabolism of Testo by the WT and mutant P450s. A, HPLC chromatograms of

the metabolites. CYP2B6 samples of WT, F206A, and V367A were incubated with Testo

and NADPH in the reconstituted system at 37°C for 15 min. The reaction mixtures were

extracted with ethyl acetate in the presence of 6β-OH-Testo (internal standard) and

analyzed by HPLC as described under Materials and Methods. B, Relative amounts of

the metabolites formed by the three CYPs. The formation of 16β-OH-Testo by the WT

was used as 100% to calculate the relative activities for the metabolites produced by WT,

F206A and V367A. The results shown represent the average from two separate

experiments.

Fig. 3. Metabolism of Andro by the WT and mutant CYPs. A, HPLC chromatograms of

the metabolites. The CYPs were incubated with Andro and NADPH in the reconstituted


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system at 37°C for 15 min. The reaction mixtures were extracted with ethyl acetate in the

presence of 16β-OH-Testo (internal standard) and analyzed by HPLC as described under

Materials and Methods. B, Relative amounts of the metabolites formed by the three

CYPs. The formation of 16β-OH-Andro by the WT was used as 100% to calculate the

relative activities for the metabolites produced by WT, F206A and V367A. The results

shown are the mean from two separate experiments.

Fig. 4. Relative amounts of 16α- and 16β-hydroxylation products of Testo (A) or Andro

(B) formed by the WT, F206A and V367A mutants of CYP2B6. The relative amounts of

the 16α-OH metabolite and the 16β-OH metabolite of Testo or Andro formed by each

CYP were calculated using the total amounts of the 16α-OH and 16β-OH metabolites as

100%. The data are taken from Fig. 2 for Testo and Fig. 3 for Andro.

Fig. 5. Loss of EFC deethylation activity of the WT and mutants after the reconstituted

reaction mixtures were incubated with NADPH in the absence (Control) or presence of

10 μM BPA or 10 μM BG for the indicated times. The EFC deethylation activity at time

zero for each condition was used as 100%. Linear regression analysis of the data (Ln %

activity remaining as a function of incubation time) was used to determine the rate

constant of inactivation (kobs). The results presented here are the average of two separate

experiments done in triplicate.


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Fig. 6. Determination of the IC50 values for BG inhibition of 100 μM EFC deethylation

activity by 2 μM of the WT (�) and F206A mutant (�). BG concentrations from 0 to 4

μM were used for the inhibition. The activity of each CYP in the absence of BG was used

as 100% control. The activity measured versus the log [BG (nM)] was fitted to the dose

response-inhibition equation using Prism software.

Fig. 7. Determination of the Ki values for inhibition of EFC deethylation activity of WT

2B6 by BG. A, A nonlinear regression plots of the data. The concentrations of EFC

ranged from 1 to 100 μM, as shown. The concentrations of BG were 0 (�), 0.2 μM (�),

0.3 μM (�), 0.5 μM (�), and 1 μM (�). The data presented were fitted to the Michaelis-

Menten equation as described in Materials and Methods. The kinetic parameters for WT

are shown in Table 2 for each concentration of BG. B, Dixon plots of the data in A. 1/V

versus BG concentration shows the inhibition of EFC deethylation activity by BG. The

concentrations of EFC were 1 (�), 2.5 μM (�), 10 μM (�), 25 μM (�), 50 μM (�),

and 100 μM (�). The apparent Ki was estimated from the intersection of the lines from

data obtained at various BG concentrations (Paine et al., 2004).

Fig. 8. Determination of the Ki values for inhibition of EFC deethylation activity of the

F206A mutant by BG. The concentrations of EFC ranged from 1 to 100 μM as indicated

in the Figure. The concentrations of BG were 0 (�), 0.003 μM (�), 0.005μM (�), 0.01

μM (�), and 0.02 μM (�). The data presented were fitted to the Michaelis-Menten

equation as described in Materials and Methods. The kinetic parameters are shown in

Table 2 for each concentration of BG. B, Dixon plot of data in A. 1/V versus BG


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concentration shows the inhibition of EFC deethylation activity by BG. The

concentrations of EFC were 1 (�), 2.5 μM (�), 10 μM (�), 25 μM (�), 50 μM (�),

and 100 μM (�). The apparent Ki was estimated from the intersection of the lines from

data obtained at various BG concentrations (Paine et al., 2004).

Fig. 9. Metabolism of BG by WT 2B6 and the F206A mutant. The P450 reconstituted

systems were incubated with BG at 37°C for 15 min and the reaction mixtures were

extracted with ethyl acetate. The metabolites of BG were analyzed by HPLC as described

in Materials and Methods. The internal standard, 5-methoxypsoralen, eluted at 15 min.


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Table 1. Kinetic constants for P450 inactivation by BPA and BGa

Inactivator P450 KI kinact kinact/KI

μM min-1 min-1mM-1

BPA WT 2.7 0.20 74

F206A 141 0.28 2

V367A 44 0.26 6

BG

WT

11.6

0.15

13

F206A 2.2 0.13 59

V367A 5.7 0.10 18

aThe catalytic activities of WT, F206A, and V367A were assessed using the EFC

deethylation activity assay and the kinetic values were determined as described in

Materials and Methods. The data shown represent the mean values of two independent

experiments performed in triplicate.


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Table 2. Kinetic constants for BG inhibition of EFC deethylation activity catalyzed by

WT and the F206A mutanta

P450 BG (μM) Kmb

(μM)

kobsb

(min-1)

Kic

(μM)

WT

0

1.7

0.80

0.12

0.2 4.2 0.60

0.3 5.0 0.54

0.5 14 0.50

1 20 0.41

F206A

0

3.6

0.56

0.004

0.003 4.6 0.50

0.005 4.9 0.44

0.01 7.3 0.39

0.02 13 0.34

aThe catalytic activities were determined using EFC concentrations from 1 μM to 100

μΜ with different BG concentrations as described under Materials and Methods.

bThe data from Fig. 7A (WT) and Fig. 8A (F206A) were fitted to the Michaelis-Menten

equation using Prism software (GraphPad software Inc., San Diego, CA).

cThe data were obtained by global fitting to a mixed model inhibition equation using

Prism software. The values are the mean of two separate experiments run in triplicate.


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Table 3. Comparison of Testo and Andro metabolism by CYP2B6 to the results

previously published results for CYP2B1 and CYP2B4a.

P450

Testo

Andro

2B1

WT

16β:16α

1.0

Total (%)b

100

16β:16α

8.0

Total (%)b

100

F206A mutant 0.2 73 0.8 22

V367A mutant 1.6 12 24 62

2B4 WT 0.1 100 6.0 100

F206A mutant ND ND ND ND

V367A mutant 1.7 98 10.3 340

2B6 WT 5.3 100 16.6 100

F206A mutant 0.1 15 0.1 34

V367A mutant 1.2 108 12.6 320

aThe data for CYP2B6 were calculated from Fig. 2 and Fig. 3. The data for CYP2B1 (He

et al., 1994; He et al., 1995) and CYP2B4 (Szklarz et al., 1996; Hernandz et al., 2006)

were previously published.

bThe catalytic activity of the WT was designated as 100% for each of the P450s.

ND: not determined


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Phe206

Val367

Thr302

Fig 1

tert-butylphenylacetylene (BPA) bergamottin (BG)

testosterone (Testo) androstenedione (Andro)

7-ethoxy-4-(trifluoromethyl)coumarin (EFC)

A B


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0.000

0.015

0.030

8.00 12.00 16.00 20.00 24.00

WT F206A V367A

16a-OH-Testo

16b-OH-Testo

Andro

Testo

0.000

0.015

0.030

8.00 12.00 16.00 20.00 24.00

0.000

0.015

0.030

8.00 12.00 16.00 20.00 24.00

Internal standard: 6b-OH-Testo

Time (min)

8 12 16 20 24

Rel

ativ

e ab

sorb

ance

at

25

4 n

m 16b-OH-Testo in WT

as 100% control

Re

lati

ve c

atal

ytic

act

ivit

y

(% o

f co

ntr

ol)

A

WT

F206A

V36

7A

0

25

50

75

100

16a-OH-Testo

16b-OH-Testo

Andro

Fig 2

16a-OH-Testo

16a-OH-Testo

16b-OH-Testo

Andro

Andro

B


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0.00

0.09

0.18

8.00 12.00 16.00 20.00 24.00

0.00

0.09

0.18

8.00 12.00 16.00 20.00 24.00

0.00

0.09

0.18

8.00 12.00 16.00 20.00 24.00

16b-OH-Andro

WT

F206A

V367A

Andro

16a-OH-Andro

Internal standard:

16b-OH-Testo

16a-OH-Andro

16b-OH-Andro

8 12 16

Time (min)

Rela

tive a

bsorb

ance a

t 254 n

m

WT

F206A

V36

7A

0

50

100

150

200

250

300

16b-OH-Andro

16a-OH-Andro

16b-OH-Andro in WT

as 100% control

Re

lati

ve

ca

taly

tic

ac

tivit

y

(%

of

co

ntr

ol)

A

Fig 3

B


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Testo

Andro

% o

f to

tal a

ctiv

ity

%

of

tota

l act

ivit

y

WT

F206A

V367A

0

20

40

60

80

10016

16

WT

F206A

V367A

0

20

40

60

80

10016

16

Fig 4


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0 3 6 9

1

2

3

4

5

W T

T i m e ( m i n )

0 3 6 9

1

2

3

4

5

V 3 6 7 A

T i m e ( m i n )

n o i n h i b i t o r

B P A

B G

0 3 6 9

0

1

2

3

4

5

F 2 0 6 A

T i m e ( m i n )

Ln %

Act

ivit

y re

mai

nin

g

BPA: 0.16 min-1

BG: 0.15 min-1 BPA: 0.04 min-1

BG: 0.13 min-1 BPA: 0.04 min-1

BG: 0.10 min-1

Fig 5


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Log [BG (nM)]

0 1 2 3 40

20

40

60

80

100

EFC

de

eth

ylat

ion

act

ivit

y

(

% o

f co

ntr

ol )

Fig 6


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EFC (M)

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.80

0.2 mM BG

0.3 mM BG

0.5 mM BG

1.0 mM BG

BG (M)

-0.5 0.5 1.0

-5

5

10

15

20 1 EFC

2.5

10

25

50

100

Fig 7

1/k

ob

s (m

in-1

)

k ob

s (m

in-1

)

A B


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EFC (M)

0 20 40 60 80 1000.0

0.2

0.4

0.60 BG

0.003

0.005

0.01

0.02

BG (M)

-0.01 0.01 0.02

-10

-5

5

10

15

20

251 EFC

2.5

10

25

50

100

A B

k ob

s (m

in-1

)

1/k

ob

s (m

in-1

)

Fig 8


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0.000

0.025

0.050

15.00 30.00 45.00 60.00

0.000

0.025

0.050

15.00 30.00 45.00 60.00

0.000

0.025

0.050

15.00 30.00 45.00 60.00

Internal standard

M3

M3

M4

M4

Re

lati

ve a

bso

rban

ce (

31

0 n

m)

15 30 45 60

Time (min)

BG

BG

BG

WT F206A V367A

Fig 9


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roles of residues f206 and v367 in human cytochrome p450...

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