Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations

Article abstract of DOI:10.1021/acs.biochem.5b01330

Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH₃, or -CF₃ at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 ? and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 ? with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.

Full text of DOI:10.1021/acs.biochem.5b01330

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Article
Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes
P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain
Length, Halogen Substitution, and Non-Active Site Mutations
Jingbao Liu, Manish B. Shah, Qinghai Zhang, C. David Stout, James Robert Halpert, and P. Ross Wilderman
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01330 • Publication Date (Web): 16 Mar 2016
Downloaded from http://pubs.acs.org on March 24, 2016
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Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450
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B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen
Substitution, and Non-Active Site Mutations
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, ‡, *
1, ‡
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Jingbao Liu
, Manish B. Shah , Qinghai Zhang , C. David Stout , James R. Halpert , and P.
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Ross Wilderman
1
School of Pharmacy, University of Connecticut, Storrs, CT 06269, United States
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Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, United
States
*
Corresponding Author: Jingbao Liu, University of Connecticut, School of Pharmacy,
Department of Pharmaceutical Sciences, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269ꢀ
3
092. Email: jingbao.liu@uconn.edu; Tel: 860ꢀ486ꢀ3103;
‡
These authors contributed equally to this work and should be considered coꢀfirst authors.
Key words
cytochrome P450; CYMALꢀ5; coumarin derivatives; (+)ꢀαꢀpinene; Oꢀdealkylation; Xꢀray
diffraction
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Abbreviations
CYP, Cytochrome P450ꢀdependent monooxygenase; 7ꢀEC, 7ꢀethoxycoumarin; 7ꢀPrC, 7ꢀpropoxy
coumarin; 7ꢀBuC, 7ꢀbutoxycoumarin; 7ꢀEMC, 7ꢀethoxyꢀ4ꢀmethylcoumarin; 7ꢀPrMC, 7ꢀpropoxyꢀ
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ꢀmethylcoumarin; 7ꢀBuMC, 7ꢀbutoxyꢀ4ꢀmethylcoumarin; 7ꢀEFC, 7ꢀethoxyꢀ4ꢀ(trifluoromethyl)
coumarin; 7ꢀPrFC, 7ꢀpropoxyꢀ4ꢀ(trifluoromethyl)coumarin; 7ꢀBuFC, 7ꢀbutoxyꢀ4ꢀ
trifluoromethyl)coumarin; 7ꢀClEFC, 7ꢀ(2ꢀchloroethoxy)ꢀ4ꢀ(trifluoromethyl)coumarin; 7ꢀBrEFC,
(
7ꢀ(2ꢀbromoethoxy)ꢀ4ꢀ(trifluoromethyl)coumarin; 7ꢀHC, 7ꢀhydroxycoumarin; 7ꢀHMC; 7ꢀ
hydroxyꢀ4ꢀmethylcoumarin;
7ꢀHFC, 7ꢀhydroxyꢀ4ꢀ(trifluoromethyl)coumarin; RNase,
ribonuclease A; DNase, deoxyribonuclease I; BME, 2ꢀmercaptoethanol; PMSF,
phenylmethanesulfonyl fluoride; CHAPS, 3ꢀ[(3ꢀcholamidopropyl)dimethylammonio]ꢀ1ꢀ
propanesulfonate; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; 7ꢀAC, 7ꢀ
alkoxycoumarin;
7ꢀAMC,
7ꢀalkoxyꢀ4ꢀmethyl
coumarin;
7ꢀAFC,
7ꢀalkoxyꢀ4ꢀ
(trifluoromethyl)coumarin; CYMALꢀ5, 5ꢀcyclohexylꢀ1ꢀpentylꢀβꢀdꢀmaltoside; E. coli,
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Escherichia coli; Ni ꢀNTA, Nickelꢀnitrilotriacetic acid affinity resin; FAꢀ4, 3α, 7α, 12αꢀTris[(βꢀ
Dꢀmaltopyranosyl)ethyloxy]cholane; HRMS, high resolution mass spectrometry; DART, direct
analysis in real time
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Abstract
Using a combined structural and biochemical approach, the functional importance of a recently
described peripheral pocket bounded by the Eꢀ, Fꢀ, Gꢀ, and Iꢀhelices in CYP2B4 and 2B6 was
probed. Three series of 4ꢀsubstitutedꢀ7ꢀalkoxycoumarin derivatives with a ꢀH, ꢀCH , or ꢀCF at
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the 4 position of the coumarin core were used initially to monitor functional differences between
CYP2B4 and 2B6. 7ꢀEthoxyꢀ4ꢀ(trifluoromethyl)coumarin (7ꢀEFC) displayed the highest
catalytic efficiency among these substrates. Mutants were made to alter sideꢀchain polarity
(V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in
catalytic efficiency of 7ꢀEFC Oꢀdeethylation by the mutants were magnified considerably by
chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in
complex with (+)ꢀαꢀpinene was solved at 2.2 Å and showed no CYMALꢀ5 in the peripheral
pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMALꢀ5 in the
peripheral pocket. In both instances, comparison of the respective wildꢀtype and mutant CYP2B
enzymes revealed that CYMALꢀ5 occupancy of the peripheral pocket had little effect on the
topology of active site residue sideꢀchains, despite the fact that the peripheral pocket and active
site are located on opposite sides of the Iꢀhelix. Analysis of available CYP2B structures suggest
that the effect of the amino acid substitutions within the peripheral pocket derive from altered
interactions
between
the
F
and
G
helices.
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The metabolism and clearance of drugs and xenobiotic compounds depends heavily on
members of the cytochrome P450 (CYP) superfamily (1). In humans, these enzymes are the
main route for phase I metabolism of drugs (2). However, the conformational diversity and
plasticity of the active sites of drug-metabolizing CYP enzymes can limit the predictive
value of one structure of a CYP ligand complex for binding of other ligands (3, 4). To better
understand metabolism mediated by CYP enzymes, the specific interaction between the
substrate and residues within as well as outside the active site must be taken into account
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(1, 2).
Xenobiotic metabolizing CYP enzymes utilize a primarily alpha helical single domain protein
fold to accept a broad range of substrates (5, 6). Substrates oxidized by members of the CYP2B
subfamily of enzymes are generally nonꢀplanar lipophilic molecules that may be neutral or
weakly basic and possess one or two hydrogenꢀbond acceptors (7-9). Members of this subfamily
of enzymes were among the first mammalian CYP enzymes to be purified and cloned, and the
low preservation of CYP2B catalytic activities across mammalian species makes this subfamily a
good platform for CYP enzyme structureꢀfunction studies (10, 11). A substantial body of
biochemical and biophysical information regarding CYP2B proteinꢀligand and proteinꢀprotein
interactions and the catalytic mechanisms of microsomal monooxygenases is available (10-12).
Techniques including siteꢀdirected mutagenesis, isothermal titration calorimetry, highꢀpressure
spectroscopy, and hydrogen/deuterium exchange coupled to mass spectrometry have been used
to probe the functional and solution structural behavior of CYP2B enzymes (10-13).
Furthermore, Xꢀray crystallography has revealed that CYP2B enzymes adopt multiple
conformations to facilitate binding of substrates with a wide range of molecular weights (Mr =
~75 – 800) (10, 11, 14, 15).
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The representative of the CYP2B subfamily found in humans, CYP2B6, takes part in the
metabolism of 3 to 12% of all drugs (16), and a number of single nucleotide polymorphisms are
known, all of which occur outside the enzyme active site (17, 18). Some of these amino acid
differences lead to altered binding or metabolism of clinically relevant drugs (9, 18).
Furthermore, alteration of select nonꢀactive site residues in CYP2B enzymes indicated that these
changes distal from the enzyme active site can significantly affect protein stability, ligand
binding and/or catalysis (19, 20).
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CYP2B6 also displays nonꢀMichaelisꢀMenten kinetics with some substrates (21). Nonꢀ
hyperbolic kinetics in CYP enzymes has been most heavily studied in human CYP3A4;
explanations for this phenomenon include enzyme conformational heterogeneity, binding of
multiple ligand molecules in the enzyme active site, or the presence of a peripheral binding site
(22, 23). CYP3A4 was initially crystallized with progesterone bound at a peripheral site, and a
recent report utilizing isomeric testosterone dimers found that binding of a monomeric
testosterone molecule to a distal allosteric site enhanced binding when the transꢀtestosterone
dimer occupied the enzyme active site (24, 25). In CYP2B enzymes, a peripheral pocket was
recently described that is bounded by the Eꢀ, Fꢀ, Gꢀ, and Iꢀhelices and is occasionally occupied
by 5ꢀcyclohexylꢀ1ꢀpentylꢀβꢀdꢀmaltoside (CYMALꢀ5) in Xꢀray crystal structures (26). This cavity
is physically separated from the active site of CYP2B enzymes by the Iꢀhelix (Figure 1).
Previously observed rearrangements of the Iꢀhelix likely allow for optimal ligand interaction
with the heme group (27). Siteꢀdirected mutagenesis of residues within or near the surface of this
peripheral pocket revealed that CYP2B4 F202W and CYP2B6 F202W had little or no catalytic
activity with the model substrate 7ꢀethoxyꢀ4ꢀ(trifluoromethyl)coumarin (7ꢀEFC), primarily due
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to enhanced uncoupling to excess water, whereas CYP2B4 F195W showed no change in 7ꢀEFC
Oꢀdeethylation activity (26).
Alkoxycoumarin derivatives are model substrates of CYP enzymes due to their general
hydrophobicity and the production of fluorescent products. Previous studies have shown that the
length of the alkoxy chain is an important determinant of Oꢀdealkylation of 7ꢀsubstituted
coumarins by CYP2B1 and CYP3A4 and a number of active site mutants (28-31). In the present
study, the active sites of CYP2B4 and CYP2B6 were probed using three series of 4ꢀsubstitutedꢀ
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7ꢀalkoxycoumarins as substrates to assess the importance of substituents with different electronic
properties (ꢀH, ꢀCH , ꢀCF ) at the 4 position of the coumarin core along with varied length (C2ꢀ
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C4) and/or halogen substitution (ꢀCl, ꢀBr) of the alkoxy chain at the 7 position. In parallel using
siteꢀdirected mutagenesis, residues on the surface of CYP2B4 or CYP2B6 were mutated to alter
sideꢀchain polarity (V/E194Q) or bulk (F/Y244W) in an effort to block detergent access to the
peripheral pocket and assess the effect of CYMALꢀ5 binding on active site topology. For this
purpose, Xꢀray crystal structures of CYP2B4 F244W in the absence of ligand and CYP2B6
Y244W in the presence of the monoterpene (+)ꢀαꢀpinene were solved. The functional
consequences of these mutations were assessed using 7ꢀEFC and its two halogenated derivatives.
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Materials and Methods
Materials. 7ꢀethoxycoumarin (7ꢀEC), 7ꢀpropoxycoumarin (7ꢀPrC) and 7ꢀbutoxycoumarin (7ꢀ
BuC) were synthesized by Dr. E. Mash (Department of Chemistry, The University of Arizona).
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ꢀhydroxyꢀ4ꢀ(trifluoromethyl)coumarin (7ꢀHFC) was purchased from Alfa Aesar. 7ꢀ
hydroxycoumarin (7ꢀHC), 7ꢀhydroxyꢀ4ꢀmethylcoumarin (7ꢀHMC) and 7ꢀethoxyꢀ4ꢀ
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(trifluoromethyl)coumarin (7ꢀEFC) were purchased from Invitrogen (Carlsbad, CA). H and C
NMR spectrum were recorded on a Bruker AVANCE 500 MHz spectrometer using TMS as an
internal standard (chemical shifts in δ), and analysis was done using MestReNova version 8.0.0.
Peak multiplicities of NMR signals were designated as s (singlet), d (doublet), dd (doublet of
doublet), t (triplet), m (multiplet). HRMS was performed at the University of Connecticut Mass
Spectrometry Facility by Dr. YouꢀJun Fu, on a JEOL AccuTOF (Peabody, MA) mass
spectrometer using a DART (IonSense) ionization source. All chemical structures were created
using Accelrys Draw 4.1. βꢀNADPH, ribonuclease A (RNase A), and deoxyribonuclease I
(DNase I) were purchased from SigmaꢀAldrich (St. Louis, MO). Nickelꢀnitrilotriacetic acid
2+
(Ni ꢀNTA) affinity resin was from ThermoFisher Scientific (Waltham, MA), and MacroꢀPrep
CM cation exchange resin was obtained from BioꢀRad Laboratories (Hercules, CA). Amicon
Ultra and UltrafreeꢀMC centrifugal filter units were purchased from Millipore (Billerica, MA).
The QuikChange XL siteꢀdirected mutagenesis kit and Escherichia coli (E. coli) TOPP3 and
JM109 cells were obtained from Agilent (Santa Clara, CA). The molecular chaperone plasmid
pGro7 was obtained from Takara Bio (Shiba, Japan). Recombinant NADPH cytochrome P450
reductase (CPR) and cytochrome b from rat liver were prepared as described previously (32,
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was obtained from Hampton Research (Aliso Viejo, CA), and the Wizard 3 crystal screen was
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obtained from Rigaku (The Woodlands, TX). 3αꢀ, 7αꢀ, 12αꢀTris[(βꢀDꢀmaltopyranosyl)ethyloxy]
cholane (FAꢀ4) is a custom made facial amphiphile (34). All other chemicals and supplies were
obtained from standard commercial sources and are of the highest purity available.
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Site-Directed Mutagenesis. CYP2B4 mutants were created using the pKK2B4dH (H226Y)
plasmid as a template (35), and CYP2B6 mutants were created using the pKK2B6dH
(Y226H/K262R) plasmid as a template (14). Mutants were created by polymerase chain reaction
(PCR) with the QuikChange XL siteꢀdirected mutagenesis kit from Agilent and a standard siteꢀ
directed mutagenesis protocol. Forward and reverse primers used for siteꢀdirected mutagenesis
are shown in Table S1. All constructs were sequenced at Retrogen Inc. (San Diego, CA, USA) to
verify the intended mutations were present and the absence of extraneous mutations.
Protein Expression and Purification. CYP2B4 enzymes were expressed in E. coli TOPP3 cells;
CYP2B6 proteins were coꢀexpressed with the GroEL/ES chaperones (pGro7 plasmid) in E. coli
JM109 cells, as described previously (15, 36). In brief, protein expression was carried out for 72
hrs at 30 °C after induction with isopropyl βꢀDꢀ1ꢀthiogalactopyranoside (1 mM) and
supplementing the culture with δꢀaminolevulinic acid (0.5 mM, A600 ~ 0.7 at 37 °C) in the
presence of ampicillin (100ꢀꢁg/ml, CYP2B4 enzymes) or Lꢀ(+)ꢀArabinose (20 mg/ml),
ampicillin and chloramphenicol (25ꢀꢁg/ml, CYP2B6 enzymes). The cells were harvested by
centrifugation (4,000×g, 10 min). The cell pellets were resuspended in 10% of the original
culture volume in buffer containing 20 mM potassium phosphate (pH 7.4 at 4 °C), 20% (v/v)
glycerol, 10 mM BME, and 0.5 mM PMSF. The resuspended cells were treated with lysozyme
(0.3 mg/mL) and stirred for 30 min at 4 °C, and then followed by centrifugation for 30 min at
7500×g in a JAꢀ14 rotor in a Beckman Coulter Avanti Jꢀ26 XPI Centrifuge. After decanting the
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supernatant, the spheroplasts were resuspended in 5% of the original volume in the buffer
containing 500 mM potassium phosphate (pH 7.4 at 4 °C), 20% (v/v) glycerol, 10 mM BME, 0.5
mM PMSF, 10 ꢁg/mL RNase A, and 10 ꢁg/mL DNase I. The resulting suspension was then
sonicated 4 × 1 min on ice. The lysate was stirred for 90 min at 4 °C in the presence of CHAPS
at a final concentration of 0.8% (w/v). After ultracentrifugation for 1 h at 245,000×g using a
fixedꢀangle Ti 50.2 rotor in an Optima Lꢀ80 XP Ultracentrifuge, the supernatant was collected
and the concentration was measured using the reduced CO difference spectra (37).
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The supernatant was applied to a Ni ꢀNTA column and washed with buffer containing 100 mM
potassium phosphate (pH 7.4 at 4 °C), 100 mM NaCl, 20% (v/v) glycerol, 10 mM BME, 0.5 mM
PMSF, 0.5% (w/v) CHAPS, and 5 mM histidine. The protein was eluted using 50 mM histidine
in the same buffer. The highest purity fractions as measured by the A /A ratio were pooled,
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and the CYP enzyme concentration was measured using the reduced CO difference spectra.
Pooled fractions were diluted with buffer containing 5 mM potassium phosphate (pH 7.4 at 4
°C), 20% (v/v) glycerol, 1 mM EDTA, 0.2 mM DTT, 0.5 mM PMSF, and 0.5% (w/v) CHAPS
and loaded onto an equilibrated MacroꢀPrep CM cation exchange column. The column was
washed using 5 mM potassium phosphate (pH 7.4 at 4 °C), 20 mM NaCl, 20% (v/v) glycerol, 1
mM EDTA, and 0.2 mM DTT, and the protein was eluted using 50 mM potassium phosphate
(pH 7.4 at 4 °C), 500 mM NaCl, 20% (v/v) glycerol, 1 mM EDTA, and 0.2 mM DTT. The
fractions containing the highest purity protein as measured by the A /A ratio were collected,
4
17 280
and the concentration was measured using the reduced CO difference spectra.
Chemical Synthesis. All compounds were synthesized in similar manner according to published
1
methods (38, 39), and the purity of the compounds exceeds 95% by H NMR.
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-ethoxy-4-methylcoumarin (7-EMC). To a solution of 7ꢀhydroxyꢀ4ꢀmethyl coumarin (100.00
mg, 0.57 mmol, 1 equiv) in CH CN (5 mL), was added K CO (78.70 mg, 0.57 mmol, 1 equiv)
3
2
3
and CH CH Br (123.06 mg, 1.14 mmol, 2 equiv). The reaction mixture was refluxed at 80 °C for
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overnight. The crude mixture was filtered and washed the residue using CH CN (10 mL). The
3
combined organic layer was dried over MgSO , and concentrated in vacuo, and then purified
4
with flash chromatography (SiO , 5ꢀ20% Ethyl Acetate in nꢀHexanes) to afford 7ꢀethoxyꢀ4ꢀ
2
1
methylcoumarin as a colorless solid (80 mg, 69% yield). H NMR (500 MHz, CDCl , 25 °C): δ
3
7.46ꢀ7.48 (d, 1H, J=10 Hz), 6.83ꢀ6.85 (d, 1H, J=10 Hz), 6.78 (s, 1H), 6.11 (s, 1H), 4.06ꢀ4.10 (q,
13
2
H, J=5 Hz, 5 Hz), 2.38 (s, 3H), 1.43ꢀ1.46 (t, 3H, J=7.5 Hz). C NMR (125 MHz, CDCl , 25
3
°
C): δ 162.3, 161.6, 155.6, 152.8, 125.8, 113.7, 112.9, 112.1, 101.6, 64.4, 18.9, 14.8. DARTꢀ
+
HRMS: m/z calcd. for C H O [M+H] : 205.08; Found: 205.08.
1
2
12
3
1
7
-propoxy-4-methylcoumarin (7-PrMC). Colorless solid, 48% yield. H NMR (500 MHz,
CDCl , 25 °C): δ 7.45ꢀ7.46 (d, 1H, J=5 Hz), 6.82ꢀ6.83 (d, 1H, J=5 Hz), 6.77 (s, 1H), 6.09 (s,
3
13
1H), 3.94ꢀ3.96 (t, 2H, J=5 Hz), 2.37 (s, 3H), 1.80ꢀ1.84 (m, 2H), 1.02ꢀ1.05 (t, 3H, J=7.5 Hz). C
NMR (125 MHz, CDCl , 25 °C): δ 162.2, 161.3, 155.3, 152.5, 125.4, 113.4, 112.6, 111.8, 101.3,
3
+
70.1, 22.3, 18.6, 10.4. DARTꢀHRMS: m/z calcd. for C H O [M+H] : 219.10; Found: 219.10.
13 14
3
1
7-butoxy-4-methylcoumarin (7-BuMC). Colorless solid, 23% yield. H NMR (500 MHz,
CDCl , 25 °C): δ 7.47ꢀ7.48 (d, 1H, J=5 Hz), 6.84ꢀ6.85 (d, 1H, J=5 Hz), 6.80 (s, 1H), 6.12 (s,
3
1H), 4.00ꢀ4.03 (t, 2H, J=7.5 Hz), 2.39 (s, 3H), 1.78ꢀ1.81 (m, 2H), 1.48ꢀ1.52 (m, 2H), 0.97ꢀ1.00
13
(t, 3H, J=7.5 Hz). C NMR (125 MHz, CDCl , 25 °C): δ 162.5, 161.6, 155.6, 152.7, 125.6,
3
1
13.6, 112.9, 112.1, 101.6, 68.5, 31.2, 19.4, 18.9, 14.04. DARTꢀHRMS: m/z calcd. for C H O
14 16 3
+
[
M+H] : 233.11; Found: 233.11.
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-propoxy-4-(trifluoromethyl)coumarin (7-PrFC). Colorless solid, 50% yield. H NMR (500
MHz, CDCl , 25 °C): δ 7.59ꢀ7.61 (d, 1H, J=10 Hz), 6.88ꢀ6.90 (d, 1H, J=10 Hz), 6.84 (s, 1H),
3
13
6
.59 (s, 1H), 3.98ꢀ4.00 (t, 2H, J=5 Hz), 1.82ꢀ1.86 (m, 2H), 1.03ꢀ1.06 (t, 3H, J=7.5 Hz). C NMR
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(
125 MHz, CDCl , 25 °C): δ 163.4, 159.7, 156.7, 141.7, 126.6, 120.5, 114.1, 112.4, 107.1, 102.1,
3
+
70.6, 22.5, 10.7. DARTꢀHRMS: m/z calcd. for C H F O [M+H] : 273.07; Found: 273.07.
1
3
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3
3
1
7
-butoxy-4-(trifluoromethyl)coumarin (7-BuFC). Colorless solid, 81% yield. H NMR (500
MHz, CDCl , 25 °C): δ 7.59ꢀ7.60 (d, 1H, J=5 Hz), 6.88ꢀ6.89 (d, 1H, J=5 Hz), 6.84 (s, 1H), 6.58
3
(s, 1H), 4.01ꢀ4.04 (t, 2H, J=7.5 Hz), 1.78ꢀ1.81 (m, 2H), 1.47ꢀ1.51 (m, 2H), 0.96ꢀ0.99 (t, 3H,
13
J=7.5 Hz). C NMR (125 MHz, CDCl , 25 °C): δ 163.1, 159.4, 156.3, 141.6, 126.1, 120.4,
3
1
13.6, 111.9, 106.7, 101.7, 68.5, 30.8, 19.1, 13.6. DARTꢀHRMS: m/z calcd. for C H F O
14 13 3 3
+
[M+H] : 287.08; Found: 287.08.
1
7
-(2-chloroethoxy)-4-(trifluoromethyl)coumarin (7-ClEFC). Colorless solid, 17% yield. H
NMR (500 MHz, CDCl , 25 °C): δ 7.64ꢀ7.66 (d, 1H, J=10 Hz), 6.94ꢀ6.96 (d, 1H, J=10 Hz), 6.88
3
13
(s, 1H), 6.64 (s, 1H), 4.30ꢀ4.33 (t, 2H, J=7.5 Hz), 3.85ꢀ3.87 (t, 2H, J=5 Hz). C NMR (125
MHz, CDCl , 25 °C): δ 162.1, 159.2, 156.3, 141.7, 126.6, 120.5, 113.6, 112.9, 107.7, 102.2,
3
+
6
8.6, 41.4. DARTꢀHRMS: m/z calcd. for C H ClF O [M+H] : 293.01; Found: 293.01.
1
2
8
3
3
1
7-(2-bromoethoxy)-4-(trifluoromethyl)coumarin (7-BrEFC). Colorless solid, 36% yield. H
NMR (500 MHz, CDCl , 25 °C): δ 7.64ꢀ7.66 (d, 1H, J=10 Hz), 6.94ꢀ6.96 (d, 1H, J=10 Hz), 6.88
3
1
3
(s, 1H), 6.64 (s, 1H), 4.36ꢀ4.39 (t, 2H, J=7.5 Hz), 3.66ꢀ3.69 (t, 2H, J=7.5 Hz). C NMR (125
MHz, CDCl , 25 °C): δ 162.2, 159.4, 156.5, 141.6, 126.8, 120.5, 113.8, 113.1, 107.9, 102.5,
3
+
6
8.6, 28.4. DARTꢀHRMS: m/z calcd. for C H BrF O [M+H] : 336.96; Found: 336.96.
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Enzymatic Assays. The standard NADPHꢀdependent assays for Oꢀdealkylation of 7ꢀ
alkoxycoumarin, 7ꢀalkoxyꢀ4ꢀmethylcoumarin, and 7ꢀalkoxyꢀ4ꢀ(trifluoromethyl)coumarin series
were performed as described previously (30, 40, 41). The reconstituted system contained the
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purified CYP enzyme with rat NADPHꢀcytochrome P450 reductase and rat cytochrome b at a
5
molar ratio of 1:4:2. Reactions using 7ꢀalkoxyꢀ4ꢀ(trifluoromethyl)coumarin series substrates
were carried out in a 100 ꢁL final volume with substrate at varying concentrations (0 ~ 300 ꢁM).
Reactions were preincubated for 3 min at 37 °C in buffer containing 50 mM HEPES (pH 7.4)
and 15 mM MgCl before initiation of the reaction by addition of NADPH to a final
2
concentration of 1 mM. After incubation for 5 min at 37 °C, the reaction was quenched by the
addition of cold acetonitrile (50 ꢁL). For detection of 7ꢀHFC, a 50 ꢁL aliquot of the reaction
mixture was transferred into 950 ꢁL of 0.1M Tris (pH 9.0), and fluorescence was determined
with λ =410 nm and λ =510 nm using a Hitachi F2000 fluorescence spectrophotometer
ex
em
(Tokyo, Japan).
Reactions using 7ꢀalkoxycoumarin and 7ꢀalkoxyꢀ4ꢀmethylcoumarin series substrates were
performed in the buffer containing 50 mM HEPES (pH 7.4) and 15 mM MgCl in a 100 ꢁL final
2
volume and substrate at varying concentrations (0 ~ 300 ꢁM). After a similar preincubation and
initiation by addition of NADPH, the reaction proceeded for 10 min at 37 °C and was quenched
by addition of 25 ꢁL 2M HCl. The entire reaction was extracted with 450 ꢁL of chloroform and
centrifuged at 3000×g for 5 min. After transferring 300 ꢁL of the organic layer to a clean test
tube, the mixture was extracted with 1 mL 30 mM sodium borate (pH 9.2) followed by
centrifugation for 5 min at 3000×g. A 900 ꢁL aliquot of the aqueous layer was transferred to a
clean test tube, and the fluorescence was measured at λ =370 nm for 7ꢀHC and λ =360 nm for
ex
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ꢀHMC and λ =450 nm for both products using a Hitachi F2000 fluorescence
em
spectrophotometer.
In all assays, calibration was performed daily by measuring the intensity of fluorescence in the
same reaction buffer containing 7ꢀHC, 7ꢀHMC or 7ꢀHFC at concentrations from 0 to 2 ꢁM. The
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K and k values were determined by regression analysis with Igor Pro 6.35 (WaveMetrics)
M
cat
using the MichaelisꢀMenten or Hill equation.
Crystallization and Data Collection. Following purification, pooled protein was diluted to a final
concentration of 18 ꢁM by addition of buffer containing 50 mM potassium phosphate (pH 7.4 at
4 °C), 500 mM sucrose, 500 mM NaCl, 1 mM EDTA, and 0.2 mM DTT. (+)ꢀαꢀpinene dissolved
in acetone was added to 2B6 Y244W to a final concentration of 180 ꢁM. The diluted enzymes
were incubated overnight at 4 °C on ice. Protein was concentrated by centrifugation using 50ꢀ
kDa cutoff Amicon ultrafiltration devices (Millipore). The procedure was repeated twice before
concentrating the protein to the final concentration of 300 ꢁM for 2B6 Y244W and 500 ꢁM for
2B4 F244W. The concentrated CYP2B6 Y244W proteinꢀligand complex was then supplemented
with 4.8 mM CYMALꢀ5, 300 ꢁM (+)ꢀαꢀpinene, and 0.028% (w/v) FAꢀ4. CYP2B4 F244Wꢀ
ligand free was supplemented with 4.8 mM CYMALꢀ5, and 0.056% (w/v) FAꢀ4. Prior to
screening, samples were filtered using 0.22 µm UltrafreeꢀMC centrifugal filters (Millipore), and
crystal screening was performed by the sittingꢀdrop vapor diffusion method. Crystals of the
CYP2B6 Y244W (+)ꢀαꢀpinene complex were obtained from the Hampton Research Crystal
Screen 2 in 0.1 M HEPES pH 7.5 and 20% w/v polyethylene glycol 10,000 after 3ꢀ4 days.
Crystals of the CYP2B4 F244W in the absence of ligand were obtained from the Rigaku Wizard
3
screen in 0.1 M HEPES pH 7.0 and 10% w/v polyethylene glycol 6,000 after 3ꢀ4 days. Crystals
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were transferred to the mother liquor containing 20% (v/v) sucrose for cryoprotection before
flash freezing them in liquid nitrogen. Crystallographic data for CYP2B6 Y244Wꢀ(+)ꢀαꢀpinene
were collected remotely at Stanford Synchrotron Radiation Lightsource (SSRL) on beam line 12ꢀ
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using 1° oscillations over 120 frames and 1 s exposures with a ADSC Quantum 315r CCD
detector at 100 K (42). CYP2B4 F244W ligand free data were collected remotely at SSRL beam
line 7ꢀ1 using 1° oscillations over 240 frames and 10 s exposures with a ADSC Quantum 315r
CCD detector at 100 K. Crystals of CYP2B6 Y244Wꢀ(+)ꢀαꢀpinene diffracted to 2.2 Å resolution
and CYP2B4 F244W ligand free diffracted to 3.0 Å resolution. Data were integrated using
iMOSFLM (43) and scaled using SCALA in CCP4 (44).
Structure Determination and Refinement. The structure of CYP2B6 Y244W in complex with
(+)ꢀαꢀpinene was determined using the coordinates of the CYP2B6ꢀ(+)ꢀαꢀpinene structure (PDB
ID: 4I91) as a starting model in the molecular replacement program Phaser (45) from the CCP4
software suite (44). The solvent content of 61.7% was determined by the Matthews coefficient
analysis, assuming the presence of one molecule in the asymmetric unit. The output model from
Phaser, with the space group P3 21, was subjected to rigid body and restrained refinement in
2
REFMAC5 (46) in CCP4 (44). The model was built manually in COOT (47) using 2F ꢀF and
o
c
F ꢀF electron density maps contoured at 1ꢀσ and 3ꢀσ, respectively. PRODRG server was used to
o
c
make the library description for (+)ꢀαꢀpinene (48). Iterative refinement was continued until the
Rꢀfactor and Rfree of 0.17 and 0.23, respectively, were achieved. The model was validated by
th
MOLPROBITY (49), which ranked the structure in the 98 percentile among structures of
comparable resolution. There were no Ramachandran outliers or bad bond lengths or angles in
the final model. The crystal structure contained a total of 675 molecules of water, two molecules
of CYMALꢀ5 detergent, and protein residues from 28 to 492 with the terminal residue being the
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first histidine of the Cꢀterminal 4ꢀHisꢀtag. Coordinates and structure factors were deposited in the
Protein Data Bank (PDB ID: 4ZV8). In parallel, the structure of CYP2B4 F244W in the absence
of ligand was determined at 3.0 Å resolution, and Matthews coefficient analysis indicated the
presence of two molecules in the asymmetric unit. The CYP2B4 ligand free structure (PDB ID:
3MVR) was used as a starting model for molecular replacement in Phaser. Iterative refinement in
REFMAC5 and model building in COOT was continued until the Rꢀfactor (0.23) and Rꢀfree
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(0.28) stopped improving. The crystal structure contained a total of 14 molecules of water, four
molecules of CYMALꢀ5 detergent, and protein residues from 28 to 492 with the terminal residue
being the first histidine of the Cꢀterminal 4ꢀHisꢀtag. MOLPROBITY analysis ranked the
th
structure in the 98 percentile among structures of comparable resolution. Coordinates and
structure factors were deposited in the Protein Data Bank (PDB ID: 5EM4). The refinement
statistics
for
both
structures
are
summarized
in
Table
S2.
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Results
Determinants of CYP2B4 and CYP2B6 Coumarin Substrate Preference. A series of alkyl ethers
of 7ꢀHC with varying alkyl chain length was previously used to probe the active sites of
CYP2B1 and CYP3A4 (28, 29, 31). Furthermore, 4ꢀsubstitutedꢀ7ꢀethyoxycoumarins, including
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7ꢀEC and 7ꢀEFC, have been used as model substrates of CYP2B enzymes. In the present
investigation three series of 4ꢀsubstituted 7ꢀalkoxycoumarins (50) were used as potential probes
of the functional potential importance of the peripheral pocket in CYP2B4 and CYP2B6 (Figure
2). These allowed assessment of the functional effect of altering the length of the 7ꢀalkoxy chain
and varying the substituent located at the 4ꢀposition of the coumarin moiety. Tables 1 and 2
summarize the steadyꢀstate kinetics results for Oꢀdealkylation of these substrates by CYP2B4
and CYP2B6, respectively. In general, increased 7ꢀalkoxy chain length decreased k and K in
cat
M
concert, so that catalytic efficiency (k /K ) remained similar within each set of 4ꢀsubstituted
cat
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compounds. The exception occurred in the 4ꢀtrifluoromethyl substituted series, where
dealkylation of 7ꢀBuFC was undetectable with either enzyme, and catalytic efficiency of 7ꢀPrFC
O-dealkylation by CYP2B6 was 7ꢀfold lower than that of 7ꢀEFC.
With the 7ꢀethoxy compounds, altering the 4ꢀsubstitutent on the coumarin core from proton (ꢀH)
to electronꢀdonating group (ꢀCH ) decreased the k and K , resulting in little change in catalytic
3
cat
M
efficiency of Oꢀdealkylation by CYP2B4 and CYP2B6. Conversely, substitution of the electronꢀ
donating 4ꢀmethyl with an electronꢀwithdrawing 4ꢀtrifluoromethyl group greatly increased kcat
for Oꢀdealkylation coupled with a further reduction in K . The 4ꢀtrifluoromethyl substitution for
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the 4ꢀmethyl increases the bulk of this group, increases the lipophilicity of the molecule, and
alters the hydrogen bonding character of the group from donor to weak acceptor (51). The
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increase in bulk at the 4ꢀcarbon may preferentially orient the 7ꢀalkoxy group toward the heme
iron for productive Oꢀdealkylation. This combination of changes leads to ~20ꢀ and 65ꢀfold
increases in catalytic efficiency of Oꢀdeethylation of 7ꢀEFC versus 7ꢀEC or 7ꢀEMC for CYP2B4
and 2B6, respectively. For 7ꢀpropoxy compounds, an increase in catalytic efficiency is seen
across the set of compounds. In CYP2B4, this increase in catalytic efficiency is solely due to
decreases in KM upon addition of the 4ꢀmethyl and 4ꢀtrifluoromethyl groups. However, in
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CYP2B6 the addition of a 4ꢀmethyl group increases kcat with only a small decrease in K , but
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changing the 4ꢀmethyl to a 4ꢀtrifluoromethyl group leads to a sharp decrease in K and virtually
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no change in k . With both enzymes, the lowest K among all the compounds tested is
cat
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observed with 7ꢀPrFC. The catalytic efficiency of CYP2B4 catalyzed Oꢀdebutylation is virtually
unchanged with the addition of the 4ꢀmethyl moiety, whereas CYP2B6 is more efficient due to a
lower KM.
Effect of Substrate Halogenation on CYP2B Substrate Specificity. To develop even more potent
ligands, the effect of halogenating the terminus of the 7ꢀethoxy chain of 7ꢀEFC was analyzed
(Figure 2). Chlorine and bromine were selected since 1) CYP2B enzymes appear to form
halogen bonds readily, especially –Clꢀπ bonds (15), and 2) nonꢀcoplanar congeners from two
groups of persistent halogenated organic pollutants, polychlorinated biphenyls and
polybrominated diphenyl ethers, are preferentially metabolized by CYP2B enzymes (52, 53).
The strength of halogen bonds is roughly correlated with the electrostatic potential on the
halogen atom. Fluorine was not used, since organofluorines generally do not form halogen bonds
relevant to medicinal chemistry (54, 55). As shown in Tables 3 and 4, the K for Oꢀdealkylation
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by both enzymes was decreased 3ꢀfold upon bromination, and 11ꢀfold upon chlorination in the
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case of CYP2B4. However, catalytic efficiency decreased in all cases due to 10ꢀ20 fold
decreased in kcat.
Effect of Modifying Residues 194 and 244 on CYP2B6 and CYP2B4 Function. Mutants were
made in CYP2B4 and 2B6 in an attempt to block off the peripheral pocket by altering sideꢀchain
polarity at residue 194 (V/Eꢀ>Q) or bulk at residue 244 (F/Yꢀ>W) (Figures 3A, 4A). Oꢀ
dealkylation of 7ꢀEFC and its halogenated derivatives was used to characterize function (Tables
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and 4). With the mutants at position 194, relatively little effect was observed on 7ꢀEFC Oꢀ
dealkylation, whereas CYP2B4 F244W and CYP2B6 Y244W showed 4ꢀand 2ꢀfold increases in
catalytic efficiency of 7ꢀEFC Oꢀdealkylation, respectively, primarily due to increased k . The
cat
effects of the mutations were even more striking with 7ꢀClEFC and 7ꢀBrEFC. With CYP2B4 the
increased catalytic efficiency of both mutants is due to 2ꢀ10 fold increases in kcat along with
essentially unaltered K . Conversely in CYP2B6 7ꢀClEFC and 7ꢀBrEFC Oꢀdealkylation, the 4ꢀ9
M
fold increase in catalytic efficiency is due to much lower K values with little change in k .
M
cat
Structures of CYP2B6 Y244W and CYP2B4 F244W. Attempts to crystallize CYP2B6 or CYP2B6
Y244W in the absence of ligand or in the presence of the most potent coumarin derivatives were
unsuccessful. Therefore, CYP2B6 Y244W was crystallized in the presence of the monoterpene
(+)ꢀαꢀpinene, for which binding affinity was unaltered [CYP2B6 WT (K =0.38 ꢁM) (27) and
S
CYP2B6 Y244W (K =0.43 ꢁM)]. Importantly, the mutant structure showed no CYMALꢀ5 in the
S
peripheral pocket. An overlay of the new CYP2B6 Y244Wꢀ(+)ꢀαꢀpinene complex (PDB ID:
4ZV8) reported here with the previously solved structure of the CYP2B6ꢀ(+)ꢀαꢀpinene complex
(PDB ID: 4I91) revealed significant differences in the orientation of residue side chains in the
peripheral pocket presumably due to the lack of detergent (Figure 3A). Despite the significant
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differences in the peripheral pocket residues, the structures of CYP2B6 WT and CYP2B6
Y244W in complex with (+)ꢀαꢀpinene revealed little difference in the orientation of active site
side chains (Figure 3B). In parallel, structure determination of ligand free CYP2B4 F244W
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(
PDB ID: 5EM4) revealed binding of CYMALꢀ5 in the peripheral pocket, similar to that
observed in the ligand free structure of CYP2B4 WT (PDB ID: 3MVR), with little difference in
the orientation of active site residues (Figure 4AꢀB). These results suggest that introduction of
tryptophan at position 244 in CYP2B6 or CYP2B4 may or may not prevent CYMALꢀ5 binding,
respectively, in the peripheral pocket of these enzymes. However, binding of CYMALꢀ5 in the
peripheral pocket located in close vicinity of the active site likely does not influence the active
site architecture. In addition to the similarity of the active site residue orientations, the similarity
in orientation of the bound (+)ꢀαꢀpinene in the CYP2B6 WT and Y244W complexes with and
without CYMALꢀ5 in the peripheral pocket is indicative of little effect on binding of ligands in
the
catalytic
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Discussion
CYP enzymes involved in mammalian detoxification generally metabolize a broad range of
substrates. These enzymes are often relatively flexible and are capable of binding multiple ligand
molecules simultaneously. This combination of properties can lead to nonꢀMichaelisꢀMenten
kinetics of substrate oxidation (56). Structurally, multiple substrate molecules can bind at distinct
locations within the distal heme pocket, as seen in the CYP2C8ꢀcisꢀretinoic acid complex (PDB
ID: 2NNH) (57) or ketoconazole complex of CYP3A4 (PDB ID: 2V0M) (58). Alternatively, a
ligand binding event may occur at a site outside the enzyme active site, as seen in the location of
progesterone in complex with CYP3A4 (24). Many of the previously solved Xꢀray crystal
structures of CYP2B4 and CYP2B6 demonstrated binding of CYMALꢀ5 in a peripheral pocket
surrounded by the Eꢀ, Fꢀ, Gꢀ, and Iꢀhelices (14, 27, 59-62). Mutants within this peripheral pocket
can greatly reduce enzyme activity (26). The current study focused on elucidating the structural
consequences of CYMALꢀ5 binding in this peripheral pocket on the CYP2B4 or 2B6 active site
and further exploring the effect of substrate halogenation on CYP2B enzyme activity.
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Ligand binding at a peripheral site in CYP enzymes could alter substrate metabolism via several
possible processes. Binding of ligand A could alter the turnover of ligand B, as seen in the
interaction of testosterone with CYP3A4 for two molecules of one substrate or in the interaction
of progesterone and carbamazepine with CYP3A4 for multiple substrates (63-65). Binding of
ligand A could alter interactions between CYP enzymes, and modify oxidation of ligand B, as
has been suggested in the case of CYP3A4, 3A5 or 2E1 (24, 66). Finally, binding of ligand A
could physically impede structural compensations necessary for optimal binding of ligand B in
the CYP enzyme active site. In this case, the cyclohexyl group of CYMALꢀ5 bound in the
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CYP2B4 or 2B6 peripheral pocket would likely prevent changes in the orientation of the Iꢀhelix,
preventing optimal ligand orientation for oxidation (27). Furthermore, introduction of extra bulk
via mutation of select residues to tryptophan severely hampered enzyme activity in both
CYP2B4 and 2B6 (26). The snapshots from comparison of the Xꢀray crystal structures of ligand
free CYP2B4 and CYP2B4 F244W and of the (+)ꢀαꢀpinene bound complexes of CYP2B6 and
CYP2B6 Y244W indicated that neither CYMALꢀ5 nor the distal mutation alters active site
topology. These results do not rule out differences in solution structural dynamic behavior
between the respective WT CYP2B enzymes and the corresponding F/Y244W mutant, either in
altering the structural response of the protein upon ligand binding or in altering the equilibrium
between different CYP2B conformations in solution.
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In Xꢀray crystal structures of CYP2B4 and CYP2B6 in complex with 4ꢀCPI or amlodipine,
residues 202 and 241 appear to form a C–H/π bond between the aromatic ring of F202 and the
I241 δꢀcarbon (Figure 5AꢀB). This type of weak hydrogen bond is involved in multiple
phenomena in organic and biological chemistry, from reaction selectivity to protein
conformational stabilization (67). In the CYP2B4 or CYP2B6 F202W mutants the geometry of
this interaction is likely altered due to the increased steric bulk of the tryptophan indole ring, and
the indole ring of the I241W mutant in CYP2B4 could interact with the phenyl ring of F202 via
areneꢀarene πꢀπ interactions. In either case the distance between the Fꢀ and Gꢀhelices would be
altered, and any resultant interaction between residues 202 and 241 would change the relative
orientations of the Fꢀ and Gꢀhelices. In contrast, substitution of tryptophan at residue 244 could
allow for further stabilization of the FꢀG cassette by creating additional hydrogen bonding
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interaction involving the pairs V194/F244W and V194Q/F244 in CYP2B4 and E194/Y244W
and E194Q/Y244 in CYP2B6.
While hydrogen bonding (C–H/π) appears to provide structural stability to CYP2B enzymes,
halogen bonding is implicated in selectivity of ligand binding to CYP2B enzymes. Structures of
multiple proteins in complex with their respective ligand demonstrate the prevalence of halogenꢀ
π bonding in Xꢀray crystal structures (15, 50, 68). In this light the implications of the modest
effects of the CYP2B4 F244W (4ꢀfold) and CYP2B6 Y244W (2ꢀfold) mutations on the catalytic
efficiency of 7ꢀEFC metabolism become clearer. With both mutants the efficiency of Oꢀ
dealkylation of the halogenated substrates increased to between 5ꢀ and 8ꢀfold greater than the
respective WT enzyme. Interestingly, in other enzyme systems, introduction of –F does not alter
ligand affinity, while heavy halogens (–Cl, –ꢀBr, –I) do increase affinity (69, 70). Additionally,
introduction of heavy halogens into drug leads has led to greater likelihood of launching a
compound as an approved therapeutic than inclusion of a –F does, possibly as a result of
improved pharmacokinetic properties (71).
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In conclusion, a comparison of (+)ꢀαꢀpinene bound CYP2B6 Y244W and ligand free CYP2B4
F244W with the previously solved CYP2B6 WTꢀ(+)ꢀαꢀpinene and the ligand free CYP2B4 WT
structures, respectively, revealed that the presence or absence of CYMALꢀ5 binding in the
peripheral pocket does not alter active site topology. However, the absence of CYMALꢀ5 in the
CYP2B6 Y244W complex led to significant differences in the orientation of residue side
chains in the peripheral pocket and near its access region. The side chain of F296 protruded into
the pocket, while residues E194 and Y244W contributed to block off the pocket. Additionally,
we investigated the utility of 7ꢀalkoxycoumarin derivatives as substrate probes of CYP2B
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enzyme function. The results demonstrated that the substituent at the 4ꢀposition and the side
chain length at the 7ꢀposition have a significant influence on the metabolism of coumarin
derivatives by CYP2B enzymes. Altering the bulk at residue 244 increased catalytic efficiency of
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7
ꢀEFC Oꢀdealkylation in both CYP2B4 and CYP2B6 as reflected in an increase in kcat.
Introduction of a halogen at the 2ꢀposition of the 7ꢀethoxy side chain of 7ꢀEFC led to a decrease
in catalytic efficiency of Oꢀdealkylation by both enzymes and accentuated the positive effects on
activity of the amino acid substitutions at positions 194 and 244. Intriguingly, the increases in
catalytic efficiency observed with these mutants are diametrically opposite to the detrimental
effects of peripheral pocket mutations at residues 202 and 241 and lack of effect of a substitution
at position 195 observed previously (26). The substrate preference profiles obtained in this study
provide insights into the effects of substrate size and halogen substitution on CYP2B catalysis.
Further studies using a combination of siteꢀdirected mutagenesis, solution biophysical methods,
and substrate metabolism will improve our understanding of halogenated substrate recognition
by CYP2B enzymes and possibly other xenobiotic metabolizing CYP enzymes.
Supplementary Material
The supplementary material includes a table of the primers used for site directed mutagenesis, a
table of crystallographic refinement information, a protocol for spectral binding titrations used
for measuring the interaction of (+)ꢀαꢀpinene with CYP2B6 Y244W, and 1HꢀNMR and 13Cꢀ
NMR spectra of synthesized compounds described in this manuscript. This material is available
free of charge via the Internet at http://pubs.acs.org.
Author Contributions
Participated in research design: Liu and Halpert.
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Conducted experiments: Liu.
Contributed new reagents: Zhang.
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Performed data analysis: Liu, Shah, Halpert, and Wilderman.
Wrote or contributed to the writing of the manuscript: Liu, Shah, Stout, Halpert, and Wilderman.
Funding Sources
This research was supported by the National Institutes of Health [Grant R01ꢀES003619] (to
J.R.H.), and [Grant R01ꢀGM098538] (to Q.Z.).
Footnotes
1
In this manuscript, CYP2B4 wildꢀtype will refer to CYP2B4dH H226Y and CYP2B6 wildꢀtype
will refer to CYP2B6dH Y226H/K262R unless otherwise indicated. These are an Nꢀterminally
truncated and modified and Cꢀterminally Hisꢀtagged forms of CYP2B4dH and CYP2B6dH,
respectively.
2
Accession Codes
Atomic coordinates and structure factors for the P450 2B4 F244W ligand free and 2B6 Y244Wꢀ
(+)ꢀαꢀpinene complexes are deposited in the Protein Data Bank as entries 5EM4 and 4ZV8,
respectively.
Acknowledgments
We thank Dr. M. Kyle Hadden and Dr. Albert M. DeBerardinis (Division of Medicinal
Chemistry, Department of Pharmaceutical Sciences, University of Connecticut) for kindly
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providing and setting up the synthetic apparatus. We also thank the staff at Stanford Synchrotron
Radiation Light source, operated by Stanford University on behalf of the United States
Department of Energy, Office of Basic Energy Sciences, for assistance with the beam line.
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