Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-oxidation and coumarin 7-hydroxylation

Article abstract of DOI:10.1016/j.dmpk.2017.11.004

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.

Full text of DOI:10.1016/j.dmpk.2017.11.004

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Functional characterization of 9 CYP2A13 allelic variants by
assessment of nicotine C-oxidation and coumarin 7-hydroxylation*
Masaki Kumondai a, Hiroki Hosono a, Masamitsu Maekawa b, Hiroaki Yamaguchi b,
Q6
, b, d, *
Nariyasu Mano b, Akifumi Oda c, Noriyasu Hirasawa a, Masahiro Hiratsuka a
a Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan
b Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, 980-8574, Japan
c Faculty of Pharmacy, Meijo University, Nagoya, 468-8503, Japan
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d Tohoku Medical Megabank Organization, Tohoku University, Sendai, 980-8575, Japan
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a r t i c l e i n f o
a b s t r a c t
Article history:
Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as
nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized
as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-
individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a
decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this
study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic
variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant
proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation
and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal
fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-
difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be
determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2,
CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward
both substrates. These findings provide insights into the mechanism underlying inter-individual differ-
ences observed in genotoxicity and cancer susceptibility.
Received 20 August 2017
Received in revised form
1 November 2017
Accepted 13 November 2017
Available online xxx
Keywords:
Cytochrome P450
CYP2A13
Genetic polymorphisms
Nicotine
Coumarin
© 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
1. Introduction
in DNA-damage-response protein expression was reported in
BEAS-2B cells, which express CYP2A13 [7]. The CYP2A13 mRNA is
Q2
Cytochrome P450 (CYP) 2A13 is an enzyme involved in the
metabolism of several substrates, including coumarin, as well as
nicotine and tobacco-specific nitrosoamines 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone (NNK) and N0-nitrosonornicotine (NNN)
(Fig.1) [1e4]. CYP2A13 also contributes to the genotoxicity caused by
metabolic activation of aflatoxins and NNK, which is a representative
procarcinogen included in tobacco [5]. In A549 cell transfectants,
highly expressed in lung and bladder tissues (approximately 20 and
5 ꢀ 10ꢁ5 copies/0.1
mg RNA described as CYP2A13/GAPDH, respec-
tively) [8]. In these tissues, bioactivation of procarcinogen caused by
CYP2A13 had a much larger contribution to genotoxicity.
Several CYP2A13 genetic polymorphisms have been identified in
Japanese and French Caucasian populations [9e11]. The frequency
of CYP2A13*2, specifically, has been reported with a frequency of
4.8e7.3% in Japanese individuals [9,11,12]. The Arg257Cys substi-
tution caused by mutations in CYP2A13*2 is located at the carboxyl
end of the G helix, resulting in a 2e3-fold reduction in CYP2A13
enzymatic activity [13,14]. CYP2A13*2 was associated with a
decreased incidence of lung adenocarcinoma in smokers [15]. We
previously found that the presence of CYP2A13*2 was associated
with a reduced risk for bladder cancer, given that it decreases DNA
damage caused by CYP2A13 [12]. Therefore, the enzymatic activity
of CYP2A13 plays an important role in cancer development.
CYP2A13 mediated NNK metabolism to induce g-H2AX production, a
sensitive marker of DNA adducts [6]. An aflatoxin-mediated increase
*
This study was supported by grants from the Smoking Research Foundation and
Japan Research Foundation for Clinical Pharmacology.
Q5
Q1
* Corresponding author. Laboratory of Pharmacotherapy of Life-Style Related
Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba,
Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
E-mail address: mhira@m.tohoku.ac.jp (M. Hiratsuka).
https://doi.org/10.1016/j.dmpk.2017.11.004
1347-4367/© 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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A
B
CYP2A13
Aldehyde oxidase
nicoꢀne-Δ1’,(5’)-iminium ion
nicoꢀne
coꢀnine
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CYP2A13
coumarin
7-hydroxycoumarin
Fig. 1. Metabolic pathways from nicotine to cotinine catalyzed by CYP2A13 and aldehyde oxidase (A). Coumarin 7-hydroxylation metabolism catalyzed by CYP2A13 (B).
As shown in Table 1, several polymorphisms have been identi-
dinucleotide phosphate oxidized form (NADPþ), glucose-6-
phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-
fied for the CYP2A13 gene in addition to CYP2A13*2 (http://www.
cypalleles.ki.se/cyp2a13.htm) [Accessed Aug 9th, 2017]. The
in vitro enzymatic activity of several of these variants has been
characterized using a recombinant enzyme system such as E. coli
cells [14,16,17]. Schlicht et al. reported that
(Asp158Glu, Arg257Cys, and Val323Leu) caused 2e3-fold decrease
in enzymatic NNK -hydroxylation activity [14]. CYP2A13*4, which
did not show an absorbance increase near 450 nm, did not have the
ability to metabolize 5-methoxypsoralen [16]. These poly-
morphisms may affect individual variations in cancer development.
To characterize the currently known 8 CYP2A13 variants, except
for CYP2A13*7, which contains an immature stop codon (Arg101-
Stop) that causes the disappearance of enzymatic activity, we eval-
uated their activity using nicotine as a substrate, following protein
expression in 293FT cells. In addition, coumarin 7-hydroxylation
catalyzed by CYP2A13 was measured to determine whether any
functional changes observed were substrate-dependent.
PDH), and
b-nicotinamide-adenine dinucleotide phosphate
reduced form (NADPH) were purchased from Oriental Yeast (Tokyo,
Japan). The following reagents were purchased from the indicated
sources: polyclonal anti-human CYP2A13 antibody (ab58740,
Abcam, CB, UK); polyclonal anti-calnexin antibody (Enzo Life Sci-
ences, Farmingdale, NY, USA); horseradish peroxidase (HRP)-con-
jugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz,
CA, USA). Triton N-101 and sodium cholate hydrate were obtained
from Sigma-Aldrich. Sodium cyanide and cytochrome c from horse
heart were purchased from Nacalai Tesque (Kyoto, Japan).
3 substitutions
a
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2.2. CYP2A13 cDNA cloning and construction of expression vectors
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Plasmids carrying full-length CYP2A13 cDNA fragments (GenBank
accession no. AF209774.1) were obtained from GenScript (USA).
CYP2A13 cDNA fragments from plasmid DNA were PCR-amplified
using a forward primer (50-CACCATGCTGGCCTCAGGGCTGCTTC-30)
and a reverse primer (50-TCAGCGGGGCAGGAAGCTCATGGTGTAG-30)
and PfuUltra High-Fidelity DNA Polymerase (Agilent Technologies,
Santa Clara, CA, USA), the underlined sequence in the forward primer
were introduced for directional TOPO cloning. Following amplifica-
tion, the wild-type CYP2A13 fragments were subcloned into the
pENTR/D-TOPO vector (ThermoFisher Scientific, Waltham, MA). A
plasmid containing CYP2A13*1 cDNA was used as a template to
generate various CYP2A13 allelic variant constructs (CYP2A13*2,
CYP2A13*4eCYP2A13*6, CYP2A13*8, and CYP2A13*9), using the
primer set for site-directed mutagenesis (Supplemental Table 1) and
the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla,
CA, USA) following the manufacturer's instructions. Other CYP2A13
constructs were generated from plasmids carrying other cDNA
templates: CYP2A13*3 from CYP2A13*8 cDNA, and CYP2A13*10 from
CYP2A13*2 cDNA. All wild-type and variant cDNAs prepared were
confirmed by Sanger sequencing. Wild-type and CYP2A13 variant
cDNAs were subcloned into the mammalian expression vector
pcDNA3.4 (ThermoFisher Scientific).
Q3
2. Materials and methods
2.1. Chemicals
(ꢁ)-Nicotine, (ꢁ)-cotinine, ( )-cotinine-(methyl-d3) derivatives,
coumarin, 7-hydroxycoumarin, and 4-methyl-7-hydroxycoumarin
were purchased from Sigma-Aldrich (Tokyo, Japan). The UltraPool
Human Liver Cytosol, 150-Donor Pool was purchased from Corning
Incorporated (Corning, NY, USA). Oxidized b-nicotinamide-adenine
Table 1
Cytochrome P450 2A13 (CYP2A13) allelic variants characterized in this study.
Variants
Protein
Nucleotide changes
Amino acid changes
CYP2A13*1
CYP2A13*2
CYP2A13.1
CYP2A13.2
74G > A
Arg25Gln
3375C > T
1634_1635insACC
1706C > T
579G > A
7343T > A
7465C > T
1706C > T
5294G > T
74G > A
Arg257Cys
133_134insThr
Asp158Glu
Arg101Gln
Phe453Tyr
Arg494Cys
Asp158Glu
Val323Leu
Arg25Gln
CYP2A13*3
CYP2A13.3
CYP2A13*4
CYP2A13*5
CYP2A13*6
CYP2A13*8
CYP2A13*9
CYP2A13*10
CYP2A13.4
CYP2A13.5
CYP2A13.6
CYP2A13.8
CYP2A13.9
CYP2A13.10
2.3. Expression of CYP2A13 variants in 293FT cells
293FT cells, purchased from ThermoFisher Scientific, were
cultured in Dulbecco's modified Eagle's medium (Nacalai Tesque)
containing 10% fetal bovine serum at 37 ꢂC under 5% CO2. Cells were
3375C > T
5792T > C
Arg257Cys
Ile331Thr
transfected with a plasmid (5 mg) encoding CYP2A13 cDNA, using
Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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the TransFectin lipid reagent (Bio-Rad Laboratories, Hercules, CA,
USA) according to the manufacturer's instructions. After incubation
for 24 h at 37 ꢂC, the 293FT cells were scraped off, and the micro-
somal fractions were prepared as previously described. Protein
concentrations were determined using the BCA Protein Assay Kit
(ThermoFisher Scientific).
CPR towards cytochrome c was calculated using the extinction
coefficient of cytochrome c (21 mMꢁ1 per cm at 550 nm).
2.7. Nicotine C-oxidation assay
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CYP2A13 nicotine C-oxidation activity was determined as pre-
viously described methods, with several modifications [21,22]. The
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reaction mixture, in a total volume of 100
following: the microsomal fraction (20 g), UltraPool Human Liver
Cytosol, 150-Donor Pool as a source of aldehyde oxidase (10 g),
nicotine (0.5, 1, 2, 5, 10, 20, 50, or 100 M), and 50 mM potassium
mL, consisted of the
2.4. Determination of microsomal P450 contents
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m
The concentrations of CYP2A13 variant holoenzymes were
spectrophotometrically measured according to previously reported
methods, with several modifications [18,19]. The microsomal frac-
tion (approximately 1.5 mg of microsomal proteins) was diluted to
m
phosphate buffer (pH 7.4). Following pre-incubation at 37 ꢂC for
3 min, reactions were initiated by the addition of an NADPH-
generating system, consisting of 0.5 mM NADPþ, 5 mM G-6-P,
5 mM MgCl2, and 1 U/mL G-6-PDH, with incubation at 37 ꢂC for
200 mL in 100 mM potassium phosphate buffer (pH 7.4) containing
1.0 mM EDTA, 20% glycerol (v/v), 0.5% sodium cholate (w/v), and
0.4% Triton N-101 (w/v). One hundred microliter of protein samples
was added to sample and reference cuvettes. A baseline between
400 and 500 nm was recorded using a Cary 300 UVeVis Spectro-
photometer (Agilent Technologies). CO gas was bubbled through
the mixture in the sample cuvette at a flow rate of 1 bubble/s for
20 s. Samples were reduced in the presence of approximately
0.2 mg of solid Na2S2O4. The absorbance spectra of both cuvettes
were then recorded between 400 and 500 nm using a spectro-
photometer. The CO-difference spectra were recorded three times
for each CYP2A13 variant. Data analysis was conducted using a
Jasco Spectra Manager (JASCO Corporation, Sendai, Japan). The
cytochrome P450 concentration was calculated using the
maximum absorbance of three records normalized to the baseline
spectrum because the spectrum baseline showed a difference in
absorbance between 400 and 500 nm. Cuvettes (Sub-Micro Cells,
16.50-Q-10/Z20) were purchased from Starna Scientific, Ltd. (Lon-
don, UK).
20 min. Reactions were terminated by adding 100
containing 1 M cotinine-(methyl-d3) as an internal standard.
Determination of nicotine C-oxidation in 20 g of the microsomal
mL of acetonitrile
m
m
fraction (containing wild-type CYP2A13 and CYP2A13 variants)
showed that cotinine formation was linear for incubations of up to
20 min. Moreover, when the reaction was performed for 20 min with
0e20
presence of up to 20
After centrifuging the reaction tube at 15,400ꢀg for 10 min,
150 L of the supernatant was diluted twice using water. The
resulting solution (10 L) was injected into a liquid chromatog-
mg of microsomal protein, cotinine formation was linear in the
mg of microsomal protein (data not shown).
m
m
raphy tandem mass spectrometry system, according to previously
described methods [21,22]. Enzymatic activity was normalized to
the amount of CYP2A13 holo-protein.
2.8. Coumarin 7-hydroxylation assay
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CYP2A13-dependent coumarin 7-hydroxylation was measured
as previously reported, with several modifications [22,23]. The re-
2.5. Western blot analysis
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action mixture (100
mL) contained the microsomal fraction (30
mg),
coumarin (2, 4, 6, 8, 10, 15, or 20
m
M), and 50 mM potassium
293FT microsomal fractions (5
mg microsomal protein) were
phosphate buffer (pH 7.4). Following pre-incubation at 37 ꢂC for
3 min, reactions were initiated by the addition of an NADPH-
generating system (described above). After incubating at 37 ꢂC for
separated by 10% sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE). Western blotting was performed accord-
ing to standard procedures. CYP2A13 was detected using
a
10 min, reactions were terminated by adding 100 mL acetonitrile
containing 200 nM 4-methyl-7-hydroxycoumarin as an internal
standard. Coumarin 7-hydroxylation activity was determined in the
polyclonal anti-human CYP2A13 antibody (diluted 1:5000). Cal-
nexin was detected using a polyclonal anti-calnexin antibody
(1:5000), as a loading control. Detection was achieved using a
secondary HRP-conjugated goat anti-rabbit IgG (1:10,000). West-
ern blots were visualized using SuperSignal West Dura Extended
Duration Substrate (ThermoFisher Scientific). Chemiluminescence
was quantified using a ChemiDoc XRSþ system with Image Lab
Software (Bio-Rad Laboratories).
microsomal fractions containing 1 of each CYP2A13 variant (30 mg
of microsomal protein). Data obtained using coumarin revealed
that 7-hydroxycoumarin formation was linear for incubations of
up to 10 min. Under these conditions, the formation of 7-
hydroxycoumarin was linear in the presence of up to 30
microsomal protein (data not shown).
mg of
After centrifuging the reaction tube at 15,400ꢀg for 10 min,150
of the supernatant was diluted twice in 50 mM potassium phosphate
buffer (pH 7.4) and 50 L of the diluted solution was subjected to
mL
2.6. Measurement of NADPH-cytochrome c reduction activity
m
The activity of NADPH-cytochrome P450 reductase (CPR) was
evaluated according to previous methods described by Omura and
HPLC. Quantitative determination of 7-hydroxycoumarin was con-
ducted as previously described [22,23]. The enzymatic activity was
normalized to the amount of CYP2A13 holo-protein.
Takesue (1970) [20]. The microsomal fraction (100
somal proteins) was diluted to 180 L in 100 mM potassium
phosphate buffer (pH 7.4) containing 1.0 mM sodium cyanide.
Protein samples (90 L) were added to sample and reference cu-
mg of micro-
m
2.9. Data analysis
m
vettes. A blank was recorded at 550 nm using a Cary 300 UVeVis
spectrophotometer (Agilent Technologies). Reactions were initiated
by addition of 1.0 mM NADPH diluted in 100 mM potassium
phosphate buffer (pH 7.4) containing 1.0 mM sodium cyanide. The
control sample contained all of the reagents except for NADPH in a
reference cuvette. The absorbance was recorded for each micro-
somal fraction expressing CYP2A13 variant protein until the
absorbance versus time plot was no longer linear. The activity of
Kinetic data, including the Km, maximum velocity (Vmax), and
intrinsic clearance (CLint ¼ Vmax/Km) values, were determined using
the Enzyme Kinetics Module of SigmaPlot 12.5 (Systat Software,
Inc., Chicago, IL, USA), a curve-fitting program based on nonlinear
regression analysis. All values are expressed as the mean SD of
experiments performed in triplicate. Statistical analyses of enzy-
matic activity and kinetic parameters were performed through
variance analysis by Dunnett's T3 test or the KruskaleWallis
Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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method (IBM SPSS Statistics Ver. 22, International Business Ma-
chines, Armonk, NY, USA). Differences or correlations with P < 0.05
were considered statistically significant.
wild-type CYP2A13, the Vmax and CLint values for the 5 CYP2A13
variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and
CYP2A13.9) were significantly lower. For 3 variants (CYP2A13.3,
CYP2A13.4, and CYP2A13.10), the kinetic parameters could not be
determined because nicotine C-oxidation and coumarin 7-
hydroxylation activity were not detected.
2.10. Three-dimensional (3D) structural modeling of CYP2A13
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The 3D structural modeling of CYP2A13 was based on the
CYP2A13 X-ray structure of DeVore et al. (2012) (Protein Data Bank
code 3T3S) [24]. After removal of the substrate, nicotine or
coumarin was docked with the CYP2A13 X-ray structure according
to the CDOCKER protocol of Discovery Studio 2.5 (BIOVIA, CA, USA).
Docking iterations were conducted taking into consideration the
binding orientation and binding energies under the following
conditions that the volume of the space was defined as 9 and the
4. Discussion
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CYP2A13 is one of the most potent metabolic activators of NNK
among human CYPs [1]. Nicotine and coumarin are also represen-
tative substrates metabolized by CYP2A13 [2e4]. CYP2A13 genetic
polymorphisms can affect individual responses to these substrates,
which may result in observed differences in smoking behavior and
cancer risk. In this study, we characterized the enzymatic activity of
9 CYP2A13 variant alleles, using the variant proteins overexpressed
in 293FT cells. We determined the kinetic parameters for wild-type
heme iron was charged to Fe3þ
.
3. Results
and
5 CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6,
CYP2A13.8, and CYP2A13.9) in terms of nicotine C-oxidation and
coumarin 7-hydroxylation. The results demonstrated that after CO-
treatment, 3 CYP2A13 variant proteins (CYP2A13.3, CYP2A13.4, and
CYP2A13.10) showed no increase in absorbance near 450 nm,
reflecting failed heme incorporation.
The CYP2A13 protein levels in 293FT cells were assessed by
Western blotting with a polyclonal CYP2A13 antibody. As shown in
Fig. 2, CYP2A13 protein was detected for all variants, but not in the
mock-treated cells. Calnexin, used as an endoplasmic reticulum-
resident protein, showed constant expression levels in the micro-
somes of transfected cells.
We measured the reduced CO-difference spectra with the
microsomal fractions for each CYP2A13 variant (1.66 0.24 mg).
We found an increase in the maximum absorption wavelength after
CO treatment of wild-type CYP2A13 and 5 CYP2A13 variants
(CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9).
The results indicated the presence of functional CYP enzymes and
the expression levels of CYP2A13 variants (Fig. 3). Three CYP2A13
variants (CYP2A13.3, CYP2A13.4, and CYP2A13.10) showed no in-
crease in their absorption maxima at 450 nm.
The CLint values obtained for nicotine C-oxidation versus those
for coumarin 7-hydroxylation for CYP2A13.2, CYP2A13.5, and
CYP2A13.8 indicated a high correlation. The Arg257Cys substitution
located on the carboxyl-terminal end of the G helix caused a
reduction in enzymatic activity. The repeating pattern of hydro-
phobic residues in the G helix is conserved in CYP2 families [25].
Lehnerer et al. (2000) reported that the Arg253Ala substitution,
which is located near the end of the G helix, led to decreased
enzymatic activity due to interference in the interaction between
rabbit CYP2B4 and P450 reductase [26]. Therefore, a reduction in
the binding between CYP2A13 and P450 reductase, caused by the
Arg257Cys substitution, could reduce CYP2A13.2 activity. In
contrast, the Arg25Glu substitution, present in CYP2A13.2 and
located in the membrane anchor region, may not influence the
enzymatic activity of CYP2A13 because the enzymatic activities for
We evaluated whether the CPR activity was different for each
CYP2A13 variant. The CPR activity for the wild type was
25.2
2.71 nmol minꢁ1 mg proteinꢁ1. For all CYP2A13 variants,
there was no significant difference in CPR activity compared to that
for the wild type enzyme (Supplemental Fig. 1).
NNK a-hydroxylation and coumarin 7-hydroxylation were reduced
The Km, Vmax, and CLint values for nicotine C-oxidation by
in case of the Arg257Cys substitution [14]. CYP2A13.5, which con-
tains a Phe453Tyr substitution, showed 20% less activity than
CYP2A13.1 did. The Phe453 residue, located in the hydrophobic
region of the L helix alongside Leu449, interacts with the distal I
helix [25]. The Phe453Tyr substitution causes a conformational
change in the K and I helices, which in turn causes a reduction in
enzymatic activity. CYP2A13.8 has a substitution in residue Asp158
located in the D helix, which interacts with residues in the G and H
loop. This interaction between the C/D and G/H loops could provide
CYP2A13.1 were 4.60
m
M, 31.5 pmol minꢁ1 pmolꢁ1 CYP2A13, and
6.86
m
L minꢁ1 pmolꢁ1 CYP2A13, respectively (Table 2). The Km
values for CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and
CYP2A13.9 were not significantly different from that of CYP2A13.1.
For the 5 CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6,
CYP2A13.8, and CYP2A13.9), the Vmax and CLint values were signif-
icantly lower than that of CYP2A13.1. The Km, Vmax, and CLint values
for coumarin 7-hydroxylation by CYP2A13.1 were 2.34
mM,
442 fmol minꢁ1 pmolꢁ1 CYP2A13, and 183 nL minꢁ1 pmolꢁ1
CYP2A13, respectively. Relative to that of wild-type CYP2A13, the
Km values for CYP2A13.2, CYP2A13.5, and CYP2A13.8 were signifi-
cantly higher. In contrast, the Km value for CYP2A13.9 was signifi-
cantly lower than that of wild-type CYP2A13. Relative to those of
a
physical coordination mechanism underlying the observed
ligand-induced conformational changes in the electron-delivery
system of the protein, located on the proximal side of the
mammalian CYP2B6 structure [27]. As reported previously, the
CYP2A6.2 variant with a Leu160His substitution located in the D
helix, led to a loss of coumarin 7-hydroxylation activity [28]. In the
case of CYP2A13.8, Asp158Glu could also cause interactions be-
tween the C/D and G/H loops. Thus, the reduced enzymatic activity
of these variants may influence intra-individual differences found
in cancer risk and genotoxicity.
Overall, all 8 CYP2A13 variants showed similar expression levels
relative to the wild-type enzyme; however, CYP2A13.3, CYP2A13.4,
and CYP2A13.10 were functionally inactive, despite showing
expression levels comparable to that of the wild-type enzyme. In
the case of CYP2A13.3, the addition of a Thr residue between 133Ala
and 134Thr results from the insertion of 3 nucleotides in the
carboxyl end of the C helix. Schlicht et al. (2007) reported that it
Mock .1
.2
.3
.4
.5
.6
.8
.9 .10
CYP2A13
Calnexin
Fig. 2. Western blots showing immunoreactive CYP2A13 proteins (upper panel) and
calnexin (lower panel). Western blotting was performed according to standard pro-
cedures using 10% SDS-PAGE. Five micrograms of microsomal fractions containing
CYP2A13 variant proteins was loaded onto each lane. CYP2A13 variants and calnexin
were detected using polyclonal antibodies against the respective proteins. The
numbers correspond to each CYP2A13 variant.
Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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Fig. 3. CO-difference spectra of CYP2A13 proteins expressed in 293FT cells.
Table 2
Kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation.
Variants Nicotine C-oxidation
Km M) Vmax (pmol/min/pmol CLint (Vmax/Km) (
Coumarin 7-hydroxylation
L/min/pmol CYP2A13) Km M) Vmax (fmol/min/pmol CLint (Vmax/Km) (nL/min/pmol CYP2A13)
(
m
m
(m
CYP2A13) (% of wild-type)
CYP2A13)
(% of wild-type)
CYP2A13.1 4.60 0.32 31.5 1.79
CYP2A13.2 7.34 0.96 14.9 1.79***
CYP2A13.3 N.D.
CYP2A13.4 N.D.
CYP2A13.5 6.43 0.49 5.98 0.42**
CYP2A13.6 10.9 1.84 15.4 1.62***
CYP2A13.8 3.48 1.33 5.52 0.14**
CYP2A13.9 20.8 13.1 16.9 5.11***
6.86 0.10
2.07 0.50* (30%)
N.D.
2.34 0.06
3.20 0.27*
N.D.
442 12.7
191 7.00***
N.D.
183 4.91
59.9 4.02*** (33%)
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
0.93 0.05*** (14%)
1.43 0.14*** (21%)
1.78 0.76* (26%)
0.91 0.25** (13%)
N.D.
3.88 0.69*** 127 10.1***
1.97 0.27 184 7.69***
3.48 0.29** 120 4.83***
33.2 3.28*** (18%)
94.3 8.37*** (52%)
34.7 3.66*** (19%)
137 10.2*** (75%)
N.D.
1.35 0.10*
N.D.
184 1.92***
N.D.
CYP2A13.10 N.D.
N.D.
These data represent the mean SD of three independently performed catalytic assays.
*P < 0.05, **P < 0.01, and ***P < 0.005 compared to CYP2A13.1. N.D. represents not determined.
The kinetic parameters of nicotine C-oxidation of CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because the enzymatic activity of CYP was not detected at
the highest substrate concentration assayed (100 M nicotine).
The kinetic parameters of CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because the enzymatic activity of CYP was not detected at the highest substrate
concentration assayed (20 M coumarin).
m
m
was not possible to obtain the CYP2A13.3 protein in active form,
due to degradation during purification [14]. In agreement with
their findings, the protein instability of CYP2A13.3 prevented us
from observing an increase in the maximum absorption wave-
length at 450 nm CYP2A13.4 has a substitution in a heme pocket-
forming amino acid, in the substrate-recognition site-1, which
causes a loss of catalytic activity and inappropriate heme binding in
CYP2A13.4 prepared from Sf9 cells [16]. Our results also demon-
strated a dramatic decrease in the enzymatic activity of CYP2A13.4
in a heterologous expression system using mammalian cells. This
Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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decrease resulted from the decrease in CYP2A13 holo-protein levels
caused by disruption of the interaction between Arg101 and heme
(Fig. 4). CYP2A13*10 was identified in the Japanese population [11],
although the catalytic efficiency of its protein product is still un-
known. CYP2A13.10 has 3 nucleotide mutations leading to the
Arg25Glu, Arg257Cys, and Ile331Thr substitutions. Ile331, located
in the J helix of CYP2A13, may affect D-E loop formation. The
negatively charged D-E loop region interacts with the positively
charged heme-proximal cavity, which influences the electrostatic
status of the neighboring active site heme [29]. The Ile331Thr
substitution, found in CYP2A13.10, causes hydrogen bond forma-
tion between Thr331 and Cys494, and then the conformation of the
D-E loop region is dramatically changed (Fig. 5). This conforma-
tional change may influence the stability of the CYP2A13 holo-
protein. The decreased CYP2A13.10 stability caused by the
Ile331Thr substitution (in addition to the Arg25Glu and Arg257Cys
substitutions), leads to a complete loss of enzymatic activity.
Therefore, CYP2A13.3, CYP2A13.4, and CYP2A13.10 also appear to
contribute to a decreased risk of bladder cancer, since this risk was
significantly decreased in individuals who harbor the CYP2A13*2
variant allele [12], which has been associated with reduced meta-
bolic activity of tobacco-specific nitrosamines, including NNK.
The nicotine C-oxidation CLint values for CYP2A13.6 and
CYP2A13.9 decreased by 21% and 13% respectively, relative to that
for wild-type CYP2A13, whereas coumarin 7-hydroxylation CLint
values decreased by 52% and 75% respectively, relative to that for
wild-type CYP2A13. Arg494, located at the CYP2A13 carboxy-
terminal domain, forms hydrogen bonds with His328, Asp332,
Met457, Asn459, and Phe460. The Arg494Cys substitution, found in
CYP2A13.6, causes hydrogen bond formation between Cys494 and
Lys337, while disrupting hydrogen bond formation between
Arg494 and His328, and Asp332 and Phe460 (Fig. 6A). Several
channels have been identified in bacterial, archaeal, and mamma-
lian P450 enzymes [30]. A solvent channel, which could serve as a
product exit channel, is located between the E and I helices and the
b5 sheet. No remarkable conformational changes (relative to the
wild-type enzyme) were observed during nicotine coordination by
CYP2A13.6 (Fig. 6B). In contrast, the Arg494Cys substitution present
in CYP2A13.6 resulted in widening of its solvent channel (Fig. 6C).
CYP2A13.9 follows the same trend, and substrate-dependent dif-
ferences in CYP2A13.6 and CYP2A13.9 activities could stem from
different 3D structures imposed by different interacting substrates.
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The coumarin 7-hydroxylation and NNK
a-hydroxylation CLint
values for CYP2A13.9 expressed in E. coli decreased by 57% and 39%,
respectively, relative to that for wild-type CYP2A13, whereas (S)-
NNN 50-hydroxylation CLint values increased by 123% relative to that
for wild-type CYP2A13. However, the CLint values for several
CYP2A13 variants were lower than that of wild-type CYP2A13
when evaluated using coumarin, NNK, and (S)-NNN [14]. Further
studies using additional substrates such as tobacco-specific nitro-
samines are needed to confirm the substrate-dependent differ-
ences in the activities of CYP2A13 variants.
In conclusion, wild-type and
8 CYP2A13 variants were
expressed in 293FT cells and their enzymatic activities were char-
acterized in vitro. These data reveal several functional alterations of
CYP2A13 allelic variants caused by genetic polymorphisms. In
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Fig. 4. Diagram of a portion of the crystal structure of CYP2A13.1 (left panel) and CYP2A13.4 (right panel). The residues Arg101 in CYP2A13.1 and Gln101 in CYP2A13.4 are shown in
yellow. Heme is shown in orange. The interactions between Arg101 and heme are shown in light blue.
Fig. 5. Diagram showing a fragment of the crystal structures of CYP2A13.2 (left panel) and CYP2A13.10 (right panel). a-Helices and b-strands are shown in red and light blue,
respectively. Ionic bonding was found to differ between CYP2A13.2 (Arg25Gln, Arg257Cys) and CYP2A13.10 (Arg25Gln, Arg257Cys, Ile331Thr).
Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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Fig. 6. Diagram showing a fragment of the crystal structures of CYP2A13.1 (left panel) and CYP2A13.6 (right panel). The residues Arg494 in CYP2A13.1 and Cys494 in CYP2A13.6 are
shown in yellow (A). Hydrogen binding shown in light green was found to differ between wild-type CYP2A13 and CYP2A13.6. Diagram showing a fragment of the crystal structures
of CYP2A13.1 and CYP2A13.6 complexed with nicotine (B) and coumarin (C).
the cluster in yellow.
a-Helices and b-strand are shown in red and light blue, respectively. The channels are represented by
particular, alteration of nicotine C-oxidation activity caused by
CYP2A13 allelic variants was demonstrated for the first time.
CYP2A13 is an important P450 isoform involved in bioactivation of
several procarcinogens, including NNK, as well as nicotine and
coumarin metabolism. These findings provide mechanistic insights
underlying inter-individual differences associated with genotox-
icity and cancer susceptibility.
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oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004

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Please cite this article in press as: Kumondai M, et al., Functional characterization of 9 CYP2A13 allelic variants by assessment of nicotine C-
oxidation and coumarin 7-hydroxylation, Drug Metabolism and Pharmacokinetics (2017), https://doi.org/10.1016/j.dmpk.2017.11.004