Human cytochrome P450 4F11: Heterologous expression in bacteria, purification, and characterization of catalytic function

Article abstract of DOI:10.1016/j.abb.2009.11.017

Human cytochrome P450 (P450) 4F11 is still considered an "orphan" because its function is not well characterized. A bacterial expression system was developed for human P450 4F11, producing ～230 nmol P450 from a 3-l culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic and isotopic labeling approach (Tang et al., 2009 [19]). Four fatty acids-palmitic, oleic, arachidonic, and docosahexaenoic-were identified in human liver and verified as substrates of P450 4F11. The products were characterized as ω-hydroxylated fatty acids by gas chromatography-mass spectrometry analysis of their trimethylsilyl derivatives. Kinetic analysis of the oxidation products confirmed that the fatty acids are substrates oxidized by P450 4F11. P450 4F11 also exhibited low activity for some drug N-demethylation reactions but none for activation of several pro-carcinogens.

Full text of DOI:10.1016/j.abb.2009.11.017

Archives of Biochemistry and Biophysics 494 (2010) 86–93
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Human cytochrome P450 4F11: Heterologous expression in bacteria,
purification, and characterization of catalytic function
1
*
Zhongmei Tang, Sandra Giovanna Salamanca-Pinzón, Zhong-Liu Wu , Yi Xiao, F. Peter Guengerich
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Human cytochrome P450 (P450) 4F11 is still considered an ‘‘orphan” because its function is not well
characterized. A bacterial expression system was developed for human P450 4F11, producing ꢀ230 nmol
P450 from a 3-l culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate
searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic
and isotopic labeling approach (Tang et al., 2009 [19]). Four fatty acids—palmitic, oleic, arachidonic,
and docosahexaenoic—were identified in human liver and verified as substrates of P450 4F11. The prod-
Received 3 November 2009
and in revised form 13 November 2009
Available online 20 November 2009
Keywords:
Cytochrome P450 4F11
Heterologous expression
Enzyme purification
Liquid chromatography–mass spectrometry
Metabolomics
ucts were characterized as
x-hydroxylated fatty acids by gas chromatography–mass spectrometry anal-
ysis of their trimethylsilyl derivatives. Kinetic analysis of the oxidation products confirmed that the fatty
acids are substrates oxidized by P450 4F11. P450 4F11 also exhibited low activity for some drug N-
demethylation reactions but none for activation of several pro-carcinogens.
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
clinically active drugs, at least in animal models [7–9]. The cata-
lytic activity of P450 4F11 expressed in Saccharomyces cerevisiae
has been investigated, with low rates of oxidation of some endog-
enous eicosanoids (e.g., leukotriene B4) and somewhat higher rates
P4502 enzymes are important heme-containing monooxygenases
that play roles in the metabolism of a large number of endogenous
compounds (including sterols, fatty acids, eicosanoids, and vitamins)
as well as xenobiotics [2]. With the successful completion of the
human genome project, 57 functional genes and 58 pseudogenes
of human P450 have been identified [3], of which ꢀ13 members
are termed ‘‘orphans” because of their unknown function [3,4].
Human P450 4F11 is one of the orphan P450s, with limited
information regarding heterologous expression and functional
characterization [4,5]. P450 4F11 has been reported to be located
in a cluster with the other five members of the P450 4F subfamily
on chromosome 19p13.1-2, and its mRNA is found mainly in hu-
man liver [6]. The P450 4F gene subfamily is known to be involved
in the metabolism of endogenous eicosanoids as well as some
for some drugs (e.g., erythromycin) [10]. The
x-oxidation of two
b-hydroxy fatty acids has been demonstrated with human P450
4F11 expressed in Spodoptera frugiperda insect cells [11]. In order
to elucidate the function of human P450 4F11, an untargeted met-
abolomic strategy as well as a more robust expression system is
required and was the focus of our study.
MS, performed either directly or coupled with chromatographic
methods, is one of the most widely used analytical tools for
metabolomic analysis [12–14]. In recent years, LC–MS—in combi-
nation with bioinformatic tools for data analysis—has proven to
be a powerful approach for assigning enzyme function, with the
advantages of high sensitivity and selectivity, wide dynamic range,
and direct analysis of complex biological samples with minimal ion
suppression [15–17]. Recently we developed a general strategy for
identification of endogenous substrates of human P450s in tissue
extracts using LC–MS assays and the program DoGEX [18,19]. In
vitro incubation of a P450 enzyme system and tissue extract is
done under a mixture of 18O2/16O2 gas (1:1, v/v). LC–MS assay of
an organic extract of the reaction mixture is performed, and the
isotopic labeling products appearing as M/M + 2 doublets are
directly identified using DoGEX [18,19]. The strategy has been
validated using both targeted and untargeted searches to identify
candidate substrates and products in human liver for some hepatic
human P450s (1A2, 2C8, 2C9) previously characterized for their
oxidations of xenobiotics [19]. Thus, this strategy has been demon-
* Corresponding author. Address: Department of Biochemistry, Vanderbilt
University School of Medicine, 638 Robinson Research Building, 2200 Pierce
Avenue, Nashville, TN 37232-0146, USA. Fax: +1 615 322 3141.
E-mail address: f.guengerich@vanderbilt.edu (F.P. Guengerich).
Present address: Chengdu Institute of Biology, Chinese Academy of Sciences,
1
Chengdu 610041, People’s Republic of China.
2
Abbreviations used: C16:0, palmitic acid; 3-OH C16:0, 3-hydroxypalmitic acid; 16-
OH C16:0, 16-hydroxypalmitic acid; 3,16-diOH C16:0, 3,16-dihydroxypalmitic acid;
C18:1, oleic acid; C20:4, arachidonic acid; C22:6 docosahexaenoic acid; DMSO,
dimethylsulfoxide; GC–MS, gas chromatography–mass spectrometry; LC–MS, liquid
chromatography–mass spectrometry; MS, mass spectrometry; NTA, nitrilotriacetate;
P450, cytochrome P450 (also termed heme-thiolate P450 [1]; PDAM, 1-pyrenyldia-
zomethane; SPE, solid phase extraction; TB, terrific broth (medium); TMS,
trimethylsilyl.
0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2009.11.017

Z. Tang et al. / Archives of Biochemistry and Biophysics 494 (2010) 86–93
87
Table 1
strated to be potentially useful in identifying reactions in tissue ex-
tracts with orphan human P450s.
Selection of N-terminal sequences of human P450 4F11 for heterologous expression.
Amino acid changes are underlined.
The aim of the present work was to establish a bacterial system
for human P450 4F11 expression and purification and to character-
ize its catalytic function using the strategy we developed [19]. The
purified enzyme was utilized for untargeted substrate searches in
human liver samples using the LC–MS metabolomic and isotopic
labeling approach. Four fatty acids were identified in liver and sub-
sequently verified as substrates of P450 4F11. The products were
Constructs(and basis)
N-Terminal amino
acid sequences
4F11 native
MPQLSLSWLGLGPVAASPWLLLLLVGGSWLLARVLA
WTYTFYDNCRRLQCFPQPP
#1 2C3 rabbit
#2 2C3 rabbit
#3 2E1 human
#4 17A1 bovine
#5 2E1 human
#8 full length
MAKKTSSKGKLPQPP
MAKKTSSKGKLQCFPQPP
MARQVHSSWNLQCFPQPP
characterized as
x-hydroxylated fatty acids, and steady-state
MALLLAVFLAWTYTFYDNCRRLQCFPQPP
MARQVHSSWNLYDNCRRLQCFPQPP
kinetic measurements of the P450 reactions were done.
MAQLSLSWLGLGPVAASPWLLLLLVGGSWLLAR
VLAWTYTFYDNCRRLQCFPQPP
Materials and methods
#11 17A1 bovine
MALLLAVFLGPVAASPWLLLLLVGGSWLLARV
LAWTYTFYDNCRRLQCFPQPP
Chemicals
Human liver samples (from organ donors) were obtained from
Tennessee Donor Services and stored at ꢁ70 °C. Rat NADPH-P450
reductase [20] and cytochrome b5 [21] were expressed in Esche-
at 190 rpm in a New Brunswick Innova 4300 shaker (New Bruns-
wick Scientific, Edison, NJ).
richia coli and purified as previously reported. L-a-1,2-Dilauroyl-
sn-glycero-3-phosphocholine, C16:0, 3-OH C16:0, 16-OH C16:0,
C18:1, C20:4, C22:6, PDAM, benzphetamine, erythromycin, ethyl-
morphine, and dansylhydrazine were purchased from Sigma–Al-
drich (St. Louis, MO). [1-14C] C16:0 (specific activity 56
mCi mmolꢁ1), [1-14C] C18:1 (specific activity 55 mCi mmolꢁ1),
[1-14C] C20:4 (specific activity 55 mCi mmolꢁ1), and [1-14C]
C22:6 (specific activity 55 mCi mmolꢁ1) were purchased from
American Radiolabeled Chemicals (St. Louis, MO). N,O-bis-(Tri-
methylsilyl)-trifluoroacetamide:trimethylchlorosilane:trimethylsi-
lylimidazole:pyridine (3:2:3:10, v/v/v/v) was purchased from Regis
Technologies (Morton Grove, IL). All other reagents and solvents
were obtained from general commercial suppliers.
Purification of recombinant P450 4F11
All purification steps were performed at 4 °C. E. coli membranes
(with the bicistronic plasmid of P450 4F11 construct #11) were
prepared as described previously [24,27]. The P450 4F11 mem-
brane fractions from 3 l of culture were diluted in buffer A
(20 mM potassium phosphate (pH 7.4) buffer containing 20% glyc-
erol (v/v), 0.5% sodium cholate (w/v), 0.5% Tergitol NP-10 (w/v),
and 10 mM 2-mecaptoethanol). The mixture was stirred for 2 h
and centrifuged at 105 g for 30 min. The resulting supernatant,
with NaCl added to a final concentration of 0.5 M, was loaded onto
a Ni2+–NTA agarose column (Qiagen, Valencia, CA; 5 ml packed-
bed volume, equilibrated with 10 column volumes of buffer A plus
0.5 M NaCl and 5 mM imidazole (buffer B)). The column was
washed with 10 column volumes of buffer B, two column volumes
of buffer B containing 25 mM imidazole, and one column volume of
buffer B containing 50 mM imidazole, and P450 4F11 was then
eluted (at a flow rate of 0.5 ml minꢁ1) with buffer B containing
200 mM imidazole.
In order to remove the detergents, the eluted reddish fractions
were applied to a Biogel HTP hydroxylapatite column (10 ml
packed-bed volume, Bio-Rad, Richmond, CA) using methods
described previously [25,28]. The column was equilibrated with
five column volumes of 20 mM potassium phosphate buffer (pH
7.7) containing 20% glycerol (buffer C) and, after loading the
P450, subsequently washed with buffer C until the A280 of the
effluent was <0.02. P450 4F11 was then eluted by increasing the
potassium phosphate concentration to 0.7 M, with the addition of
sodium cholate to a final concentration of 0.2% (w/v). The eluted
P450 4F11 fractions were pooled and dialyzed overnight against
100 volumes of 100 mM potassium phosphate (pH 7.4) containing
20% glycerol (v/v) and 0.1 mM EDTA to remove cholate.
Construction of P450 4F11 expression plasmids
In order to find an appropriate N-terminal sequence for heterol-
ogous expression of human P450 4F11 in E. coli, several N-terminal
mutations were introduced into the native construct (pCRÒ2.1
4F11, gift from H.W. Strobel, Univ. Texas, Houston) by PCR-based
mutagenesis (Supplementary Table S1). A C-terminal (His)6 tag
was introduced into the P450 4F11 cDNA at the same time, using
a 30-primer containing sequence encoding histidine residues and
an XbaI site. PfuUltra™ High-Fidelity DNA polymerase (Stratagene,
La Jolla, CA) was used for the PCR amplification, at an annealing
temperature of 55 °C. The products were purified, double-digested,
and ligated into the monocistronic pCW vector [22] (not coding for
NADPH-P450 reductase). The modifications were confirmed by
sequencing the open reading frame regions of the new constructs.
A bicistronic plasmid of 4F11 construct #11 (Table 1) was also con-
structed by ligating the corresponding cDNA with a bicistronic
pCW vector (containing the cDNA for NADPH-P450 reductase
[23]) using the NdeI and XbaI restriction sites.
Expression of P450 4F11 in E. coli BL21
Other methods
Expression trials of all P450 4F11 constructs were performed at
28 °C in E. coli BL21 competent cells using methods described else-
where [24,25]. Large-scale expressions for the bicistronic plasmid
of P450 4F11 construct #11 were performed in 2.8-l Fernbach
UV–visible spectra were recorded using an Aminco DW-2a/OLIS
spectrophotometer (On-Line Instrument Systems, Bogart, GA).
P450 concentrations were estimated spectrally as described previ-
ously [29].
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was
done using Ready Gel Tris–HCl gels (10%, w/v; Bio-Rad, Hercules,
CA) and Laemmli buffer (Bio-Rad) [30]), and stained with Bio-Safe
Coomassie stain (Bio-Rad) following the instructions provided by
the manufacturer.
flasks, with 500 ml TB media supplemented with 100
l
g mlꢁ1
ampicillin, 340
l
g mlꢁ1 thiamine, and 0.025% (v/v) of a mixture
of trace elements [26] and incubated for 4 h at 37 °C with gyroro-
tary shaking at 220 rpm. After the addition of 1.0 mM isopropyl-b-
D
-1-thiogalactoside and 1.0 mM 5-aminolevulinic acid, cultures
were further incubated at 27 °C for 40 h with gyrorotary shaking

88
Z. Tang et al. / Archives of Biochemistry and Biophysics 494 (2010) 86–93
Separation of human liver extract using SPE columns
HPLC-UV method [11] with some modifications. The PDAM deriv-
ative of the product was separated using a 4.6 mm ꢂ 250 mm YMC
A CHCl3/CH3OH (2:1, v/v) extract of human liver homogenate
was prepared, based on a procedure described in our previous
work [19,31]. Fractions of total human liver extract were separated
using an aminopropyl SPE column (Thermo Scientific, Bellefonte,
PA) based on a procedure previously described, with some modifi-
cations [32]. The aminopropyl column (0.5 g) was washed twice
(gravity pressure) with 2 ml of hexane. The entire dry human liver
extract (pooled from 0.6 g of each human liver sample from four
different individuals, dissolved in 0.5 ml of CHCl3) was applied to
the column. The column was then eluted (in order) with 4 ml of
CHCl3/(CH3)2CHOH (2:1, v/v), 4 ml of 2% C2H5CO2H in (C2H5)2O,
and 4 ml of CH3OH. The eluates recovered with each solvent mix-
ture were collected and labeled as liver extract fractions 1 (neutral
lipids), 2 (fatty acids), and 3 (phospholipids), respectively. All
fractions were dried under a stream of N2, stored at ꢁ70 °C, and
dissolved in 1.0 ml of DMSO as stock solutions prior to incubations
with P450 4F11.
ODS-AQ octadecylsilane (C18) column (5 lm; Waters, Milford, MA)
with a linear gradient increasing from 95% to 100% CH3CN in H2O
(v/v) over 12 min and held at 100% CH3CN for 5 min at a flow rate
of 1.0 ml minꢁ1. The UV absorbance (275 nm) was monitored using
an Agilent 1200 series diode-array detector (Agilent Technologies,
Palo Alto, CA). 16-OH C16:0 was used for a standard curve, as pre-
viously described [11].
For incubations of P450 4F11 and drugs (benzphetamine, eryth-
romycin, and ethylmorphine), the mixture of purified human P450
4F11 and NADPH-P450 reductase was first dialyzed for 3 h against
100 volumes of 100 mM phosphate buffer (pH 7.4) containing
0.1 mM EDTA to remove glycerol, which contains trace amounts
of interfering formaldehyde. The incubation was done in 200
of 100 mM phosphate buffer (pH 7.4) containing purified human
P450 4F11 (1.0 M), NADPH-P450 reductase (2.0 M), -1,2-dil-
auroyl-sn-glycero-3-phosphocholine (75 M), and the drug
ll
l
l
L-a
l
substrate (1.0 mM). Reactions were initiated with the NADPH-gen-
erating system described above and terminated by the addition of
In vitro P450 incubations and steady-state kinetic analysis of P450
4F11 reactions
200
l
l of CH3CN after incubation at 37 °C for 20 min. Each mixture
was then reacted with 600
l
l of a 0.5 mg mlꢁ1 solution of dans-
ylhydrazine (freshly prepared in CH3CN containing 0.3% (v/v)
CH3CO2H), incubated at room temperature for 15 min, and dried
under an N2 stream. For measuring drug metabolism activity (N-
demethylation) of P450 4F11, the residue of the reaction mixture
In vitro P450 incubations were done in 1.0 ml of 100 mM phos-
phate buffer (pH 7.4) containing purified human P450 4F11
(1.0
lM), NADPH-P450 reductase (2.0
lM),
L-a-1,2-dilauroyl-sn-
glycero-3-phosphocholine (75
l
M), and an aliquot of the DMSO
(after dansylhydrazine derivatization) was redissolved in 100 ll
solution of human liver extract fraction (2%, v/v). For 16O2/18O2 iso-
topic labeling experiments, reactions were performed using the
method described previously [18,19]. The enzyme reaction was ini-
tiated by the addition of an NADPH-generating system including
of CH3CN and (an aliquot was) separated using the same HPLC col-
umn as described for 3-OH C16:0, with a linear gradient increasing
from 50% to 100% CH3CN in H2O (v/v) over 12 min and held at 100%
CH3CN for 5 min, at a flow rate of 1.0 ml minꢁ1. Fluorimetric mea-
surement of the formaldehyde adduct product was performed
using a FL-750 Spectrofluorescence detector (McPherson Instru-
ments, Acton, MA), with kexcitation 340 nm and kemission > 418 nm
(filter).
100
and 2
l
l of 100 mM glucose 6-phosphate, 50
l
l
l of 10 mM NADP+,
l of a 1 mg mlꢁ1 solution of yeast glucose 6-phosphate
dehydrogenase [33]. All reaction mixtures were incubated at
37 °C for 20 min and quenched by adding 2 ml of a mixture of
CHCl3/CH3OH (2:1, v/v). After centrifugation at 2 ꢂ 103 g for
40 min, the (lower) organic phase was carefully separated, dried
LC–MS/MS metabolomics and data analysis
under an N2 stream, and redissolved in 100 ll of CH3CN/H2O
(2:1, v/v) mixture for LC–MS analysis (vide infra).
The procedures for LC–MS/MS assays and data analysis using
the program DoGEX were as described in our previous work [19].
Briefly, the approach involves incubating a reconstituted P450 sys-
tem with an extract of the tissue where it is normally expressed
and a 1:1 ratio of 18O2/16O2. The 18O2/16O2 oxidation products
appear as M and M + 2 doublets and are identified using the pro-
gram DoGEX, which searches for the doublets. In principle all such
doublets (above the noise level) are products derived from oxida-
tion of endogenous substrates. Further experiments are used to
validate the positive hits.
14C-Radiolabeled substrates were used for analysis of rates of
oxidation of fatty acids, as previously described [19]. For steady-
state kinetic studies, the concentration of the substrate containing
radioactive fatty acid varied, ranging from 0 to 200
organic extract of the P450 reaction mixture was dissolved in
100 l of CH3OH for HPLC analysis. HPLC separation was per-
formed using a 4.6 mm ꢂ 150 mm Prodigy octadecylsilane (C18)
column (5 m; Phenomenex, Torrance, CA) with an isocratic mix-
ture of 90% CH3OH/10% H2O (v/v) at a flow rate of 1.0 ml minꢁ1
lM. The dried
l
l
.
Radioactivity was determined in line with a b-RAM flow counter
LC separation was done using a Waters Acquity UPLC system
(Waters, Milford, MA) with an Acquity UPLC BEH octadecylsilane
(IN/US Systems, Tampa, FL), with the scintillation cocktail pumped
at a rate of 3.0 ml minꢁ1
.
(C18) column (1.7
l
m; 1.0 mm ꢂ 100 mm) at 50 °C. The sample
In the incubations of P450 4F11 with 3-OH C16:0, the reaction
mixtures were prepared in 1.0 ml of 100 mM phosphate buffer
(10 l) was subjected to the UPLC column and separated with a
l
linear gradient from 95% mobile phase A, 10 mM NH4CH3CO2 in a
5:95 (v/v) CH3CN/H2O mixture, to 100% mobile phase B, 10 mM
NH4CH3CO2 in a 95:5 (v/v) CH3CN/H2O mixture, over 20 min and
(pH 7.4) containing purified human P450 4F11 (0.1
P450 reductase (0.2 M), -1,2-dilauroyl-sn-glycero-3-phospho-
choline (35 M), cytochrome b5 (0.2 M), and 3-OH C16:0
(100 M). For kinetic analysis, the concentration range of the sub-
strate was from 0 to 150 M. Reactions were initiated with the
lM), NADPH-
l
L-a
l
l
held at 100% mobile phase B for 5 min at a flow rate of
l
0.1 ml minꢁ1. MS/MS analysis was performed on a ThermoFinnigan
LTQ ion trap mass spectrometer (ThermoFisher, Watham, MA)
equipped with an electrospray ionization source, using the same
instrument parameters as previously defined [19]. Data were ac-
quired using a Finnigan Xcalibur software package. Analysis of
the resulting LC–MS data was done with the Matlab-based com-
puter program DoGEX, generating a matrix of integration areas
versus retention time and molecular mass of all the detected chro-
matographic peaks [18]. The 18O2/16O2 oxidation products appear-
ing as M and M + 2 doublets were identified using the program
l
NADPH-generating system described above, incubated at 37 °C
for 10 min, and terminated by addition of 2 ml of ethyl acetate.
After centrifugation (2 ꢂ 103 g for 10 min), the (upper) organic
phase was carefully collected and dried under an N2 stream. The
residues were dissolved in 80 ll of CH3CN, mixed with 20 ll of a
1 mg mlꢁ1 PDAM solution (freshly prepared in ethyl acetate), and
kept at room temperature for 90 min. The PDAM derivative of
3-OH C16:0 and its product were directly detected using an

Z. Tang et al. / Archives of Biochemistry and Biophysics 494 (2010) 86–93
89
Table 2
DoGEX, with an isotopic ratio range of 0.95–1.9 via spectral filter-
Purification of human P450 4F11 from E. coli membrane fractions.
ing [18].
Purification step
P450 (nmol)
Yield (%)
Spectral binding titrations
Membranes
227
214
106
73
100
94
47
Solubilized fractiona
Ni2+–NTA column fractionb
Hydroxylapatite column fractionc
Binding assays of fatty acids and human P450 4F11 were done
[19,34] using an OLIS/DW-2a spectrophotometer. Varying concen-
trations of fatty acids (dissolved in C2H5OH) were added to a solu-
32
a
Solubilization using a mixture of sodium cholate plus Tergitol NP-10, followed
by centrifugation to remove insoluble material.
P450 eluted with 200 mM imidazole.
Eluted with 0.7 M potassium phosphate buffer (pH 7.7) containing 0.2% sodium
cholate (w/v).
tion of purified P450 (2 lM) in 1.0 ml of 100 mM phosphate buffer
b
(pH 7.4). The reference cuvette, containing the same concentration
of enzyme in buffer, was titrated with an equal volume of the vehi-
cle solvent (C2H5OH). The final concentration of C2H5OH was <2%
(v/v) in all cases. Visible spectra (350–500 nm) were recorded after
each addition of fatty acid.
c
Following purification using a Ni2+–NTA column, the detergent
Tergitol NP-10 and some other residual proteins were removed
using chromatography on hydroxylapatite [25,28]. Purified P450
4F11, showing a single band at ꢀ50 kDa (Fig. 1), was obtained with
an overall recovery of 32% (Table 2).
Purified P450 4F11 exhibited typical spectra for P450 enzymes
(Fig. 2A). Second-derivative analysis of the ferric enzyme indicated
that ꢀ98% of the ferric iron was in the low-spin state (judged by
the relative intensities of the bands at 390 and 418 nm in the spec-
trum [38,39]) (Fig. 2B). The Fe2+ꢃCO vs. Fe2+ difference spectrum
showed a kmax at 451 nm, with a small amount of (inactive) cyto-
chrome P420 (Fig. 2C).
Characterization of oxidation products
Characterization of the oxidation products was performed by
GC–MS as previously described [19] after preparing the corre-
sponding TMS ethers. Each fatty acid (50 nmol generally, or
100 nmol of 3-OH C16:0) was used in each 1.0 ml reaction mixture
for in vitro P450 incubations. Because only one product was formed
from each fatty acid (as judged by initial LC–MS analysis), silylation
was done directly on the in vitro incubation residue. For the unsat-
urated fatty acids, hydrogenation was performed prior to silylation
[19]. The resulting TMS derivatives were analyzed by GC–MS in the
electron impact mode.
Searches for P450 4F11 substrates in human liver extract
Metabolic activation of pro-carcinogens by P450 4F11
Substrate searches were done using an LC–MS metabolomic and
isotopic labeling approach (developed in our previous work [19])
with purified P450 4F11 and human liver extract fractions 1, 2,
and 3. Each reaction mixture was analyzed using UPLC-MS in both
the negative and positive ion modes in order to profile as many
metabolites as possible. However, no peaks with the characteristic
16O/18O isotopic signatures (derived from O2) were detected for the
incubations of P450 4F11 with liver extract fraction 1 (neutral lip-
ids) or 3 (phospholipids). The DoGEX program results for the anal-
ysis of liver extract fraction 2 (fatty acids) incubated with P450
4F11 showed several potential products in the negative ion mode
(Fig. 3). Four different doublets (m/z 273/271, 299/297, 321/319,
and 345/343) were identified and verified by comparisons of the
P450 4F11/liver extract fraction 2 samples from experiments with
and without NADPH using Xcalibur (Waters UPLC-MS software
The umu assay was carried out using a modification of the
procedure described by Oda et al. [35,36] in a 96-well microplate
version (Supplementary Fig. S3). Salmonella typhimurium NM2009
(TA1535/pSK1002/pNM12) [37] was used as the bacterial test
strain. The incubation mixture included the bacteria, a recombi-
nant P450 system, procarcinogen, and an NADPH-generating sys-
tem. As a positive control E. coli Top10 bicistronic membrane
fractions containing P450 1B1 (0.01
tase were used.
lM) and NADPH-P450 reduc-
The induction of the umuC gene expression (as a response to
DNA damage) was determined by measuring cellular b-galactosi-
dase activity (absorbance at 405 and 490 nm) using following
equation:
1000ðA405 ꢁ A490
t ꢂ V ꢂ A595
Þ
umu units ðU mlꢁ1Þ ¼
where t is the time of the assay and V is the volume of the culture
used in the assay (in ml).
Results
Expression and purification of recombinant P450 4F11
Expression trials with seven P450 4F11 constructs (with differ-
ent N-terminal sequences, Table 1) were done in different E. coli
strains (DH5a, DH5a plus the chaperon groEL/ES, TOP10, XL10-
gold, XL-Blue, and BL21) (data not shown). The longer version of
P450 4F11, construct #11 (with the hydrophobic sequence
preserved), yielded the highest expression level, ꢀ230 nmol of cor-
rectly folded P450 from a 3-l E. coli BL21 culture (Table 2).
Purification of human P450 4F11 (containing a C-terminal (His)6
tag) is summarized in Table 2. The detergents sodium cholate and
Tergitol NP-10 were selected for solubilization. The recovery of
spectrally measured P450 was 94% after solubilization and separa-
tion from the unsolubilized membranes by ultracentrifugation.
Fig. 1. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of recombinant
P450 4F11. See Table 2. Lane 1, solubilized membrane fraction (10 pmol P450); lane
2, Ni2+–NTA agarose-purified fraction (66 pmol P450); lane 3, hydroxylapatite-
purified fraction (23 pmol P450); lane 4, protein Mr markers.

90
Z. Tang et al. / Archives of Biochemistry and Biophysics 494 (2010) 86–93
Fig. 3. DoGEX results of substrate search with liver extract fraction 2 and human
P450 4F11. The green regions indicate that the ratio of chromatographic peaks (m/z
and m/z + 2) is close to 0.95 (the lower limit of the target ratio) and the yellow area
shows all the values beyond the threshold value. Positive values (approaching the
target of 0.95, and circled with broken ovals) were found for four different pairs of
peaks, subsequently identified, in the tR 7–9 min region and are shown in the
expanded sections.
Spectral binding titrations
3-OH C16:0 has been reported to be catalyzed by P450 4F11 to
Fig. 2. Spectra of purified P450 4F11. (A) Absorbance spectra of P450 4F11 (ferric
and ferrous spectra). The P450 concentration was 2.0 lM, and the ferrous spectrum
form the
x-hydroxylation product [11]. Therefore, interactions of
the fatty acids C16:0, C18:1, C20:4, C22:6, and also 3-OH C16:0
with P450 4F11 were investigated. The spectral changes induced
by binding of the fatty acids to P450 4F11 are shown in Supple-
mentary Fig. S1. Kd values were estimated from the titration curves
was recorded following reduction with Na2S2O4. (B) Second derivative of absor-
bance spectrum (ferric state). (C) Fe2+ꢃCO vs. Fe2+ difference spectrum. The P450
concentration was 2.2 lM (kmax 451 nm).
of
DAmax versus the concentrations of fatty acids (Table 3). C16:0
package) (Fig. 4, i.e. a set of LC–MS data showing typical peaks and
control data, not directly related to the kinetic analysis, vide infra).
All of the peaks (Fig. 4, marked with ovals) were found in the sam-
ple incubated with NADPH but not in the sample without NADPH.
The results were further confirmed by comparison with the incu-
bation with no addition of the P450 (results not shown). All four
m/z doublets identified by the DoGEX program were finally judged
to be oxidation products (formed from the reaction of liver extract
fraction 2 with human P450 4F11), on the basis of their tR and m/z
values being identical in Xcalibur and the DoGEX program.
and C18:1 exhibited typical ‘‘Type I” and ‘‘Type II” difference spec-
tra3 [34] with P450 4F11, respectively, with micromolar affinity.
Titration of P450 4F11 with C20:4 or C22:6 yielded no real spectral
changes (DA390 < 0.005 at a substrate concentration of 50 lM, only a
decrease at 418 nm). 3-OH C16:0 seemed to exhibit an unusual but
reproducible mix of Type I and II binding to P450 4F11, with appar-
ent Kd values of 69 14 and 98 16
lM, respectively.
Characterization of oxidation products
The molecular masses of the potential substrates corresponding
to the identified doublets were deduced as 256, 282, 304, and 328,
based on the method described previously [19]. Moreover, MS frag-
mentation analysis (Supplementary Table S2), knowledge of the
nature of the components of each liver extract fraction obtained
from SPE columns (as described elsewhere [32]), and the LIPIDM-
APS database (http://www.lipidmaps.org) suggested that the fatty
acids C16:0, C18:1, C20:4, and C22:6 were the most likely sub-
strates in the P450 4F11 reactions. All four fatty acids were con-
firmed as substrates in human liver for P450 4F11 by incubations
of authentic fatty acids with purified P450 4F11 (vide infra).
GC–MS assays of TMS derivatives were used to identify the
oxidation products of P450 4F11 reactions. All products were char-
acterized as
x-hydroxylated fatty acids (Table 4) based on a promi-
nent ion fragment at m/z 103 (CH2–O–SiMeþ) considered specific to
3
3
Ligands displacing a distal water molecule from the heme iron of P450 enzymes
change the state of the heme iron from low-spin to high-spin, which gives rise to a
‘‘Type I” change in the spectral properties of the enzyme, with an increase in
absorbance at ꢀ390 nm and a decrease at ꢀ420 nm. ‘‘Type I” binding (ligands that
bind directly to the heme iron of P450 enzymes) give rise to a difference spectrum
with a maximum at ꢀ430 nm and a minimum at ꢀ418 nm [34].

Z. Tang et al. / Archives of Biochemistry and Biophysics 494 (2010) 86–93
91
Fig. 4. Comparison of LC–MS analysis for incubation of P450 4F11 and liver extract fraction 2 with (A) and without (B) the NADPH-generating system. The y-axis shows the
trace at each indicated m/z value, and the sensitivity is indicated on each trace. The tR values (in ovals) indicate the ( -) hydroxylated fatty acid products.
x
Table 3
Table 4
Spectral titration of human P450 4F11 with fatty acids.
EI GC–MS analysis of TMS derivatives of the oxidation products of fatty acids
produced by human P450 4F11.
a
Fatty acid
D
Amax
Ks (lM)
Substrate
[M+Å
]
Major fragment ions
Product
C16:0
C18:1
C20:4
0.047 0.006b
0.037 0.003c
—
40 11
71 14
—
C16:0
C18:1
C20:4
C22:6
416
444
472
500
504
103/311/385/401a
103/339/413/429
103/367/441/457
103/395/469/485
103/399/489
x
x
x
x
x
-OH
-OH
-OH
-OH
-OH
d
C22:6
3-OH C16:0
—
—
0.062 0.006b
0.065 0.006c
69 14
98 16
3-OH C16:0
a
a
See Supplemental data, Fig. S1. Estimated from the hyperbolic fitting plot of
Amax versus the fatty acid concentration using a hyperbolic equation in GraphPad
A GC–mass spectrum of the hydroxylation product is shown in Supplemental
D
data Fig. S2.
Prism software (GraphPad Software, San Diego, CA).
b
Difference in absorbance between the absorbance maximum at 388 nm and
minimum at 418 nm,
D
A388–A418
.
Table 5
c
Difference in absorbance between the absorbance maximum at 438 nm and
Steady-state kinetics analysis of oxidations of fatty acids by human P450 4F11.
minimum at 418 nm,
D
A438–A418
.
d
kcat (minꢁ1
)
Km
(l
M)
kcat/Km (minꢁ1 Mꢁ1
l )
—, no detectable binding (
D
A388 < 0.005 at 50 lM).
Fatty acid
C16:0
C18:1
C20:4
C22:6
1.8 0.1
1.0 0.1
0.54 0.06
0.35 0.04
27.5 4.5
47
4
0.038 0.004
0.012 0.002
0.007 0.002
0.007 0.002
0.37 0.14
x
-hydroxylation of fatty acids [11,40]. The GC-mass spectrum of the
85 10
80 21
48 14
74 26
hydroxylation product of the incubation of C16:0 with P450 4F11 is
shown as an example (Supplementary Fig. S2). These results are con-
sistent with previous literature on P450 4F11 and 3-hydroxy C16:0,
which was also analyzed as the TMS derivative [11].
3-OH C16:0
Km = 0.37 0.14 minꢁ1 Mꢁ1 (10-fold higher than for C16:0), com-
l
parable with a previous result [11].
Steady-state kinetic analysis of P450 4F11 reactions
P450 4F11 was previously reported [10] to be a catalyst for
some drug N-demethylation reactions. N-Demethylation rates with
benzphetamine, erythromycin, and ethylmorphine were 0.83, 0.91,
and 0.87 minꢁ1, respectively, which are higher than reported
previously (0.25, 0.81, and 0.18 minꢁ1, respectively) [10].
Analyses for all the products (Fig. 4, plus 3,16-diOH C16:0) were
done as described in Materials and Methods, and the kinetic
parameters kcat and Km are summarized (Table 5). The catalytic
efficiency (kcat/Km) measured for the oxidation of C16:0 catalyzed
by P450 4F11 was ꢀ3-fold higher than that measured for the reac-
tion of P450 4F11 with C18:1. With the reactions of P450 4F11
with the longer carbon chains and more unsaturation (C20:4 and
C22:6), the kcat/Km values were lower than for C16:0 and C18:1.
Lack of metabolic activation of pro-carcinogens by P450 4F11
In order to investigate a potential role of P450 4F11 in the met-
abolic activation of pro-carcinogens, genotoxicity was evaluated by
The
x-hydroxylation reaction with 3-OH C16:0 yielded kcat/

92
Z. Tang et al. / Archives of Biochemistry and Biophysics 494 (2010) 86–93
measuring b-galactosidase activity in S. typhimurium tester strain
NM2009, using compounds previously reported to be pro-carcino-
gens bioactivated by other P450s, e.g., 1B1, 1A1, and 3A4 [37,41].
Following general screening of the umuC gene induction (Supple-
mentary Fig. S3A), some of the compounds that showed an appar-
ent induction of b-galactosidase activity were evaluated at
different concentrations (benzo[a]pyrene and aflatoxin B1) (Sup-
plementary Fig. S3B). P450 4F11 did not bioactivate any of these
pro-carcinogens. Responses with P450 1B1 (positive control [41])
were observed under these experimental conditions.
Based on our previous work [19], fatty acids are major compo-
nents in human liver but they may affect the detection of other
trace but interesting compounds during searches for substrates.
An aminopropyl SPE column [32] was used to isolate the fatty acid
fraction from other classes of lipids in human liver extract prior to
in vitro P450 incubation. However, the results of untargeted
searches in each liver extract fraction indicate that only the fatty
acids were detected as substrates for human P450 4F11. It is un-
clear if fatty acids are actually the main substrates for human
P450 4F11 or whether they are just more readily detected than
other compounds during LC–MS assays and metabolomics. Chem-
ical derivatization methods are being developed to improve the
sensitivity of MS detection of small molecules in biological
samples.
Discussion
An expression system has been developed for human P450 4F11
in E. coli BL21, with a reasonable expression level (Table 2). The
P450 was purified and showed typical spectral properties, with a
kmax at 451 nm for the reduced P450-CO complex (Fig. 2). Untar-
geted substrate searches for P450 4F11 were done using an LC–
MS metabolomic and isotopic labeling approach [19] in different
human liver extract fractions (Figs. 3 and 4). The fatty acids
C16:0, C18:1, C20:4, and C22:6 were identified as substrates in hu-
man liver for P450 4F11. Binding assays were done for human P450
4F11 with the four identified fatty acids, as well as the previously
identified substrate 3-OH C16:0 [11]. We presently have no expla-
nation for the unusual behavior of the spectral binding for some of
the fatty acids with P450 4F11 (Table 3 and Supplementary Fig. S1),
especially in the cases of C18:1 and 3-OH C16:0, which show
repeatable Type II binding3 to P450 4F11. The oxidation products
of the fatty acids formed by P450 4F11 were all characterized as
Acknowledgments
This work was supported in part by Grants R37 CA090426 and
P30 ES00267 from the United States Public Health Service. We
thank D.L. Hachey and M.W. Calcutt for mass spectrometry assis-
tance and discussions and A.R. Brash for use of a hydrogenation
system.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.abb.2009.11.017.
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