Stereoselective formation and metabolism of 4-hydroxy-retinoic acid enantiomers by cytochrome p450 enzymes

Article abstract of DOI:10.1074/jbc.M112.404475

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred(4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

Full text of DOI:10.1074/jbc.M112.404475

JBC Papers in Press. Published on October 15, 2012 as Manuscript M112.404475
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.404475
Stereoselective Formation and Metabolism of 4-Hydroxy-Retinoic Acid
Enantiomers by Cytochrome P450 Enzymes*
Jakob A. Shimshoni,1 Arthur G. Roberts,2,3 Michele Scian, 4 Ariel R. Topletz,1 Sean A.
Blankert,3 James R. Halpert,2 Wendel L. Nelson4 and Nina Isoherranen1
1Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA
2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego,
CA 3Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA
4Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA
* Running title: Stereoselective retinoic acid metabolism
To whom correspondence should be addressed: Nina Isoherranen, PhD, Department of
Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98195, United States. Tel.:
+1-206-543-2517; fax: +1-206-543-3204. E-mail address: ni2@u.washington.edu
Keywords: Retinoic acid, Cytochrome P450, stereoselectivity
Background: CYP26A1 is
enzyme in the metabolism of all-trans
retinoic acid (atRA).
Results: CYP26A1 metabolizes atRA
stereoselectively to (4S)-OH-RA, which
corresponds to atRA orientation within the
CYP26A1 active site.
Conclusion: Preference for (4S)-OH-RA is
conserved in atRA metabolism and in
elimination of 4-OH-RA.
a
critical
metabolite of atRA, 4-OH-RA, possesses a
chiral center, the stereochemical course of
atRA 4-hydroxylation has not been
studied previously. (4S)- and (4R)-OH-
RA enantiomers were synthesized and
separated by chiral column HPLC.
CYP26A1
predominantly
was
found
(4S)-OH-RA.
to
form
This
stereoselectivity was rationalized via
docking of atRA in the active site of a
CYP26A1 homology model. The docked
structure showed a well-defined niche for
atRA within the active site and a specific
orientation of the β-ionone ring above the
plane of the heme consistent with
Significance:
Determination
of
stereoselectivity in atRA metabolism is
important for better understanding of
retinoid
biology
and
CYP26A1
biochemistry.
stereoselective
abstraction
of
the
SUMMARY
hydrogen atom from the pro-(S)-position.
In contrast to CYP26A1, CYP3A4 formed
the 4-OH-RA enantiomers in a 1:1 ratio
and CYP3A5 preferentially formed (4R)-
OH-RA. Interestingly, CYP3A7 and
CYP2C8 preferentially formed (4S)-OH-
RA from atRA. Both (4S)- and (4R)-OH-
RA were substrates of CYP26A1 but (4S)-
OH-RA was cleared three-fold faster than
(4R)-OH-RA. In addition, 4-oxo-RA was
all-trans-Retinoic acid (atRA), the major
active metabolite of vitamin A, plays a
role in many biological processes,
including maintenance of epithelia,
immunity, and fertility and regulation of
apoptosis and cell differentiation. atRA is
metabolized mainly by CYP26A1, but
other P450 enzymes such as CYP2C8 and
CYP3As also contribute to atRA 4-
hydroxylation. Although the primary
1
Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

formed from (4R)-OH-RA but not from
(4S)-OH-RA by CYP26A1. Overall, these
findings show that (4S)-OH-RA is
preferred over (4R)-OH-RA by the
enzymes regulating atRA homeostasis.
regulate atRA metabolism in other tissues
during development and in adulthood
(11,12,14,15).
The oxidative metabolism of atRA by
CYP26A1 is believed to be the first step in
its degradation and inactivation (14).
However, the main atRA metabolites, 4-
OH-RA and 4-oxo-RA, also bind to the
three RAR isoforms with affinities similar to
atRA (16), and they induce gene
transcription, inhibit proliferation and
induce differentiation in various atRA
susceptible cell lines and models (17,18).
Formation of metabolites from atRA by
CYP26A1 also appears to be required for
differentiation of P19 EC cells into neurons,
and to atRA induced differentiation of
embryonic stem cells (19,20). These data
suggest that metabolites of atRA may be
important in retinoid function when
sufficient concentrations are reached in vivo.
However, transgenic animal studies have
shown that CYP26 enzymes are required for
removal of atRA rather than for formation
of active metabolites during mouse
development (21).
The hydroxylation of atRA to 4-OH-RA
results in the formation of a chiral center
(Figure 1) but all studies characterizing
formation of 4-OH-RA and its activity have
been conducted using racemic 4-OH-RA.
Since formation of 4-OH-RA could be
stereospecific and the biological activity
may reside in a single 4-OH-RA enantiomer,
understanding of the functional role(s) of
atRA metabolites requires characterization
of the stereochemical course of metabolism.
In addition, stereospecificity of the enzymes
involved in RA biotransformation may help
in better understanding of the structure-
function of the proteins involved in
regulating RA homeostasis.
The
stereoselectivity
observed
in
CYP26A1 function will aid in better
understanding of the active site features
of the enzyme and the disposition of
biologically active retinoids.
all-trans-Retinoic acid (atRA) is the major
biologically active metabolite of vitamin A
(retinol). It is an essential component of
signaling networks in chordates. During
embryonic and fetal development atRA
regulates cell differentiation, and during
adult life it is important for maintaining
healthy epithelia, immunity, fertility and in
regulating cell proliferation (1). atRA also
plays a role in neurogenesis in adults and in
cell survival (2), and it contributes to
regulation of glucose homeostasis (3). These
effects of atRA result mainly from its
binding to the three nuclear retinoic acid
receptors (RARα, RARβ and RARγ) that
are activated by atRA resulting in induction
of gene transcription (4).
Therapeutically atRA (Tretinoin®), 13-cis-
retinoic acid (Isotretinoin®) and 9-cis-
retinoic acid (Alitretinoin®) are used in the
treatment of various cancers and skin
diseases (4). All three RA isomers are
cleared mainly by metabolic oxidation and
glucuronidation (5-8). The main circulating
metabolite of 13-cis-RA and atRA detected
in human plasma is 4-oxo-RA (9,10), the
subsequent metabolite of 4-OH-RA. 4-OH-
RA is the primary oxidized metabolite of
atRA. It is formed by several cytochrome
P450 enzymes including CYP26s, CYP3As
and CYP2C8 (6-8,11-13). Of these P450
enzymes, CYP26A1 appears to be the main
isoform responsible for both atRA clearance
and for formation of 4-OH-RA in human
liver (13). CYP26A1 and CYP26B1 have
also been identified as the key enzymes that
Stereoselectivity appears to be a key
characteristic of retinoid biotransformation.
Hydroxylation of retinol in F9 cells results
predominantly in the formation of (4S)-OH-
2

retinol (22), and in the firefly squid, the
visual pigment is (4R)-OH-retinal (23). The
mouse and zebrafish retinol saturase
determining the UV absorbance at 360 nm.
Protein expression and enzymatic
formation of 4-OH-RA enantiomers-
CYP26A1 microsomes were prepared and
rat reductase was expressed and purified as
previously described (13,25). CYP3A4,
CYP3A5, CYP3A7 and CYP2C8 were
purchased from BD Gentest and were co-
expressed with P450 reductase and
cytochrome b5. All incubations were
conducted in 100 mM potassium phosphate
buffer (pH=7.4) at 37 ºC and initiated by
addition of NADPH (1 mM final
concentration) (Sigma-Aldrich, St. Louis,
MO). The reactions were quenched and
metabolites extracted using ethyl acetate as
selectively
dihydroretinol, and the biological activity
(adipocyte differentiation and RAR
forms
(R)-all-trans-13,14-
activation) of this product resides in the (R)-
enantiomer (24).
Based on the observed stereoselectivity
associated with other retinoids and the tight
binding of atRA to CYP26A1, it was
hypothesized that formation of 4-OH-RA by
CYP26A1 would be stereoselective. To test
this hypothesis, a chiral column HPLC
method was developed to separate the
4-OH-RA enantiomers, standards of the
(4S)- and (4R)-OH-RA enantiomers were
synthesized, and the formation of 4-OH-RA
enantiomers by CYP26A1, CYP2C8 and
CYP3A4 isoforms was characterized. The
formation of 4-OH-RA by CYP26A1 was
stereoselective resulting in formation of
(4S)-OH-RA whereas the other P450s
formed both 4-OH-RA enantiomers with
varying stereoselectivities. (4S)-OH-RA was
also a better substrate of CYP26A1 than
(4R)-OH-RA resulting in formation of
different metabolites and suggesting that the
observed stereoselectivity may have broader
biological importance.
described
previously
(25,26).
The
stereoselective formation of 4-OH-RA by
P450 enzymes (5 nM) was characterized
with 10 nM-40 µM atRA as a substrate.
P450 reductase (10 nM) was added to
incubations with CYP26A1. The product
formation data were analyzed and the
Michaelis-Menten equation was fitted to the
data using GraphPad Prism v.5 (La Jolla,
CA).
Metabolism of (4R)- and (4S)-OH-RA by
CYP26A1- The metabolites formed from
(4R)-OH-RA
and
(4S)-OH-RA
by
CYP26A1 were identified using LC-MS/MS
conditions identical to those previously
reported for racemic 4-OH-RA (15). Five
pmoles of CYP26A1 and 10 pmoles of P450
reductase were incubated with 1 µM of each
enantiomer in 1 mL of potassium phosphate
(100 mM, pH 7.4) buffer, and samples were
extracted using ethyl acetate as described
(15).
The time course of (4R)-OH-RA and (4S)-
OH-RA depletion by CYP26A1 was
determined at six substrate concentrations
between 2 and 250 nM using a P450
reductase to CYP26A1 ratio of 2:1 (2
pmol/mL P450 in each incubation), in 100
mM potassium phosphate buffer (pH=7.4) at
37 ºC. All incubations were conducted under
EXPERIMENTAL PROCEDURES
Separation of (4S)- and (4R)-OH-RA
enantiomers- The 4-OH-RA enantiomers
were
separated
using
an
Agilent
Technologies (Palo Alto, CA) 1200 series
HPLC system equipped with a Chiralcel
OD-RH column (5 µm, 2.1 x 150 mm), an
Agilent temperature controlled auto-
sampler, a multiple wavelength detector and
HP Chemstation software. The mobile phase
flow was 0.17 ml/min. A linear gradient
from initial 50:50 water:acetonitrile for 14
min to 20:80 over 10 min was used,
followed by 20:80 water:acetonitrile for 10
min. Analytes were quantified by
3

red light in triplicate and the experiments
were repeated three times on separate days.
The reactions were initiated by addition of
NADPH (1 mM final concentration). At
0.5, 1, 2, and 5 min, 0.5 mL aliquots were
collected and quenched with 3 mL of cold
ethyl acetate. For a separate experiment at
depletion rate constant as described
previously (25). The Km was obtained using
the depletion data according to equation 1
(27),
[S]
(1)
kdep = kdep,max ×(1−
)
[S]+ Km
in which the substrate concentration [S] was
corrected for depletion during the incubation
(25). The maximum efficiency of depletion,
the intrinsic clearance (Vmax/Km), was
calculated as kdep,max divided by the protein
concentration as described (25). All
nonlinear fits were done using GraphPad
Prism v.5 (La Jolla, CA) and values are
reported as mean ± standard error.
2.5nM substrate concentration,
1
mL
aliquots were collected at 0.5, 1, 1.5 and 2
min. As an internal standard, 20 µL of a 2.5
µM solution of 4-oxo-RA-D3 (Toronto
Research Chemicals, North York, Ontario)
in ethanol was added, and samples were
extracted as previously described (15).
4-OH-RA enantiomers were analyzed using
an AB Sciex API 5500 Q/LIT mass
spectrometer equipped with an Agilent 1290
Infinity UHPLC and an Agilent Zorbax C18
column (3.5 µm, 2.1 mm x 100 mm). The
analytes were separated using a 10 min
linear gradient from initial 5:95 acetonitrile:
0.1% aqueous formic acid to a final 95:5
acetonitrile: 0.1% aqueous formic acid.
Analytes were detected using positive ion
electrospray mass spectrometry with the
declustering potential set to 80, collision
energy to 33 (4-OH-RA) and 35 (4-oxo-RA-
D3), and collision exit potential to 13 (4-OH-
RA) and 2 (4-oxo-RA-D3). The parent-
fragment MS/MS transitions of m/z
299.2→121.2 Da for (4R)-OH-RA and (4S)-
OH-RA and m/z 300.2→226.2 Da for 4-
oxo-RA-D3 were used. The data were
analyzed using Analyst software, the 4-OH-
RA concentration was plotted as a function
of time. The concentration versus time data
was log-linear for two minutes and only data
up to two minutes was used. Points for
which concentration was below limit of
quantification of the assay (0.1nM) were not
included in the analysis. The depletion rate
The kinetics of the formation of 4-oxo-RA
and 4,16-OH2-RA were analyzed from (4R)-
OH-RA and (4S)-OH-RA incubations
respectively
using
identical
LC-MS
conditions as described above. 2 pmoles of
CYP26A1 and 4 pmoles of P450 reductase
were incubated with 5-150 nM of each
enantiomer in 1 mL of potassium phosphate
(100 mM, pH 7.4) buffer, and samples were
extracted using ethyl acetate. For 4-oxo-RA
quantification a standard curve using a 4-
oxo-standard was used and formation
velocity measured. The formation of 4,16-
OH2-RA from (4S)-OH-RA was determined
using relative quantification of 4,16-OH2-
RA peak height to internal standard ratio as
a function of (4S)-OH-RA concentration.
The affinity (Kd) of the 4-OH-RA
enantiomers to CYP26A1 was determined
by fitting the Morrison equation (tight
binding equation, equation 2) to the data as
previously described for atRA binding to
CYP26A1 (25).
([E]+[S]+ Km ) − ([E]+[S]+ Km )2 − 4[E][S]
V =Vmax
•
2[E]
constant was obtained from the log-linear fit
C = C0e−k
t in which C
dep
to the equation:
Significance of differences between
determined kinetic parameters for the two
enantiomers were evaluated using the Z-test
equals 4-OH-RA enantiomer concentration
at a given time t, C0 the initial concentration
of 4-OH-RA enantiomers and kdep the
4

and a p-value <0.05 was considered
significant.
Chemical synthesis- The overall pathway
for synthesis of 4-OH-RA enantiomers from
methyl 4-oxo-retinoate is shown in Figure 1.
amino acid sequence of human CYP26A1
(NCBI Sequence No. NP_000774.2). Two
cytochrome P450 X-ray crystal structures,
cyanobacterial CYP120A1 (PDB ID: 2VE3
(31)) with atRA bound and mammalian
CYP2C8 (PDB ID: 2NNH, (32)) with two 9-
cis-RAs bound, were used as structural
templates. The CYP120A1 structure and the
homology model were aligned and heme and
atRA in the CYP120A1 structure were
roughly docked to the homology model
using PyMOL v1.2 (DeLano Scientific LLC,
Palo Alto, CA, USA). To model the
predominant structure in solution, molecular
dynamics (MD) simulations were performed
of the complex using GROMACS Version
4.07 (33). The topology files were modified
to reflect the cysteinyl ligation as described
previously (34,35). atRA was used to form
the active site of the homology model of
CYP26A1. The atRA molecule was
obtained from PubChem (Compound
Identifier CID 444795), and the protein was
energy minimized by steepest descent
approach using the MMFF94 force field (36)
in Avogadro (Version 1.01). Since the
primary site of metabolism is at the C-4
carbon, a time-averaged distance restraint of
3.4 and 7.4 Å was applied between the heme
iron and this carbon. The complex was
immersed in a simulated water box with 120
Å sides and ~50,000 waters, corresponding
to roughly twice the length of the diagonal
of the protein, or ~50 Å. Next, the complex
was energy minimized by the method of
steepest descent in order to remove van der
Waals contact between overlapping waters
and the amino acids of the protein. The
simulations were run with Berendsen
temperature and pressure coupling (37) at
300K, using the GROMOS 53a6 force field
(38,39) and periodic boundary conditions in
all directions. Electrostatics of the system
were measured using the particle-mesh
Ewald method (40). The MD simulation
reached an equilibrium at >10 ns, but was
The
synthesis
of
the
4-
dimethylaminocinnamoate esters of methyl
4-OH-retinoate for CD spectroscopy is also
shown. All syntheses are described in detail
in supplemental data.
Assignment of absolute stereochemistry
using CD spectroscopy- CD samples were
prepared by dissolving weighed amounts of
4-dimethylaminocinnamoate esters (Figure
1) of the enantiomeric methyl 4-
hydroxyretinoates in acetonitrile to make
200 µM solutions (100 µg/ml). Spectra were
recorded at 25°C on a JASCO J715
spectropolarimeter (JASCO Corporation,
Tokyo, Japan) using a 0.10 cm path length
cell with a nitrogen flow rate of 4 L/min.
The wavelength and degree ellipticity scales
were calibrated using a reference (1S)-(+)-
10-camphorsulfonic acid ammonium salt
(ACS) sample assuming that the ACS
maximum corresponds to [Θ]290.5 = + 7910
(28). To minimize the effects of a possible
photo-isomerization, only a single scan for
each spectrum was collected at a scan rate of
100 nm/min with a 2 nm band width and a
0.2 nm step resolution over the wavelength
range of 200–500 nm. The collected spectra
were trimmed at a dynode voltage of 650 V
(where required) prior to baseline
subtraction
(acetonitrile)
and
computationally smoothed using a two point
fast Fourier transformation filter function
(FFT filter) included within JASCO
software (Spectra Manager 1.53.04). The
raw ellipticity data (mdeg) were normalized
by dividing them by the UV-Vis absorbance
of the solutions at λmax (356 nm).
Generation and validation of a CYP26A1
Homology Model- A CYP26A1 homology
model was made using Modeller (29,30).
The homology model was built using the
5

continued to 18.75 ns. The root mean square
deviation (RMSD) of the backbone over the
course of the simulation is shown in
Supplemental Figure S1. The structure at the
18.75 ns time point was energy minimized
by method of conjugate gradient energy and
steepest descent minimization methods. The
secondary structure assignments of this
CYP26A1 homology model are shown in
supplemental Table S1.
The CYP26A1 homology model with
atRA restrained at C-4 was validated as
described for other homology models of
CYP26A1 (41-43) and the results are
presented in supplemental Table S2. The
backbone φ and ψ angles of the homology
model were evaluated from Ramachandran
plot obtained on the RAMPAGE server
(http://mordred.bioc.cam.ac.uk/~rapper/ram
page.php). The Ramachandran plot is shown
with 20 Å sides was searched with the
software. The maximum number of potential
binding sites was set to 10. Binding sites
with ~100 fold weaker binding affinity (i.e.
3 kcal/mol) than the tightest binding site
were excluded as potential binding sites.
The dissociation constant (Kd) was
calculated for comparative purposes from
the free energy (ΔG) in kcal mol-1 using the
ΔG
equation: Kd = eRT where
T
is the
temperature in K and R is the gas constant in
kcal mol-1 K-1.
RESULTS
Separation of the 4-OH-RA enantiomers
and stereoselective formation of 4-OH-RA
by CYP26A1- A method for the separation
of 4-OH-RA enantiomers using a Chiralcel
OD-RH column was developed, and the
separation of (4S)- and (4R)-OH-RA is
shown in Figure 2a. To determine whether
formation of 4-OH-RA by CYP26A1 is
stereoselective, atRA was incubated with
recombinant human CYP26A1 microsomes.
A chromatogram of an incubation of atRA
with CYP26A1 is shown in Figure 2b.
CYP26A1 formed preferentially a single 4-
OH-RA enantiomer. Other hydroxylation
products were also detected in the CYP26A1
incubations that eluted after the 4-OH-RA
enantiomers (Figure 2b), as previously
reported (11,25). Based on previous studies
(15) these products include 18-OH-RA and
16-OH-RA.
in
Supplemental
Figure
S2.
The
compatibility of the homology models three-
dimensional fold with the amino acid
sequence was compared using Verify 3D
(44). The program ERRAT was used to
analyze the non-bonded interactions
between different atom types (45). The
quality of the homology model was overall
better than previously built homology
models (41-43).
Molecular Docking of atRA to CYP3A4.
atRA was docked into the active site of
CYP3A4 using Autodock Vina 1.1.1 (46).
To
sample
the
known
CYP3A4
conformations, atRA was docked into the
published X-ray crystal structures of the
enzyme (PDBID: 2J0D (47), 3NXU (48),
3TJS (49), 1TQN (50), 3UA1 (49), 2V0M
(47), 1W0E (51), 1W0F(51) and 1W0G
(51)). AutoDockTools 1.4.6 (The Scripps
Research Institute, La Jolla, CA) was used
to put Gasteiger charges on the homology
model and atRA. During the simulation, the
protein was kept rigid and the carboxylic
acid group of atRA was allowed to rotate. A
box centered 10 Å away from the heme iron
Determination of absolute stereochemistry
of 4-OH-RA enantiomers- To determine the
absolute stereochemistry of the predominant
4-OH-RA formed by CYP26A1, authentic
standards of the (4S)- and (4R)-OH-RA
enantiomers were synthesized (Figure 1)
from 4-oxo-RA ester. Diborane (dimethyl
sulfide) reduction in the presence of the
methylated (S)- and (R)-Corey-Bakshi-
Shibata (CBS)-oxazaborolidine catalysts
(52), yielded the enantiomeric methyl (4S)-
and (4R)-OH-retinoates. The corresponding
6

free acids were obtained by hydrolysis of the
methyl esters with aqueous methanolic
KOH, followed by acidification with citrate
buffer (pH=6). The enantiomeric excess (ee
%) of the 4-OH-RA enantiomers was
determined using chiral HPLC. The
enantiomeric excesses (ee %) of the
concentration dependence in the formation
(Supplemental Figure S3).
Stereoselectivity in 4-OH-RA formation by
other P450 isoforms- The stereoselective
formation of 4-OH-RA enantiomers by
CYP3A4, CYP3A5, CYP3A7 and CYP2C8
was determined, as these enzymes have been
previously shown to metabolize atRA in
human liver. The chromatograms of the
separation of the products formed are shown
in Supplemental Figure S4. The other P450
enzymes were much less stereoselective in
the 4-hydroxylation of atRA than
CYP26A1. CYP3A4 formed (4R)-OH-RA
and (4S)-OH-RA in approximately 1:1 ratio
with equal Km and kcat values (Table 1).
CYP3A5 formed (4R)-OH-RA twice as
efficiently as (4S)-OH-RA, whereas
CYP3A7 formed (4S)-OH-RA nearly twice
as efficiently as (4R)-OH-RA (Table 1). The
determined kinetic constants summarized in
Table 1 show that stereoselective product
ratios are mainly due to different kcat values
and not differences in Km for formation of
the (4S)- and (4R)-OH-RA.
Metabolism of 4-OH-RA enantiomers by
CYP26A1- Racemic 4-OH-RA is a substrate
of CYP26A1, and several sequential
metabolites are formed from 4-OH-RA by
CYP26A1 (15). Mass spectrometric analysis
of the metabolic products formed from (4S)-
OH-RA showed that the dihydroxylated
product, 4,16-OH2-RA, was the predominant
metabolite formed from (4S)-OH-RA
(Figure 4) and 4-oxo-RA was a minor
metabolite. In contrast, 4-oxo-RA was the
major product formed from (4R)-OH-RA
synthesized
(4S)-
and
(4R)-OH-RA
enantiomers were 95% and 96%,
respectively (Figure 2c and 2d). The
retention times of the synthetic standards
show that CYP26A1 forms predominantly
the (4S)-OH-RA.
To confirm the absolute stereochemistry of
the products, the 4-hydroxyl group of each
methyl retinoate enantiomer was coupled to
the
4-(dimethylamino)cinnamoyl
chromophore (Figure 1), and the exciton
circular dichroism (CD) spectra of the
4-(dimethylamino)cinnamoate esters were
obtained for the coupled products (Figure 3).
The CD-spectra are mirror images of each
other and show exciton split CD Cotton
effects. For the (4R)-enantiomer, a negative
first (longer wave length) and positive
second Cotton effects were expected, since
the 4-(dimethylamino)cinnamoyl and the
pentaenacyl chromophores constitute
a
counterclockwise screw (Figure 3), while for
the (4S)-enantiomer the opposite Cotton
effects are expected (53). The obtained CD-
spectra were compared to the CD spectra of
related 4-(dimethylamino)cinnamoyl esters
of (4S)- and (4R)-OH-retinal (23) and the
absolute configuration of the 4-OH-RA
enantiomers confirmed. These data show
that
CYP26A1
hydroxylates
atRA
predominantly to the (4S)-OH-RA.
along
metabolites
with
multiple
including
dihydroxylated
4,16-OH2-RA,
The Michaelis-Menten kinetic values of
(4S)-OH-RA formation by CYP26A1 were
determined (Supplemental Figure S3). The
4,18-OH2-RA, 4-oxo-16-OH-RA and 4-oxo-
18-OH-RA. These products were also
detectable from (4S)-OH-RA but were
quantitatively minor (Figure 4). The
identification of the metabolites was similar
to those shown previously from racemic 4-
OH-RA (15).
k
cat for (4S)-OH-RA formation was 2.1 ± 0.2
pmol/min/pmol P450 and Km 10 ± 6 nM.
The Km and kcat of (4R)-OH-RA formation
could not be determined due to the very low
formation of this enantiomer and the lack of
7

To determine the affinity of (4S)-OH-RA
and (4R)-OH-RA to CYP26A1, both
enantiomers were incubated with CYP26A1
and the formation of 4-oxo-RA from (4R)-
OH-RA and 4,16-OH2-RA from (4S)-OH-
RA was measured as a function of 4OH-RA
concentration (Figure 5a and b). Although
the Km value for (4S)-OH-RA (37.4 ± 9.7
nM) was lower than that for (4R)-OH-RA
(45.8 ± 12.3 nM), the Km values were not
significantly different from each other (p >
0.25). The kcat value for 4-oxo-RA formation
6a and 6b show the orientation of atRA in
the CYP26A1 active site.
According to the model of atRA bound
with CYP26A1, the β-ionone ring is
positioned over the heme allowing oxidation
of C4 as well as C18 and C16. The residues
surrounding the β-ionone ring (F299, W112,
L120, T304, and S126, Figure 6a and 6b)
position the (4S)-hydrogen 1.5 Å closer to
the heme than (4R)-hydrogen (4S-hydrogen
distance 4.6 Å, (4R)-hydrogen distance 6.1
Å). The distance between C-4 and the heme
iron was 5.6 Å. Overall the CYP26A1 active
site shown in the model is small and consists
of a network of hydrophobic residues that
surround the nonatetraene chain of atRA.
The nonatetraene chain of atRA directly
interacts with several residues, including
F84, W112, P113, V116, F374, and the
hydrophobic parts of E303 and V370. The
terminal carboxylic acid group of atRA
interacts with R64, the positively charged
residue in the active site.
To investigate the potential binding
orientations of atRA within the CYP3A4
active site, atRA was docked in the
published CYP3A4 crystal structures
(Supplemental Figure S5). atRA was found
in a number of orientations within the
CYP3A4 active site as shown in Figure 6c
and 6d. The distances between the heme iron
and the hydrogen atoms at C-4 varied
between 3.1 and 16.5 Å. The average
distance was not different for (4S) and (4R)
position (Table 2), in agreement with the
similar rates of formation of (4R)- and (4S)-
OH-RA by CYP3A4. Consistent with a
large flexible active site of CYP3A4, the
predicted affinity of the preferred orientation
of atRA within the CYP3A4 active site
varied 50-fold among the known crystal
structures. The average affinity of 2.5 µM is
similar to the experimentally measured Km
of 3 µM.
from (4R)-OH-RA was 11.3
pmol/min/pmol P450.
±
1.3
To determine the catalytic efficiency of
CYP26A1 in metabolizing 4-OH-RA
enantiomers substrate depletion experiments
were conducted. When (4S)-OH-RA and
(4R)-OH-RA
were
incubated
with
CYP26A1 at 2.5 nM concentrations (<<Km)
the depletion of (4S)-OH-RA was
significantly faster (p<0.005, kdep=1.8 ± 0.5
1/min) than that of (4R)-OH-RA (kdep=0.6 ±
0.02 1/min) (Figure 5). To further confirm
the different kinetics of (4R)-OH-RA and
(4S)-OH-RA depletion by CYP26A1 the
depletions rates were analyzed at different
concentrations. The depletion curves as well
as the relationship between kdep and 4-OH-
RA concentration are shown in Figure 5e
and f. The (4S)-OH-RA had significantly
higher (p < 0.005) intrinsic clearance (1.4 ±
0.1 mL/min/pmol P450) than (4R)-OH-RA
(0.7 ± 0.1 mL/min/pmol P450). The affinity
of (4S)-OH-RA (Km 5.2 ± 1.3 nM) was not
significantly different from that of (4R)-OH-
RA (Km 11.0 ± 5.2 nM).
Homology model of atRA bound CYP26A1
and Molecular Docking of atRA to CYP3A4-
To explore the stereoselectivity in 4-OH-RA
formation by CYP26A1 in comparison to
CYP3A4, a homology model of CYP26A1
with atRA in the active site was constructed
and energy minimized. atRA was also
docked to the CYP3A4 active site. Figure
The orientation of atRA docked in the
1TQN crystal structure of CYP3A4 (Figure
8

6c and 6d) corresponds best to the average
orientation of atRA in the different CYP3A4
structures. In this orientation, the β-ionone
ring is located between residues R105, F108,
I120, F215 and S119 positioning the ring in
an angle that allows hydrogen atom
abstraction from both the pro-S and pro-R
position. The orientation of the β-ionone
ring within the CYP3A4 active site in all
structures (Supplemental Figure S5) has the
C16 located away from the heme, which is
consistent with the lack of formation of the
16-OH-RA by CYP3A4. The nonatetrene
chain of atRA interacts with hydrophobic
residues, F215, M371, L482, L483 and I369,
in addition to the hydrophobic regions of
R212 and T309 in the atRA docked 1TQN
structure. The carboxylic acid group of
atRA hydrogen bonds with S312 and E308.
Repulsion from the hydrophilic residues
within the active site might explain the large
differences in the atRA orientations between
the docked structures (Table 2).
enzymes that evolved to mediate atRA
homeostasis.
Despite the fact that CYP26A1 forms
preferentially (4S)-OH-RA, both 4-OH-RA
enantiomers are good substrates of
CYP26A1. This is plausible as the (4R)-OH-
RA can be formed by the fetal liver
CYP3A7, and by CYP3A4, CYP3A5 and
CYP2C8 in the adult liver, and hence an
elimination pathway for this enantiomer is
also needed. However, the metabolism of 4-
OH-RA by CYP26A1 was steresoselective
with (4S)-OH-RA having significantly
higher (p<0.005) intrinsic clearance than
(4R)-OH-RA. While the Km-value for (4S)-
OH-RA depletion was consistently lower
than that of (4R)-OH-RA the Km values
between the enantiomers were not
significantly different. As such, the greater
CLint of (4S)-OH-RA is likely due to higher
kcat for the formation of the diol metabolite
observed from (4S)-OH-RA than for the
formation of 4-oxo-RA from the (4R)-OH-
enantiomer. Interestingly, as shown before
for metabolism of atRA by CYP26A1, the
Km-values measured via product formation
were higher than those measured via
substrate depletion. This could, in part be
due to the fact that the determination of the
affinity via product formation allows
correction for the depletion of free substrate
due to binding to CYP26A1. The Clint and
Km values obtained for the 4-OH-RA
enantiomers were similar to the Clint values
obtained for 4-OH-RA formation and atRA
depletion by CYP26A1 (15,25).
DISCUSSION
The physiological effects of RA
demonstrate high stereoselectivity, as atRA
is believed to be the biologically active
geometric isomer with less activity residing
in the 13-cis-RA, 9-cis-RA and 9,13-dicis-
RA stereoisomers. Generally, atRA has a
higher affinity for many proteins such as
RARs, CYP26A1 and CRABPs than the
other RA isomers (16,26,54). However, the
stereoselective metabolism of atRA and the
role of stereoselectivity in the activity and
biological fate of the metabolites have not
been previously reported. This study shows
for the first time that the hydroxylation of
atRA by CYP26A1 is highly stereoselective.
In contrast, the P450 enzymes that are not
believed to be critical in atRA metabolism,
CYP3A4, CYP3A5, CYP3A7 and CYP2C8,
showed limited stereoselectivity suggesting
that this is an inherent property of the
Different metabolites were formed from
(4S)- and (4R)-OH-RA by CYP26A1,
suggesting that the binding orientations of
the two enantiomers in CYP26A1 active site
are different. The profile of metabolites
formed from (4S)-OH-RA by CYP26A1 is
similar to that observed following
incubations of atRA with CYP26A1,
whereas the products formed from (4R)-OH-
RA do not agree well with the product
9

profile from atRA (15). These results
support the finding that CYP26A1
metabolizes atRA predominantly to (4S)-
OH-RA, which is sequentially metabolized
to 4,16-OH2-RA product. In addition, the
fact that only 4,16-OH2-RA was formed
from (4S)-OH-RA suggests that the other
diols observed in atRA incubations with
CYP26A1 are formed by 4-hydroxylation of
the primary metabolites 18-OH-RA and 16-
OH-RA. These data also suggest that in vivo
4-oxo-RA is formed via different enzymatic
pathway from atRA, and not by CYP26A1.
The high binding affinity of (4S)-OH-RA
determined here also partially explains the
observed non-dissociative metabolism of
atRA by CYP26A1 (25). Whether
metabolism of 4-OH-RA by UGT enzymes
and by the enzymes forming 4-oxo-RA is
also stereoselective is currently not known.
The stereoselectivity of hydroxylation of
atRA by CYP26A1 offers a unique
opportunity to enhance our understanding of
the molecular interactions between atRA
and CYP26A1, and to refine the
understanding of the structure of the
CYP26A1 active site. The stereoselective
formation of (4S)-OH-RA by CYP26A1
likely requires that the hydroxyl group be
delivered from the si-face of the molecule to
give (4S)-OH-RA. This is well illustrated in
the homology model of CYP26A1, which
shows a very well defined niche for atRA
within the CYP26A1 active site and a
distinct orientation of the β-ionone ring
above the plane of the heme. The orientation
of atRA within the CYP26A1 active site is
consistent with stereoselective hydrogen
atom abstraction from the pro-(S)-position
and subsequent hydroxyl radical rebound.
P450 mediated hydroxylations usually occur
with retention of the stereochemistry at the
reacting carbon, although occasionally
products arising from both retention and
inversion are reported (55).
In terms of the active site residues, the
presented homology model is in good
agreement
with
previously
reported
homology models of CYP26A1 (41-43). The
previous models are also consistent with the
stereoselective 4S hydroxylation of atRA.
The presented model also explains both the
stereoselectivity of 4-hydroxylation of atRA
and the observed lack of regioselectivity in
hydroxylation (15,26). It shows that the
hydrogen atoms in C16 and C18 lie in a
plane above the heme together with the pro-
S hydrogen atom at C4. As such, the model
suggests that hydroxylation at the C16
position would also be stereoselective as
only one of the gem-dimethyl groups is
close to the heme. The inferred limited
mobility of atRA within the CYP26A1
active site is in contrast with the apparent
greater mobility within the CYP3A4 active
site. The multiple binding modes found for
atRA with the CYP3A4 structures together
with the similar average distances of the
pro-S and pro-R hydrogen atoms is in
agreement with the observed equal rates of
(4S)-OH-RA and (4R)-OH-RA products
formed by CYP3A4.
In summary, this study reports for the first
time the synthesis and the determination of
the absolute configuration of 4-OH-RA
enantiomers and shows that CYP26A1
forms the (4S)-OH-RA stereoselectively.
The observed product stereoselectivity and
metabolism of both 4-OH-RA enantiomers
by CYP26A1 will be useful in better
modeling of CYP26A1 and aid in the
structural design of novel CYP26A1
inhibitors as therapeutic agents. These
results show that CYP26A1 accepts various
biologically active retinoids as substrates,
but has a conserved preference for the (4S)-
OH-RA enantiomer over (4R)-OH-RA.
These results show that the two 4-OH-RA
enantiomers are physiologically distinct due
to their different formation and elimination
kinetics resulting in likely different cellular
10

exposures to these enantiomers and
formation of different downstream active
metabolites such as 4-oxo-RA. Further
studies are needed to determine the role of
stereoselectivity in the activity of the 4-OH-
RA enantiomers and in the formation of 4-
oxo-RA from the individual stereoisomers.
R01-GM081569-S1 and GM054995-16. The
authors wish to thank Dr. Alex Zelter and
Dr. Jayne E. Thatcher for their assistance in
enzyme expression, preparation of
microsomes and conduct of preliminary
experiments, Dr. David R. Goodlett for use
of his Linux cluster for the MD simulations
and Dr. Justin Lutz for his assistance in
obtaining the accurate mass measurements.
Acknowledgements. This work was
supported by NIH grants R01-GM081569,
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Figures Legends
Figure 1. Synthesis of (+)-(4S)- and (-)-(4R)-all-trans-RA and the 4-dimethylaminocinnamoate
esters of methyl 4-OH-retinoiate. Reaction conditions were: (a) (R)-2-methyl-CBS-
oxazaborolidine ((R)-CBS), BH3·S(CH3)2, THF, 16 hr, yield 85%. (b) (S)-2-methyl-CBS-
oxazaborolidine ((S)-CBS), BH3·S(CH3)2, THF, 16 hr, yield 79%. (c) 1M KOH in water:MeOH
(8:1) room temp, 4 hr; citrate buffer (pH 6) (d) 4-(dimethylamino)cinnamoyl-1H-1,2,4-tetrazole
amide, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
Figure 2. HPLC separation of the 4-OH-RA enantiomers and stereoselective formation of
(4S)-OH-RA by CYP26A1. The 4-OH-RA enantiomers were separated from the racemic mixture
using a Chiralcel OD-RH column (5 µm, 2.1 x 150 mm) and a water-acetonitrile gradient as
described in Experimental section. Panel (a) shows the separation of (4R)- and (4S)-OH-RA in a
racemic mixture, and panel (b) shows the stereoselective formation of (4S)-OH-RA from atRA (200
nM) by CYP26A1 (5 pmol/mL). The determination of enantiomeric excess (ee) for the synthetic
(-)-(4R)-OH-RA (ee 96.3%, rt 11.4 min) and (+)-(4S)-OH-RA (ee 95.1%, rt 9.8 min) and the
identification of the two enantiomers is shown in panels (c) and (d).
Figure 3. Determination of the absolute configuration of 4-OH-RA enantiomers by exciton
circular dichroism (CD) spectroscopy. CD spectra (top) of the 4-(dimethylamino)cinnamoyl
esters of methyl (4S)- and (4R)-OH-retinoate in acetonitrile. The bottom panels show the structures
of the 4-(dimethylamino)cinnamoyl esters. The UV-vis spectrum of the enantiomers is shown in
green in the top panel.
Figure 4. Identification of the metabolites formed from (4S)-OH-RA (a) and (4R)-OH-RA (b)
by CYP26A1. The MS/MS transitions monitored are shown next to each trace and the identified
14

metabolites are indicated for each peak. Incubations and analysis were carried out as described in
the experimental section and metabolic products were identified as described previously (15).
Figure 5. Metabolism of (4S)-OH-RA (left panel) and (4R)-OH-RA (right panel) by
CYP26A1. The formation of specific metabolites was measured using (4S)-OH-RA (panel a) and
(4R)-OH-RA (panel b) as substrates, and the Km value determined according to equation 2. The
relative formation rate of 4,16-OH2-RA was quantified from peak height ratio whereas 4-oxo-RA
formation was quantified using a standard curve. Error bars show the standard deviations of
triplicate measurements for each point. Panels c and d show the depletion of (4S)-OH-RA and (4R)-
OH-RA by CYP26A1 at 2.5 nM initial concentration (closed circles). The closed squres show the
depletion of 4OH-RA enantiomers in no-enzyme control incubations. No NADPH controls were
also done and they were identical to the no enzyme controls (not shown). Error bars show the
standard deviation of triplicate incubations. Representative determinations of the concentration
dependent depletion of (4S)-OH-RA and (4R)-OH-RA by CYP26A1 are shown in panels e and f,
respectively. The depletion rates were measured at six different concentrations and the depletion
rate constants determined (insets in panels e and f). All depletion experiments were conducted on
multiple days and in triplicate on each day. Standard deviations for the individual measurements are
shown by error bars. Equation 1 was fitted to the data of depletion constants as a function of 4-OH-
RA concentration to determine the Km and kdep,max of (4S)-OH-RA and (4R)-OH-RA depletion by
CYP26A1. The kinetic values for (4S)-OH-RA were Km = 5.2 ± 1.3 nM and kdep,max = 2.9 ± 0.3 min-
1. For (4R)-OH-RA the values were Km = 11.0 ± 5.2 nM and kdep,max = 1.5 ± 0.2 min-1.
Figure 6. The binding of atRA to CYP26A1 (a and b) and CYP3A4 (c and d). The
orientation of atRA within the energy minimized CYP26A1 homology model is shown from two
different angles in panels a and b. In panels c and d, only the 1TQN X-ray crystal structure of
15

CYP3A4 is shown for clarity. The atRA molecule bound to the 1TQN X-ray crystal structure is
highlighted, since it is roughly average from all the bound orientations which are shown semi-
transparent. Stick models of atRA (orange), interacting residues (green) and the heme (red) with
a cartoon (gray) representation of protein structures in the background. Only the interacting
amino acid side chains are shown.
16

Tables
Table 1. Michaelis-Menten kinetic parameters for (4S)-OH-RA and (4R)-OH-RA formation by
CYP3A isoforms
CYP3A4
CYP3A5
CYP3A7
(4S)-OH-RA (4R)-OH-RA (4S)-OH-RA (4R)-OH-RA (4S)-OH-RA (4R)-OH-RA
Km
(µM)
kcat
4.7 ± 0.6
5.1 ± 0.2
1.1
4.6 ± 1.1
5.3 ± 0.6
1.2
6.0 ± 1.2
3.6 ± 0.2
0.6
6.0 ± 1.1
7.0 ± 0.4
1.1
3.5 ± 1.3
5.1 ± 0.6
1.5
3.1 ± 0.6
2.7 ± 0.2
0.9
(pmol/min/pmol P450)
kcat /Km
(µL/min/pmol P450)
17

Table 2. Kd and distance parameters for docking of atRA to the available Crystal Structures of
CYP3A4.
PDB ID
Kd (µM)
Distance of
Distance of
4R-H from heme (Å)
4S-H from heme (Å)
1TQN
1W0E
1W0F
1W0G
2J0D
2V0M
3TJS
3UA1
3NXU
Average
0.93
0.93
0.93
3.00
2.15
0.30
0.40
13.52
0.67
7.3
5.3
4.3
8.2
6.3
4.0
5.3
9.9
4.0
11.3
16.5
12.0
3.1
9.1
8.1±4.2
16.4
11.9
3.5
9.5
8.3±3.9
2.54±3.97
18

Figure 1
O
R
O
methyl 4-oxo-retinoate
O
b
a
R = CH3
c
R = CH3
c
R = H
R = H
H
HO
H
OH
(-)-(4R)-OH-RA
d
(+)-(4S)-OH-RA
d
R = CH3
R = CH3
H
H
OR'
R'O
R' = dimethylaminocinnamoyl
R' = dimethylaminocinnamoyl

Figure 2.
a.
b.
c.
d.

Figure 3

Figure 4
a.
b.

Figure 5
(4S)-OH-RA
(4R)-OH-RA
a.
b.
d.
f.
c.
e.

Figure 6
b.
a.
c.
d.