Stereospecific reduction of 5β-reduced steroids by human ketosteroid reductases of the AKR (aldo-keto reductase) superfamily: Role of AKR1C1-AKR1C4 in the metabolism of testosterone and progesterone via the 5β-reductase pathway

Article abstract of DOI:10.1042/BJ20101804

Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in the liver to terminate hormone action. One main metabolic pathway, the 5β-pathway, involves 5β-steroid reductase (AKR1D1, where AKR refers to the aldo-keto reductase superfamily), which catalyses the reduction of the 4-ene structure, and ketosteroid reductases (AKR1C1-AKR1C4), which catalyse the subsequent reduction of the 3-oxo group. The activities of the four human AKR1C enzymes on 5β-dihydrotestosterone, 5β-pregnane-3,20-dione and 20α-hydroxy-5β-pregnan-3-one, the intermediate 5β-dihydrosteroids on the 5β-pathway of testosterone and progesterone metabolism, were investigated. Product characterization by liquid chromatography-MS revealed that the reduction of the 3-oxo group of the three steroids predominantly favoured the formation of the corresponding 3α-hydroxy steroids. The stereochemistry was explained by molecular docking. Kinetic properties of the enzymes identified AKR1C4 as the major enzyme responsible for the hepatic formation of 5β-tetrahydrosteroid of testosterone, but indicated differential routes and roles of human AKR1C for the hepatic formation of 5β-tetrahydrosteroids of progesterone. Comparison of the kinetics of the AKR1C1-AKR1C4-catalysed reactions with those of AKR1D1 suggested that the three intermediate 5β-dihydrosteroids derived from testosterone and progesterone are unlikely to accumulate in liver, and that the identities and levels of 5β-reduced metabolites formed in peripheral tissues will be governed by the local expression of AKR1D1 and AKR1C1-AKR1C3. The Authors Journal compilation 2011 Biochemical Society.

Full text of DOI:10.1042/BJ20101804

Biochem. J. (2011) 437, 53–61 (Printed in Great Britain) doi:10.1042/BJ20101804
53
Stereospecific reduction of 5β-reduced steroids by human ketosteroid
reductases of the AKR (aldo-keto reductase) superfamily: role of
AKR1C1–AKR1C4 in the metabolism of testosterone and progesterone via
the 5β-reductase pathway
Yi JIN, A. Clementina MESAROS, Ian A. BLAIR and Trevor M. PENNING1
*Department of Pharmacology and Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA
19104-6084, U.S.A.
Active sex hormones such as testosterone and progesterone
are metabolized to tetrahydrosteroids in the liver to terminate
hormone action. One main metabolic pathway, the 5β-pathway,
involves 5β-steroid reductase (AKR1D1, where AKR refers to the
aldo-keto reductase superfamily), which catalyses the reduction
of the 4-ene structure, and ketosteroid reductases (AKR1C1–
AKR1C4), which catalyse the subsequent reduction of the 3-oxo
group. The activities of the four human AKR1C enzymes on 5β-
dihydrotestosterone, 5β-pregnane-3,20-dione and 20α-hydroxy-
5β-pregnan-3-one, the intermediate 5β-dihydrosteroids on the
5β-pathway of testosterone and progesterone metabolism, were
investigated. Product characterization by liquid chromatography–
MS revealed that the reduction of the 3-oxo group of the three
steroids predominantly favoured the formation of the correspond-
ing 3α-hydroxy steroids. The stereochemistry was explained
by molecular docking. Kinetic properties of the enzymes
identified AKR1C4 as the major enzyme responsible for the
hepatic formation of 5β-tetrahydrosteroid of testosterone, but
indicated differential routes and roles of human AKR1C for
the hepatic formation of 5β-tetrahydrosteroids of progesterone.
Comparison of the kinetics of the AKR1C1–AKR1C4-catalysed
reactions with those of AKR1D1 suggested that the three
intermediate 5β-dihydrosteroids derived from testosterone
and progesterone are unlikely to accumulate in liver, and that
the identities and levels of 5β-reduced metabolites formed in
peripheral tissues will be governed by the local expression of
AKR1D1 and AKR1C1–AKR1C3.
Key words: dihydrosteroid, hydroxysteroid dehydrogenases,
liquid-chromatography–MS (LC–MS), steroid metabolism,
tetrahydrosteroid.
(pregnane X receptor) and the constitutive androstane receptor
[7]. Activation of the hepatic PXR leads to induction of CYP3A4
(cytochrome P450 3A4), which is responsible for the metabolism
of approximately 50% of drugs consumed. Additionally, 5β-
reduced pregnanes are important neuroactive steroids [8] and
have been implicated in vasorelaxation [9] and human parturition
[10,11].
Enzymes most implicated in the metabolism of 5α/β-DHS
to the corresponding isomeric 3α/β,5α/β-THS are the NADPH-
dependent HSDs (hydroxysteroid dehydrogenases) of the AKR1C
subfamily [12,13]. The four human isoforms are: AKR1C1 (also
known as 20α-HSD), AKR1C2 (also known as human type 3
3α-HSD), AKR1C3 (also known as human type 2 3α-HSD
and type 5 17β-HSD) and AKR1C4 (also known as human
type 1 3α-HSD). These enzymes are highly homologous in
sequence (identity >86%), but have different tissue expression
properties. All isoforms are abundantly expressed in liver
at comparable levels, but AKR1C4 is found only in liver,
whereas AKR1C1–3 are differentially expressed in the lung,
prostate, mammary gland and testis [13]. Individual human
AKR1C enzymes catalyse ketosteroid reduction at the 3-,
17-, or 20-position to varying degrees depending on the substrate.
Individual isoforms can reduce the same steroid with different
INTRODUCTION
The levels of active steroid hormones are tightly regulated through
a balance of synthesis and metabolism. Liver is the primary
site of metabolism for steroid hormones containing a ꢀ4-3-one
functionality, which include active hormones such as testosterone
and progesterone. These active hormones are converted into THS
(tetrahydrosteroids), which are conjugated in phase 2 reactions
leading to elimination [1,2]. The metabolic sequence for the
formation of THS involves the pathways shown in Scheme 1.
In the first step, the 4-ene moiety of the steroid is reduced
by 5α- or 5β-steroid reductases to form the respective DHS
(dihydrosteroids). In the subsequent step, the 3-oxo group of DHS
is reduced by ketosteroid reductases to form THS. 5α-Steroid
reductases and the 5α-pathway have been a focus since the potent
androgen 5α-DHT (5α-dihydrotestosterone, also called 17β-
hydroxy-5α-androstan-3-one) is formed from testosterone [3,4].
In contrast, less is known about the 5β-pathway since 5β-steroid
reductase, which is a member of the AKR (aldo-keto reductase)
family (see http://www.med.upenn.edu/akr), named as AKR1D1,
is historically believed to be only involved in steroid inactivation
[5,6]. It is now recognized that the 5β-reduced steroids are not
inactive. 5β-Pregnane-3,20-dione is a potent ligand for the PXR
Abbreviations used: AKR, aldo-keto reductase; APCI, atmospheric pressure chemical ionization; HSD, hydroxysteroid dehydrogenase; DHS,
dihydrosteroid(s); 5α/β-DHT, 5α/β-dihydrotestosterone; LC, liquid chromatography; MRM, multiple reaction monitoring; PXR, pregnane X receptor; THS,
tetrahydrosteroid(s).
1To whom correspondence should be addressed (email penning@upenn.edu).
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the 5β-pathway of progesterone metabolism, i.e. 5β-pregnane-
3,20-dione and 20α-hydroxy-5β-pregnan-3-one.
In the present study, we have characterized the stereochemical
outcomes and kinetic properties of the reduction of three
intermediate 5β-DHS, i.e. 5β-DHT, 5β-pregnane-3,20-dione
and 20α-hydroxy-5β-pregnan-3-one, catalysed by AKR1C1–4
enzymes. We demonstrate that the 3-oxo reduction of these
three 5β-reduced steroids by all of the human AKR1C enzymes
stereospecifically favoured the formation of 3α,5β-THS. Our
results support differential roles of human AKR1C enzymes in
the hepatic metabolism of active testosterone and progesterone.
In addition, the activities of AKR1C1–4 enzymes were compared
with that of AKR1D1 in the preceding step of the pathway (i.e.
the 5β-reduction). Results suggest that the intermediate 5β-DHS
are unlikely to accumulate in the liver and enter the circulation.
Local formation of bioactive 5β-reduced steroids would thus be
dependent upon the presence of AKR1D1 when it co-exists with
extrahepatic AKR1C1–3 isoforms.
Scheme 1 Pathways of steroid metabolism leading to THS formation (A)
and structures of the intermediate 5β-DHS in the pathways of testosterone
and progesterone metabolism (B)
S, steroid.
EXPERIMENTAL
Materials
stereochemical outcomes and the stereochemistry of the reaction
is often steroid-dependent [4,14,15]. Although the four human
AKR1C enzymes are implicated in reducing both 5α- and 5β-
DHS, only their involvement in the reduction of 5α-DHT has
been thoroughly investigated [4]. Incomplete kinetic data
have been reported for the reduction of 5β-reduced steroids by
human AKR1C enzymes [16–20], but there has not been any
rigorous metabolite identification (i.e. by MS). Nor has there been
any attempt to distinguish the role of individual human AKR1C
isoforms on reducing 5β-DHS in testosterone and progesterone
metabolism.
It is known that the major metabolites of testosterone include
androstanediol (or 5α-androstane-3α,17β-diol, a 3α,5α-THS)
and etiocholanolone (5β-androstane-3α,17β-diol, a 3α,5β-
THS) [1]. Differential stereochemical and kinetic behaviour
of human AKR1C enzymes established their different roles
in the formation of 5α-THS during testosterone metabolism
[4]. Specifically, with 5α-DHT, the intermediate steroid on
the 5α-pathway, AKR1C1 acts as a reductive 3β-HSD to yield
5α-androstane-3β,17β-diol as the main product, AKR1C2 and
AKR1C4 catalyse the efficient formation of 5α-androstane-
3α,17β-diol, and AKR1C3 catalyses the slow formation of
a mixture of 3α,5α- and 3β,5α-products. The liver-specific
expression of AKR1C4 limits this enzyme to the clearance of 5α-
DHT in that organ. However, AKR1C1 and AKR1C2 are believed
to be responsible for the formation of 5α-androstane-3β,17β-
diol [a pro-apoptotic ligand of ERβ (oestrogen receptor β)] and
5α-androstane-3α,17β-diol (an inactive androgen) respectively in
androgen target tissues, such as the prostate.
A major metabolite of the potent progestin progesterone is
pregnanediol (or 5β-pregnane-3α,20α-diol), which is a 3α,5β-
THS, indicating the importance of the 5β-pathway in the meta-
bolism of progesterone [1]. Progesterone metabolism via the
5β-pathway can occur similarly to testosterone metabolism, i.e.
progesterone is reduced to 5β-pregnane-3,20-dione by AKR1D1,
which in turn is reduced at the 3-oxo position by AKR1C1–
4 (Scheme 1). However, since human AKR1C enzymes also
have 20-ketoreductase activity, formation of 20-hydroxy steroids
is also possible. Thus, alternatively, progesterone can be first
reduced at the 20-oxo position to form 20α-hydroxyprogesterone,
a reaction that is known to be catalysed by AKR1C1 [13]. 20α-
Hydroxyprogesterone can then be reduced by AKR1D1 to form
20α-hydroxy-5β-pregnan-3-one, which in turn can be reduced by
AKR1C1–4 enzymes to isomeric 5β-pregnane-3,20-diols. Thus,
for progesterone, there are two possible intermediate 5β-DHS on
All steroids were obtained from Steraloids. Pyridine nucleotides
were purchased from Roche Applied Science. All other
reagents were purchased from Sigma–Aldrich and were
of ACS (American Chemical Society) grade or better.
Recombinant AKR1C1–4 enzymes were overexpressed and
purified to homogeneity as described previously [21,22]. Under
standard assay conditions, the specific activities of AKR1C1–3
were determined to be 1.8 μmol/min per mg for AKR1C1,
2.3 μmol/min per mg for AKR1C2 and 1.3 μmol/min per mg
for AKR1C3 for the NADP+ -dependent oxidation of 0.2 mM
1-acenaphthenol, the specific activity of AKR1C4 was
0.3 μmol/min per mg for the NAD+ -dependent oxidation of
75 μM androsterone and the specific activity of AKR1D1 was
80 nmol/min per mg for the NADPH-dependent reduction of
10 μM testosterone [14,21,23].
LC (liquid chromatography)–MS analysis
Products for the reduction of ketosteroids catalysed by AKR1C1–
4 enzymes were prepared as follows. Reaction mixtures contained
100 mM potassium phosphate buffer (pH 7.0), 20 μM steroid,
0.5 mM NADPH and 4% methanol in a total volume of 500 μl.
The reaction was initiated by the addition of purified enzyme
(7.3 μg of AKR1C1, 9.0 μg of AKR1C2, 12.3 μg of AKR1C3 or
5.6 μg of AKR1C4), and for the no-enzyme control buffer was
substituted. Systems were incubated at 37◦C and incubation times
were 20 min and 90 min for the enzymatic reactions and 90 min
for non-enzymatic controls. Reaction mixtures were extracted
twice with 1 ml of water-saturated ethyl acetate. The pooled
organic extracts were vacuum-dried and the residues were re-
dissolved in 200 μl of methanol.
Normal-phase chiral chromatography for LC–MS experiments
was performed using a Waters Alliance 2690 HPLC system.
Gradient elution was performed in the linear mode. A Chiralpak
IA column (250 mm long × 4.6 mm diameter, internal diameter
5 μm; Daicel Chemical Industries) was employed with a flow rate
of 1 ml/min. Solvent A was hexanes (Fisher, Optima grade) and
solvent B was ethanol/hexanes (1:1, v/v). A linear gradient was
used starting with 98.5% solvent A to 50% solvent A in 17 min
and maintained for 5 min to elute all of the polar compounds
and prevent overpressure. At 25 min the gradient was changed to
98.5% solvent A and kept at 98.5% to re-equilibrate the column
to the initial conditions. The separation was performed at 30◦C.
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Reduction of 5β-reduced steroids by human AKR1C enzymes
55
MS was conducted using a Finnigan TSQ Quantum Ultra
AM spectrometer (Thermo Fisher) equipped with an APCI
(atmospheric pressure chemical ionization) source operated in
the positive-ion mode. Full-scan analysis was conducted in Q3
only in the mass interval 150–400. For MRM (multiple reaction
monitoring) analyses, unit resolution was maintained for both
parent and product ions. Operating conditions were as follows:
vaporizer temperature 450◦C, heated capillary temperature
300◦C with a discharge current of 6 μA applied to the corona
needle, and nitrogen was used for the sheath gas, auxiliary
gas and ion sweep gas, set at 25, 5 and 3 (in arbitrary units)
respectively. CID (collision-induced dissiociation) was performed
using argon as the collision gas at 1.5 mTorr in the second
[r.f. (radio frequency)-only] quadrupole. An additional D.C.
offset voltage was applied to the region of the second multipole
ion guide (Q0) at 10 V to impart enough translational kinetic
energy to the ions so that solvent adduct ions dissociate to
form sample ions. Full scanning analyses were performed in the
range of m/z 100 to m/z 600. Products of the reactions were
identified on the basis of their LC retention times and mass
spectra relative to those observed with the authentic standards.
LC–APCI/MRM/MS analyses were conducted using m/z 291
([M + H]+ )→273 ([M + H–H2O]+ ) (collision energy, 26 eV)
for 5β-DHT; m/z 275 ([M + H–H2O]+ )→259 ([M + H–2H2O]+ )
(collision energy, 26 eV) for 5β-androstane-3α/β,17β-diol;
m/z 317 ([M + H]+ )→299 ([M + H–H2O]+ ) (collision energy,
23 eV) for 5β-pregnane-3,20-dione; m/z 301([M + H–H2O]+ )
→283 ([M + H–H2O]+ ) (collision energy, 23 eV) for 3α/β-
hydroxy-5β-pregnan-20-one and 20α/β-hydroxy-5β-pregnan-3-
one; and m/z 303 ([M + H–H2O]+ )→285 ([M + H–H2O]+ )
(collision energy, 23 eV) for 5β-pregnane-3α/β,20α/β-diols.
or eqn (2):
kcat[E]
v =
(2)
1 + (Km/[S]) + [S]/Ki
where v is the initial velocity, [E] and [S] are the total molar
concentrations of the enzyme and steroid substrate respectively,
kcat (s−1) is the turnover number, Km (μM) is the apparent
Michaelis–Menten constant for the steroid substrate and Ki (μM)
is the apparent dissociation constant for the presumed inhibitory
enzyme–substrate complex [24].
Molecular modelling
Conformations of 5α-DHT bound to AKR1C1 (PDB code 1MRQ)
and AKR1C2 (PDB code 1IHI) found previously using the auto-
mated docking program AUTODOCK were used as the templates
to model the binding of 5β-DHT [25]. The structure of 5β-
DHT was retrieved from the co-ordinates of the ternary complex
AKR1D1–NADP+ –5β-DHT (PDB code 3DOP). Two templates
were used for the manual docking of 5β-DHT. Template 1 is
the productive docking position of 5α-DHT in AKR1C2 for the
formation of the 3α-reduced product, i.e. the β-face of the steroid
is presented to NADPH. Template 2 is the productive docking
position of 5α-DHT in AKR1C1 for the formation of the 3β-
reduced product, i.e. the α-face of the steroid is presented to
NADPH. Docking of 5β-DHT was performed as follows. With
each template, 5β-DHT was manually docked in position by
overlaying the 3-oxo group of 5β-DHT to that of the template.
The docking of 5β-DHT on to the template was examined with
the steroids in a similar facial orientation and also in a reversed
facial orientation. Positions of 5β-DHT were then adjusted to
avoid steric clashing with the surrounding residues if possible
without significantly changing the position and orientation of the
3-oxo group. The four docked models were evaluated on the
basis of distances to surrounding residues as either ‘allowed’ or
‘disallowed’ (<2 Å; 1 Å = 0.1 nm).
TLC analysis
Samples were prepared in
a
similar fashion as for
LC–MS analysis and applied to Partisil LK6D Silica
TLC plates (Whatman). Chromatograms were developed in
cyclohexane/ethyl acetate (5:13, v/v). Products were identified
by co-migration with authentic standards applied to the same
plate following visualization by spraying with acetic acid/sulfuric
acid/anisaldehyde (100:2:1, by vol.) and heat.
RESULTS
Reduction of 5β-DHT catalysed by AKR1C1–4
The 3-oxo reduction of 5β-DHT can yield two possible products,
5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol.
Both synthetic standards are available and could be well separated
by TLC (results not shown) and LC–MS methods (Figure 1).
The NADPH-dependent reductions of 5β-DHT catalysed by
AKR1C1–4 were analysed. All reactions were run to completion,
where the relative intensities of the 5β-DHT peak in samples
containing AKR1C1–4 were less than 2% of the 5β-DHT peak
in the non-enzymatic control sample. In all samples containing
AKR1C enzymes, 5β-androstane-3α,17β-diol was found to be
the predominant product. Only with AKR1C2 did the reaction
yield a low but significant amount of 5β-androstane-3β,17β-diol
(11.4% of the total products) (Table 1). In contrast, reaction
samples with AKR1C1, AKR1C3 and AKR1C4 contained less
than 1.5% of the total products as 5β-androstane-3β,17β-diol.
No significant epimerase activity was observed with AKR1C3 and
AKR1C4, where epimerase activity was seen during the reduction
of 5α-DHT and 5α-DHT-17β-sulfate by these enyzmes [4,14].
Individual human AKR1C enzymes are known to have different
kinetic properties with the same steroid substrate [4,13–15].
This was also true with 5β-DHT. kcat and Km values for the
reduction of 5β-DHT catalysed by AKR1C1–4 isoforms were
determined by continuous spectrofluorimetric assays where the
Determination of steady-state kinetic parameters
Initial rates of the NADPH-dependent reduction of ketosteroids
catalysed by AKR1C1–4 enzymes were measured with a Hitachi
F-2500 fluorescence spectrophotometer by monitoring the change
in fluorescence emission of NADPH. λex and λem wavelengths
were set at 340 nm and 450 nm respectively. Changes in
fluorescence units were converted into nanomoles of cofactor by
using standard curves of fluorescence emission against known
NADPH concentrations. Typical reaction samples contained
enzyme (1.4 μg of AKR1C1, 1.2 μg of AKR1C2, 1.6 μg of
AKR1C3, 0.8 μg of AKR1C4 or 2.1 μg of AKR1D1), NADPH
(saturating concentration 8 μM or 25 μM), various steroid
concentrations (0.1 μM–50 μM), 100 mM potassium phosphate
buffer (pH 7.0) and 4% methanol in a total volume of 1 ml.
Reaction mixtures were run at 25◦C. Analysis of kinetic data
was carried out as described previously [14]. Data were fitted by
non-linear least-squares regression to eqn (1):
kcat[E][S]
v =
(1)
Km + [S]
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Table 1 Product distribution for the reduction of 5β-DHT and 5β-pregnane-
and a Km that was 26 times higher compared with AKR1C4. The
kinetic behaviours of AKR1C2 and AKR1C3 were similar for the
reduction of 5β-DHT, both showing a low catalytic efficiency and
moderate substrate inhibition.
3,20-dione catalysed by human AKR1C enzymes
For 5β-DHT reduction, 3α- and 3β- products were 5β-androstane-3α,17β-diol and
5β-androstane-3β,17β-diol.Therewasnosignificantdifferenceinproductdistributionbetween
samples from 20 min and 90 min incubations. For 5β-pregnane-3,20-dione reduction, 3α-
and 3α,20α- products were 3α-hydroxy-5β-pregnan-20-one and 5β-pregnane-3α,20α-diol.
Values are shown as percentage of total.
Reduction of 5β-pregnane-3,20-dione and
20α-hydroxy-5β-pregnan-3-one catalysed by AKR1C1–4
Substrate . . . 5β-DHT
Product. . . 3α-
5β-pregnane-3,20-dione
5β-Pregnane-3,20-dione and 20α-hydroxy-5β-pregnan-3-one are
the intermediate 5β-DHS on the 5β-pathway of progesterone
metabolism, which are subject to metabolic transformation by
AKR1C enzymes for the formation of 5β-THS.
For 5β-pregnane-3,20-dione, both the 3- and 20-oxo groups
can potentially be reduced by AKR1C enzymes. There are
eight possible products for the reaction, which include four
monohydroxy products (3α-hydroxy-5β-pregnan-20-one, 3β-
hydroxy-5β-pregnan-20-one, 20α-hydroxy-5β-pregnan-3-one
and 20β-hydroxy-5β-pregnan-3-one) and four dihydroxy
products (5β-pregnane-3α,20α-diol, 5β-pregnane-3β,20α-diol,
Enzyme
3β-
3α-
3α,20α-
AKR1C1
AKR1C2
AKR1C3
AKR1C4
98.5
88.6
99.6
99.3
1.5
11.4
0.4
14.2
66.2
76.6
22.4
85.8
33.8
23.4
77.6
0.7
5β-pregnane-3α,20β-diol
and
5β-pregnane-3β,20β-diol).
Synthetic standards for all possible products were available
for product authentication by TLC and LC–MS. In LC–MS
analysis, 5β-pregnane-3α,20β-diol and 5β-pregnane-3β,20β-
diol could not be separated. However, lack of separation of
these two standards by LC–MS had no effect on the results of
the experiment, since no product peaks around their retention
times were detected in the reaction samples. The retention time
of 5β-pregnane-3α,20α-diol was close to 20β-hydroxy-5β-
pregnan-3-one in LC–MS analysis; however, they could be well
separated with TLC.
It was found by LC–MS analysis and TLC that, for the reduction
of 5β-pregnane-3,20-dione catalysed by AKR1C enzymes, 3α-
hydroxy-5β-pregnan-20-one and 5β-pregnane-3α,20α-diol were
detected as the main products at the end point of the reaction
(Figure 2). The distribution between the two products for
each AKR1C1–4 enzyme varies, with 5β-pregnane-3α,20α-
diol as the major product for the AKR1C1 and AKR1C4
reactions and 3α-hydroxy-5β-pregnan-20-one as the major
product for AKR1C2 and AKR1C3 (Table 1). Interestingly,
significant transient formation of 20α-hydroxy-5β-pregnan-3-one
was observed at earlier time points of the 5β-pregnane-3,20-
dione reaction catalysed by AKR1C1 (Supplementary Figure S2
at http://www.BiochemJ.org/bj/437/bj4370053add.htm). Thus, in
the reaction catalysed by AKR1C1, two monohydroxy products,
3α-hydroxy-5β-pregnan-20-one and 20α-hydroxy-5β-pregnan-
3-one, were formed in comparable amounts, each of which
was subsequently further converted into 5β-pregnane-3α,20α-
diol. In contrast, only one monohydroxy product, 3α-hydroxy-
5β-pregnan-20-one, was observed in the reaction catalysed by
AKR1C2–4. The distribution between the the monohydroxy and
dihydroxy products suggests that formation of the dihydroxy
product was much slower for AKR1C2 and AKR1C3 than for
AKR1C4.
Figure 1 Product characterization by LC–MS for the reduction of 5β-DHT
catalysed by human AKR1C isoforms
(A) Ion chromatograms (m/z 250–320) of authentic standards. 3α-ol, 5β-androstane-
3α,17β-diol; 3β-ol, 5β-androstane-3β,17β-diol. (B–F) Corresponding ion chromatograms
of reaction samples containing no enzyme (B) and AKR1C1–4 (C–F). Samples were prepared as
described in the Experimental section. The disappearance of substrate in each reaction sample
containing enzyme indicated that the reaction had reached the end point.
For the reduction of 20α-hydroxy-5β-pregnan-3-one catalysed
by AKR1C1–4 enzymes, there are two possible products: 5β-
pregnane-3α,20α-diol and 5β-pregnane-3β,20α-diol. Product
identification by LC–MS and TLC showed only the formation
of 5β-pregnane-3α,20α-diol for the reaction catalysed by all four
AKR1C enzymes.
Kinetic properties for the reductions of 5β-pregnane-3,20-
dione and 20α-hydroxy-5β-pregnan-3-one catalysed by each
AKR1C1–4 enzyme are shown in Table 2 (also see Supplementary
Figure S1). With 5β-pregnane-3,20-dione as the substrate,
AKR1C4 was moderately more efficient: its catalytic efficiency
was 1.5-, 2.4- and 5.4-fold higher than those of AKR1C1,
depletion of NADPH was monitored (see the Supplementary
Figure S1 at http://www.BiochemJ.org/bj/437/bj4370053add.htm
for representative results). Thus, for the reduction of 5β-DHT
catalysed by AKR1C2, the activity measured represented the
total activity, since a mixture of stereoisomeric products were
formed. Results are shown in Table 2 (see Supplementary Figure
S1 for representative results). AKR1C4 was found to be the most
efficient enzyme for the reduction of 5β-DHT with the highest
kcat and the lowest Km values. The catalytic activity of AKR1C1
displayed a kcat value that was approximately three times lower
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57
Figure 3 Docking of 5β-DHT into AKR1C active sites
Details of the model building are given in the Experimental section. Residues in the enzyme
active sites are shown in blue. The cofactor is shown in purple, and the 5α-DHT template
is shown in white. Docked 5β-DHT is shown in green (sterically allowed position) or red
(sterically disallowed position). In the upper panels, the 5α-DHT template was positioned for
3α-reduction in AKR1C2. Upper left-hand panel, 5β-DHT docked on the template position so
that the facial orientation of the A-ring was the same. Upper right-hand panel, 5β-DHT docked
on the template position so that the facial orientation of the A-ring was inverted. In the lower
panels, the 5α-DHT template was positioned for 3β-reduction in AKR1C1. Lower left-hand
panel, 5β-DHT docked on the template position so that the facial orientation of the A-ring was
the same. Lower right-hand panel, 5β-DHT docked on the template position so that the facial
orientation of the A-ring was inverted. The Figures were prepared using Swiss-PDBViewer.
Figure 2 Product characterizations by LC–MS for the reduction of 5β-
pregnane-3,20-dione catalysed by human AKR1C isoforms
(A) and (B) Ion chromatograms (m/z 250–320) of authentic standards. 3,20-dione, 5β-
pregnane-3,20-dione; 3α-ol, 3α-hydroxy-5β-pregnan-20-one; 3β-ol, 3β-hydroxy-5β-
pregnan-20-one; 20α-ol, 20α-hydroxy-5β-pregnan-3-one; 20β-ol, 20β-hydroxy-5β-
pregnan-3-one; 3α,20α-diol, 5β-pregnane-3α,20α-diol; 3β,20α-diol, 5β-pregnane-3β,
20α-diol; 3α,20β-diol, 5β-pregnane-3α,20β-diol; 3β,20β-diol, 5β-pregnane-3β,20β-diol.
(C–G)Ionchromatogramsofreactionsamplescontainingnoenzyme(C)andAKR1C1–4(D–G).
Samples were prepared as described in the Experimental section. The disappearance of substrate
in each reaction sample containing enzyme indicated that the reaction had reached the end point.
Reduction of testosterone, progesterone and
20α-hydroxyprogesterone catalysed by AKR1D1
Kinetic properties for the reduction of testosterone, progesterone
and 20α-hydroxyprogesterone catalysed by AKR1D1 were
examined (Table 2). The common feature of these reactions
was the observation of potent substrate inhibition. For both
testosterone and 20α-hyroxyprogesterone, Ki values were
determined to be significantly lower than their respective Km
values. Thus the observed reaction rates were significantly lower
than their estimated Vmax values. In the reaction of progesterone,
strong substrate inhibition was also observed. The data point
of the lowest progesterone concentration displayed the highest
reaction rate, so the kinetic parameters for the reaction could
not be accurately determined. The rate constant reported was
calculated on the basis of highest observed rate.
AKR1C2 and AKR1C3 respectively. This difference in catalytic
efficiency stemmed mostly from different kcat values, since similar
Km values were obtained for AKR1C1–4. With the exception
of AKR1C4, low degrees of substrate inhibition (Ki/Km>16)
were observed for the reduction of 5β-pregnane-3,20-dione
catalysed by AKR1C1–3. With 20α-hydroxy-5β-pregnan-3-one
as the substrate, the differences in catalytic efficiency between
AKR1C4 and AKR1C1–3 were more pronounced such that the
catalytic efficiency of AKR1C4 was 8.9-, 12- and 15-fold higher
than those of AKR1C1, AKR1C2 andAKR1C3 respectively. Low
degrees of substrate inhibition (Ki/Km>15) were also observed
for the reduction of 20α-hydroxy-5β-pregnan-3-one catalysed by
AKR1C1, AKR1C3 and AKR1C4, but not for that by AKR1C2.
Kinetic properties for the reduction of 3α-hydroxy-5β-
pregnan-20-one were also examined to compare the 20-oxo
reduction activity of AKR1C enzymes with the 3-oxo reduction
activity of 5β-steroids (Table 2). As expected, AKR1C1 displayed
the highest kcat value. Activities of AKR1C2 and AKR1C3 for this
reaction were low. Although the value of kcat for AKR1C4 was
4-fold lower than that for AKR1C1, a relatively smaller Km of
AKR1C4 resulted in a slightly better catalytic efficiency for the
enzyme than AKR1C1.
Molecular modelling of 5β-DHT binding in AKR1C1–4
Using an automated docking method we have previously found
‘productive’ docked positions of 5α-DHT in AKR1C1 and
AKR1C2 to account for the stereospecific formation of 3β-
and 3α-reduced products by the two enzymes respectively [25].
These productive positions of 5α-DHT were used as templates
to model 5β-DHT binding. 5β-DHT was manually docked in
the productive position of 5α-DHT in AKR1C2 using a similar
facial orientation. The resulting model placed the 3-oxo group
in position for a reaction in which the β-face of the steroid
faces the 4-pro-R-hydride of the cofactor (Figure 3, upper left-
hand panel). This model was deemed as ‘allowed’ since no steric
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58
Y. Jin and others
Table 2 Kinetic parameters for the reactions catalysed by AKR in the 5β-pathway for the metabolism of testosterone and progesterone
+
Steady-state parameters were determined using a fluorimetric assay. Values are given as means S.D. (n>2). n.a., not available.
−
Substrate (reaction)
Enzyme
kcat (min− 1
)
Km (μM)
kcat/Km (min− 1 · M− 1) × 106
Ki (μM)
+
+
5β-DHT (3-oxo reduction)
AKR1C1
AKR1C2
AKR1C3
AKR1C4
AKR1C1
AKR1C2
AKR1C3
AKR1C4
AKR1C1
AKR1C2
AKR1C3
AKR1C4
AKR1C1
AKR1C2
AKR1C3
AKR1C4
AKR1D1
AKR1D1
AKR1D1
1.7 0.1
0.9 0.1
5.2 0.6
0.8 0.1
0.33
1.1
0.8
23.5
10.8
6.7
3.0
16.3
1.6
–
−
−
+
+
+
−
4.0 0.2
5.5 1.0
–
24
4.7 1.2
5.7 1.3
–
28.5 0.9
–
26
8.2 2.5
−
−
−
+
+
+
1.2 0.2
1.4 0.3
−
−
+
+
4.7 0.3
0.2 0.05
−
−
+
+
+
5β-Pregnane-3,20-dione (3- and 20-oxo reduction)
20α-Hydroxy-5β-pregnan-3-one (3-oxo reduction)
3α-Hydroxy-5β-pregnan-20-one (20-oxo reduction)
4.3 0.2
0.4 0.1
3
−
−
−
+
+
+
2.0 0.2
0.3 0.1
−
−
−
+
+
+
0.9 0.3
0.3 0.1
−
−
−
+
+
4.9 0.1
0.3 0.06
−
−
+
+
+
2.1 0.1
1.3 0.1
−
−
−
+
+
0.6 0.1
0.5 0.1
1.2
−
−
+
+
+
1.6 0.3
1.7 0.2
0.94
14.3
2.9
0.75
0.31
3.8
3.9
n.a.
8.8
3
−
−
−
+
+
+
4.3 0.6
0.3 0.1
−
−
−
+
+
4.0 0.2
1.4 0.2
–
−
−
+
+
+
0.9 0.1
1.2 0.4
22 10
–
–
0.4 0.2
n.a.
0.06 0.02
−
−
−
+
+
0.4 0.04
1.3 0.2
−
−
+
+
1.5 0.05
0.4 0.07
−
−
+
+
+
Testosterone (5β-reduction)
Progesterone (5β-reduction)*
20α-Hydroxyprogesterone (5β-reduction)
4.7 2.1
1.2 0.7
−
−
−
+
(0.21 0.03)
(< 0.1)
−
+
+
+
12.3 3.1
1.4 0.4
−
−
−
*For the reduction of progesterone catalysed by AKR1D1, the observed maximum rate constant was reported.
conflict with surrounding residues were observed. Using the same
template, the facial orientation of the 5β-DHT was inverted to
make the α-face of the steroid presented to the cofactor for 3β-
HSD reaction (Figure 3, upper right-hand panel). However, the
resulting model is clearly disallowed since the bent structure in
5β-DHT caused the steroid nucleus to clash with the side chain
of Trp227. Similar docking experiments were performed using the
productive position of 5α-DHT in AKR1C1 as the template (the
steroid was positioned for the formation of 3β-reduced product)
(Figure 3, lower panels). 5β-DHT could not be accommodated
in the position of this template in either facial orientation. Our
docking experiments revealed that the steroid-binding channel of
all human AKR1C enzymes is narrow between residues 54 and
227, and cannot accommodate a 5β-steroid in positions that lead
to a 3β-HSD reaction. Of the four isoforms, only AKR1C2 has a
valine residue at position 54, whereas the others have the slightly
bulky Leu54. This would explain why 5β-androstane-3β,17β-diol
was observed as a minor product only with AKR1C2.
Stereospecificity of 3-oxoreduction of 5β-DHS catalysed by human
AKR1C isoforms
The 3-oxo reduction of 5β-DHT, 5β-pregnane-3,20-dione
and 5β-pregnan-20α-3-one catalysed by all four human
AKR1C enzymes yielded respective steroid products in the
3α-configuration as the sole or predominant product. This
stereospecificity is in stark contrast with the previous observation
that for the 3-oxo reduction of 5α-DHT and the hormone-
replacement therapeutic tibolone (a ꢀ5-3-one steroid) the
distribution between 3α- and 3β-reduced products can vary
significantly with each enzyme–substrate combination [4,15].
A comparison between 5α-DHT and 5β-DHT as substrate for
AKR1C1–4 enzymes highlights the effect of the configuration
of A/B-ring junction on stereochemical outcome of 3-oxo
reduction. When 5α-DHT was used as the substrate, the reactions
catalysed by AKR1C1, 3 and 4 enzymes yielded a mixture of
3α- and 3β-hydroxy androstanediols in comparable amounts
(the ratios between the two products were 1:2.9, 1.6:1 and
3.6:1 respectively), whereas the reaction catalysed by AKR1C2
generated the 3α-reduced steroid as the predominant product
(the 3α/3β ratio was 20:1). In contrast, when 5β-DHT was
used as the substrate, the reactions catalysed by all four
isoforms predominantly formed the 3α-reduced steroid (the
3α/3β ratios were 66:1, 7.8:1, 271:1 and 137:1 respectively).
Again the AKR1C2 reaction was an exception in that a small but
significant amount of 3β-product was now observed. These results
confirmed that the structure of the A-ring significantly affects the
stereochemical outcome of the reaction.
DISCUSSION
Active sex hormones such as testosterone (a potent androgen)
and progesterone (a potent progestin) are extensively metabolized
to THS in liver for the termination of hormone action. This is
achieved by the sequential reduction of the 4-ene moiety and
the 3-oxo group (Scheme 1). The four human AKR1C enzymes
are implicated to perform reactions following either 5α-reductase
or AKR1D1 to form 5α- and 5β-THS products respectively.
Historically, 5β-reduced steroids have been mostly regarded as
inactive steroids and thus received less attention than their 5α-
reduced counterparts. The formation of 5β-reduced steroids is
inarguably catabolically important since it is believed to account
for two-thirds of the mass of steroids that are inactivated [26].
In the present study, the role of individual AKR1C1–4 enzymes
on the 5β-pathways of testosterone and progesterone metabolism
was elucidated.
Molecular modelling of the binding of 5β-DHT provided
the structural basis for the effect of A-ring structure on
stereochemistry. 5β-Reduced steroids are structurally distinct in
that the 5β-configuration causes a 90◦ bend in the A/B-ring
junction. It was found that a steroid with a bent structure can
only be accommodated without steric clashing when its β-face is
presented to the cofactor so that only the 3α-axial alcohol will
form. This restriction is largely caused by the narrow channel
formed between the side chains of residues 54 and 227 on the
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Reduction of 5β-reduced steroids by human AKR1C enzymes
59
opposite sides of the steroid-binding pocket. The steroid is less
restricted in AKR1C2 since the enzyme has a less bulky side chain
at position 54 compared with other isoforms.
Substrate preferences of AKR1C1–4
All four human AKR1C enzymes are able to catalyse ketosteroid
reduction at 3-, 17-, and 20-positions of steroids [13]. AKR1C1 is
considered mainly as a 20-ketoreductase on the basis of its pref-
erence to reduce progesterone, whereas AKR1C2 and AKR1C4
are efficient 3-oxosteroid reductases and AKR1C3 acts as a 17-
ketosteroid reductase. Our results in the present study indicate
that these assignments can change on the basis of the substrate
examined. When 5α- and 5β-DHT are compared as substrates,
with the exception of AKR1C2, human AKR1C enzymes in
general displayed higher turnover numbers with the 5β-reduced
steroid substrates. Increased reactivity with 5β-reduced steroids
at the 3-position resulted in comparable reactivity for reduction
at the 3-position and the 20-position for AKR1C1. AKR1C2,
however, is a poor enzyme with 5β-reduced steroids for reaction
at either the 3- or 20-positions, due to its decreased activity for 3-
oxo reduction. Interestingly, AKR1C3 displayed better turnover
numbers for 3-ketoreduction (5β-DHT and 20α-hydroxy-5β-
pregnan-3-one) than that for 20-ketoreduction (3α-hydroxy-5β-
pregnan-20-one), and these turnover numbers were also higher
than that for its known 17-ketoreduction activity [13]. AKR1C4
retains its strong preference for reaction at the 3-position
with 5β-reduced substrates as seen with 5α-reduced substrates.
These substrate preferences suggest different roles of individual
AKR1C1–4 enzymes in systemic (liver) and peripheral steroid
hormone metabolism.
Scheme 2 Proposed roles of AKR enzymes on the 5β-pathways of (A)
testosterone and (B) progesterone metabolism
different ligand may be responsible for the activation of PXR in
this organ.
Expression of human AKR1C isoforms in fresh
liver biopsies were found to follow the rank order of
AKR1C4>AKR1C1>>AKR1C2>AKR1C3 [29]. In addition,
AKR1C4 has displayed the highest catalytically efficiency
for 3-oxo reduction with almost all steroid substrates studied
previously [4,13,14]. This is also the case with 5β-DHS
studied in the present work, although the difference is more
pronounced with 5β-DHT and 20α-hydroxy-5β-pregnan-3-one,
but less so with 5β-pregnane-3,20-dione. Thus, being the most
abundant and catalytically efficient enzyme, AKR1C4 is set
to be the most important 3-ketoreductase for the formation
of 3α,5α/β-THS in this organ. More specifically for the 5β-
metabolic pathways, AKR1C4 is clearly the principal enzyme
responsible for the formation of 3α,5β-THS of testosterone,
whereas all AKR1C1–4 isoforms are likely to contribute to the
formation of 3α-hydroxy-5β-pregnan-20-one and 5β-pregnane-
3α,20α-diol from 5β-pregnane-3,20-dione and the formation of
5β-pregnane-3α,20α -diol from 20α-hydroxy-5β-pregnan-3-one
(Scheme 2).
Implications for 5β-THS formation in liver
AKR1D1 is known to be predominantly expressed in liver [27] and
is now shown to efficiently catalyse the reduction of testosterone,
progesterone and 20α-hydroxyprogesterone. Strikingly, all three
reactions displayed potent substrate inhibition. With testosterone
and 20α-hydroxyprogesterone, the values of Ki, estimated by
fitting the data to the kinetic equation with a substrate inhibition
term, were significantly less than their respective values of Km. As
a result of the potent substrate inhibition, the value of kcat predicted
by data analysis could not be effectively reached in these reactions.
In fact, the observed maximum rate constants were 5- and 10-fold
lower than their predicted kcat values for testosterone and 20α-
hydroxyprogesterone respectively. With progesterone, values of
kcat and Km could not be accurately determined; the available
data indicated strong substrate inhibition that resulted in low
observed reaction rates. When the kinetics of the step catalysed
by AKR1D1 were compared with the subsequent step catalysed by
AKR1C1–4, the catalytic efficiencies of AKR1D1 were found
to be much lower than those of AKR1C4, which displayed
the highest catalytic efficiency among human AKR1C isoforms
for 3-oxo reduction with the 5β-reduced substrates. This would
suggest that the formation of 5β-DHS (the reaction catalysed
by AKR1D1) occurs less efficiently than the disappearance of
5β-DHS (the reaction catalysed by AKR1C1–4 enzymes). Taken
together, it appears that the intermediate 5β-DHS metabolites
are prevented from accumulating in the liver by strong substrate
inhibition in the AKR1D1 reaction and by the fast reaction
catalysed by AKR1C4 leading to their metabolism to 5β-THS.
This is consistent with the observation of very low levels of 5β-
pregnane-3,20-dione in circulation [28]. Since the build-up of
5β-pregnane-3,20-dione is predicted not to occur in the liver, a
Implications for peripheral formation of 5β-reduced steroids
In liver, 5β-DHS are unlikely to accumulate to high levels and
enter the circulation, since they would be rapidly metabolized
by AKR1C4. However, 5β-DHS may accumulate in tissues
in which AKR1D1 and AKR1C1–3 are co-expressed since
these extrahepatic human AKR1C isoforms displayed lower
catalytic efficiencies for 5β-THS formation. The expression
levels of AKR1D1 and AKR1C1–3 in a given peripheral
tissue will control the identity and level of the 5β-reduced
steroid metabolites formed. 5β-Reduced steroids are increasingly
being recognized as active compounds with important regulating
functions [8,9,11,30,31]. It is most likely that the active 5β-
steroids are formed locally by AKR1D1 and AKR1C1–3
enzymes. Indeed, AKR1D1 has been found to be highly expressed
in the placenta and myometrium where 5β-pregnane-3,20-dione
has been implied to act as a tocolytic hormone and prevent
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60
Y. Jin and others
10 Sheehan, P. M. (2006) A possible role for progesterone metabolites in human parturition.
Aust. N.Z. J. Obstet. Gynaecol. 46, 159–163
11 Sheehan, P. M., Rice, G. E., Moses, E. K. and Brennecke, S. P. (2005)
5β-Dihydroprogesterone and steroid 5β-reductase decrease in association with human
parturition at term. Mol. Hum. Reprod. 11, 495–501
parturition [11]. 5β-Pregnane-3,20-dione can decrease uterine
sensitivity to the uterotonic peptide hormone oxytocin by binding
directly to the uterine oxytocin receptor [30], and also can act
through PXR in regulating uterine contractility [31].
It is well established that progesterone and its metabolites
are important neurosteroids. 3α-hydroxy-5β-pregnan-20-one and
its 5α-isomer 3α-hydroxy-5α-pregnan-20-one are highly potent
positive modulators of the GABAA receptor and exert differential
effects on the GABAC receptor (where GABA is γ -aminobutyric
acid) [8,32]. It is believed that biosynthesis of these steroids occurs
in the central nervous system. Although it is not known whether
AKR1D1 is expressed in human brain, expression and activities
of 5α-reductase and AKR1C1–3 in human brain have been
demonstrated previously [33,34]. However, 5β-reductase and its
products have been found in the quail brain, and progesterone
was found to be metabolized to 5β-pregnane-3,20-dione and 3α-
hydroxy-5β-pregnan-20-one in the brains of 1-day-old chicks
[35,36]. Thus it is highly probable that thses two 5β-pregnane
steroids are also formed locally in human brain by AKR1D1 and
AKR1C1–3.
AKR expression can be regulated by factors such as hormonal
status and oxidative stress [37,38], which may explain the
changes in circulating levels of progesterone metabolites during
female menstrual cycle and pregnancy [28,39]. Dysregulation in
AKR expression may also contribute to the abnormal plasma
concentrations of neurosteroids observed in diseases such as the
premenstrual dysphoric disorder [40], depression disorder [41]
and chronic fatigue syndrome [42].
12 Penning, T. M. (2003) Hydroxysteroid dehydrogenases and pre-receptor regulation of
steroid hormone action. Hum. Reprod. Update 9, 193–205
13 Penning, T. M., Burczynski, M. E., Jez, J. M., Hung, C. F., Lin, H. K., Ma, H., Moore, M.,
Palackal, N. and Ratnam, K. (2000) Human 3α-hydroxysteroid dehydrogenase isoforms
(AKR1C1–AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and
tissue distribution reveals roles in the inactivation and formation of male and female sex
hormones. Biochem. J. 351, 67–77
14 Jin, Y., Duan, L., Lee, S. H., Kloosterboer, H. J., Blair, I. A. and Penning, T. M. (2009)
Human cytosolic hydroxysteroid dehydrogenases of the aldo-ketoreductase superfamily
catalyze reduction of conjugated steroids: implications for phase I and phase II steroid
hormone metabolism. J. Biol. Chem. 284, 10013–10022
15 Steckelbroeck, S., Jin, Y., Oyesanmi, B., Kloosterboer, H. J. and Penning, T. M. (2004)
Tibolone is metabolized by the 3α/3β-hydroxysteroid dehydrogenase activities of the
four human isozymes of the aldo-keto reductase 1C subfamily: inversion of
stereospecificity with a ꢀ5(10)-3-ketosteroid. Mol. Pharmacol. 66, 1702–1711
16 Deyashiki, Y., Taniguchi, H., Amano, T., Nakayama, T., Hara, A. and Sawada, H. (1992)
Structural and functional comparison of two human liver dihydrodiol dehydrogenases
associated with 3α-hydroxysteroid dehydrogenase activity. Biochem. J. 282, 741–746
17 Higaki, Y., Usami, N., Shintani, S., Ishikura, S., El-Kabbani, O. and Hara, A. (2003)
Selective and potent inhibitors of human 20α-hydroxysteroid dehydrogenase (AKR1C1)
that metabolizes neurosteroids derived from progesterone. Chem.–Biol. Interact.
143–144, 503–513
18 Matsuura, K., Shiraishi, H., Hara, A., Sato, K., Deyashiki, Y., Ninomiya, M. and Sakai, S.
(1998) Identification of a principal mRNA species for human 3α-hydroxysteroid
dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D2 11-ketoreductase
activity. J. Biochem. 124, 940–946
19 Trauger, J. W., Jiang, A., Stearns, B. A. and LoGrasso, P. V. (2002) Kinetics of
allopregnanolone formation catalyzed by human 3α-hydroxysteroid dehydrogenase
type III (AKR1C2). Biochemistry 41, 13451–13459
AUTHOR CONTRIBUTION
20 Usami, N., Yamamoto, T., Shintani, S., Ishikura, S., Higaki, Y., Katagiri, Y. and Hara, A.
(2002) Substrate specificity of human 3(20)α-hydroxysteroid dehydrogenase for
neurosteroids and its inhibition by benzodiazepines. Biol. Pharm. Bull. 25, 441–445
21 Burczynski, M. E., Harvey, R. G. and Penning, T. M. (1999) Expression and
characterization of four recombinant human dihydrodiol dehydrogenase isoforms:
oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo. Biochemistry 38, 10626
22 Ratnam, K., Ma, H. and Penning, T. M. (1999) The arginine 276 anchor for NADP(H)
dictates fluorescence kinetic transients in 3α-hydroxysteroid dehydrogenase, a
representative aldo-keto reductase. Biochemistry 38, 7856–7864
Yi Jin and Trevor Penning developed the overall experimental strategy and wrote the
paper. Yi Jin performed the majority of the experiments. Clementina Mesaros and Ian Blair
performed LC–MS analysis.
FUNDING
ThisworkwassupportedbytheNationalInstitutesofHealth[grantnumbersR01-DK47015,
R01-CA90744 and P30 ES015857 (to T.M.P)] and by a FOCUS Junior Faculty Investigator
Award (to Y.J.) for Research in Woman’s Health funded by the Edna G. Kynett Memorial
Foundation.
23 Di Costanzo, L., Drury, J. E., Penning, T. M. and Christianson, D. W. (2008) Crystal
structure of human liver ꢀ4-3-oxosteroid 5β-reductase (AKR1D1) and implications for
substrate binding and catalysis. J. Biol. Chem. 283, 16830–16839
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Received 3 November 2010/20 April 2011; accepted 26 April 2011
Published as BJ Immediate Publication 26 April 2011, doi:10.1042/BJ20101804
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Biochem. J. (2011) 437, 53–61 (Printed in Great Britain) doi:10.1042/BJ20101804
SUPPLEMENTARY ONLINE DATA
Stereospecific reduction of 5β-reduced steroids by human ketosteroid
reductases of the AKR (aldo-keto reductase) superfamily: role of
AKR1C1–AKR1C4 in the metabolism of testosterone and progesterone via
the 5β-reductase pathway
Yi JIN, A. Clementina MESAROS, Ian A. BLAIR and Trevor M. PENNING1
Department of Pharmacology and Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA
19104-6084, U.S.A.
Figure S1 Representative kinetic data for the reaction catalysed by AKR1 enzymes
◦
(A) Reduction of 5β-DHT catalysed by AKR1C1, and (B) reduction of 5β-pregane-3,20-dione catalysed by AKR1C1. Initial velocities of reactions were measured fluorimetrically at 25 C and data
were analysed as described in the Experimental section of the main paper. Results are mean values of incubations performed in duplicate or triplicate. Non-linear fitting analysis of the data using the
program GraFit yielded the curves and estimates of Vmax, Km, Ki and associated S.E.M. (Std. Error) values.
1To whom correspondence should be addressed (email penning@upenn.edu).
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Y. Jin and others
Figure S2 TLC chromatograms of the reduction of 5β-pregnane-3,20-dione
catalysed by AKR1C1 and AKR1C4
Comparable amounts of 3α-hydroxy-5β-pregnan-20-one and 20α-hydroxy-5β-
pregnan-3-one were formed from a 30 min reaction of 5β-pregnane-3,20-dione catalysed by
AKR1C1, each of which was subsequently further converted into 5β-pregnane-3α,20α-diol.
In contrast, only one monohydroxy product 3α-hydroxy-5β-pregnan-20-one was observed
in the 15 min reaction of 5β-pregnane-3,20-dione catalysed by AKR1C4. Formation of
5β-pregnane-3α,20α-diol was also observed in the 60 min incubation of AKR1C4. Experiments
were performed as described in the Experimental section of the main paper. Reactions were
conducted in 1 ml systems containing 100 mM potassium phosphate (pH 7.0), 0.4 mM NADPH,
39.3 μM steroid and purified enzyme (4.6 μg of AKR1C1 or 5.6 μg of AKR1C4). Buffer was
used for the no-enzyme (No E) control. P-3,20-dione, 5β-pregnane-3,20-dione; P-20α-ol,
20α-hydroxy-5β-pregnan-3-one; P-3α-ol, 3α-hydroxy-5β-pregnan-20-one; P-3α, 20α-diol,
5β-pregnane-3α,20α-diol.
Received 3 November 2010/20 April 2011; accepted 26 April 2011
Published as BJ Immediate Publication 26 April 2011, doi:10.1042/BJ20101804
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