Role of microsomal steroid hydroxylases in Δ7-steroid biosynthesis

Article abstract of DOI:10.1134/S0006297913030103

CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ⁴- steroids and Δ⁵-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ⁵-Δ⁷ steroids as a substrate.

Full text of DOI:10.1134/S0006297913030103

ISSN 0006ꢀ2979, Biochemistry (Moscow), 2013, Vol. 78, No. 3, pp. 282ꢀ289. © Pleiades Publishing, Ltd., 2013.
Published in Russian in Biokhimiya, 2013, Vol. 78, No. 3, pp. 375ꢀ384.
Role of Microsomal Steroid Hydroxylases
7
∆
in ꢀSteroid Biosynthesis
T. A. Sushko*, A. A. Gilep, A. V. Yantsevich, and S. A. Usanov
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, 220141 Minsk,
Belarus; fax: +375 (172) 637ꢀ274; Eꢀmail: sushkoꢀtatjana@rambler.ru; agilep@iboch.basꢀnet.by
Received July 5, 2012
Revision received October 31, 2012
Abstract—CYP17 (steroid 17αꢀhydroxylase/17,20ꢀlyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two
independent reactions at the same active center and has a unique ability to differentiate ∆4ꢀsteroids and ∆5ꢀsteroids in the
17,20ꢀlyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of
7ꢀdehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using
7ꢀdehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7ꢀdehydropregnenolone have been
identified. Catalytic activity of CYP17 from different species with 7ꢀdehydropregnenolone has been estimated. It is shown
that CYP21 cannot use ∆5ꢀ∆7 steroids as a substrate.
DOI: 10.1134/S0006297913030103
Key words: cytochrome P450, CYP17, CYP21, 7ꢀdehydrosteroids, steroid hormone biosynthesis
Steroid hormone biosynthesis pathways vary greatly
Cytochrome CYP17 (17αꢀsteroid hydroxylase/
among different species of vertebrate animals. 17,20ꢀlyase) is a key enzyme of steroid hormone biosynꢀ
Differences in catalytic activity and expression profiles of thesis that is unique due to its ability to catalyze two indeꢀ
cytochrome P450s involved in steroid hormone biosynꢀ pendent reactions in the same active center. CYP17 catꢀ
thesis supply needs of the particular animal species.
alyzes either selective 17αꢀhydroxylation of P5 and P4
CYP11A1 catalyzes the first, rateꢀlimiting for with the formation of 17αꢀhydroxyꢀderivatives serving as a
steroid hormone biosynthesis reaction of conversion of precursors for mineralocorticoids and glucocorticoids
cholesterol to pregnenolone (P5) (Fig. 1) [1]. P5 might biosynthesis, or 17,20ꢀlyase reaction, where 17αꢀhydroxyꢀ
be further converted by the 3βꢀhydroxysteroid dehydroꢀ progesterone (17OHP4) and 17αꢀhydroxypregnenolone
genase/∆5ꢀ∆4 isomerase to progesterone (P4) or be (17OHP5) are transformed to androstendione (AND) and
metabolized by cytochrome CYP17 in the endoplasmic dehydroepiandrosterone (DHEA), which are precursors
reticulum.
for sex hormone biosynthesis [2, 3] (Fig. 1). The ratio
between the 17αꢀhydroxylase and 17,20ꢀlyase activities is
physiologically important and may direct steroid hormone
biosynthesis to the production of either corticoids or sex
hormones [2]. Cytochrome CYP21 (steroid 21ꢀhydroxyꢀ
lase), in turn, catalyzes the reaction of P4 and 17OHP4
conversion to 11ꢀdeoxycorticosterone (DOC) and 11ꢀ
deoxycortisol (DCS) accordingly, providing an important
step in aldosterone and cortisol synthesis [4]. Cytochrome
CYP17 is characterized by the unique ability to differentiꢀ
ate ∆4ꢀsteroids (P4 derivatives) and ∆5ꢀsteroids (P5 derivꢀ
atives) [5, 6]. There are significant speciesꢀspecific differꢀ
ences in the metabolism of P4 and P5 [7ꢀ15].
Abbreviations: ALA, 5ꢀaminolevulinic acid; AND, androstenꢀ
dione; CPR, NADPHꢀdependent cytochrome P450ꢀreducꢀ
tase; CYP17, steroid 17αꢀhydroxylase/17,20ꢀlyase; CYP21,
steroid 21ꢀhydroxylase; DCS, 11ꢀdeoxycortisol; DHEA,
dehydroepiandrosterone; DOC, deoxycorticosterone; DTT,
dithiothreitol; 7dhC, 7ꢀdehydrocholesterol; 7dhP5, 7ꢀdehyꢀ
dropregnenolone; 7dh17OHP5, 7ꢀdehydroꢀ17αꢀhydroxyꢀ
pregnenolone; IPTG, isopropylꢀβꢀDꢀthiogalactopyranoside;
Kd, dissociation constant; 17OHP4, 17αꢀhydroxyprogesꢀ
terone; 17OHP5, 17αꢀhydroxypregnenolone; P4, progesꢀ
terone; P5, pregnenolone; PMSF, phenylmethylsulfonyl fluoꢀ
ride.
Until recently, it was suggested that the main routes
of steroid biosynthesis in mammals had been studied in
* To whom correspondence should be addressed.
282

METABOLISM OF 7ꢀDEHYDROPREGNENOLONE BY CYP17
283
Fig. 1. Scheme of steroid hormone biosynthesis pathway in Chordata.
detail and the chemical structures of the final products
Furthermore, there are indications of the metaboꢀ
and intermediates were known. However, investigations lism of steroids with unsaturated bonds in the Bꢀring not
of the metabolism of alternative steroids and their physiꢀ only under pathological conditions, but also under norꢀ
ological significance are now very relevant. For example, mal conditions. Therefore, a significant amount of 7dhC,
it was shown that 7ꢀdehydrocholesterol (7dhC) might be which is localized at the plasma membranes of kerꢀ
effectively metabolized in vitro by CYP11A1, which catꢀ atinocytes, is accumulated in skin, where the precursors
alyzes the conversion of cholesterol to P5, with the proꢀ of vitamin D3 is formed from 7dhC by exposure to ultraꢀ
duction of 7ꢀdehydropregnenolone (7dhP5) [16, 17]. The violet radiation [20]. Vitamin D3, like vitamin D2, is
kinetic parameters of this process are comparable with the effectively metabolized by cytochrome CYP11A1 [21,
oxidation of cholesterol by the same enzyme [17]. This 22]. Evidence for the expression of steroid hormone
fact indicates the possible existence of an alternative biosynthesis enzymes such as CYP17 and CYP21 in skin
pathway of biosynthesis of steroid hormones with unsatuꢀ [23] suggests the possibility of subsequent metabolism of
rated bonds in the Bꢀring, which begins from the converꢀ 7ꢀdehydrosteroids.
sion of 7dhC by cytochrome CYP11A1.
Increased level of ∆7ꢀsteroids is typical for some aniꢀ
The presence of conjugated double bonds in posiꢀ mals. High content of estrogens with unsaturated bonds
tions 5 and 7 of the steroid Bꢀring is a characteristic feaꢀ in the Bꢀring is found in pregnant mares [24]. It seems
ture of 7ꢀdehydrosteroids. The enzyme 7ꢀdehydrocholesꢀ that estrogens of this type play a role only in a certain
terol reductase reduces unsaturated bonds in the Bꢀring of stage of ontogenesis [25]. Additionally, it was demonstratꢀ
the molecule and converts 7dhC, which is an intermediꢀ ed ex vivo that 7dhC is effectively converted to 7dhP5 by
ate in a cholesterol biosynthesis pathway, to cholesterol. adrenal glands of various animals [22]. Furthermore,
Insufficiency of 7ꢀdehydrocholesterol reductase catalytic steroidal ∆5ꢀ∆7 and ∆4ꢀ∆7ꢀdienes are formed in adrenal
activity causes significant decrease or lack of conversion tissues.
of 7dhC to cholesterol and accumulation of steroidal 5,7ꢀ
Therefore, the goal of the present study was to eluciꢀ
dienes. This pathological condition is described as date the role of human microsomal cytochrome P450s
Smith–Lemli–Opitz syndrome (SLOS) [18]. More than involved in steroid hormone biosynthesis in the metaboꢀ
a half of the steroids circulating in a fetus with SLOS are lism of ∆7ꢀsteroids. In the present work, the possibility of
estrogens with unsaturated bonds in the Bꢀring [19]. This metabolism of 7ꢀdehydrosteroids is shown for the first
fact suggests the participation of enzymes involved in time, and the products of the reaction are identified. The
steroid biosynthesis in the metabolism of ∆7ꢀsteroids in features of the metabolism of 7ꢀdehydrosteroids by
the case of this pathology.
CYP17 from different species have been studied.
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284
SUSHKO et al.
A. The column was washed with 2ꢀ3 volumes of buffer A
MATERIALS AND METHODS
containing 0.2% Emꢀ913 and then with 10 volumes of
Materials. The following chemicals were used in the buffer A containing 0.2% Emꢀ913 and 100 mM glycine
present work: 7ꢀdehydrocholesterol, P4, 17OHP4, P5, (buffer B). Proteins were eluted from the column with
17OHP5, sodium cholate, Triton Xꢀ100, NADPH, buffer B containing 50 mM histidine and 0.2% Emꢀ913
phenylmethylsulfonyl fluoride (PMSF), aminolevulinic (0.5% for CYP21). Eluted fractions were applied to a colꢀ
acid (ALA was synthesized in the Laboratory of Lipid umn with hydroxyapatite equilibrated with 10 mM potasꢀ
Chemistry of the Institute of Bioorganic Chemistry of the siumꢀphosphate buffer, pH 7.4. The column was washed
National Academy of Sciences of Belarus and kindly proꢀ with 10 volumes of 50 mM potassiumꢀphosphate buffer,
vided by Prof. M. A. Kisel), 2ꢀhydroxypropylꢀβꢀ pH 7.4, containing 20% glycerol, 50 µM P4, 0.2% Emꢀ
cyclodextrin, sodium dodecyl sulfate, Coomassie brilliant 913 (0.5% for CYP21), and 0.1 mM DTT. The proteins
blue Rꢀ250, Hepes, Tris buffer, arabinose, ampicillin, were eluted from the column with 0.3 M potassiumꢀphosꢀ
kanamycin, cholesterol oxidase (from Cellulomonas phate buffer, pH 7.4, containing 20% glycerol, 50 µM P4,
species) (Sigma, USA); Ni2+ꢀNTA agarose (Qiagen, 0.2% Emꢀ913 (0.5% for CYP21), and 0.1 mM DTT. The
USA); Emulgen 913 (Emꢀ913) (Kao Atlas, Japan); highly purified heme proteins were stored at –73°C.
agarose, isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG), Highly purified recombinant NADPHꢀcytochrome P450
and dithiothreitol (DTT) (Gibco BRL, USA); Bactoꢀ reductase (CPR) from Rattus norvegicus was heteroloꢀ
Tryptone, BactoꢀPeptone, and BactoꢀYeast extract gously expressed in E. coli and purified using affinity
(Difco Laboratories, USA); 7ꢀdehydropregnenolone chromatography as previously described [28].
(Institute of Bioorganic Chemistry, National Academy of
Analytical methods. Cytochrome P450 concentration
Sciences of Belarus), BioꢀGel HTP (BioꢀRad, USA); was determined spectrophotometrically using coefficient
methylene chloride (Ekosꢀ1, Russia). Restriction ε450ꢀ490 = 91 mM–1·cm–1 for the absorbance of the comꢀ
enzymes and other enzymes for DNA modification were plex of reduced hemoprotein with CO [29]. The CPR
from New England Biolabs (USA) and Promega (USA).
concentration was determined from the absorption specꢀ
Expression of CYP17 and CYP21. Escherichia coli trum using molar extinction coefficient ε456
=
JM109 competent cells were transformed with plasmid 21.4 mM–1·cm–1 [30]. Protein homogeneity was deterꢀ
pCWori containing CYP17A1 (or pTrc99 containing mined electrophoretically using SDSꢀPAGE.
human CYP21A2) and plasmid pGro12 containing
Interaction of cytochrome P450 with substrate
molecular chaperones GroES–GroEL [26, 27]. Transꢀ induces changes in conformation of the active center of
formed cells were screened on Petri dishes with LBꢀagar the molecule and leads to the transition from sixꢀcoordiꢀ
containing ampicillin (100 µg/ml) and kanamycin nated lowꢀspin hemeꢀiron species towards fiveꢀcoordiꢀ
(40 µg/ml). Overnight culture (3 ml) was used to inocuꢀ nated highꢀspin complex [31] that is visualized by
late 0.5 liter of TBꢀmedium containing 100 mM potassiꢀ increase in absorbance at wavelength 390 nm and
umꢀphosphate buffer, pH 7.4, rare salt solution, ampiꢀ decrease in absorbance at 420 nm. These changes are
cillin (100 µg/ml), and kanamycin (40 µg/ml). The mixꢀ referred to as the type I spectral response. Titrations with
ture was incubated in a thermostated orbital shaker at the steroid substrates were conducted in 50 mM potassiꢀ
37°C and 180 rpm. After reaching absorbance A600 ~ 0.4, umꢀphosphate buffer, pH 7.4. Each cuvette contained
protein expression was stimulated by adding IPTG 1 µM of protein solution. Ligands, dissolved in ethanol,
(0.5 mM), also adding ALA (0.65 mM), arabinose were added to the final concentration of 50 µM. The
(4 mg/ml), ampicillin (100 µg/ml), and kanamycin same volume of ethanol was added to the control cuvette.
(40 µg/ml). After 48 h of incubation in the orbital shaker The titration was stopped if light scattering increased and
at 26°C and 140 rpm, the cells were cooled for 1 h at 4°C the baseline changed as a result of decrease in solubility of
and collected by centrifugation (5000g, 10 min). The pelꢀ the steroid with increase in concentration. When the disꢀ
let was suspended in buffer A (50 mM TrisꢀHCl buffer, sociation constant (Kd) was calculated, ligands with valꢀ
pH 7.4, containing 20% glycerol, 0.3 M NaCl) containꢀ ues more than 25 µM were not further used. The maximal
ing 0.5 mM PMSF and 50 µM P4 (1 volume cells/4 volꢀ concentration of steroid (50 µM) was used to determine
umes buffer). The cells were frozen to the temperature of the fullness of the enzyme active center occupation with
–73°C.
Isolation and purification of recombinant proteins.
the steroid. The Kd value was calculated using SigmaPlot:
Cells were thawed and treated using an Emulsiflex C5
apparatus (Avestin, Canada). Recombinant CYP17 and
CYP21 were solubilized from membranes by adding drops
of Emꢀ913 to final concentration 1%. The suspension was
A = (∆Amax/2Et)⋅(S + Et + Kd) − 2 (S + Et + Kd)2 − (4⋅S⋅Et)
[32, 33],
centrifuged 1 h at 100,000g to remove nonꢀsolubilized where A – amplitude of the spectral response, Amax
–
membrane structures. The supernatant was applied to a amplitude of the spectral response under saturating conꢀ
column with Ni2+ꢀNTAꢀagarose equilibrated with buffer centration of ligand, Et – total protein concentration, S –
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METABOLISM OF 7ꢀDEHYDROPREGNENOLONE BY CYP17
285
total ligand concentration, Kd – dissociation constant of in a system containing 0.05 µM P450 and 0.1 µM CPR at
the enzyme–substrate complex.
temperature 37°C.
Determination of CYP17 catalytic activity. The 17αꢀ
hydroxylase activity of the recombinant CYP17 was
determined in a reconstituted system containing CYP17
and CPR. Aliquots of concentrated proteins (0.5 µM
CYP17 and 1 µM CPR) were mixed and incubated at
RESULTS
Estimation of parameters of interaction of CYP17
room temperature for 5 min. The steroid substrates were with steroid substrates. Cytochromes P450 involved in
added to the reaction mixture to final concentration steroid hormone biosynthesis are characterized by high
50 µM and incubated 10 min at temperature 37°C. The substrate specificity. A distinctive feature of cytochromes
reaction was started by adding NADPH to final concenꢀ P450 is a characteristic change in spectral properties of
tration 0.25 mM. Aliquots (0.5 ml) were taken from the the heme protein occurring on ligand binding. Substrate
incubation mixture at selected time intervals. The reacꢀ binding induces type I spectral response, which is characꢀ
tion was stopped by adding 5 ml of methylene chloride. terized by decrease in absorbance at 420 nm and increase
The mixture was vigorously shaken, and the layers were in absorbance at 390 nm [31]. The experiment for estimaꢀ
separated by centrifugation at 5000g for 10 min. The tion of catalytic activity of CYP17 was preceded by comꢀ
aqueous layer was carefully removed, and the organic parative study of the binding of the different substrates to
layer was dried under a flow of argon at temperature 40°C. CYP17. Spectral studies show that H. sapiens CYP17 has
The dry residue was dissolved in 100 µl of methanol and a characteristic type I spectrum resulting from increase in
analyzed using an Accella HPLC equipped with diodeꢀ highꢀspin enzyme content occurring on interaction with
array and massꢀspectrometer detector LCQꢀFleet steroid substrates. The maximal value of Kd was deterꢀ
(ThermoSci, USA).
mined for the reaction products (AND and DHEA). It
Determination of CYP21 catalytic activity with was established that H. sapiens CYP17 has high affinity
7dhP5. The catalytic activity of CYP21 was determined in towards 7dhP5 (Kd = 1.75 0.25 µM) (Fig. 2), comparaꢀ
a reconstituted system containing CYP21 and CPR. ble with the affinity of CYP17 to natural steroid substrates
Activity was measured as described above for the CYP17 (Table 1). Therefore, we have shown that 7dhP5 might
0.02
0
0.05
0.03
–0.02
0.01
0
350
400
500
10
20
25
7dhP5, µM
Wavelength, nm
Fig. 2. Spectral changes induced by 7dhP5 binding to the active center of CYP17. The titration was conducted in 50 mM potassiumꢀphosꢀ
phate buffer, pH 7.4. Each cuvette contained 1 µM of CYP17.
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SUSHKO et al.
7dhP5, the catalytic activity of H. sapiens CYP17, E. cabalꢀ
lus CYP17, and C. porcellus CYP17 demonstrating different
types of activity (∆5, ∆4–5, and ∆4, accordingly) with 7dhP5
was analyzed (Table 2). It was found that H. sapiens CYP17
and E. caballus CYP17 efficiently metabolize 7dhP5, catꢀ
alyzing either the 17αꢀhydroxylase or 17,20ꢀlyase reacꢀ
tions. CYP17 from C. porcellus belonging to ∆4ꢀtype of
activity accomplishes only 17αꢀhydroxylation of 7dhP5,
and products of 17,20ꢀlyase activity cannot be detected in
the reaction mixture. Therefore, the presence of the unsatꢀ
urated bond in the steroid Bꢀring does not influence the
steroid recognition in the active center of CYP17.
Cytochrome b5 influences catalytic activity of CYP17.
Analysis of the catalytic activity in the presence of
cytochrome b5, which is a natural effector of CYP17,
demonstrates that cytochrome b5 predominantly stimuꢀ
lates 17,20ꢀlyase activity of the enzyme in the reaction
with “classical” steroids and does not influence signifiꢀ
cantly the reaction with ∆7–∆5 steroids.
Table 1. Interaction of human CYP17 with steroid subꢀ
strates
Substrate
∆Amax
Kd, µM
P5
0.07
0.05
0.05
0.02
0.05
–
0.86 0.12
0.63 0.05
1.92 0.09
2.32 0.35
1.75 0.25
–
17OHP5
P4
17OHP4
7dhP5
DHEA
AND
–
–
Note: The titrations were conducted in 50 mM potassiumꢀphosphate
buffer, pH 7.4. Each cuvette contained 1 µM CYP17. Ligand
concentration was changed in the range of concentration
0.1ꢀ50 µM by the addition of the ligand dissolved in ethanol.
Catalytic activity of CYP21 with 7dhP5. Cytochrome
CYP21 catalyzes important steps in steroid hormone
biosynthesis. Investigation of the possibilities of biosynꢀ
effectively bind to the substrate recognition site and to the thesis of corticosteroids with a double bond in the 7,8
active site of CYP17.
position is of particular interest. In the present study, the
Catalytic activity of CYP17 with 7dhP5. Efficient catalytic activity of CYP21 with 7dhP5 was analyzed to
binding demonstrated by spectral methods does not always elucidate whether ∆7–∆5ꢀsteroids might serve as precurꢀ
mean that a substance can be effectively metabolized by sors in corticoid hormone biosynthesis. Despite the fact
the enzyme. We evaluated the catalytic activity of the that CYP21 predominantly hydroxylates ∆4ꢀsteroids, we
recombinant highly purified human CYP17 using 7dhP5 have suggested that 7dhP5 might be used as a substrate due
as a substrate in a reconstituted system. It was found that to changes in conformation occurring because of the presꢀ
CYP17 efficiently metabolizes 7dhP5 (Fig. 3). Mass specꢀ ence of an unsaturated bond in the Bꢀring. Analysis of the
trometric analysis identified the first product with maxiꢀ 3D structure of CYP21 [34] showed that there is no spatial
mal quantity as 7ꢀdehydroꢀ17αꢀhydroxypregnenolone restriction for 7dhP5 metabolism. However, analysis of the
(7dh17OHP5), and the second product as androstaꢀ5,7ꢀ enzymatic activity of CYP21 in the reconstituted system
dienꢀ3βꢀolꢀ17ꢀone (7dhDHEA) (Fig. 3). These results containing CPR as electron donor demonstrates that
indicate siteꢀspecific hydroxylation of either ∆4ꢀ and ∆5ꢀ CYP21 cannot metabolize 7dhP5. Presumably, the presꢀ
steroids, or ∆7–∆5ꢀsteroids by H. sapiens CYP17.
ence of the keto group in the 3 position of the steroid molꢀ
Studies of speciesꢀspecificity of catalytic activity of ecule is the most important factor for binding to CYP21.
CYP17 with 7dhP5. To study speciesꢀspecificity of the catꢀ Further studies are needed to investigate the possibility of
alytic activity of CYP17s from different organisms with metabolism of ∆7ꢀsteroids by cytochrome CYP21.
Table 2. Enzymatic activity of CYP17 from different species with 7dhP5
Enzymatic activity, min–1
17αꢀhydroxylase
17,20ꢀlyase
CYP17
17ОНP5
7dh17ОНP5
+b5
P5
7dhP5
–b5
+b5
–b5
H. sapiens
C. porcellus
E. caballus
2.4
6
5.2
1.2
6.2
0.28
–
1.2
–
1.4
–
1.8
–
15.5
2.7
10.5
1.9
3.1
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METABOLISM OF 7ꢀDEHYDROPREGNENOLONE BY CYP17
287
a
12
10
2
8
6
1
4
2
0
3
0
5
10
15
20
25
t, min
b
60.42
269.14
297.16
100
90
313.12
80
70
60
1
2
3
60.40
50
40
30
20
295.26
251.29
200
315.17
287.14
367.29
400
10
0
330.91
400
200
200
400
m/z
Fig. 3. a) HPLC profile of products formed during metabolism of 7dhP5 by cytochrome CYP17; b) mass spectra of the main reaction prodꢀ
ucts: 1) 7dhDHEA, m/z ([MH]+) = 287; 2) 7dh17OHP5, m/z ([MH]+) = 331; 3) 7dhP5, m/z ([MH]+) = 315.
DISCUSSION
in the metabolism of alternative substrates has been
shown [36ꢀ42] (Fig. 4).
It has been previously postulated that CYP17 is a
Recently, investigations of ∆7ꢀsteroid biosynthesis
highly specific enzyme having a small active center that have become very intense. The ∆7ꢀsteroids are predomiꢀ
restricts the spatial orientation of substrates [35]. Despite nant for certain species. In humans, ∆7ꢀsteroids can be
this fact, apart from the catalysis of reactions with “clasꢀ detected in large amounts in certain pathologies. Their
sical” substrates, the possibility of involvement of CYP17 accumulation might cause numerous disorders including
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288
SUSHKO et al.
a
b
c
Fig. 4. a) Alternative positions of steroid hydroxylation by CYP17; b) 16ꢀen synthase activity of CYP17. Species: 1) CYP17 H. sapiens;
2) CYP17 S. scrofa; 3) CYP17 B. taurus; 4) CYP17 C. porcellus; 5) CYP17 E. caballus; c) scheme of conversion of 7dhP5 catalyzed by CYP17
H. sapiens.
abnormalities in the functioning of the nervous system. the products of the reaction were identified using high perꢀ
Furthermore, wide use of estrogen replacement therapy formance liquid chromatography coupled with mass specꢀ
containing steroids with unsaturated bonds in the Bꢀring trometry. The 7dh17OHP5 product of the 17αꢀhydroxyꢀ
in correction of menopausal disorders is an important lase reaction is consequently converted to 7dhDHEA by
factor that stipulates the interest in the metabolism of ∆7ꢀ the 17,20ꢀlyase reaction (Fig. 4). It has been shown that
steroids. It has been demonstrated that long usage of addition of “classical” substrate P5 leads to decrease in
drugs containing equiline and equilinine are risk factors catalytic activity of CYP17 with 7dhP5. This fact can be
for endometrial and breast cancer. Currently, the funcꢀ explained by the effect of competitive inhibition.
tional importance of ∆7ꢀsteroid circulating in the human
The species specificity of this reaction has been studꢀ
organism is not elucidated. There are indications that ∆7ꢀ ied. It has been determined that the presence of an unsatꢀ
steroids might be substrates for CYP11A1 [16, 17]. In the urated bond in the Bꢀring does not influence steroid
present work, the possibility of the subsequent metaboꢀ recognition in the active center of CYP17. The CYP17s
lism of steroids with unsaturated bonds in the Bꢀring by from different species belonging to different types of
the microsomal steroid hydroxylases CYP17 and CYP21, enzymatic activity (∆5, ∆4–5, ∆4), metabolize ∆5–∆7ꢀ
which catalyze key steps of corticoid and sex hormones steroids in the same way as “classical” ∆5ꢀsteroid subꢀ
biosynthesis, has been studied.
strate.
We have shown that cytochrome CYP21 cannot
The data obtained in the present work indicate the
possibility of involvement of CYP17 in an alternative pathꢀ metabolize ∆5–∆7ꢀsteroids; this fact indicates the major
way of biosynthesis of estrogen with unsaturated bonds in significance of the structure of the Aꢀring of CYP21 subꢀ
the Bꢀring using 7ꢀdehydropregnenolone as a precursor; strates.
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METABOLISM OF 7ꢀDEHYDROPREGNENOLONE BY CYP17
289
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Vreken, P., PollꢀThe, B. T., Dorland, L., Duran, M., Jira,
P. E., Smeitink, J. A., Wevers, R. A., and Wanders, R. J.
(1998) Am. J. Hum. Genet., 63, 329ꢀ338.
19. Shackleton, C. H., Roitman, E., Kratz, L. E., and Kelley,
R. I. (1999) J. Clin. Endocrinol. Metab., 84, 1157ꢀ1159.
20. Slominski, A., and Wortsman, J. (2000) Endocr. Rev., 21,
457ꢀ487.
Our results have important fundamental significance
in updating information about steroid metabolism by
CYP17. The possibility of CYP17 metabolizing steroid
derivatives with two unsaturated bonds in the Bꢀring indiꢀ
cates the likely metabolism of steroid hormones of this
type. Investigations of possible involvement of other
enzymes of steroid hormone biosynthesis pathways in
metabolism of such types are needed.
21. Slominski, A., Semak, I., Wortsman, J., Zjawiony, J., Li, W.,
Zbytek, B., and Tuckey, R. C. (2006) FEBS J., 273, 2891ꢀ
2901.
22. Slominski, A. T., Zmijewski, M. A., Semak, I., Sweatman,
T., Janjetovic, Z., Li, W., Zjawiony, J. K., and Tuckey, R. C.
(2009) PLoS One, 4, e4309.
This work was conducted in the context of task 2.09
of the government program of directed fundamental
research “Physiologically Important Substances”.
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BIOCHEMISTRY (Moscow) Vol. 78 No. 3 2013