22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting oxidoreductases

Article abstract of DOI:10.1016/j.jsbmb.2012.09.035

Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β- ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH₂) formation as a dominant fluorescent product. Basing on LC-MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3β-ol were deduced to be steroidal co-products of NBD-NH₂, indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors.

Full text of DOI:10.1016/j.jsbmb.2012.09.035

Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
Contents lists available at SciVerse ScienceDirect
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2
2-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting
oxidoreductases
a
b
a
b
Yaroslav V. Faletrov , Katsiarina I. Bialevich , Irina P. Edimecheva , Dzmitry G. Kostsin ,
a
b
a,c,∗
Elena V. Rudaya , Ekaterina I. Slobozhanina , Vladimir M. Shkumatov
Research Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya str. 14, 220030 Minsk, Belarus
Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Academicheskaya str. 27, 220072 Minsk, Belarus
Department of Chemistry, Belarusian State University, Leningradskaya str. 14, 220030 Minsk, Belarus
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2012
Received in revised form
Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-
yl)amino)-23,24-bisnor-5-cholen-3␤-ol (22-NBD-cholesterol), a common fluorescent sterol analog,
binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases
1
2 September 2012
(CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent
Accepted 23 September 2012
system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-
nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH2) formation as a dominant fluorescent product. Basing
on LC–MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregneno-
lone and 20-formyl-pregn-5-en-3␤-ol were deduced to be steroidal co-products of NBD-NH2, indicating
intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were
defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was
also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas
aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some
microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent
probe for steroid hormone-binding enzymes or receptors.
Keywords:
Cytochrome P450scc
Cholesterol dehydrogenase
Cholesterol oxidase
2
2-NBD-cholesterol
Docking
Mass spectrometry
©
2012 Elsevier Ltd. All rights reserved.
1
. Introduction
a terminal oxidase of mitochondrial monooxygenative system,
which includes additionally two electron-transferring proteins –
adrenodoxin (Adx) and NADPH-dependent adrenodoxin-reductase
(AdR). Pregnenolone has no hormonal activity. Like other 3␤-
hydroxy-5-ene, pregnanes and androstanes, it should be first
converted into the corresponding 3-keto-4-en derivative by the
Cholesterol (cholest-5-en-3␤-ol) is an important lipid com-
ponent of plasma membranes of most vertebrates. It is also a
biosynthetic precursor of bile acids, vitamin D and steroid hor-
mones, which are active participants of cell signaling and other
regulation processes, including cell homeostasis, proliferation and
apoptosis. Moreover, steroid hormones are involved in control of
whole organism functions, such as sexual differentiation, repro-
duction, fertility and blood pressure [1,2]. Biosynthesis of steroid
hormones is performed by two groups of oxidoreductases, namely,
cytochromes P450 and hydroxysteroid-dehydrogenases. The first
rate-limiting step of mammalian steroidogenesis is a conversion
of cholesterol into pregnenolone catalyzed by P450scc (CYP11A1).
This enzyme consists of a single polypeptide [3,4] and acts as
+
NAD(P) -dependent 3␤-hydroxysteroid dehydrogenase/ꢀ5–ꢀ4
isomerase (3␤-HSD) to form the active steroid hormone [2]. On the
other hand, cholesterol-metabolizing oxidoreductases also play
essential roles in the cholesterol assimilation by some microbes
(e.g., genera Pseudomonas, Nocardia, Mycobacterium, etc.). Notably,
that the pathogenicity and persistence of Mycobacterium tuber-
culosis – a pathogen causing about 2 million deaths annually –
are closely associated with the utilization of host cell cholesterol.
An initial step of the microbial degradation of the cholesterol
and other 3␤-hydroxy-5-ene steroids is their conversion into
corresponding 3-keto-4-en derivatives. This step is catalyzed by
+
∗ Corresponding author at: Research Institute for Physical Chemical Problems,
either cholesterol oxidases (CHOXs, EC 1.1.3.6) or NAD -dependent
Belarusian State University, Leningradskaya str. 14, 220030, Minsk, Belarus.
Tel.: +37517 200 82 72; fax: +37517 209 54 61.
cholesterol dehydrogenases (CHDHs, EC 1.1.1.145), more often
referred in the literature as “microbial 3␤-HSD” [5–11].
E-mail addresses: yaroslav82@tut.by (Y.V. Faletrov), catherina bel @tut.by
2
2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bis-
(K.I. Bialevich), shadyro@open.by (I.P. Edimecheva), kostsindzmitry3@gmail.com
(D.G. Kostsin), lusova@mail.ru (E.V. Rudaya), slobozhanina@ibp.org.by
(E.I. Slobozhanina), biopharm@bsu.by (V.M. Shkumatov).
nor-5-cholen-3␤-ol (22-NBD-cholesterol) is a fluorescent choles-
terol analog, which has been used widely for investigations of the
0
960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jsbmb.2012.09.035

6
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Y.V. Faletrov et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
sterol traffic in cells, structural organization of lipid membranes
and some cholesterol-binding proteins (sterol carrier protein-2,
steroidogenic acute regulatory protein, etc.) [12,13]. To the best of
our knowledge, the enzymatic conversion of 22-NBD-cholesterol
into the corresponding 3-O-esters has been reported only [1,14,15].
However, there are practically no publications about interaction
of the fluorescent steroid with cholesterol-converting oxidore-
ductases of the aforesaid pathways. Previously, we announced the
ability of the bacterial CHOXs to convert 22-NBD-cholesterol into
its 3-keto-4-one derivative [16].
acetate; corresponding organic phases were combined, evaporated
to dryness and then dissolved in 0.5 ml of ethanol.
2.4. 22-NBD-cholesterol conversion by CHDH
The stock solution of the 22-NBD-cholesterol in propanol-2 was
added to 50 mM potassium phosphate buffer (pH 7.4) with 0.05%
sodium cholate, 0.2% Polysorbate 80 and then sonicated for 0.5 min
at 35 kHz, resulting in 2–100 ␮M initial concentrations of the fluo-
rescent steroid. NAD+ was added up to 500 ␮M. Unless otherwise
In this article we show the results of computer simulations
supported by further experimental data, demonstrating that the
fluorescent steroid is a substrate of two cholesterol-metabolizing
oxidoreductases, namely, the mammalian P450scc and the bacte-
rial CHDH. We establish structures of the corresponding fluorescent
products of these reactions and also propose possible applications
of the newly discovered processes.
stated, enzymatic oxidation was initiated by the addition of the
CHDH up to 0.04 U/ml. The conversion went at 30 C for various
time periods and stopped by ethyl acetate extraction. Each sample
◦
(1 ml) was extracted twice with 4 ml of ethyl acetate. The corre-
sponding organic phases were combined, evaporated to dryness
and then dissolved in 0.5 ml of ethanol. Further processing of the
probes was carried out as described earlier (see Section 2.3).
2
. Materials and methods
2.5. 22-NBD-cholesterol incubation with growing opportunistic
bacteria
2.1. Enzymes and chemicals
Opportunistic strains of Escherichia coli, Pseudomonas aeruginosa
and Staphylococcus aureus were taken from the collection of the
Republican Research and Practical Center for Epidemiology and
Microbiology (Republic of Belarus). The bacteria were cultivated for
The following reagents were used: 22-NBD-cholesterol (Molec-
ular Probes), cholesterol, pregnenolone, progesterone, hydroxy-
+
lamine hydrochloride, NADPH and NAD (Sigma), Polysorbate 80
◦
and sodium cholate (Fluka). CHOX from Brevibacterium sterolicum
and CHDH from Nocardia sp. (≥18 U/mg solid) were obtained from
Sigma. P450scc, adrenodoxin (Adx) and adrenodoxin-reductase
24 h at 37 C on Petri dishes as described elsewhere [22] with 22-
◦
NBD-cholesterol added up to 10 ␮M into molten medium (45 C)
prior to the cultivation. After the cultivation corresponding zones
(medium + bacteria) were cut out and placed into different tubes
containing 10 ml ethanol. Then the tubes were sonicated (15 min
at 35 kHz) and centrifuged (10 min at 3000 × g). The supernatant
aliquots (1 ml) were transferred into vials and analyzed by HPLC
with fluorescent detection (see Section 2.8).
(
AdR) were purified from bovine adrenal cortex as described pre-
viously [4]. Spectral purity indexes for P450scc, Adx and AdR were
A391/A280 0.83, A414/A280 0.83 and A272/A455 8.5, respectively. The
P450scc preparation was more than 90% in high-spin form and
contained less than 5% inactive P420 fraction.
2.2. Homology search and molecular docking simulations
2.6. Spectrofluorimetry
The homology search was carried out by BLAST 2.2.22+ pro-
Fluorescence measurements were conducted with the spec-
trofluorimeter SM2203 (Solar). The slits of excitation and emission
were set at 5 nm for all the fluorescence measurements. The
fluorometric registration of the CHDH-mediated conversion of 22-
NBD-cholesterol was performed using excitation at 350 nm (close
to the NADH absorption maximum); fluorescence was registered
at 470 nm and 530 nm (close to emission maxima of NADH and
22-NBD-cholesterol, respectively).
gram using BLASTP algorithm [17]. The docking simulations
were conducted with Autodock 4.0; the Graphical User Inter-
face “AutoDockTools” (The Scripps Research Institute) was used
to prepare, run, and analyze the docking simulations [18,19] with
the 3D-structure of bovine P450scc (PDB ID: 3MZS) [20]. Gasteiger
partial charges [21] were calculated and assigned to the atoms of
heme and amino acid residues. The docking space was defined as
a 60 A˚ × 60 A˚ × 60 A˚ box with its center close to redox-active heme
of the protein. Small molecules were created and prepared using
HyperChem 7.01 (Hypercube). The Lamarckian genetic algorithm
with default parameters was applied for rigid docking calcula-
tions. The binding energy values were calculated automatically by
Autodock.
2.7. TLC
TLC was performed on silica aluminum foils (Fluka) with flu-
orescent indicator (254 nm). TLC plates were developed with
benzene:acetone mixture (4:1 (v:v)). Spots of substances were
monitored visually under 365 nm UV-light. Individual spots were
cut out and extracted by ethanol in the necessary cases.
2
.3. 22-NBD-cholesterol conversion by P450scc enzymatic system
The stock solution of 22-NBD-cholesterol in propanol-2 was
2.8. HPLC
added to 50 mM potassium phosphate buffer (pH 7.4) with 0.02%
sodium cholate, 0.1% Polysorbate 80 and then sonicated. The
initial concentration of the fluorescent steroid was 2–20 ␮M.
Then P450scc, Adx and AdR were added from stock solutions up
to 0.2 ␮M, 2 ␮M and 0.2 ␮M, respectively, and mixtures were
HPLC was conducted using either the LC-10AT (Shimadzu)
system with photodiode array detector SPD-M10A (operated at
200–600 nm range) or the LC10-AD (Shimadzu) system with fluo-
rometric detector RF-10Axl (medium sensitivity, wavelengths of
excitation and emission 466 and 530 nm, respectively). In both
cases the column was LiChroCART C18 (250 mm × 4 mm, 5 ␮m)
(Merck) with the elution conducted at a flow rate of 1 ml/min using
either eluent A comprising acetonitrile:water:propanol-2 (84:16:5
(v:v:v)) or eluent B comprising acetonitrile:water:propanol-2
(80:20:5 (v:v:v)).
◦
pre-incubated for 5 min at 30 C. NADPH was then added from a
◦
stock solution up to 250 ␮M and incubation proceeded at 30 C
for 30 min. Control experiments were conducted without either
P450scc or Adx and AdR as well as with hydrogen peroxide instead
of NADPH. The conversions were stopped by ethyl acetate extrac-
tion. Each sample (1 ml) was extracted twice with 4 ml of ethyl

Y.V. Faletrov et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
61
2
.9. Purification of products
could be localized in the proximity of the protein’s heme (Fig. 1).
Two different enzyme–steroid complexes were predicted (Fig. 1A
and B) with 22-NBD-cholesterol binding energy values of approx-
imately −14.0 ± 0.2 kcal/mol, showing high affinity of the steroid
to the protein. In these complexes the NBD-group of 22-NBD-
cholesterol is primarily surrounded by the hydrophobic residues
comprising Trp88, Leu102, Phe203, etc. Polar groups of Gln377
and Thr354 are close to the 3␤-hydroxyl of the steroid, enabling
of a direct or a water-mediated hydrogen bond formation [20].
In the complexes A and B, the distances from Fe to each atom
Definite reaction products were firstly separated from the reac-
tion mixtures by TLC as described above. The corresponding TLC
fluorescent yellow spots were cut out and the substances extracted
with ethanol. The ethanolic extracts were concentrated to 200 ␮L
by evaporation under argon stream and then applied on the HPLC
column, which was equilibrated with 20% eluent A in water. Subse-
quently the column was washed by 20% eluent A in water followed
by 100% eluent A. Fractions with absorbance maxima at 470 nm
were collected. The recovery was 60–75% according to photometric
data.
of the C17 N23 fragment of the steroid side-chain are within
3.4–4.8 A˚ , but the complex A geometry favors the oxidation of
C20 and C21 (Fig. 1A), whereas the complex B geometry – C22
and N23 (Fig. 1B). Taking previous data [25–27] into account, the
predicted distances allowed us to propose the oxidation of 22-NBD-
cholesterol by P450scc. It should be noted that P450scc oxidizes
most of its steroidal substrates at positions 20 and 22; however,
the oxidations at 17, 23 and 24 positions of substrates other than
cholesterol were also reported [28–31]. Thus, our results confirm
the potential ability of the 22-NBD-cholesterol to bind the active
site of the P450scc, enabling the oxidation of the steroid side-chain.
2
.10. Liquid chromatography–mass spectrometry (LC–MS)
LC–MS measurements were performed using the LC-2020 (Shi-
madzu) system, equipped with a photodiode array detector and a
mass spectrometer. The last one was operated in either electrospray
(
ESI) or atmospheric pressure ionization (APCI) mode at a detector
◦
voltage of 1.2 kV, a heat block temperature of 400 C and a desolva-
◦
tion line temperature of 250 C. Negative (m/z 150–650) or positive
m/z 250–450) ions were monitored. Nitrogen was used as a drying
(
and nebulizer gas at flow rates 1.5 L/min and 15 L/min, respectively.
The column was Shim-pack VP-ODS (2 mm × 150 mm, 5 ␮m). The
elution was performed by the mixture of acetonitrile:water:formic
3.2. P450scc-catalyzed conversion of 22-NBD-cholesterol
It was established, that the addition of 22-NBD-cholesterol
to P450scc solutions decreased the pregnenolone-induced spec-
tral changes (A420 − A390), indicating a competitive binding of the
steroids near the heme of P450scc [32]. An increase in 22-NBD-
cholesterol fluorescence intensity after addition of P450scc was
also monitored, suggesting that 22-NBD-cholesterol binds into a
lipophilic site of the P450scc [12,13]. The TLC and HPLC analy-
ses of the samples where 22-NBD-cholesterol had been incubated
with the complete P450scc-dependent side-chain cleavage system
(NADPH-AdR-Adx-P450scc) revealed a selective formation of a flu-
orescent substance as a dominant product (Fig. 2). There was no
formation of this product when incomplete variants of the side-
chain cleavage system were used as well as when the NADPH was
substituted by a hydrogen peroxide. These data indicate that the
substance formation is due to the specific P450scc enzymatic activ-
ity and that hydrogen peroxide, which could be generated in the
P450scc-dependent system [33], does not seem to be a participant
of the reaction monitored. According to TLC and HPLC mobility of
the product, the substance was concluded to be more polar than the
substrate (Fig. 2A and B). The LC–MS analysis of 22-NBD-cholesterol
conversion by P450scc allowed us to establish that the fluorescent
◦
acid (50:50:0.05 (v:v:v)) at 0.4 ml/min and at 40 C.
Additionally, aliquots of the probes of P450scc-catalyzed
conversion of 22-NBD-cholesterol were analyzed by LC–MS as
described above after additional derivatization with the cholesterol
oxidase according to [16,23] (for 5-en-3␤-ol steroids conversion
into 4-en-3-oxo steroids) as well as with hydroxylamine according
to [24] (for conversion of oxosteroids into oximes).
2.11. Calculations of kinetic parameters
Maximal velocity (Vmax) and apparent Michaelis constant (Km)
values were estimated by hyperbolic regression of the data using
Origin 7.0 software. Initial data were obtained from three indepen-
dent experiments.
3
. Results
3.1. Interaction of 22-NBD-cholesterol with cytochrome P450scc
revealed by docking simulations
Docking simulations with the crystal structure of bovine
P450scc [20] and 22-NBD-cholesterol demonstrated that the latter
product is the 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH ,
[M−H] ion with m/z 179) (Fig. 3). The structure was additionally
2
−
Fig. 1. Conformations of 22-NBD-cholesterol in the active site of bovine P450scc (PDB ID: 3MZS) predicted by docking simulations. The steroid is drawn with bold lines.
Gray, blue, red, aquamarine and green fragments correspond to C, N, O, H and Fe atoms, respectively. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

6
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Y.V. Faletrov et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
Fig. 2. Analysis of 22-NBD-cholesterol conversion by the P450scc-dependent side chain cleavage system. (A) TLC plate in 365 nm UV-light (shown as black and white),
reflecting selective formation of the product IIP in the case of 22-NBD-cholesterol (I) conversion by complete P450scc-dependent system: a – P450scc + Adx + AdR + NADPH,
b – Adx + AdR + NADPH, c – P450scc + NADPH, d – P450scc + Adx + AdR + H2O2, e – P450scc. (B) A representative chromatographic curve (fluorescence at 530 nm) showing the
separation of I and IIP (eluent B) (see Section 2.10 for further details). (C) Mass spectrum with the electron impact ionization of the main fluorescent product IIP, showing the
product is 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (identity with the compound mass spectrum from Wiley8 library). The analysis was performed as described [34] with
direct injection of the purified substance in our case.
+
+
+
confirmed by the mass spectrometry with the electron impact ion-
ization of the purified product (M ion with m/z 180) (Fig. 2C).
H O] , [M+H] and [M+H+CH CN] ) (Fig. 4A, C, D), which may
2 3
+
be attributed to the 3-keto-4-en derivative of 20-formyl-pregn-
5-en-3␤-ol. Moreover, after derivatization of the probes with
hydroxylamine, formation of two isomeric 20-oximes of pregneno-
lone (MW 331 Da) was detected (RT 4.8 and 5.4 min; m/z 332 and
373) as well as two additional more hydrophobic compounds (RT
6.3 and 6.8 min), which had identical mass spectra consistent with
To determine a steroidal co-product, forming in pair with
NBD-NH2 during P450scc-dependent 22-NBD-cholesterol conver-
sion, we first performed LC–MS analysis using APCI+ mode. This
allowed registering the formation of compounds, giving cations
with m/z values, which are attributable to supposed products of
the reaction under consideration, namely, 20-formyl-pregn-5-en-
+
+
MW 345 (m/z 346, 369 and 387, i.e. [M+H] , [M+H+CH CN–H O]
and [M+H+CH CN] ) of 22-oximes of 20-formyl-pregn-5-en-3␤-ol
3
2
+
3
␤-ol (molecular weight (MW) 330 Da; m/z 295, 313, 331) as
3
well as pregnenolone (MW 316 Da; m/z 299, 317). To improve
detection of steroidal analytes, derivatization with CHOX [16,23]
or with hydroxylamine [24] was used. The CHOX treatment of
the probes resulted in the appearance of three new compounds,
which possessed absorption maximum at 242 nm. Two of the
compounds were identified as the 3-keto-4-en derivative of 22-
NBD-cholesterol [16] and progesterone (retention times (RT) 45.3
and 8.3 min, respectively) using comparison with authenticated
standards. The third substance peak (RT 10.1 min) gave a mass
spectrum consistent with MW 328 Da (m/z 311, 329, 370, i.e. [M+H-
(Fig. 4B, E, F). In total, our data is in accordance with the formation
of NBD-NH , pregnenolone and 20-formyl-pregn-5-en-3␤-ol dur-
2
ing the P450scc-catalyzed conversion of 22-NBD-cholesterol. Vmax
and Km values for 22-NBD-cholesterol conversion in the reconsti-
−
1
tuted enzymatic system was determined to be 1.0 ± 0.3 min and
12.2 ± 2.5 ␮M, respectively. For comparison, using the same con-
ditions for natural cholesterol conversion yielded Vmax and Km of
−
1
8.0 min and 10 ␮M, respectively. Thus, Vmax/Km ratio for the arti-
ficial substrate was estimated to be approximately 10 times less
than for the natural one.
Fig. 3. 22-NBD-cholesterol conversion by complete P450scc-dependent system. (A) LC–MS chromatographic curves showing NBD-NH2 (IIP) formation after 22-NBD-
cholesterol (I) (retention times 2.6 and 42.3 min, respectively) conversion by complete P450scc-dependent system: 1 and 2 – 40-times multiplied signals of selected anions
with m/z 493 and 179, respectively; 3 – the signal due to total anions with m/z 150–650. (B) The mass spectrum of the fluorescent product of P450scc-dependent conversion
of 22-NBD-cholesterol, its interpretation (tfa is “trifluoracetic acid” – a concomitant from the solvents) and the established structure of the product.

Y.V. Faletrov et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
63
Fig. 4. 22-NBD-cholesterol conversion by complete P450scc-dependent system: detection of derivatives of non-fluorescent steroidal products. (A) LC–MS chromatographic
curves (APCI+ detection mode) showing progesterone (IIIa–c) and another compound (IIIb–c) formation after the cholesterol oxidase treatment [16,23] of probes of the
P450scc-dependent 22-NBD-cholesterol conversion: 1 – TIC, 2 and 3 – multiplied signals of selected cations with m/z 356 and 370, respectively. (B) LC–MS chromatographic
curves (ESI+ detection mode) showing pregnenolone 20-oximes (IIIa–h) and two other compounds (IIIb–h) formation after the hydroxylamine treatment [24] of probes of
the P450scc-dependent 22-NBD-cholesterol conversion: 1 – TIC, 2 and 3 – multiplied signals of selected cations with m/z 387 and 373, respectively. (C and D) Mass spectra of
the IIIa–c and IIIb–c and their interpretation based on the progesterone and 20-formyl-pregn-4-en-3-one structures, respectively. (E and F) Mass spectra of the IIIa–h and
IIIb–h and their interpretation based on structures of oximes of pregnenolone and 20-formyl-pregn-5-en-3␤-ol, respectively. Acn is acetonitrile (CH3CN).
3
sp.
.3. 22-NBD-cholesterol conversion by the CHDH from Nocardia
seemed to be identical to the mass spectrum of 22-NBD-cholest-
4-en-3-one, whereas its absorbance spectrum was shown to be
similar with the absorbance spectrum of 22-NBD-cholesterol. Thus,
the dominant product of the CHDH-mediated 22-NBD-cholesterol
conversion was concluded to be the 3-oxo-5-en derivative of the
substrate (formally, “22-NBD-cholest-5-en-3-one”). Vmax and Km
values for the 22-NBD-cholesterol conversion were estimated to
During 22-NBD-cholesterol incubation with the CHDH in the
+
presence of NAD , an increase in the absorbance at 340 nm (Fig. 5)
as well as in the fluorescence emission at 470 nm were observed,
pointing out NAD+ conversion into NADH [35]. Initial slopes of the
corresponding absorbance kinetic curves obtained during choles-
−
1
be 2.1 ± 0.4 min and 143 ± 13 ␮M, respectively, whereas for the
+
terol and 22-NBD-cholesterol incubation with the CHDH and NAD
CHDH-mediated natural cholesterol conversion the parameters
−
1
let us conclude that the natural substrate is about four times
more preferable than the fluorescent substrate. In the case of 22-
NBD-cholesterol conversion, the fluorescence emission at 530 nm
increased 5 times as compared to emission at 470 nm, whereas in
the case of natural cholesterol the increase of fluorescence emission
at 470 nm was higher than at 530 nm. This was due to effective reso-
nance energy transfer from NADH (emission maximum at 470 nm)
to the NBD-fluorophore (excitation maxima at 470 nm and 350 nm
were 2.5 ± 0.5 min and 49 ± 5 ␮M, respectively.
3.4. 22-NBD-cholesterol conversion by opportunistic bacteria
P. aeruginosa
A possibility of using the established activities of CHOXs and
the CHDH toward the fluorescent substrate for the detection of
some pathogenic microbes [5–11] represents an interest for med-
ical analysis. To elucidate an applicability of CHDHs (microbial
3␤-HSDs) and CHOXs as reporter enzymes for selective detection
of some bacteria, opportunistic strains of Escherichia coli, S. aureus
and P. aeruginosa were cultivated on the 22-NBD-cholesterol-
containing medium. The HPLC analysis of the ethanolic extracts
from the bacterial cells that had been cultured for 24 h demon-
strated that the formation of the 22-NBD-cholest-4-en-3-one took
place in the case of P. aeruginosa only (Fig. 6). The homology
search allowed establishing that among all these bacteria only
(
minor); an emission maximum at 530 nm) (data not shown). TLC
and HPLC analysis of the samples after 22-NBD-cholesterol incuba-
tion with the CHDH in the presence of NAD revealed the formation
of a new fluorescent compound together with 22-NBD-cholest-4-
en-3-one – the single product of the steroid oxidation by CHOXs
+
[
16]. This new product seemed to be more lipophilic than 22-
NBD-cholest-4-en-3-one according to Rf values (0.62 and 0.46,
respectively) and HPLC retention times (7.6 and 6.4 min, respec-
tively) (Fig. 5B and C). The mass spectrum of the new product

6
4
Y.V. Faletrov et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
Fig. 5. CHDH-mediated conversion of 22-NBD-cholesterol. (A) Kinetic curves showing NADH formation during conversion of cholesterol and 22-NBD-cholesterol (I) (20 ␮M)
dashed and solid line, respectively) with the CHDH (2.5 U/ml) and NAD+ (200 ␮M) in buffer A. (B) TLC plate in 365 nm UV-light (depicted in black and white) showing
(
separation of I and the products of its conversion by the CHDH – 22-NBD-cholest-4-en-3-one (II) and 22-NBD-cholest-5-en-3-one (IID): 1 – I (standard), 2 and 4 – a mixture
of I and II, 3 – a probe of the CHDH-mediated conversion of I. (C) a HPLC chromatogram, demonstrating separation of I and products of its oxidation by CHDH (II and IID). (D)
Proposed scheme of the CHDH-mediated conversion of 22-NBD-cholesterol.
strong reversible inhibitor of P450scc, which forms Fe
N
coordination bond in the active site of the enzyme [36]. The
NBD-substituent hinders such Fe N coordination in the case of
2
2-NBD-cholesterol due to its planarity and electron-accepting
properties. Quantum-chemical calculations, on the other hand,
show that the negative partial charge on the N-atom of the flu-
orescent steroid is still sufficient for effective Fe N coordination
(data not shown). The ability of P450scc to incorporate steroids
with bulky substituents into its active site was shown previously
for 25-doxyl-27-nor-cholesterol [37] and (20R)-20-phenyl-5-
pregnen-3,20-diol [38] (the substrate and the inhibitor of P450scc,
respectively). Shape and volume of P450scc active site [39] also
allow the binding of 22-NBD-cholesterol in it. In this work we
showed that the 22-NBD-cholesterol is converted by reconstituted
NADPH-AdR-Adx-P450scc system, resulting in oxidative C22 N23
bond cleavage and selective NBD-NH2 formation. It should be
noted that another artificial 23,24-bisnorcholesterol derivative,
bearing a 7-hydroxyresorufin moiety at the C22 atom, has been
previously reported to undergo P450scc-dependent conversion
in Leydig tumor cell mitochondria [40], resulting in formation of
pregnenolone and highly fluorescent 7-hydroxyresorufin. Pregne-
nolone formation was postulated based on radioimmunoassay
(RIA) data only. Because of RIA assays may demonstrate cross-
reactivity toward structurally related steroids and the reaction
mentioned [40] is not in accordance with common P450-catalyzed
O-dealkylation mechanism, another steroidal product may be
also formed. We have supposed that the P450scc-catalyzed 22-
NBD-cholesterol conversion proceed via a 22-hydroxy derivative
rearrangement as common P450-mediated N-dealkylation [41].
Alternative ways of the P450scc-catalyzed NBD-NH2 formation
from 22-NBD-cholesterol, resulting in pregnenolone formation,
are possible via oxidation of the presumed 20␣-hydroxy interme-
diate at the 20-hydroxyl [39] or N23 (analogous to oxidation of
sphingolipids described [34]). Indeed, basing on our data of the
LC–MS analysis, pregnenolone was confirmed to be a major non-
Fig. 6. The superimposition of the HPLC curves obtained by the analysis of 22-NBD-
cholesterol incubation: a – with bacteria P. aeruginosa, b – with E. coli, c – with S.
aureus, d – without any bacteria (negative control), e – with the pure CHOX from
B. sterolicum (positive control). I – 22-NBD-cholesterol, II – 22-NBD-cholest-4-en-
3
-one. Peaks of others fluorescent substances with lower retention times may be
attributed to further 22-NBD-cholesterol biodegradation by the bacteria.
P. aeruginosa possesses the 3␤-HSD (GenBank ID: YP 001350609,
AAM27844) and CHOXs (YP 002438395, ABJ13412, etc.). The
obtained data, including established detection limits (about
0.2 pmol) of 22-NBD-cholesterol and 22-NBD-cholest-4-en-3-one,
and the other common advantages of fluorescent substrates-based
assays (selectivity, convenience, etc.) [14], may allow scientists to
use this novel 22-NBD-cholesterol conversion for the precise detec-
tion of various microbes, expressing CHOXs and/or CHDHs.
fluorescent steroidal co-product of NBD-NH . Another co-product
2
of NBD-NH2 was also detected and it was similarly deduced to
be 20-formyl-pregn-5-en-3␤-ol. Minor content of 20-formyl-
pregn-5-en-3␤-ol (10–12% from the pregnenolone formation
level) in the probes may be attributed to natural specificity of
the interaction between the P450scc and 22-NBD-cholesterol.
Moreover, an insufficient extraction of the steroidal aldehyde
due to Schiff bases formation with NH2-groups of the proteins
(P450scc and others) can take place. Additionally, an oxidative
conversion (deformylation) of 20-formyl-pregn-5-en-3␤-ol into
pregnenolone and/or other steroids seems to be possible [42,43].
4
. Discussion
In this work a putative binding of 22-NBD-cholesterol into
the catalytically active site of bovine P450scc has been shown
for the first time. It should be noted that 22-NBD-cholesterol is
a NBD derivative of 20(S)-aminomethylpregn-5-en-3␤-ol – a

Y.V. Faletrov et al. / Journal of Steroid Biochemistry & Molecular Biology 134 (2013) 59–66
65
Thus, these circumstances can bring about diminishing of final
content of the 20-formyl steroid in the probes.
The N-dealkylation of arylalkylamines has been well charac-
terized primarily for drug-metabolizing P450s [41], although an
ability of P450scc to catalyze the cumene hydroperoxide-driven
N-dealkylation of N,N-dimethylaniline was previously described
aliphatic chains. Because real 3D-structures of 3␤-HSDs have not
fully revealed yet, the established properties of 22-NBD-cholesterol
may provide opportunities to study structural features of the
enzymes’ active centers using fluorescence-based techniques.
In summary, the conversion of 22-NBD-cholesterol by three
sterol-converting oxidoreductases, namely, CHOX, CHDH and
cytochrome P450scc, as well as structures of the specific fluorescent
products have been established. Our results unambiguously prove
the substrate-like localization of 22-NBD-cholesterol in the active
sites of the enzymes, which provides opportunities for their inves-
tigations using fluorescence-based techniques. This is particularly
interesting in the cases of the cholesterol dehydrogenase or homol-
ogous 3␤-HSDs with partially undefined 3D-structures and P450scc
– with slightly uncertain catalytic mechanism. Moreover, the estab-
lished conversions of 22-NBD-cholesterol by microbial enzymes
create prerequisites for creation of new selective and sensitive
methods for detection of the CHOX- or CHDH-expressing microbes.
The 3-keto-4-en derivative of 22-NBD-cholesterol was obtained
and, being interpreted as a fluorescent 3-ketosteroid hormone ana-
log, it proposed to be useful for structure-function investigations of
various steroid hormone-related enzymes and receptors.
[
44]. It is known that a P450-catalyzed N-dealkylation proceeds
according to either hydrogen-atom transfer (HAT) or single electron
transfer (SET) mechanisms [40,41]. It should be mentioned, that the
SET variant of the 22-NBD-cholesterol oxidation may lead to a non-
fluorescent NBD-derivative formation via the N23-centered radical
rearrangement described [45] for NBD-labeled cardiolipin. Ability
of P450scc to catalyze various oxidations at C22 of such “unusual”
substrates as 25-doxyl-27-nor-cholesterol, vitamin D3 and ergo-
sterol (pro-vitamin D2) [28–31] confirms possibility of formation
a 22-hydroxylated 22-NBD-cholesterol as an intermediate during
P450scc-catalyzed conversion of the artificial steroid.
In this work 22-NBD-cholesterol was also shown to undergo the
+
NAD -dependent oxidation of its 3␤-hydroxy group, catalyzed by
the bacterial CHDH, followed by the ꢀ5–ꢀ4 isomerization. Similar
reaction was previously shown for two bacterial CHOXs–FAD-
containing enzymes, using oxygen as an oxidative co-substrate
[
16]. The catalytic properties of the CHDH and in particular CHOX
Acknowledgments
enzymes have revealed practical applications for (1) the measure-
ment of cholesterol in clinical analysis and (2) biotechnological
synthesis of the fluorescent 3-keto-4-en-steroid, which may be
interpreted as a fluorescent 3-ketosteroid hormone analog – a
new probe for steroid hormone-binding enzymes or receptors.
Upon exhaustive bioconversions of 22-NBD-cholesterol by CHOX or
CHDH the corresponding 3-keto-4-en-derivative (22-NBD-cholest-
The authors thank Prof. O.I. Shadyro and Mrs. A.G. Lisovskaya
from the Department of Chemistry (Belarusian State University)
for the assistance with mass spectrometry experiments, Prof. L.P.
Tytov and Mr. F. Lebedev (The Belarusian Republican Research and
Practical Center for Epidemiology and Microbiology) for the assis-
tance with bacteriological experiments. The authors acknowledge
Dr. M. Guenther from Whitehead Institute/MIT (Cambridge, MA,
USA) and Dr. A.V. Shkumatov from the Department of Biological
Chemistry and Molecular Pharmacology at Harvard Medical School
4
-en-3-one) was purified with high yields (70–80%). It should also
be noted that both CHOX and 22-NBD-cholesterol have been used
separately as molecular tools for investigations of cholesterol’s role
in structure-function properties of biological membranes. In mem-
brane bilayer the fluorophore of 22-NBD-cholesterol was reported
to be partially exposed to aqueous media, suggesting lateral or
even “up-side down” orientation of the fluorescent steroid in
comparison with cholesterol [12,13]. In the latter orientation, the
CHOX-mediated oxidation of 22-NBD-cholesterol in membranes
seems to be strongly hindered. Thus, the reactions established may
provide an additional approach to elucidate differences in orienta-
tions of the artificial sterol in biological membranes.
(Boston, USA) for proof reading and comprehensive revision of the
manuscript. This research was supported by Belarusian State Pro-
grams “Innovative Biotechnology” and “Applied and Fundamental
Medicine”.
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