Evaluation of the fluorescent probes Nile Red and 25-NBD-cholesterol as substrates for steroid-converting oxidoreductases using pure enzymes and microorganisms

Article abstract of DOI:10.1111/febs.12265

The fluorescent probes Nile Red (nonsteroidal dye) and 25-{N-[(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)-methyl]amino}-27-norcholesterol (25-NBD-cholesterol) (a cholesterol analog) were evaluated as novel substrates for steroid-converting oxidoreductases. Docking simulations with autodock showed that Nile Red fits well into the substrate-binding site of cytochrome P450 17α-hydroxylase/ 17,20-lyase (CYP17A1) (binding energy value of -8.3 kcal·mol ^-1). Recombinant Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found to catalyze the conversion of Nile Red into two N-dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron-donating partners of CYP17A1. The highest specific activity value (1.30 ± 0.02 min^-1) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeast's NADPH-cytochrome P450 reductase. The dye was also metabolized by pure CYP17A1 into the N-dealkylated derivatives, and gave a type I difference spectrum when titrated into low-spin CYP17A1. Analogously, docking simulations demonstrated that 25-NBD-cholesterol binds into the active site of the microbial cholesterol oxidase (CHOX) from Brevibacterium sterolicum (binding energy value of -5.6 kcal·mol^-1). The steroid was found to be converted into its 4-en-3-one derivative by CHOX (K_m and k_cat values were estimated to be 58.1 ± 5.9 μm and 0.66 ± 0.14 s^-1, respectively). The 4-en-3-one derivative was also detected as the product of 25-NBD-cholesterol oxidation with both pure microbial cholesterol dehydrogenase (CHDH) and a pathogenic bacterium, Pseudomonas aeruginosa, possessing CHOXs and CHDHs. These results provide novel opportunities for investigation of the structure-function relationships of the aforementioned oxidoreductases, which catalyze essential steps of steroid bioconversion in mammals (CYP17A1) and bacteria (CHOX and CHDH), with fluorescence-based techniques. Docking simulations show that fluorescent substances Nile Red and 25-NBD-cholesterol fit well the substrate-binding sites of CYP17A1 and the cholesterol oxidase (CHOX), respectively. Recombinant yeasts, expressing CYP17A1, as well as pure CYP17A1 catalyze the Nile Red conversion into N-dealkylated derivatives. 25-NBD-cholesterol is converted into its 4-en-3-one derivative by the CHOX and cholesterol dehydrogenase as well as by bacteria Pseudomonas aeruginosa.

Full text of DOI:10.1111/febs.12265

Evaluation of fluorescent probes Nile Red and 25-NBD-cholesterol as substrates for steroid-converting
oxidoreductases using pure enzymes and microorganisms
Yaroslav V. Faletrov1, Nina S. Frolova1, Hanna V. Hlushko1, Elena V. Rudaya1, Irina P. Edimecheva1, Stephan
Mauersberger2, Vladimir M. Shkumatov1,3*
1
Research Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya str. 14,
220030, Minsk, Belarus
2 Institute of Microbiology, Dresden University of Technology, Dresden, Germany
3 Department of Chemistry, Belarusian State University, Leningradskaya str. 14, 220030, Minsk, Belarus
*Corresponding author: Vladimir M. Shkumatov, Research Institute for Physical Chemical Problems,
Belarusian State University, Leningradskaya str. 14, 220030, Minsk, Belarus; phone: +37517-200-82-72; fax:
+37517-209-54-61; e-mail: biopharm@bsu.by
Running title: New conversions of Nile Red and 25-NBD-cholesterol
Article type: Original Article
Abbreviations:
25-NBD-cholesterol,
25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-
norcholesterol; 22-NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-
3β-ol; Nile Red, 9-(diethylamino)-5H-benzo[a]phenoxazin-5-one; CHOX, microbial cholesterol oxidase;
CHDH, microbial cholesterol-dehydrogenase; CYP17A1, cytochrome P450 17α-hydroxylase/17,20-lyase; Adx,
adrenodoxin; AdR, adrenodoxin reductase; CPR, NADPH-cytochrome P450 reductase; HSD, hydroxysteroid-
dehydrogenase.
Enzymes: cholesterol oxidase (EC 1.1.3.6), cholesterol dehydrogenase or microbial 3β-hydroxysteroid
dehydrogenase (EC 1.1.1.145), CYP17A1, cytochrome P450c17 or P450 17α-hydroxylase/17,20-lyase
(EC 1.14.99.9); NADPH-cytochrome P450 reductase (EC 1.6.2.4); adrenodoxin reductase (EC 1.18.1.2).
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/febs.12265
© 2013 The Authors Journal compilation © 2013 FEBS

Keywords: 25-NBD-cholesterol, Nile Red, cholesterol dehydrogenase, cholesterol oxidase, CYP17A1,
recombinant yeasts.
Abstract
Fluorescent probes Nile Red (non-steroidal dye) and 25-NBD-cholesterol (a cholesterol analogue) have been
evaluated as novel substrates for steroid-converting oxidoreductases. Docking simulations with AutoDock show
that Nile Red fits well the substrate-binding site of CYP17A1 (binding energy value = -8.3 kcal/mol).
Recombinant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found
to catalyze conversion of Nile Red into two N-dealkylated derivatives. The conversion by the yeasts was shown
to increase in the cases of co-expression of electron-donating partners of CYP17A1. The highest specific
activity value (1.30 0.02 min-1) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the
yeast’s P450-reductase. The dye is also metabolized by pure CYP17A1 into the N-dealkylated derivatives and
can cause a type I difference spectra when titrated into low spin CYP17A1. Analogously, docking simulations
demonstrate that 25-NBD-cholesterol binds into active sites of the cholesterol oxidase (CHOX) from
B. sterolicum (binding energy value = -5.6 kcal/mol). The steroid was found to be converted into its 4-en-3-one
derivative by the CHOX (Km and kcat values were estimated to be 58.1
5.9 μM and 0.66
0.14 s-1,
respectively). The 4-en-3-one derivative has been detected as the product of 25-NBD-cholesterol oxidation with
both pure cholesterol dehydrogenase (CHDH), and a pathogenic bacteria Pseudomonas aeruginosa, possessing
CHOXs and CHDHs. These results provide novel opportunities for investigation of structure-function
relationship of the aforementioned oxidoreductases, catalyzing essential steps of steroid bioconversion in
mammals (CYP17A1) and bacteria (CHOX and CHDH), by fluorescence-based techniques.
Introduction
Cholesterol (cholest-5-en-3β-ol) is a major mammalian steroid, functioning as a structural component of cellular
plasma membranes and as biosynthetic precursor of such important bio-regulators as bile acids, vitamin D and
steroid hormones. Essential steps of mammalian steroid hormones biosynthesis are performed by
oxidoreductases of two groups, namely, cytochromes P450 (CYPs) and hydroxysteroid-dehydrogenases
(HSDs). In particular, CYP17A1 catalyzes conversion of pregnenolone into 17α-hydroxypregnenolone and then
into dehydroepiandrosterone as well as progesterone into 17α-hydroxyprogesterone. 3β-HSD catalyzes
conversion of 3β-hydroxy-5-en steroids pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone
© 2013 The Authors Journal compilation © 2013 FEBS

into their hormonally active derivatives – progesterone, 17α-hydroxyprogesterone and androstenedione,
respectively. It is noteworthy that CYP17A1 inhibitors are potential or approved drugs against prostate cancer
[1-3].
On the other hand, some microorganisms (for example, genera Nocardia, Pseudomonas, Rhodococcus,
Mycobacterium and Vaccinia virus) also perform biotransformations of cholesterol as well as 3β-hydroxy-5-en
steroids [4-8]. An initial step of microbial bioconversions of the steroids is often initiated by cholesterol-
dehydrogenases (CHDHs), commonly cited as “microbial 3β-HSDs”, or catalytically similar cholesterol
oxidases (CHOXs). These enzymes are likely involved in pathogen-induced suppression of inflammatory and
immune responses to infection [4-8]. It is important to note that oxidoreductases of steroid catabolism by
Mycobacterium tuberculosis (a severe pathogen) and, probably, other microorganisms are interesting as
potential targets for new antibiotics [4, 6].
Fluorescent non-steroidal dye Nile Red and a fluorescent cholesterol analogue 25-NBD-cholesterol are
commonly used as unalterable probes in various biochemical and biophysical studies. In particular, 25-NBD-
cholesterol is used mainly for studies of lipid membranes, but differences between 25-NBD-cholesterol and
natural cholesterol concerning membrane orientation and intracellular distribution limit the utility of the probe
[9, 10]. 25-NBD-cholesterol was used also for functional characterization of the yeast ABC-transporter Aus1p
[11]. To the best of our knowledge, no enzymatic conversion of the fluorescent steroid has been described to
date, but its etherification by acyl-CoA-acyl transferases may be proposed [9, 10, 12] according to results of
previous studies. Analogously, Nile Red is used as a probe for intracellular neutral lipid droplets [13] and
hydrophobic sites of proteins [14, 15]. Nile Red was also identified as a substrate of the Candida albicans ATP-
binding cassette (ABC) transporters Cdr1p and Cdr2p as well as the major facilitator superfamily (MFS)
transporter Mdr1p [16]. Recently, Nile Red has been shown to be a substrate of drug-metabolizing human liver
CYP3A4 [17]. To our knowledge, this is the only enzymatic conversion of the dye known so far. Thus,
possibilities of enzymatic conversions of both 25-NBD-cholesterol and Nile Red seem to be not fully
investigated. In general, only few fluorescent substrates for steroid-converting oxidoreductases have been
discovered [18-23]. Hence, the aim of this study was to evaluate Nile Red and 25-NBD-cholesterol as new
fluorescent substrates for steroid-converting oxidoreductases mentioned above, i.e. CYP17A1 and
CHOX/CHDH, respectively.
© 2013 The Authors Journal compilation © 2013 FEBS

Results and Discussion
Docking simulations suggest Nile Red as a possible substrate of CYP17A1
Docking simulations with the crystal structure of human CYP17A1 [24] and Nile Red demonstrated that the dye
can bind into the active center of the P450 (Fig. 1А). The predicted binding energy value of the enzyme-ligand
complex was -8.1 kcal/mol, which is considerably smaller as compared to a natural substrate of CYP17A1
progesterone (binding energy value -9.4 kcal/mol). The calculated distance from the heme’s iron atom of
CYP17A1 to the N atom of the diethylamino- substituent of the dye was 4.8 Å. This points out to a possibility of
oxidative N-dealkylation of Nile Red by CYP17A1 [25, 26]. Arg239 was calculated to form a hydrogen bond
with the oxo-group of the dye, stabilizing conformation of the complex. Another type of the P450-dye complex,
where the oxo-group of Nile Red was located close to the heme of CYP17A1 with the binding energy value
-8.3 kcal/mol, was also predicted (Fig. 1B). This indicates a possibility of an unproductive binding of the dye in
the active site of CYP17A1.
Nile Red is converted into two N-dealkylated products by the recombinant yeasts expressing CYP17A1
During the incubation of Nile Red with the strains of S. cerevisiae and Y. lipolytica, expressing CYP17A1, a
successive formation of two colored fluorescent products was detected according to the TLC (RF for Nile Red,
products M1 and M2 were 0.51, 0.40 and 0.26, respectively) and HPLC-FL (corresponding retention times were
10.0, 6.8 and 4.9 min, respectively) data (Fig 2). LC-MS analysis confirmed that the products M1 and M2 were
mono- and di-N-dealkylated derivatives of Nile Red (dominant [M+H]+ ions with m/z 291 and 263,
respectively) (Fig. 3), which were firstly described as products of the dye bioconversion by pure CYP3A4 from
human microsomes [17]. According to HPLC-FL data, relative total 24 h conversion of Nile Red by Y. lipolytica
DE8-84 and S. cerevisiae YEp5117α [27-29] was estimated to be 79 2 % and 16 3 %, respectively, whereas
the minor conversion (less than 3 1 %) was observed in the cases of the control strains Y. lipolytica DE7-71.1
and S. cerevisiae, lacking CYP17A1 (Fig. 2B and Fig. 4). The formation of products M1 and M2 were also
detected when Nile Red was converted by pure CYP17A1. The yields were about 5.2 0.2 and 0.9 0.3 %,
respectively, for 1 h biotransformation of 25 μM Nile Red at 30 nM CYP17A1. Moreover, Nile Red was found
to cause type I difference spectral changes when titrated into low-spin CYP17A1 with apparent Kd value about 5
1 μM, respectively. All these results demonstrate a specific impact of CYP17A1 in the dye’s N-dealkylation.
A minor impact of the yeasts’ homologues of CYP3A4 (for example, sterol 22-desaturase Erg5p of S. cerevisiae
and alkane-hydroxylase ALK5p of Y. lipolytica) in the bioconversion of Nile Red should also be noted. The
© 2013 The Authors Journal compilation © 2013 FEBS

specific activity values of bioconversions of Nile Red into M1 as well as progesterone (a natural substrate of
CYP17A1) into 17α-hydroxyprogesterone are presented in Table 1.
The specific activity towards Nile Red for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeast’s
CPR, was determined to be 1.30 min-1, which was comparable with the activity of the N-dealkylation, catalyzed
by pure CYP3A4 (1.4 min-1) [17]. In contrast, in this strain progesterone 17α-hydroxylation proceeds about 10
times more efficiently, than Nile Red N-dealkylation, emphasizing strong preference of CYP17A1 towards the
natural substrate versus the artificial one. This is in accordance with our docking results. In the case of 24 h
bioconversion of Nile Red by Y. lipolytica DC5, the product M2 was found to be dominant (about 85 5 %),
whereas the major product for the other strains was M1. As it is shown in Table 1, an additional co-expression
of electron-donating partners of CYP17A1 in the cases of Y. lipolytica strains (Adx together with AdR [30] for
DE8-81 and, especially, CPR for DC5) enhanced specific activity values considerably (about 5 and 13 times,
respectively). It should be noted here that the CYP17A1:CRR ratio in recombinant yeasts increases by co-
expression of CPR from 3:1 to 1:6 approximately [31], overcoming limitations of the P450 activity due to
electron transport (optimum ratio is about 1:2 [17, 30]). In contrast to the conversion of Nile Red, there were no
significant differences in the progesterone 17α-hydroxylation specific activities by the yeasts, in spite of the co-
expression of the electron-donating partners (Table 1), indicating a high affinity of the substrate towards
CYP17A1. S. cerevisiae YEp5117α demonstrated the smallest specific activity value with respect to the Nile
Red bioconversion among the other CYP17A1-expressing strains tested. Besides electron-transport efficiency,
possible reasons for such difference are higher quantity of neutral lipid droplets, comprised mainly by
triacylglycerols [32-34], and less amount of ABC transporters, putatively involved in Nile Red efflux from the
cells [16, 35] in the case of Y. lipolytica. In this connection it is worth mentioning that the Nile Red conversion
should be considered when this dye is used as a lipid droplet marker or a substrate of ABC-transporters to study
recombinant yeasts, expressing CYP3A4 or CYP17A1 [28, 36]. Also the dye’s conversion is interesting as a
rare example of N-dealkylation catalyzed by specific steroidogenic cytochromes P450 [23], which are
characterized primarily with respect to natural steroidal substrates without N atoms in their structures. CYP17A1
is a molecular target for drugs against prostate cancer. Moreover, recombinant yeasts, expressing mammalian
steroidogenic cytochromes P450, have been used as whole-cell test systems for screening of inhibitors of
CYP17A1, CYP19A1 and CYP11B2 [2, 3, 24, 27]. Thus, the Nile Red bioconversion reveals a perspective for
using of advantages of fluorescent substrates-based assays (selectivity, convenience etc.) for this purpose.
© 2013 The Authors Journal compilation © 2013 FEBS

Docking simulations predict 25-NBD-cholesterol to be a substrate of the CHOX from B. sterolicum
Docking simulations of the interactions between the CHOX from B. sterolicum (PDBcode: 1COY) and
25-NBD-cholesterol demonstrated that the latter could be localized in the proximity of the protein’s flavin
(Fig. 5). The calculated binding energy value for 25-NBD-cholesterol interaction was shown to be about
-5.6 kcal/mol, whereas for 22-NBD-cholesterol it was -11.9 kcal/mol [22], indicating a worse affinity to the
CHOX of the former steroid as compared to the latter one with a shorter side chain. In the complex the NBD-
group of 25-NBD-cholesterol is primarily surrounded by the hydrophobic residues comprising Ile85, Gly84,
Phe83, Met59, Leu60, which is a prerequisite for increase of the steroid fluorescence during its binding to the
CHOX. In the predicted complex the distance between the C3 atom of the steroid and Nε atom of the enzyme’s
flavin was within 3.0 Ǻ, indicating a possibility of specific substrate-like conversion of 25-NBD-cholesterol by
the CHOX. Thus, these results confirm the potential ability of the 25-NBD-cholesterol to bind the active site of
the CHOX and to be oxidized by the enzyme at C3 position.
25-NBD-cholesterol conversion by the CHOX and CHDH: confirmation of the ultimate product structure.
LC-MS analysis of the 25-NBD-cholesterol conversion by the CHOX from B. sterolicum revealed the formation
of a product (II) with a molecular weight of 2 Da less than the substrate (I) (signals from ions [M+K]+ with m/z
601 versus 603, respectively). Moreover, appearance of a strong signal with m/z 563 from the [M2+H]+ ion in
the spectrum of the product instead of a signal with m/z 547 from the [M1+H-H2O]+ ion in the spectrum of 25-
NBD-cholesterol reflects a typical difference between mass-spectra of 4-en-3-one steroids and their 3β-hydroxy-
5-en precursors, respectively [37] (Fig. 6A and 6B). 4-en-3-one steroid nature of the product was also confirmed
by the marked absorbance maximum at 240 nm (Fig. 6C). These data together with our previous results
concerning the 22-NBD-cholesterol oxidation by CHOXs [22] confirm that the product is the 4-en-3-one
derivative of 25-NBD-cholesterol (formally, “25-NBD-cholest-4-en-3-one”) (Fig. 6D). Together with this 4-en-
3-one derivative another product was detected to be formed during the 25-NBD-cholesterol incubation with the
CHDH from Nocardia sp. and NAD+. This new product demonstrated higher mobility than the 4-en-3-one
derivative in the TLC system used (Rf values 0.62 and 0.46, respectively). Based on this data and our previous
results concerning the 22-NBD-cholesterol oxidation by the CHDH [23], we concluded that this new product is
the 5-en-3-one derivative of 25-NBD-cholesterol.
Kinetic parameters of the CHOX-driven 25-NBD-cholesterol oxidation together with the similar data for 22-
NBD-cholesterol and cholesterol are summarized in Table 2. As shown, kcat/Km values decrease in accordance
© 2013 The Authors Journal compilation © 2013 FEBS

with increasing volume and planarity of steroids’ side chains (substituents at C17) [22]. This is in agreement
with the docking results. Affinity to the detergent micelles may influence the kinetics of the oxidation [9, 10].
NADH formation rates during incubation of the CHDH and NAD+ with cholesterol, 22-NBD-cholesterol and
25-NBD-cholesterol were estimated to be 83, 39 and 6 nmol NADH per second, respectively. Thus, the
25-NBD-cholesterol conversion proceeded about 7 times less effectively than 22-NBD-cholesterol at the
conditions. 25-NBD-cholesterol seems to be the worst substrate for the CHDH among the steroids tested. The
same is also true for the CHOX . It should be noted that there is a higher affinity of the CHDH (3β-HSD) from
M. tuberculosis to pregnenolone as compared to cholesterol with the longer side chain [38], whereas it is vise
versa for the CHDH from Nocardia [39].
25-NBD-cholesterol was also shown to be converted by cells of a pathogenic bacteria Pseudomonas aeruginosa,
resulting in the formation of “25-NBD-cholest-4-en-3-one” and some others fluorescent compounds (Fig. 7).
Other fluorescent products formation may be proposed to be due to catabolism of the substrate and the product
by alternative steroid-converting enzymes of the bacteria. The discovered conversion provides perspectives for
elucidation of bacteria, expressing either CHOXs or CHDHs, in complex biological samples. Also CHOX- and
CHDH-mediated conversions of NBD-cholesterol may be suitable for an enzymatic synthesis of the
corresponding fluorescent 4-en-3-one steroids - potential ligands for steroid hormones receptors and other
steroid-converting enzymes. It is important to note, that the CHDH from Nocardia is highly homologous to the
3β-HSD from M. tuberculosis as well as to mammalian 3β-HSDs. Because the 3D-structures of the 3β-HSDs
have not been completely revealed yet, the established properties of 25-NBD-cholesterol may provide additional
opportunities to study the enzymes’ active centers using fluorescence-based techniques [23]. 25-NBD-
cholesterol was shown to be worse substrate than 22-NBD-cholesterol. But it should be noted that the former
steroid is a true cholesterol derivative with the full length side chain in contrast to the later steroid, being
actually a pregnane derivative. Thus, the established conversions of 25-NBD-cholesterol suggest a perspective
usage of the steroid and it’s 4-en-3-one derivative to study of steroid-converting oxidoreductases, operating with
sterols’ side chains (for, instance mammalian CYP11A1 or mycobacterial CYP125) [1, 4, 23].
Concluding remarks
Selective N-dealkylation of the fluorescent dye Nile Red by recombinant yeasts Y. lipolytica and S. cerevisiae,
expressing CYP17A1, has been described for the first time. The N-dealkylation is catalyzed by CYP17A1
© 2013 The Authors Journal compilation © 2013 FEBS

according to the results of docking simulations, showing that Nile Red binds properly to the active site of the
enzyme, and the experimental data. It should be mentioned that, to the best of our knowledge, no fluorescent
substrate has been described for CYP17A1 so far. The N-dealkylation of Nile Red by steroidogenic CYP17A1,
which differs from drug-metabolizing CYP3A4, was shown. It is an additional rare example on N-dealkylations
by steroidogenic cytochromes P450. Similarly, 25-NBD-cholesterol has been established to undergo conversion
into its 4-en-3-one derivative, catalyzed by the pure bacterial CHOX and CHDH as well as cells of bacteria
P. aeruginosa, expressing these enzymes. The established bioconversions of Nile Red and 25-NBD-cholesterol
provide opportunities for studies of the structure-function relationships of the steroid-converting
oxidoreductases in whole-cell biological systems using fluorescence-based techniques.
Materials and Methods
Docking simulations
Docking simulations were conducted using AutoDock 4.0 and the Graphical User Interface “AutoDockTools”
(The Scripps Research Institute) [40, 41] as described [23]. 3D-structures of human CYP17A1 (PDBcode
3RUK) [24] and the CHOX of Brevibacterium sterolicum (PDBcode 1COY) [42] were used for modeling of the
ligand-protein interactions of Nile Red and 25-NBD-cholesterol, respectively.
Enzymes and chemicals
22-NBD-cholesterol was from Molecular Probes (Eugene, OR). 25-NBD-cholesterol was from Avanti Polar
Lipids (Birmingham, AL). Nile Red (suitable for fluorescence, ≥ 98 %), cholesterol, progesterone, NADPH,
NAD+, CHOX from Brevibacterium sterolicum (8 U/mg solid) and CHDH from Nocardia sp. (30 U/mg solid)
were obtained from Sigma-Aldrich (St. Louis, MO). L-α-dilauroyl-sn-glycerophosphocholine L-α-dioleoyl-sn-
glycero-3-phosphocholine and CHAPS were from Serva. Polysorbate 80 and sodium cholate were from Fluka.
Yeast extract, peptone, D-galactose and D-glucose were from Difco (USA). All other chemicals used were
purchased from Sigma-Aldrich. Pure bovine CYP17A1 was obtained as described previously [43], but
progesterone was not added to the final CYP17A1 preparation. Second derivative spectrophotometry in Soret
region confirmed that the preparation contains more than 90 % of the native CYP17A1 in a low spin state [44].
Nile Red bioconversion by pure CYP17A1 and spectrophotometric titration of CYP17A1 by Nile Red
© 2013 The Authors Journal compilation © 2013 FEBS

The bioconversion of Nile Red by CYP17A1 and spectrophotometric titration of CYP17A1 by Nile Red were
conducted as described earlier for CYP3A4 [17].
Yeast strains and cultivation
In this work the following recombinant yeasts were used: S. cerevisiae YEp5117α, expressing CYP17A1;
Y. lipolytica E129A15, expressing CYP17A1; Y. lipolytica DC5, expressing CYP17A1 together with the yeast’s
CPR; Y. lipolytica DE8-84.1, expressing CYP17A1 together with Adx and AdR. Wild types of S. cerevisiae and
the strain Y. lipolytica DE7-74, expressing Adx and AdR, were used as controls. To obtain S. cerevisiae
YEp5117α, the parental strain S. cerevisiae GRF18 (Mat α his3-11 his3-15 leu2-3 leu2-112 cir+ canR) was
transformed with the shuttle YEp51 vector, encoding bovine CYP17A1 under the control of GAL10 promoter.
The strain Y. lipolytica E129A15 was obtained by crossing the strain A1−5 (MATB met Alk+) with the strain
E129 (MATA leu2-270 Alk+), containing an expression cassette with the CYP17A1 sequence. The strain
Y. lipolytica DC5 was obtained by crossing the strain E150 (MATB leu2-270 his-1 Alk+), expressing CYP17A1,
and the strain CXAU1 (MATA ade1 Alk+), bearing a p67RY1 plasmid with inserted sequence of the yeast’s
CPR. The strains Y. lipolytica DE series were obtained by crossing the strain E129L (MATA leu2-270 lys11-23
Alk+), expressing Adx with or without CYP17A1, with the strain E150 (MATB leu2-270 his-1 Alk+), expressing
AdR. All Y. lipolytica strains were transformed with the p67-based plasmids with the expression of the aforesaid
proteins under the control of the isocytrate lyase promoter ICL1 [27-29, 45].
The yeast strains were grown at 30 °C with shaking at 200 rpm on standard YPD (1 % yeast extract, 2 %
peptone and 2 % glucose) medium during 24 h. After this time D-galactose (up to 2 % w/v) or ethanol (up to 1
% v/v) was added to the S. cerevisiae and Y. lipolytica strains, respectively, to induce CYP17A1 expression [27-
29].
Nile Red bioconversion by the recombinant yeasts, expressing CYP17A1
A freshly grown suspension of one of the yeasts was adjusted dropwise with water up to cellular density of
1 × 107 cells/ml. To initiate biotransformations, 10 ml of the cell's suspension were mixed with proper aliquots
of ethanolic stock solutions (2 mM Nile Red). Such mixtures were shaken at 200 rpm and 30 °C during 24 h.
The bioconversion was stopped by the extraction of the probe with ethyl acetate (twice with a double volume of
the solvent and one more with an equal volume after 2 freeze-thaw cycles for additional cells destruction). Then
the combined extracts were centrifuged (3000 × g, 5 min). Subsequently the organic extracts were transferred
© 2013 The Authors Journal compilation © 2013 FEBS

into fresh flasks and evaporated to dryness. Dried extracts were re-dissolved in 1 ml of ethanol each. The probes
were analyzed using TLC, HPLC-FL and LC-MS as described below. Initial velocities of the dye N-dealkylation
by the yeasts were estimated from the data of HPLC-FL analysis (see below) of the probes of Nile Red (25 μM)
bioconversion within 1 h. Progesterone (100 μM) was treated in the same manner as a control. All
bioconversions were done in duplicate. Concentration of CYP17A1 in the cells was determined according to
protocol described [46].
25-NBD-cholesterol conversion by CHDH and CHOX
The stock solution of 25-NBD-cholesterol (1 mM) in propanol-2 was added to 25 mM potassium phosphate
buffer (pH 8.5) with 0.05 % sodium cholate, 0.1 % Polysorbate 80 (buffer A) and then sonicated for 0.5 min at
35 kHz, resulting in 2-100 μM initial concentrations. NAD+ was additionally used in the case of the CHDH.
Enzymatic oxidations were initiated by the addition of the CHDH or the CHOX. The conversion went at 30 ºС
for various time periods. In some cases 22-NBD-cholesterol and cholesterol conversions at the similar
conditions were conducted simultaneously for comparison. The reactions were stopped by extraction of the
probes (1 ml) with ethyl acetate (twice with 4 ml). The organic extracts were clarified by centrifugation (3000 ×
g, 5 min), evaporated to dryness and re-dissolved in 0.4 ml of ethanol. The resulting probes were analyzed by
TLC, HPLC-FL and HPLC-MS (see below). For estimation of kinetic parameters of 25-NBD-cholesterol
conversion by the CHOX, the reaction was conducted using 5-100 μM of the steroid. The conversions were
done in triplicate. Catalytic constant (kcat) and apparent Michaelis constant (Кm) values were estimated by
hyperbolic regression of the HPLC-FL data using Origin 7.0 software (OriginLab Corp.). The conversion rates
of cholesterol, 22-NBD-cholesterol and 25-NBD-cholesterol (initial concentrations were 20 μM) by the CHDH
(2.5 U/ml) at the presence of NAD+ (200 μM) were measured, based on initial slopes of spectrophotometric
kinetic curves of absorption at 340 nm, using ε340 6200 M-1×cm-1 to estimate NADH formation. The reactions
were conducted in buffer A at 25 °C.
Thin layer chromatography (TLC)
TLC was performed on silica aluminum foils (Fluka) with fluorescent indicator (254 nm). TLC plates were
developed with benzene:acetone mixture (4:1). Spots of substances were monitored visually under 365 nm UV-
light. If it was necessary, individual spots were cut out and extracted by ethanol for the additional analysis.
© 2013 The Authors Journal compilation © 2013 FEBS

High performance liquid chromatography with fluorometric detection (HPLC-FL)
HPLC-FL was conducted using the LC10-AD (Shimadzu) system with spectrophotometric and fluorometric
detector RF-10Axl, operated at the medium sensitivity. The column was LiChroCART C18 (250×4 mm, 5 μm)
(Merck). Elution conducted at 30 °C with the eluent, comprising acetonitrile:water:propanol-2 (84:16:5), using
flow rates of 1 ml/min and 1,5 ml/min for analysis of Nile Red and 25-NBD-cholesterol conversions,
respectively.
Liquid chromatography-mass spectrometry (LC-MS)
LC-MS measurements were performed using the LCMS-2020 (Shimadzu) system, equipped with a photodiode
array detector and a mass-spectrometer. For registration of mass-specrta electrospray ionization mode was used
and positive ions with m/z 150-750 were monitored. The column was Shim-pack VP-ODS (150×2 mm, 5 μm)
(Shimadzu). The elution was performed at 0.4 ml/min and at 40 °C. The eluent A, comprising
acetonitrile:water:formic acid (50:50:0.05), was used for the analysis of probes of the Nile Red bioconversions.
The eluent B, comprising acetonitrile:water:formic acid (90:10:0.05), was used for analysis of
25-NBD-cholesterol bioconversions.
Acknowledgments
The authors thank Prof. O.I. Shadyro and Dr. A.G. Lisovskaya from the Department of Chemistry of Belarusian
State University for the assistance with mass-spectrometry experiments. Dr. D.G. Kostsin and Prof. E.I.
Slobozhanina from Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus
are gratefully acknowledged for providing Nile Red and 25-NBD-cholesterol as well as for helpful discussions.
We also acknowledge Prof. L.P. Titov and Mr. F.A. Lebedev from The Belarusian Republican Research and
Practical Center for Epidemiology and Microbiology for the assistance in the experiment with bacteria
Pseudomonas aeruginosa. The authors acknowledge Dr. A.V. Shkumatov from the Department of Biological
Chemistry and Molecular Pharmacology at Harvard Medical School (Boston, USA) for proof reading and
comprehensive revision of the manuscript. This work was supported by the Belarusian State Program “Applied
and Fundamental Medicine”.
© 2013 The Authors Journal compilation © 2013 FEBS

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TABLES:
Table 1. Specific activity values for Nile Red (25 μM) and progesterone (100 μM) bioconversions by the
recombinant yeasts.
Yeast
Expressed protein(s)
Inducer
Specific activities of bioconversion of the
substrates by the yeasts,
nmol of the product */nmol CYP17/min
Nile Red
(25 μM)
0.05
Progesterone
(100 μM)
S. cerevisiae YEp5117α
S. cerevisiae (wild type,
control)
CYP17A1
-
D-Galactose
D-Galactose
13.2 0.8
< 0.02
ND**
Y. lipolytica E129A15
Y. lipolytica DE8-84.1
Y. lipolytica DC5
CYP17A1
Ethanol
Ethanol
Ethanol
Ethanol
0.25
0.35
11.1 1.3
11.8 1.1
12.5 0.9
ND
CYP17A1, Adx, AdR
CYP17A1, CPR
1.30
Y.lipolytica
DE7-71 Adx, AdR
< 0.02
(control)
*the product is the metabolite M1 in the case of the Nile Red or 17α-hydroxyprogesterone in the case of
progesterone. In the case of Nile Red specific activity values are represented as the means of duplicates whereas
in the case of progesterone – as the means standard deviations of four separate experiments;
** ND – not detected.
Table 2. Steady-state kinetic parameters of 25-NBD-cholesterol, 22-NBD-cholesterol and cholesterol
bioconversion by CHOX.
Substrate
25-NBD-сholesterol
-5.6 kcal/mol
0.40 0.14
22-NBD-сholesterol [22]
-11.9 kcal/mol
0.60 0.20
Сholesterol [22]
-11.4 kcal/mol
6.40 0.80
Binding energy
k
cat (c-1)
Km (M)
(59.1 3.9) × 10-6
0.7 × 104
(60.0 4.4) × 10-6
1.0 × 104
(19.4 2.0) × 10-6
33 × 104
kcat/Km (s-1 × M-1)
© 2013 The Authors Journal compilation © 2013 FEBS

Fig. 1. The conformation of Nile Red in the active site of human CYP17A1 (PDBcode 3RUK) predicted by
docking simulations. (A) Conformation allowing for N-dealkylation. (B) Conformation prohibiting
N-dealkylation. The dye is drawn with bold lines; gray, blue, red fragments correspond to C, N, O atoms,
respectively. The protein’s residues are all shown in blue.
© 2013 The Authors Journal compilation © 2013 FEBS

Fig. 2. Chromatographic data, showing formation of two fluorescent products (M1 and M2) at Nile Red
(NR) incubation with recombinant yeasts, expressing CYP17A1. (A) Representative HPLC-FL
chromatogram, demonstrating separation of NR, M1 and M2 from the probe of 8 h incubation of NR with Y.
lipolytica DE8-84 cells. (B) TLC plate in 365 nm UV-light (shown as black and white), reflecting selective
formation of M1 and M2 during the 24 h incubation of NR with yeasts, expressing CYP17A1: 1 and 6 –
standard of NR; 2 - Y. lipolytica DE8-84, expressing CYP17A1, Adx and AdR; 3 - Y. lipolytica DE7-74
(expressing Adx and AdR only, control); 4 – S. cerevisiae YEp5117α, expressing CYP17A1; 5 - wild type of S.
cerevisiae. See “Material and Methods” for further details.
Fig. 3. Analysis of the LC-MS data for the probe, taken after 48 h incubation of NR with Y. lipolytica
DE8-84 cells, confirmed that M1 and M2 are mono- and di-N-dealkylated derivatives of NR, respectively.
(A) A chromatographic curve, obtained by detection of absorbance at 500 nm. (B) A chromatographic curves,
obtained at mass-spectrometric detection of cations with m/z 150-750: bold black line – the signal form total
ions’ current; thin colored light-green, blue and purple lines – signals from [M+H]+ cations with m/z 263, 291
and 319, respectively (multiplication factors used are shown in brackets). For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.
© 2013 The Authors Journal compilation © 2013 FEBS

Fig. 4. HPLC-FL chromatograms reflecting selective formation of Nile Red N-dealkylated products in
strains expressing CYP17A1. A - Y. lipolytica DE7-71, expressing Adx and AdR; B - Y. lipolytica DE8-84,
expressing CYP17A1, Adx and AdR; C - S. cerevisiae (wild type); D - S. cerevisiae YEp5117α, expressing
CYP17A1. Probes were taken after 24 h biotransformation. NR – Nile Red, M1 and M2 – mono- and di-N-
dealkylated derivatives of the dye, respectively. See “Material and Methods” for further details.
© 2013 The Authors Journal compilation © 2013 FEBS

Fig. 5. The conformation of the 25-NBD-cholesterol in the active site of the bacterial CHOX (PDBcode
1COY) predicted by docking simulations. The dye is drawn with bold lines; gray, blue, red fragments
correspond to C, N, O atoms, respectively. The residues of aminoacids and the flavin are shown as blue and
green thin lines, respectively.
Fig. 6. Mass-spectra and absorbance spectra of 25-NBD-cholesterol (I) and the product of its oxidation by
CHOX (II). A – the mass-spectrum of 25-NBD-cholesterol (I), obtained at LC-MS analysis (electrospray
ionization, positive ions mode), and its interpretation; B - the mass-spectrum of II at the same condition; C –
absorbance spectra of chromatographic eluates, corresponding I (dotted line) and II (firm line); retention times
are given additionally; D – structures of 25-NBD-cholesterol (I) and its 4-en-3-one derivative – the product of
the steroid oxidation by CHOX (II). See “Materials and Methods” for other details.
© 2013 The Authors Journal compilation © 2013 FEBS

Fig. 7. HPLC-FL chromatograms reflecting formation of “25-NBD-choles-4-en-3-one” (II) during
incubation of 25-NBD-cholesterol (I) with the cells of Pseudomonas aeruginosa within 6 h (A) and 24 h
(B). See “Materials and Methods” for other details.
© 2013 The Authors Journal compilation © 2013 FEBS