The primary physiological significance of
cytochrome P450c27 (CYP27) has been associated with its role in the
degradation of the side chain of C27 steroids in the
hepatic bile acid biosynthesis pathway, which begins with
7
-hydroxylation of cholesterol in liver. However, recognition that
in humans P450c27 is a widely or ubiquitously expressed mitochondrial
P450, and that there are alternative pathways of bile acid synthesis
which begin with 27-hydroxylation of cholesterol catalyzed by P450c27,
suggests the need to reevaluate the role of this enzyme and its
catalytic properties. 27-Hydroxycholesterol was thought to be the only
product formed upon reaction of P450c27 with cholesterol. However, the
present study demonstrates that recombinant human P450c27 is also able
to further oxidize 27-hydroxycholesterol giving first an aldehyde and
then 3
-hydroxy-5-cholestenoic acid. Kinetic data indicate that in a
reconstituted system, after 27-hydroxycholesterol is formed from
cholesterol, it is released from the P450 and then competes with
cholesterol for reentry the enzyme active site for further oxidation.
Under subsaturating substrate concentrations, the efficiencies of
oxidation of 27-hydroxycholesterol and 3
-hydroxy-5-cholestenal to
the acid by human P450c27 are greater than the efficiency of hydroxylation of cholesterol to 27-hydroxycholesterol indicating that
the first hydroxylation step in the overall conversion of cholesterol
into 3
-hydroxy-5-cholestenoic acid is rate-limiting. Interestingly,
3
-hydroxy-5-cholestenoic acid was found to be further metabolized by
the recombinant human P450c27, giving two monohydroxylated products
with the hydroxyl group introduced at different positions on the
steroid nucleus.
 |
INTRODUCTION |
The major pathway for the metabolism and excretion of cholesterol
in mammals is the formation of the bile acids in liver. Cholesterol can
be degraded to bile acids in mammalian liver via two different routes,
one starting with 7
-hydroxylation, catalyzed by the microsomal
cytochrome P450 cholesterol 7
-hydroxylase
(P450c7),1 and the other one
initiated by 27-hydroxylation, catalyzed by the mitochondrial
cytochrome P450c27 (1). The route that begins with 7
-hydroxylation
(the classical pathway) is well described and is believed to be
quantitatively most important, whereas the sequence of reactions
leading to bile acids after 27-hydroxylation of cholesterol
(alternative pathways) are only now being investigated (2-8).
Discovery that cultured human macrophages can efficiently convert
cholesterol into 27-hydroxycholesterol and its oxidation product
3
-hydroxy-5-cholestenoic acid and secrete both products into the
medium (9) initiated a series of experiments showing that there is a
continuous flux of these 27-oxygenated products from peripheral tissues
to the liver where they are rapidly converted into bile acids (10). The
net uptake by the human liver of circulating 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid was measured and found to correspond
to
4% of the total bile acid formation, assuming quantitative
conversion into bile acids (10). Investigation of the quantitative
importance of this pathway in extrahepatic tissues in relation to high
density lipoproteins (HDL)-mediated reverse cholesterol transport, the
most important mechanism for cholesterol removal from extrahepatic
cells, also indicates that the 27-hydroxylation pathway is an
alternative and/or a complement to HDL-mediated reverse cholesterol
transport (11, 12). Finding that P450c27 mRNA and enzyme activity
are present in most if not all tissues (13-17) indicates that this
27-hydroxylation pathway is not limited to macrophages and probably
represents a general mechanism for removal of intracellular
cholesterol.
Although P450c27 appears to be the only enzyme responsible for the
conversion of cholesterol into 27-hydroxycholesterol (18-21), it has
not been proven that P450c27 can efficiently oxidize
27-hydroxycholesterol into 3
-hydroxy-5-cholestenoic acid. One
hypothesis is that this reaction may be catalyzed by alcohol and
aldehyde dehydrogenases (22). Moreover, even though comparable amounts
of 27-hydroxycholesterol and 3
-hydroxy-5-cholestenoic acid are
formed upon incubation of human macrophages with cholesterol, purified
rabbit liver P450c27 has been found only to produce
27-hydroxycholesterol from cholesterol with no detectable
3
-hydroxy-5-cholestenoic acid (23). Recently, we overexpressed human
P450c27 in Escherichia coli yielding the highly purified
enzyme and in initial experiments we also did not observe the formation
of 3
-hydroxy-5-cholestenoic acid (24). Herein, we describe the
conditions under which 3
-hydroxy-5-cholestenoic acid is formed from
cholesterol in a reconstituted system demonstrating for the first time
that P450c27 is able to catalyze multiple oxidation reactions at the
C-27 atom of cholesterol. We also show that under identical in
vitro conditions 27-hydroxycholesterol and
3
-hydroxy-5-cholestenal are more efficiently converted into
3
-hydroxy-5-cholestenoic acid by P450c27 than is cholesterol into
27-hydroxycholesterol. Because 27-hydroxycholesterol has a number of
potent biological activities that relate to regulation of cholesterol
synthesis (25, 26), cytotoxicity to different cell types (27-30), and platelet aggregation (31), discovery of factors influencing the level
of 27-hydroxycholesterol is important. Because a single enzyme
catalyzes both the formation and the degradation of
27-hydroxycholesterol, studies identifying factors influencing the
rates of both the oxidation of cholesterol into 27-hydroxycholesterol,
and the conversion of 27-hydroxycholesterol into
3
-hydroxy-5-cholestenoic acid by P450c27, provide insight as to how
the level of these two oxygenated cholesterol metabolites and the ratio
between them may differ within extrahepatic tissues. These studies
establish the initial sequence of reactions in alternative bile acid
biosynthetic pathways and expand our knowledge of the diversity of the
substrates utilized by this enzyme.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Reagents for bacterial growth were from Difco.
[4-14C]Cholesterol was from DuPont; tritium-labeled and
unlabeled 27-hydroxycholesterol were synthesized as described
previously (32). 3
-Hydroxy-5-cholestenoic acid was synthesized via
standard Wittig-Horner reaction of
3
-tert-butyldimethylsilyloxocholest-24-al (33).
Tritium-labeled 3
-hydroxy-5-cholestenoic acid was obtained by
oxidizing the unlabeled free acid with commercial 3
-hydroxysteroid dehydrogenase (obtained from Boehringer Mannheim) under the conditions previously described (34) to yield the 3-oxo-4-unsaturated analogue of
cholestenoic acid. This compound was methylated and converted into the
corresponding enol trimethylsilyl ether (34). The crude enol ether was
immediately reduced with NaB[3H]4 (obtained
from Amersham International, UK) in isopropanol. The resultant methyl
ester of 3
-hydroxy-5-cholestenoic acid was hydrolyzed by alkaline
saponification and extracted with ether from acidified water phase. The
ether extract was washed with water until neutral, and the ether was
evaporated. The material was pure as judged by radio thin layer
chromatography with the use of toluene/ethyl acetate 3:7 (v:v) as a
moving phase, and the identity was confirmed by combined gas
chromatography-mass spectrometry (GC-MS) of the trimethylsilyl
ether-methyl ester. The specific radioactivity was 5 × 109 cpm/µg, and it was diluted with unlabeled
3
-hydroxy-5-cholestenoic acid to a specific radioactivity of 600,000 cpm/µg prior to incubation. Cholesten-27-al was prepared from
unlabeled and labeled 3
-hydroxy-5-cholestenoic acid by methylation
and acetylation followed by reduction with diisobutyluminium hydride
(35). The aldehyde was purified by preparative thin-layer
chromatography, using toluene/ethyl acetate 3:7 (v:v) as a moving
phase. The identity of the compound was confirmed by GC-MS of the
trimethylsilyl ether-methyl ester derivative. There was a continuous
degradation of the aldehyde into the corresponding acid and alcohol due
to non-enzymatic dismutation. As a consequence, only newly synthesized
and newly purified material could be used in the experiments, and in
general the purified aldehyde used contained a minimum of 4% of the
corresponding acid and 4% of the corresponding alcohol.
Recombinant human P450c27, recombinant bovine adrenodoxin (Adx), and
recombinant adrenodoxin reductase (Adr) were expressed and purified as
described previously (24). P450c27 with a heme content of 17.6 nmol/mg
protein showing a single band upon silver-stained SDS-polyacrylamide
gel electrophoresis was used in this study. Prior to enzymatic assays
P450c27 was dialyzed overnight against 100 volumes of 40 mM
phosphate buffer (pH 7.4) containing 20% glycerol to remove 1 M NaCl and 0.5% sodium cholate present in the elution
buffer used during the last purification step, and concentrated to 70 µM using ultrafiltration membrane cones (Amicon). Spectral purity indexes of recombinant Adx
(A414/A276) and
recombinant Adr
(A278/A450) were 0.95 and
7.5, respectively. Adx and Adr are considered to be pure with the
spectral purity indexes beginning from 0.86 for Adx and 7.5 for Adr
(36).
Enzyme Assays--
P450c27 activities using either cholesterol,
27-hydroxycholesterol or 3
-hydroxy-5-cholestenal as a substrate were
assayed in 40 mM phosphate buffer (pH 7.4), containing
0.1% Tween 20 (24). When 3
-hydroxy-5-cholestenoic acid was used as
a substrate, the buffer did not contain detergent. Reaction mixtures (1 ml) contained variable amounts of P450c27, Adx, Adr, and substrate.
Enzymatic assays were initiated by addition of NADPH (final 1 mM), carried out at 37 °C for different times, and
terminated by adding 2 ml of CH2Cl2. Steroids,
after extraction with CH2Cl2, were evaporated, dissolved in methanol and analyzed by HPLC. The conditions used for
separation of cholesterol from 27-hydoxycholesterol and
3
-hydroxy-5-cholestenoic acid were the same as described previously
(37). The retention times for 3
-hydroxy-5-cholestenoic acid,
27-hydroxycholesterol, and cholesterol were 9.25, 10.95, and 18.6 min,
respectively. Under these conditions, however, 27-hydroxycholesterol
and 3
-hydroxy-5-cholestenal were not separated, and the two products
appeared as one homogenous peak. To separate 27-hydroxycholesterol and
3
-hydroxy-5-cholestenal, another HPLC system was developed. Steroids
were separated using a YMC-PACK-ODS-A (4.6 × 250 mm) (YMC Co.,
Ltd.) column and isocratic elution with methanol/water/acetic acid
85:15:0.01 (v/v/v). The flow rate was 1 ml/min. The retention times for
3
-hydroxy-5-cholestenoic acid, 27-hydroxycholesterol, and
3
-hydroxy-5-cholestenal were 40, 46, and 51 min, respectively. The
products obtained after the incubation of P450c27 with different
substrates were identified using GC-MS.
 |
RESULTS |
Previous studies of enzymatic activities showed no formation of
3
-hydroxy-5-cholestenoic acid upon incubation of purified P450c27
with cholesterol (23, 24). In these experiments, relatively low
(0.1-0.2 nmol) amounts of P450c27 were used in reconstituted systems.
Results using cultured human macrophages (9) and human aortic
endothelial cells (17) demonstrating conversion of cholesterol into
3
-hydroxy-5-cholestenoic acid led us to further investigate metabolism of cholesterol by P450c27 using greater (up to 3 nmol) amounts of enzyme. These experiments were carried out in the presence of 0.1% Tween 20 because addition of non-ionic detergents
substantially increases solubility of cholesterol in aqueous solutions
(38, 39). To be able to compare catalytic properties of P450c27 toward different substrates under similar conditions, 0.1% Tween 20 was also
included in the reaction mixture when 27-hydroxycholesterol and
3
-hydroxy-5-cholestenal were used as substrates.
Product Formation upon Incubation of Cholesterol with
P450c27--
As judged by HPLC separation (Fig.
1C), two major products are
formed from cholesterol when 1.5 µM P450c27 is used in a
reconstituted system in the presence of 0.1% Tween 20. No products
were formed when either Adx or NADPH were omitted (data not shown).
GC-MS analysis performed after preliminary HPLC separation showed that the first peak is 3
-hydroxy-5-cholestenoic acid, the second peak besides 27-hydroxycholesterol may contain small amounts of
3
-hydroxy-5-cholestenal, and the third peak is cholesterol. Finding
that second peak is not homogeneous was surprising because
3
-hydroxy-5-cholestenal was not detected when lower concentrations
of P450c27 (0.04 µM or 0.2 µM) were used
(Fig. 1A). To check whether 3
-hydroxy-5-cholestenal is
really not separated from 27-hydroxycholesterol under the
chromatographic conditions used and to develop an HPLC system allowing
separation of all three reaction products, 3
-hydroxy-5-cholestenal
was synthesized (see "Experimental Procedures"). Analysis of the
chromatographic behavior of chemically synthesized
27-hydroxycholesterol and 3
-hydroxy-5-cholestenal revealed that
these two products are indeed not separated from each other under the
conditions described in Fig. 1. The new chromatographic system (see
"Experimental Procedures") separates 3
-hydroxy-5-cholestenoic acid, 3
-hydroxy-5-cholestenal, and 27-hydroxycholesterol (Fig. 2). The retention time of the chemically
synthesized 3
-hydroxy-5-cholestenal was virtually identical to that
of one of the biologically generated products. However, the amounts of
this product after preliminary HPLC separation were too small to allow
clear identification by GC-MS. The final proof that
3
-hydroxy-5-cholestenal is indeed the intermediate in the conversion
of cholesterol into 3
-hydroxy-5-cholestenoic acid catalyzed by
P450c27 was obtained from the incubations with 27-hydroxycholesterol
described below. Because the new chromatographic system does not allow
quantitation of unmetabolized cholesterol, the patterns of product
formation (Fig. 1B) were generated by first determining the
total conversion of cholesterol into 3
-hydroxy-5-cholestenoic acid
and (27-hydroxycholesterol+3
-hydroxy-5-cholestenal) using the
initial chromatographic system, and then the ratio between the three
products was determined using the second chromatographic system.

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Fig. 1.
Time course of product formation when
different amounts of human recombinant P450c27 are incubated with 50 nmol of cholesterol. The reaction conditions are as described
under "Experimental Procedures." A, formation of
27-hydroxycholesterol (27-OH) when 0.04 nmol ( ) and 0.2 nmol ( ) of P450c27 were used in the enzymatic assay. Neither
3 -hydroxy-5-cholestenal nor 3 -hydroxy-5-cholestenoic acid were
detected under the experimental conditions used. B,
formation of 27-hydroxycholesterol (27-OH, ),
3 -hydroxy-5-cholestenal (27-CHO, ) and
3 -hydroxy-5-cholestenoic acid (27-COOH, ) when 1.5 nmol of P450c27 was used in the enzymatic assay. C, HPLC
separation of products formed after 40 min of the reaction of 1.5 nmol
of human recombinant P450c27 with 50 nmol of cholesterol. Abbreviations
as in Fig. 1B. HPLC was performed using a Waters HPLC
equipped with a Nova-Pak C18 column (3.9 × 150 mm)
and a precolumn of the same type connected to a -RAM radioactivity
flow detector (INUS Systems Inc., Tampa, FL). A linear solvent gradient
between solvent A (acetonitrile/methanol/water, 40:40:20) and solvent B
(100% methanol) over 15 min was used, after which the flow was kept at
100% solvent B for another 10 min.
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Fig. 2.
HPLC separation of products formed after 10 min of reaction of 1.5 nmol of human recombinant P450c27 with 50 nmol
of 27-hydroxycholesterol. Reaction conditions and steroid
separation are described under "Experimental Procedures." The
arrow indicates a minor polar product which is a
monohydroxylated cholestenoic acid. The inset shows the
kinetics of formation of 3 -hydroxy-5-cholestenal (27-CHO,
) and 3 -hydroxy-5-cholestenoic acid (27-COOH,
).
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Kinetic analysis of the reaction using 1.5 µM P450c27 and
50 µM cholesterol reveals that there is a lag phase in
the formation of 3
-hydroxy-5-cholestenoic acid (Fig. 1B),
indicating that a certain amount of 27-hydroxycholesterol should be
accumulated before it can be further metabolized. The presence of the
lag phase and the fact that the ratio between 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid is not constant but changes during
time course of the reaction (Fig. 1B) indicates that after 27-hydroxycholesterol is formed from cholesterol, it is released from
the P450c27, and then competes with cholesterol to reenter the enzyme
active site for further oxidation. The more 27-hydroxycholesterol formed, the more efficiently it competes with cholesterol, changing a
ratio between 27-hydroxycholesterol and 3
-hydroxy-5-cholestenoic acid from 10.96 after 10 min of reaction to 1.51 after 40 min under the
conditions used in Fig. 1B. If 27-hydroxycholesterol were
not released from the P450c27 active site, the formation of
3
-hydroxy-5-cholestenoic acid would parallel the formation of
27-hydroxycholesterol.
Product Formation upon Incubation of 27-Hydroxycholesterol with
P450c27--
In the reconstituted system containing 1.5 µM P450c27 and 50 µM 27-hydroxycholesterol
as initial substrate, the major product formed was
3
-hydroxy-5-cholestenoic acid (Fig. 2). The amount of
3
-hydroxy-5-cholestenoic acid was increased with reaction time (Fig.
2, inset), no 3
-hydroxy-5-cholestenoic acid was detected when NADPH was omitted from the reaction mixture, and formation of
3
-hydroxy-5-cholestenoic acid was substantially reduced in the
presence of ketoconazole, which is an inhibitor of many P450 s
including P450c27 (data not shown). The amounts of
3
-hydroxy-5-cholestenal formed in incubations with
27-hydroxycholesterol were 2-3 times higher than in the incubations
with cholesterol. The mass spectrum of the trimethylsilyl ether of this
metabolite (Fig. 3B) was
identical to that of synthetic 3
-hydroxy-5-cholestenal (Fig.
3A). Identification of the aldehyde as an intermediate
establishes the expected reaction sequence in the overall conversion of
cholesterol into 3
-hydroxy-5-cholestenoic acid by P450c27.

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Fig. 3.
Mass spectrum of trimethylsilyl
(TMS) ether of the synthetic 3 -hydroxy-5-cholestenal
(A) and one of the metabolites eluting at 51 min (Fig. 2)
upon incubation of 27-hydroxycholesterol with P450c27
(B).
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Product Formation upon Incubation of 3
-Hydroxy-5-cholestenal
with P450c27--
Due to the instability, only small amount of
3
-hydroxy-5-hydroxycholestenal was synthesized. This amount,
however, was enough to show that synthetic
3
-hydroxy-5-hydroxycholestenal can be efficiently converted by
P450c27 into 3
-hydroxy-5-cholestenoic acid and to compare under
identical conditions the rates of conversion of
3
-hydroxy-5-hydroxycholestenal into 3
-hydroxy-5-cholestenoic acid
and cholesterol into 27-hydroxycholesterol (Table
I). Because 3
-hydroxy-5-hydroxycholestenal can dismutate nonenzymatically, the
formation of 3
-hydroxy-5-cholestenoic acid was also determined in
the absence of NADPH. As is seen from Table I, the capacity of P450c27
to convert 3
-hydroxy-5-hydroxycholestenal into
3
-hydroxy-5-cholestenoic acid is at least 5 times higher than that
to convert cholesterol into 27-hydroxycholesterol. The fact that 79%
of 3
-hydroxy-5-hydroxycholestenal was converted into
3
-hydroxy-5-cholestenoic acid within 10 min of reaction also
indicates that experimental conditions using this substrate were not
optimal for kinetic analysis and the concentration of the substrate
could be rate-limiting. The rate of conversion of
3
-hydroxy-5-hydroxycholestenal into 3
-hydroxy-5-cholestenoic acid
may be even higher than in Table I if optimal conditions are used.
Product Formation upon Incubation of 3
-Hydroxy-5-cholestenoic
Acid with P450c27--
As is seen from Fig. 2, there is a minor polar
product eluting from the column earlier than
3
-hydroxy-5-cholestenoic acid which is formed upon incubation of
P450c27 with 27-hydroxycholesterol. To check the origin of this
product, 3
-hydroxy-5-cholestenoic acid was used as substrate at very
low concentrations to maximize conversion. Fig.
4 shows that 3
-hydroxy-5-cholestenoic
acid can be metabolized by P450c27. The formation of the product is
time-dependent and requires the complete reconstituted
system consisting of P450c27, Adx, Adr, and NADPH (data not shown).
Isolation of the polar product fraction by HPLC and analysis of the
trimethylsilylated methyl ester showed that it consists of one
major and one minor monohydroxylated metabolites (Fig.
5, A and B). The
mass spectrum of both products was similar to the mass spectrum of the
corresponding derivative of 3
-hydroxy-5-cholestenoic acid with
prominent peaks at m/z 79, m/z 129 (3
-hydroxy-5-unsaturated steroid), and
m/z 213. The ions at m/z
255 (nucleus
trimethylsilyl ether), m/z
345 (M
side chain), m/z 373 (M
129), m/z 412 (M
90), and
m/z 502 (M) in the mass spectrum of cholestenoic
acid derivative were shifted in the two products by two mass units to
m/z 253, m/z 343, m/z 371, m/z 410, and
m/z 500 as shown in Fig. 5. This is consistent
with loss of one trimethylsilyl ether group from the steroid nucleus in
both products. Although it was not possible to define the specific
location of the hydroxyl group introduced in the steroid nucleus by
P450c27 in each product from the fragmentation pattern, it is possible
to exclude certain positions due to the absence of characteristic peaks
obtained with standards. P450c27 has been reported to catalyze
hydroxylations in positions 24, 25, and 1 (40, 41). All these three
positions are, however, excluded in the metabolites in Fig. 5. Because
mass spectra of cholestenoic acid derivatives are not identical to that
of 3
,7
-dihydroxy-5-cholestenoic acid or
3
,7
-dihydroxy-5-cholestenoic acid, 7
- and 7
-positions can
also be excluded as possible sites of hydroxylation.

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Fig. 4.
HPLC separation of products formed after 30 min reaction of 1.5 nmol of human recombinant P450c27 with 2.3 nmol of
3 -hydroxy-5-cholestenoic acid (27-COOH). The
reaction conditions and HPLC separation are as described in Fig.
1.
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Fig. 5.
Mass spectrum of trimethylsilyl
(TMS) ether of the major (A) and minor
(B) metabolites from the incubations of
3 -hydroxy-5-cholestenoic acid with P450c27.
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Kinetic Analysis of Cholesterol and 27-Hydroxycholesterol
Metabolism Catalyzed by P450c27--
Because accurate determination of
Km and Vmax requires linear
rates of product formation with respect to enzyme concentrations, we
determined the effect of varying P450c27 concentrations on product
formation when cholesterol and 27-hydroxycholesterol were used as
substrates. Fig. 6 shows that the rates
of formation of 27-hydroxycholesterol from cholesterol and
3
-hydroxy-5-cholestenoic acid from 27-hydroxycholesterol are linear
within P450c27 concentration ranges of 0.3-0.75 µM and
0.3-1.5 µM, respectively. As is also seen from Fig. 6,
when the concentration of the substrate is 50 µM, there
is a direct correlation between the amount of the
3
-hydroxy-5-cholestenoic acid formed either from cholesterol or
27-hydroxycholesterol and the concentration of P450c27. The fact that,
upon incubation of cholesterol with P450c27, there is a threshold
concentration of the enzyme below which no 3
-hydroxy-5-cholestenoic
acid is detected explains why this metabolite was not identified in the
previous studies in which lower concentrations of both P450c27 and
cholesterol were used.

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Fig. 6.
Effect of varying P450c27 concentrations on
product formation when 50 µM cholesterol (A)
or 50 µM 27-hydroxycholesterol (B) were used
as substrates. Incubations with cholesterol have been carried out
for 20 min, and those with 27-hydroxycholesterol for 10 min.
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We tried to determine Km and
Vmax for both cholesterol and
27-hydroxycholesterol. In kinetic experiments, the maximal concentration of steroid used was 30 µM with no turbidity
being observed after addition of the steroid to the reaction mixture. In the case of cholesterol, the reaction conditions (amount of P450c27
and reaction time) have been optimized for only one product, 27-hydroxycholesterol, with no formation of 3
-hydroxy-5-cholestenoic acid. The conversion of either cholesterol or 27-hydroxycholesterol into product did not exceed 8%. The relationship between the initial velocity (vo) of the P450c27-catalyzed reaction
and the concentration of the substrate ([S]) (Fig.
7) shows that the reactions are
first-order with respect to substrate concentration indicating that the
range of the substrate concentrations used is much lower than
Km. Because determination of Km
requires use of a range of substrate concentrations between 0.33 and
2.0 Km (42), it becomes rather problematic to
accurately determine Km for either cholesterol or
27-hydroxycholesterol because of their limited solubilities in aqueous
solutions even in the presence of detergent. From double-reciprocal
plots (data not shown), we could only make a rough estimate of
Km of 100 µM or higher for both
cholesterol and 27-hydroxycholesterol. However, in the case when [S]
Km, it is possible to determine the enzyme catalytic efficiency
(kcat/Km) using Equation 1
(43)
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(Eq. 1)
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kcat is a catalytic constant of the enzyme,
and [E] is the concentration of the enzyme. Because in our
experiments [E] is constant and known, this equation can
be rearranged into that shown in Equation 2.
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(Eq. 2)
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(kcat/Km) are determined
by plotting vo/[E] as the ordinate
and [S] as the abscissa (Fig. 7). The fact that
(kcat/Km) for cholesterol is
approximately 3 times lower than that for 27-hydroxycholesterol (Table
II) indicates that in the overall
conversion of cholesterol into 3
-hydroxy-5-cholestenoic acid the
first hydroxylation step is rate-limiting.

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Fig. 7.
Effect of varying substrate concentrations on
the formation of 27-hydroxycholesterol from cholesterol (A)
and 3 -hydroxy-5-cholestenoic acid from 27-hydroxycholesterol
(B). Reactions with both substrates have been carried
out for 10 min using 0.5 µM P450c27.
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P450c27 has a wide substrate specificity (44). The best endogenous
substrate for P450c27 is 5
-cholestane-3
,7
,12
-triol, which
is formed during the hepatic bile acid synthesis pathway initiated by
7
-hydroxylation of cholesterol. Using
5
-cholestane-3
,7
,12
-triol, rabbit and human enzymes are
able to hydroxylate the terminal methyl group three times to give at
first 5
-cholestane-3
,7
,12
,27-tetrol, the intermediate
aldehyde, and subsequently 3
,7
,12
-trihydroxy-5
-cholestanoic acid (35, 45). Because kcat is also known as the
turnover number (43), the value
(kcat/Km) for these
substrates can be calculated from previous experiments where these
kinetic parameters were determined. Comparison of the values of
(kcat/Km) for different
substrates of P450c27 as well as for cholesterol of another cytochrome
P450 (P450scc, catalyzing the conversion of cholesterol to pregnenolone
in mitochondria of steroidogenic tissues) revealed that enzyme's
catalytic efficiency
(kcat/Km) appeared to be the
highest for 5
-cholestane-3
,7
,12
-triol followed by
5
-cholestane-3
,7
,12
,27-tetrol, 27-hydroxycholesterol, and cholesterol (Table II). Comparison of the
(kcat/Km) values between the
two mitochondrial P450s that utilize cholesterol as a substrate
(P450c27 and P450scc) (Table II) indicates that at subsaturating
substrate concentrations cholesterol is a much better substrate for
P450scc than for P450c27.
 |
DISCUSSION |
The present study clearly and conclusively demonstrates for the
first time that recombinant human P450c27 is able to hydroxylate the
terminal methyl group of cholesterol three times to give at first
27-hydroxycholesterol, then 3
-hydroxy-5-cholestenal, and finally
3
-hydroxy-5-cholestenoic acid with the first hydroxylation step
being rate-limiting. In addition to the finding that at low, subsaturating, concentrations 27-hydroxycholesterol is an even better
substrate for P450c27 than cholesterol, we demonstrate that after
P450c27 finishes complete oxidation of the C-27 atom of cholesterol to
form 3
-hydroxy-5-cholestenoic acid, it can further introduce a
hydroxyl group at different positions of the steroid nucleus but not at
the steroid side chain. Such activity toward steroid substrates has not
been reported previously, and indicates that regioselectivity of
hydroxylations catalyzed by P450c27 is determined by the state of the
oxidation of the C-27 atom of the side chain, and if C-27 is completely
oxidized, P450c27 will introduce hydroxyl groups in the steroid
nucleus. Because the intracellular concentration of
3
-hydroxy-5-cholestenoic acid is very low (12) and because this acid
is easily transported out from the cells, it is not possible to ascribe
a physiological importance to these novel hydroxylations.
Finding that Km for both cholesterol and
27-hydroxycholesterol are high and that the concentration of both
substrates used in previous reconstitution experiments was much lower
than Km, provides an explanation for the inability
to detect 3
-hydroxy-5-cholestenoic acid in previous studies. When
[S]
Km, very little enzyme-substrate complex
[ES] is formed, and consequently very little product is
formed within the time of the assay. In the present study, using higher
amounts of P450c27, we increased the concentration of
[ES], thus increasing the formation of the intermediate
product, 27-hydroxycholesterol. This allowed demonstration that P450c27
can efficiently convert cholesterol into 3
-hydroxy-5-cholestenoic
acid. Because 3
-hydroxy-5-cholestenoic acid is found in human serum
at concentrations 6.72 ± 2.79 µg/100 ml (3), this reaction is
of physiological significance.
Our data also indicate that in the reconstituted system after
27-hydroxycholesterol is formed from cholesterol, it is released from
P450c27 and competes with cholesterol to reenter the enzyme active site
for further oxidation. We speculate that a similar sequence of events
occurs in vivo. Indeed, if 27-hydroxycholesterol formed from
cholesterol in vivo, does not leave P450c27 active site and
is directly oxidized to 3
-hydroxy-5-cholestenoic acid, no
27-hydroxycholesterol would be found in the serum. The concentration of
27-hydroxycholesterol in human serum ranges from 12.5 to 29.4 µg/100
ml (46). P450c27 is the only enzyme in humans responsible for the
conversion of cholesterol to 27-hydroxycholesterol (18-21). In
patients with cerebrotendinous xanthomatosis, a hereditary sterol
storage disorder disease caused by the mutations in CYP27, serum levels
of 27-hydroxycholesterol as well as 3
-hydroxy-5-cholestenoic acid
are significantly reduced if detected at all (21).
The metabolic fate of 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid formed in the alternative bile acid
pathway in nonhepatic tissues is under study. Obviously, it is not
possible to identify all the factors which determine what fraction of
27-hydroxycholesterol formed in the inner mitochondrial membrane is
further metabolized into 3
-hydroxy-5-cholestenoic acid from
reconstitution experiments. Present data do, however, indicate that the
concentration of cholesterol in the inner mitochondrial membrane and
the level of expression of P450c27 will establish the product profile
(the ratio between 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid) in a given tissue. Experiments with
different types of cultured cells (human lung alveolar macrophages,
human monocyte-derived macrophages, human umbilical endothelial cells,
bovine aortal endothelial cells, and human fibroblasts) (12) as well as
rat liver mitochondria (47) indicate that cholesterol content in the
inner mitochondrial membrane in these types of cells is low,
subsaturating for P450c27, and availability of cholesterol to the
enzyme limits synthesis of 27-hydroxycholesterol. Under these
circumstances, as it follows from our studies using reconstituted
system, the total production of 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid and the ratio between them in a given
tissue will be determined by the level of expression of P450c27.
Indeed, macrophage preparations (human lung alveolar macrophages and
human monocyte-derived macrophages) have considerably higher capacity
to secrete 27-hydroxycholesterol and 3
-hydroxy-5-cholestenoic acid
into culture media than do human umbilical endothelial cells, bovine
aortal endothelial cells, and human fibroblasts. The ratios between
27-hydroxycholesterol and 3
-hydroxy-5-cholestenoic acid secreted by
both types of macrophages were similar and range between 0.05 and 0.5, whereas for human endothelial cells this ratio ranges between 5 and 10, and for bovine endothelial cells it is found to be between 1.5 and 3 (12). No significant amounts of 3
-hydroxy-5-cholestenoic acid were formed from the human fibroblasts (12). Macrophages but not endothelial
cells or fibroblasts showed a significant amount of P450c27 by
immunoblotting analysis (12).
As it was discovered recently, human liver microsomes contain an
oxysterol 7
-hydroxylase having high specificity for side chain-hydroxylated C-27 steroids that has properties different from
those of P450c7 (5-8). A novel P450 (Cyp7b1) that has 39% of homology
to hepatic P450c7 and that can metabolize 27-hydroxycholesterol to
7
,27-dihydroxycholesterol has been cloned from rat and mouse brain
(48, 49). Besides brain, Cyp7b1 mRNA was detected in several
nonneuronal tissues, including the liver (48). Several lines of
evidence support the hypothesis that the murine Cyp7b1 gene encodes an
enzyme identical to the murine hepatic microsomal oxysterol
7
-hydroxylase (50). Presence of 3
,7
-dihydroxy-5-cholestenoic acid in human serum at concentrations comparable with that of 3
-hydroxy-5-cholestenoic acid (3) indicates that expression of human
oxysterol 7
-hydroxylase may not be limited to liver. Taken together,
these data indicate that in humans 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid may also undergo 7
-hydroxylation in
nonhepatic tissues. If so, tissue levels of 27-hydroxycholesterol and
3
-hydroxy-5-cholestenoic acid will be influenced, among other factors, by the activities of two enzymes, P450c27 and oxysterol 7
-hydroxylase.
In conclusion, the quantitative importance of the alternative pathway
leading to bile acids which begins with 27-hydroxylation of cholesterol
is not yet clear. Evidence is being accumulated that the potential
capacities of the classical and the alternative pathways may be
similar, and under circumstances in which the classical pathway is
repressed, there is a compensatory increase of the contribution of the
alternative bile acid biosynthetic pathways (4, 51-54). The present
study establishes the enzymatic basis for production of the initial
intermediates in alternative bile acid biosynthetic pathways
(27-hydroxycholesterol and 3
-hydroxy-5-cholestenoic acid) and how
the ratio of production of the intermediates may vary between different
extrahepatic cells.