(Received for publication, December 4, 1995; and in revised form, February 21, 1996)
From the
The Candida albicans sterol 14-demethylase gene
(P-450
, CYP51) was transferred to the yeast plasmid
YEp51 placing it under the control of the GAL10 promoter. The resulting
construct (YEp51:CYP51) when transformed into the yeast strain GRF18
gave a clone producing 1.5 µmol of P-450/liter of culture, the
microsomal fraction of which contained up to 2.5 nmol of P-450/mg of
protein. Two oxygenated precursors for the 14
-demethylase,
3
-hydroxylanost-7-en-32-al and 3
-hydroxylanost-7-en-32-ol,
variously labeled with
H and
O at C-32 were
synthesized. In this study the conversion of
[32-
H,32-
O]- and
[32-
H,32-
O]3
-hydroxylanost-7-en-32-al
with the recombinant 14
-demethylase was performed under
O
or
O
and the
released formic acid analyzed by mass spectrometry. The results showed
that in the acyl-carbon bond cleavage step (i.e. the
deformylation process) the original carbonyl oxygen at C-32 of the
precursor is retained in formic acid and the second oxygen of formate
is derived from molecular oxygen; precisely the same scenario that has
previously been observed for the acyl-carbon cleavage steps catalyzed
by aromatase (P-450
) and 17
-hydroxylase-17,20-lyase
(P-450
,CYP17). In the light of these results the
mechanism of the acyl-carbon bond cleavage step catalyzed by the
14
-demethylase is considered.
Our earlier studies on the removal of C-19 of androgens in the
formation of estrogens (1, 2, 3) and of the
14-methyl group of lanosterol during sterol biosynthesis (Fig. SI, Conversion 1
4)
(4) raised the
possibility that these seemingly unrelated conversions may occur by
closely related mechanisms involving three steps as shown in Fig. R1.
Scheme I:
The sequence of reactions
catalyzed by sterol 14-demethylase. Although lanosterol is the
physiological substrate for the enzyme from most sources,
dihydrolanosterol (reduced at the 24,25-double bond) as well as
lanostane derivatives containing a
-double bond also
serve as substrates. With
-substrates, 7,14-diene is
formed following the C-C bond cleavage
step.
Figure R1: Reaction 1.
These studies also indicated that in each case the
same catalyst was responsible for all three reactions, and this feature
was firmly established through genetic studies and purification to
homogeneity of the two enzymes, aromatase (P-450) (5, 6) and lanosterol 14
-demethylase
(P-450
)(7, 8) . The third step in
estrogen biosynthesis has aroused much interest (9, 10) and the current view of the mechanism is
influenced by our
O labeling
experiments(2, 3) , which highlighted the novel nature
of the process, leading to the proposal that the reaction involves an
acyl-carbon cleavage represented by Fig. R2(9) .
Figure R2: Reaction 2.
Although all the experimental findings available to date on the
C-C bond cleavage step in 14-demethylation, for example the
requirement for NADPH plus O
for the reaction and release
of the C
unit as formate, could be explained (3, 9) by the reaction of Fig. R2, the direct
scrutiny of the hypothesis has not been possible hitherto due to the
unavailability both of appropriately labeled
O substrates
and an enzyme preparation that produced sufficient formic acid for
accurate
O isotope analysis.
The present paper
describes a satisfactory resolution of these difficulties and reports
on the status of oxygen during the C-C bond cleavage step
catalyzed by lanosterol 14-demethylase (3
4).
The analysis
of benzyl formate, prepared from the enzymatically produced formic
acid, was performed by gas chromatography-mass spectrometry using a
Hewlett-Packard 5890/VG TS-250 and a 30 m 0.32 mm inner
diameter column of DB17 with splitless injection(12) .
The
experimentally determined value for C natural abundance
(12.4%) for benzyl formate was used to correct all the peaks between m/z 137-141, due to other isotopomers. The distribution
of the isotopomers in benzyl formate was measured by comparison of the
normalized ion signal areas determined by selected ion recording and
corroborated by recording the full spectrum.
Using the methods of Barton et al.(13) ,
lanost-8-en-3-ol acetate was converted to
3
-hydroxylanost-7-one from which was prepared
3
-acetoxylanost-7-en-32-onitrile (5) by the procedure
described by Batten et al.(14) . The nitrile (5) was reduced to 3
-hydroxylanost-7-en-32-al (6a)
either with lithium aluminum hydride (14) or with
diisobutylaluminum hydride as outlined below.
3-Acetoxylanost-7-en-32-onitrile (5) (800 mg, 1.66 mmol)
in dry tetrahydrofuran (25 ml) was cooled in ice and 0.5 N diisobutylaluminum hydride in toluene (20 ml) was added slowly
with stirring. The solution was allowed to stand at room temperature
for 0.5 h, after which time it was poured into 10% aqueous hydrochloric
acid (100 ml). The product was extracted in the usual manner, applied
to a silica gel column (3
45 cm), and eluted with petroleum
ether containing increasing amounts of ethyl acetate (up to 10%). The
solvent was removed in vacuo. The resulting
3
-hydroxylanost-7-en-32-al (6a) (250 mg, 0.57 mmol) was
recrystallized from methanol, m.p. 129-132 °C (literature
128-130 °C)(13) , R
, 0.5 (ethyl
acetate/petroleum ether, 3:7); IR (Nujol) 3540-3150 and 1720
cm
. Mass spectrum of its trimethylsilyl derivative
gave a molecular ion at m/z 514.
Figure 1: Strategy for the cloning of the modified CYP51 gene of C. albicans. PCR mutagenesis to change the triplet at position 263 from CTG to TCT was performed using the four primers as described under ``Experimental Procedures.'' The SalI-NsiI fragment coding for the N terminus of the protein and containing the mutation was then ligated to the C terminus encoding NsiI-HindIII fragment from pW91P, and the modified gene was inserted into SalI-HindIII cut YEp51.
Recombinant PCR was used to replace the triplet 263 (CTG) with one encoding serine (TCT) in the S. cerevisiae host. Inside primers used in the PCR mutagenesis were: 1, 5`-AAAGAAATTAAATCTAGAAGAGAA-3` and 2, 5` ACGTTCTCTTCTAGATTT AATTTCTTT-3`. In a first step two separate PCR reactions were performed using outside primer 1/inside primer 2 and inside primer 1/outside primer 2, respectively. The partially overlapping DNA fragments obtained were purified, mixed, and recombined in a subsequent PCR step using outside primers 1 and 2. PCR reactions were performed on a Perkin-Elmer DNA thermal cycler; conditions consisted of an initial 5 cycles of 1-min denaturation at 94 °C, an annealing step for 4 min at 48 °C, and an extension step for 3 min at 70 °C, followed by 25 cycles of a denaturation step for 1 min at 94 °C, an annealing step for 2 min at 55 °C, and an extension step for 3 min at 72 °C. PCR was undertaken using Pfu polymerase (Promega). Introduction of the mutation and maintenance of the authentic sequence was corroborated by DNA sequencing using Sequenase 2 (Amersham Corp.) after cloning the mutant SalI/NsiI fragment into YEp51 with ligation to the NsiI/HindIII fragment containing the C terminus and terminator regions of C. albicans CYP51. The restored CYP51 fragment was cloned directly into SalI/HindIII digested vector. All restriction enzymes and T4 DNA ligase were obtained from Promega and the recommended conditions for use were applied.
Scheme II: Structure of the key synthetic intermediate (5) and various isotopomers of the 32-oxo (6) and 32-hydroxy derivatives(7) .
The two tritiated substrates (6e) and (7d)
were used for the assay of the 14-demethylase activity by
monitoring the release, in the medium, of
HCOOH from the
aldehyde (6e) or
HCOOH plus
H
O from the hydroxy compound (7d). In
the metabolism of the hydroxy compound (7d), tritium is released
in water during the oxidation of the hydroxy into the aldehyde group
and in formic acid during the subsequent C-C bond cleavage step
converting the 32-oxo derivative (6) to the 7,14-diene (see Fig. SIII, structure of the type 11). An oxidative
activity in most preparations of 14
-demethylase converts the
initially produced formate into CO
and H
O. Our
projected mechanistic experiments required an improved enzyme activity,
free from the above oxidation reaction, in order to provide at least 4
µg of formic acid for MS analysis.
Scheme III:
Postulated mechanism for the
acyl-carbon bond cleavage reaction catalyzed by sterol
14-demethylase using 6a as the substrate. The reactions in the
sequence are: (i) adduct formation using the
Fe
-OOH species, which is formed from the resting state of
the enzyme, 2e, O
, and H
; (ii) homolytic cleavage; (iii) fragmentation; and (iv) disprop
ortionation.
Transformation of the yeast strain GRF18 with YEp51:CYP51 produced 1.5 µmol of the demethylase/liter of culture, while
the derived microsomal fraction was found to contain up to 2.5 nmol of
P-450/mg of protein. The level of expression is higher than has been
reported for other P-450 in yeast or E. coli, suggesting that
the availability of heme is not limiting. This productivity was not
dependent on the CTG to TCT mutation undertaken. Molecular modelling
studies predict that the residue at position 263 is on the surface of
the protein, thus explaining the absence of effect on the activity of
the enzyme when the unmodified gene was expressed previously. ()
Table 1
shows that the specific activity of the
enzyme in microsomes from recombinant vector, based on release of
formic acid from the H-labeled 32-oxo derivative (6e), was 0.1- 0.25 nmol/nmol of P-450/min, and, as expected, no
activity was detectable in the host strain harboring the parent vector.
The specific activity of the cloned enzyme remained unchanged when it
was purified to homogeneity and reconstituted with NADPH-cytochrome
P-450 reductase from pig liver. The activity is similar to that found
for homogeneous CYP51 obtained from a wild type strain of C.
albicans(22) , but lower than those reported from other
sources(8, 23, 24, 25) . The reason
for the low specific activity of C. albicans CYP51 is not
known but the possibility has been considered that the physiological
substrate for this enzyme may be 24-methylene dihydrolanosterol rather
than the lanosterol derivatives used in in vitro assays by us
and others(22) .
The mass spectrometric analysis,
using either full scan or selected ion recording, of benzyl formate
obtained from the incubation of the H-labeled aldehyde (6b) under
O
gave a single isotopic
peak at m/z 137 due to
HCOO-Bzl (entry 1, Table 2). The absence of molecular ions due to higher masses m/z 138-139 established that t
he important region of the
spectrum was free from background noise. Benzyl formate from the
incubation of the
H-labeled aldehyde (6b) under
O
gave a major peak at m/z 139
ascribable to
HC
OO-Bzl (entry 2, Table 2). The intensity of the la
tter peak was at least 50% of
the combined intensities of the peaks due to all the
H-containing isotopomers of benzyl formate. This result
shows that during the cleavage of the C-14-C-32 bond of the
aldehyde (6) one atom of oxygen from
O
is incorporated into the released format
e. Now the complementary
experiment was performed in which the doubly labeled aldehyde (6c), containing
H as well as
O at
C-32, was used as the substrate and incubated under
O
. Under these conditions, a predominant peak
at m/z 139 was observed (entry 3, Table 2), showing the
transfer of the carbonyl oxygen of the aldehyde into the formate. The
most significant feature of the experiment in which the same doubly
labeled precursor (6c) was deformylated under
O
was the presence of a peak at
m/z 141 for the isotopomer in which the deuterium containing benzyl
formate contained two atoms of
O. In essence, this
experiment (entry 4, Table 2) represents the summation of the
results of entries 2 and 3, showing that in the cleavage of the
C-C bond of the aldehyde by the 14
-demethylase the original
aldehydic oxygen of the substrate is retained in the released formic
acid, while its second oxygen is derived from molecular oxygen.
Attention is drawn to the presence of substantial amounts of
deuteriated benzyl formate containing either one or noO in the experiment of entry 4 (Table 2). The
formation of these species is attributed to the loss of the aldehydic
oxygen by exchange with the oxygen of H
O during the
incubation. The extent of the exchange increases in the experiments
performed under
O
because of the need to
perform time-consuming manipulations for replacing air with
O gas. Another adverse consequence of this operation is
some denaturation of the enzyme resulting in the production of lower
amounts of formate.
The notion that the C-C bond cleavage reaction in the
multistep process catalyzed by 14-demethylase occurs by the same
generic reaction that has previously been found to operate for
aromatase (CYP19) (2, 3) and
17
-hydroxylase-17,20-lyase (CYP17) (12) is supported by
the present study. The fission process corresponding to an acyl-carbon
cleavage is reducible to the stoichiometry of Fig. R2. The two
main predictions of Fig. R2, that in the overall process the
carbonyl oxygen atom of the substrate, together with an atom of oxygen
from O
, are incorporated in the expelled formate, have been
validated experimentally. We have advocated that the cleavage process
may be rationalized by assuming that in the catalytic cycle of P-450s,
the Fe
-OOH species, which is normally directed to produce
an iron-monooxygen species involved in the hydroxylation reaction, may
be trapped to give an adduct when the substrate skeleton contains a
properly juxtapositioned electrophilic
center(26, 27) .
The intermediacy of a peroxide
adduct in acyl-carbon bond cleavages (Fig. R2) has been inferred
from a range of observations, either described or reviewed in previous
publications(10, 27, 28, 29, 30) .
Several mechanistic alternatives are possible through which the
products of the reaction of Fig. R2may be formed from the
peroxide-adduct of the type 8, Fig. SIII. For example, in
the case of CYP17 evidence has been presented to show that certain
acyl-carbon bond cleavage reactions catalyzed by the enzyme occur by a
homolytic fission route producing a carbon radical that either
undergoes a disproportionation process producing an olefin or an oxygen
rebound reaction forming a hydroxy compound(29) . A similar
scenario may be envisaged for the related acyl-carbon bond cleavage
reaction, promoted by 14-demethylase, as shown in Fig. SIII. When the intermediacy of a peroxide-adduct was
originally proposed, the possibility was considered that it may
rearrange by a Baeyer-Villiger process to produce a formate ester which
then, through an elimination reaction, creates the double bond in the
final product(2) . Such a possibility was, however, excluded
for aromatase by showing that 10
-hydroxyestr-4-ene-3,17-dione
formate was not aromatizd by the enzyme(2, 9) . O-Acyl derivatives have been isolated during the reactions
catalyzed by 14
-demethylase (31) and CYP17(32) ,
but further work is required to establish whether these are bona
fide intermediates in the acyl-carbon cleavage reaction or are
formed merely as side products. The problem posed by a mechanism for
the reaction of Fig. R2, which operates through the intermediacy
of an O-acyl derivative is that it requires a single enzyme,
not only to possess the activity for three different oxidative
reactions, but also a fourth activity to promote the difficult removal
of the elements of formic acid.
In the light of these considerations, we favor the mechanism shown in Fig. SIIIfor the conversion of the 32-oxo derivative (6) into the 7,14-diene (11). In principle the initially formed peroxy adduct (8) may cleave by an ionic or a radical process. The latter cleavage mode, however, has the advantage that it gives an intermediate alkoxy radical (9), which is ideally suited to undergo fragmentation producing formate, and the substrate radical (10), which can be conveniently converted into the product (11). Furthermore, the mechanism is based on a precedent from an equivalent acyl-carbon cleavage reaction catalyzed by CYP17 for which evidence for a radical process has been obtained (29) .