(Received for publication, August 6, 1996, and in revised form, February 14, 1997)
From the Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, Sheffield University, Sheffield, S10 2UH, United Kingdom and § Zeneca Agrochemicals, Jealotts Hill Research Station, Jealotts Hill, Bracknell, Berkshire RG42 6ET, United Kingdom
Cytochrome P-45061 (CYP61) was a cytochrome P-450
revealed during the yeast genome project when chromosome XIII was
sequenced. Here we report on the properties of this second microsomal
P-450 of vegetatively growing yeast. The enzyme kinetics associated with its endogenous role in sterol 22-desaturation
revealed a Km of 20.4 µM and a
Vmax of 2.9nmol/min/nmol CYP61. The affinity of
the enzyme for antifungal drugs was characterized to investigate its
potential role in determining tolerance to these sterol
14
-demethylase (CYP51) inhibitors. Drug binding induced a type II
spectral change, which became saturated at equimolar
concentrations of azole drug and P-450. Fluconazole exhibited slightly reduced affinity in comparison to ketoconazole as indicated by carbon monoxide displacement. These and
Ki determination for fluconazole (0.14 nM) revealed CYP61 to have a similar affinity to azole
drugs when compared with data available for CYP51, and the implications
for antifungal treatment were considered.
The cytochrome P-450 (CYP) superfamily are involved in a variety
of monooxygenase reactions including xenobiotic and endogenous substrates. Genome projects are uncovering more genes encoding such
proteins, and one such instance was cyp61 (ERG5)
encoded on chromosome XIII of Saccharomyces cerevisiae (1).
Previous studies on S. cerevisiae have identified a single
form of P-450 during purification from microsomal fractions of
vegetatively growing yeast. This P-450 performed a role in sterol
14-demethylation (2), later associated with the gene,
cyp51 (ERG11; Ref. 3). This is the only P-450
activity associated with a family found in plants, animals, and fungi
(4). Other studies based on inhibition and co-factor characteristics
indicated a further P-450-mediated activity was present in vegetative
yeast (5). This was confirmed when such a protein was purified from
microsomes obtained from a vegetatively growing strain with a
cyp51 gene disruption (6). N-terminal amino acid sequence
confirmed the protein to be CYP61 from chromosome XIII (1).
Inhibitors of CYP51 are of considerable commercial importance as antifungal compounds and selectively inhibit fungal CYP51 over the mammalian and plant counterparts (7). Because they may also bind to CYP61, a potential antifungal target, we characterized the affinity of this P-450 for two of the main drugs employed, ketoconazole and fluconazole, to consider the relative potency and potential contribution CYP61 may make to azole antifungal susceptibility.
Unless specified, all chemicals were obtained
from Sigma (Poole, Dorset, UK). Ketoconazole was purchased from Janssen
Pharmaceutica, and fluconazole was from Pfizer. Microsomes were
prepared, and CYP61 was purified from semi-anaerobically grown cells of
the S. cerevisiae strain DK2, which contains a
gene disruption in cyp51 (cyp51;
erg11) as
described previously (6). Rabbit NADPH-cytochrome P-450 reductase was a
gift from Prof. M. Akhtar, University of Southampton.
Ergosta-5,7-dienol was purified from a polyene-resistant
erg5 mutant of S. cerevisiae
(
22-desaturase defective) as described previously
(6).
The standard reaction mixture contained purified CYP61
(0.5 nmol), 1 unit of rabbit NADPH cytochrome P-450 reductase, and varying concentrations of ergosta-5,7-dienol dispersed in 80 nmol dilauroylphosphatidylcholine, and the reaction volume was adjusted to
950 µl with 100 mM potassium phosphate buffer, pH 7.2. NADPH was added at a concentration of 23 mM to the mixture
to start the reaction. For experiments involving inhibition of
activity, azole drug was added prior to NAOPH. All reactions were
incubated at 37 °C for 20 min in a shaking water bath. Reactions
were stopped by the addition of 3 ml of methanol, and the sterols were
extracted using 2 ml (0.5%, w/v) of pyrogallol in methanol and 2 ml of
60% (w/v) potassium hydroxide (in water), incubated at 90 °C for 2 h in a preheated water bath. After cooling the saponified mixture was
extracted with 3 × 5 ml of hexane and dried under nitrogen. A
Hewlett/Packard gas chromatograph/mass spectrometer was used to confirm
sterol identities. An Ultra 1 capillary column was used (10 m × 0.2 i.d.) on a temperature program 50 °C (1 min) increased by
40 °C/min to 290 °C with a run time of 17 min. Injection port
temperature was 280 °C (splitless), and the carrier gas was helium
at 40 kilopascal. Trimethylsilylated derivatives of ergosta-5,7-dienol, and the 22-desaturated metabolite (ergosterol) were
clearly separated as two distinct peaks (6). The conversion ratio was
calculated from the areas of the two peaks, and the activity (nmol
ergosterol formed/min) was obtained from the amount of
ergosta-5,7-dienol added and the conversion ratio. Linear regression
was used in double reciprocal plot analysis.
Binding spectra were obtained for azole antifungals according to the method of Wiggins and Baldwin (8). Briefly, purified CYP61 (0.2 nmol/ml) was placed in both sample and reference cuvettes (1-cm path length) of a Philips PU8800/02 scanning spectrophotometer. Azole antifungals dissolved in Me2So were added direct to the sample cuvette, the contents were mixed, and the spectrum was recorded between 350 and 500 nm. By adding azole antifungal, the change in absorbance between the type II peak (420-427 nm) and the corresponding trough (390-410 nm) was related to the concentration of azole antifungal added. The maximum concentration of Me2So used (1% by volume) caused no change in the spectrum over the region scanned.
Carbon monoxide displacement studies of azole antifungal bound to CYP61 was monitored using equimolar concentrations of CYP61 (0.2 nmol/ml) and varying concentrations of azole antifungal. After 2 min the contents of both cuvettes were reduced by the addition of sodium dithionite, and a base line was recorded between 400 and 500 nm. The contents of the sample cuvette were bubbled with carbon monoxide for 30 s. The cuvette was sealed, and the reduced difference spectra were recorded.
Ergosta-5,7-dienol was aerobically metabolized to
ergosterol (Fig. 1A) by a reconstituted
monooxygenase system containing CYP61 and NADPH cytochrome 450 reductase. The purified enzyme was shown to have a
Km of 20.4 µM for ergosta-5,7-dienol and a maximal enzymatic rate (Vmax) of 2.9 nmol
ergosterol formed/min/nmol CYP61. The identity of the sterol peaks in
gas chromatography were confirmed by mass spectroscopy and showed
conversion of the sterol into ergosterol. Control experiments showed
P-450 and NADPH dependence for the reaction.
Spectral Ligand Binding Studies on CYP61
The absolute
absorption spectrum of CYP61 is shown in Fig. 2. The
oxidized cytochrome absorbed at 553 and 532 nm with a Soret maximum at
417 nm, indicating that it was in the low spin state. The Soret peak of
the reduced carbon monoxide (CO) complex was situated at 448 nm.
Addition of the azole antifungals, ketoconazole and fluconazole (Fig.
1B), gave rise to typical type II difference spectra, on
binding of N-3 of imidazole (ketoconazole) and N-4 of triazole
(fluconazole) to the CYP61 heme, with absorption maximum and minimum at
428 and 410 nm, respectively. The increase in value of absorbance (peak
maximum to peak minimum) measured after successive additions of
ketoconazole and fluconazole showed that the spectral change was
linearly dependent on the concentration of azole antifungal and was
saturated when equimolar amounts of ketoconazole and fluconazole were
added (Fig. 3). Fig. 4 shows the results
of experiments to measure displacement of azole by CO. The
concentration of ketoconazole required to inhibit CO binding to CYP61
by 50% (IC50) was 0.2 nmol. The larger IC50
value of 0.5 nmol for fluconazole suggests that this compound was less
able than ketoconazole to inhibit the formation of the CO-CYP61
complex.
Inhibition of Reconstituted CYP61
Fluconazole was compared for its inhibitory
activity against reconstituted CYP61-mediated sterol
22-desaturation, and the results were compared with its
inhibitory activity against the target enzyme, Candida
albicans CYP51 (sterol 14
-demethylase). Fluconazole was shown
to be a competitive inhibitor of CYP61 as shown in Fig.
5. The Ki for fluconazole inhibition of CYP61 was calculated to be 0.14 nM.
Previously we reported the purification of a second P-450 of
vegetatively growing yeast, CYP61, from a strain containing a gene
disruption in cyp51 (5). The enzymological properties of
CYP61 in sterol 22-desaturation were characterized here
for the first time with reference to CYP51 (sterol 14
-demethylase)
previously purified from vegetatively growing yeast (2). We also
investigated the affinity of CYP61 for azole antifungals because this
enzyme may contribute to overall azole tolerance or to resistance of
fungi, if possessing a high affinity. Resistance is a major problem in agriculture and in the clinic where fluconazole resistant
Candida sp. occur in >10% of late stage AIDS patients. It
may represent a potential target for new antifungal development in the
mounting crisis of increased fungal disease (7).
In undertaking sterol 22-desaturation the substrate
affinity constant of 20.4 µM and maximum enzymatic rate
of 2.9 nmol/min/nmol P-450 observed for CYP61 were typical of other
P-450s mediating endogenous reactions including CYP51-mediated sterol
14
-demethylation, where a Vmax of 9 nmol/min/nmol P-450 was observed (9).
The absolute absorption spectrum of CYP61 was in general similar to
CYP51 with the hemoprotein in a low spin state and the reduced carbon
monoxide complex giving a maximum at 448 nm. An exception was in the
visible region, where the peaks at 553 and 532 nm are in contrast with
those reported for sterol 14-demethylase (CYP51), which gave peaks
at 570 and 534 nm (2).
Azole binding and inhibition was examined with reference to published
data. The ability of ketoconazole (10) and
fluconazole1 to inhibit CO binding to
C. albicans CYP51, was examined and gave IC50
values of 0.1 and 0.3 nmol, respectively. Using the same quantity of
enzyme the corresponding IC50 values for CYP61 were only
slightly larger at 0.2 and 0.5 nmol, respectively. These findings
reveal only slightly reduced sensitivity of CYP61 compared with CYP51
for azole antifungal binding. Using this assay on a range of plant and
mammalian enzymes, IC50 values several orders of magnitude
higher were obtained (11). This relatively high affinity for azole
antifungals was further supported by studies on the inhibition of
activity. Competitive inhibition was observed for fluconazole and a
Ki of 0.14 nM. This value compares with
0.10 nM for C. albicans CYP51.1 Thus
inhibitors of CYP51 probably act on CYP61 at subminimum inhibitory
concentrations for yeast, but with growth arrest 14-methylated sterols predominate in which many subsequent reactions, including
22-desaturation, of the ergosterol pathway are blocked.
Other inhibitors of CYP51 have been observed to inhibit CYP61 (12), and
in some treatments of the cereal pathogen Rhynchosporium
secalis these effects seem to predominate and correlate with
growth arrest.2 Thus CYP61 may represent an
antifungal target, although gene disruption has indicated that for
growth of yeast on glucose under laboratory conditions it is not
essential (13).
Sterol 22-desaturase is an ancient P-450 activity
reflected in the presence of such desaturation in some plant sterols as
well as in fungal sterols. The plant activity may well be mediated by a
member of the same P-450 family, as for sterol 14
-demethylase undertaken by members of the CYP51 family in animals, plants, and
fungi. It might be anticipated that further studies on CYP61 will cast
light on the evolution of the P-450 superfamily and that this enzyme
may have arisen from CYP51 following a gene duplication event some time
after the separation of plants and fungi from animals. Perhaps this was
the first such development in plant and fungal P-450 evolution, and
subsequent further divergence then occurred.