(Received for publication, August 6, 1996, and in revised form, October 25, 1996)
From the Krebs Institute for Biomolecular Research,
Department of Molecular Biology and Biotechnology, University of
Sheffield, Sheffield, S10 2UH, United Kingdom, the § Max
Delbruck Centre for Molecular Medicine, Robert-Rossle-Strasse 10, D-13122 Berlin-Buch, Germany, the ¶ Department of Biochemistry,
University of Southampton, Bassett Crescent East, Southampton, SO16
7PX, United Kingdom, the
Nitrogen Fixation Laboratory, John
Innes Centre, Norwich, NR4 7UH, United Kingdom, and
Zeneca Agrochemicals, Jealott's Hill
Research Station, Bracknell,
Berkshire, RG42 6ET, United Kingdom
Sterol 14-demethylase (P45051) is the target
for azole antifungal compounds, and resistance to these drugs and
agrochemicals is of significant practical importance. We undertook
site-directed mutagenesis of the Candida albicans P45051
heterologously expressed in Saccharomyces cerevisiae to
probe a model structure for the enzyme. The change T315A reduced enzyme
activity 2-fold as predicted for the removal of the residue that formed
a hydrogen bond with the 3-OH of the sterol substrate and helped to
locate it in the active site. This alteration perturbed the heme
environment, causing an altered reduced carbon monoxide difference
spectrum with a maximum at 445 nm. The changes also reduced the
affinity of the enzyme for the azole antifungals ketoconazole and
fluconazole and after expression induced by galactose caused 4-5-fold
azole resistance in transformants of S. cerevisiae. This is
the first example of a single base change in the target enzyme
conferring resistance to azoles through reduced azole affinity.
Sterol 14-demethylase (P45051) participates in sterol
biosynthesis, is an essential requirement for yeast viability (1), and
has been identified as the only cytochrome P450 found in animals, plants, and fungi, suggesting that it represents an ancient metabolic activity within the superfamily of P450 enzymes. Despite the presence of a human equivalent of the (2), selective inhibitors of the fungal
enzyme have been developed as a central part of antifungal therapy.
Such drugs are of increasing importance, since the incidence of fungal
infection has risen dramatically due to immunocompromised conditions
after organ transplantation, cancer chemotherapy, or for patients
suffering from AIDS (3). Selective inhibitors have also been developed
that act as agrochemical fungicides for phytopathogenic fungi but are
tolerated by plants.
Most inhibitors of the enzyme contain an azole moiety, often a
triazole. The N-4 (or N-3 of imidazole) can bind to the heme of the
enzyme as a sixth ligand with the N-1 substituent group binding to the
apoprotein (4). The development of resistance to fluconazole in >10%
of late stage AIDS patients suffering candidosis has stimulated
interest on the mechanisms of resistance and how to diagnose and
overcome this problem. It is known for a few isolates of Candida
albicans that resistance can occur through phenomena related to
decreased accumulation of drug in the cell (5, 6), although for other
fungi suppressor mechanisms operate that change the 14-methylated
sterols accumulating under treatment (7, 8), and recently leaky P45051
mutants of Ustilago maydis were identified that exhibited
resistance, demonstrating altered target site as a mechanism of
resistance in a congenic background (9). Previously, a P45051-defective
strain, SG1, was observed to be azole-resistant, but such
resistance can be attributed to the second defect it has in sterol
5,6-desaturation, which suppresses the effect of a
chemical or genetic block at P45051 (10).
Information on the structure of eukaryotic P450s is lacking due to
difficulties associated with crystallizing these microsomal proteins,
but based on the known structure of soluble bacterial P450s, molecular
modeling can be attempted. One such attempt was for C. albicans P45051 (11) and suggested interactions by the substrate
with the apoprotein. The positioning of the substrate in the active
site was modeled to consider the removal of the sterol 14-methyl
group, and a hydrogen bond between sterol C3-OH and Thr315
was predicted.
This paper presents results of experiments designed to examine the effect of mutation of this residue on activity and azole antifungal binding. Alteration to Ala by a single base substitution produced an altered protein exhibiting reduced activity. Furthermore, this altered protein had reduced affinity for fluconazole and ketoconazole and produced relative resistance in transformants of Saccharomyces cerevisiae.
Our previous studies have
employed a yeast expression system to express the C. albicans P45051 using the S. cerevisiae GAL10 promoter
in the vector YEp51 (12). Recombinant PCR1
was employed to replace the codon 315 (ACT) with one encoding alanine
(GCT). The following oligonucleotides were used as outside primers: 1)
5-AAATTTGCTAAAGCTGCTTTGACTACT-3
, annealing to positions 429-456 of
the C. albicans P45051 and preceding the endogenous BglII site present in the P45051 gene, and 2)
5
-TGGCATATGCATTCTGAGAGTTTCCTT-3
, annealing to the 3
-end at positions
1098-1125 of the P45051 and containing the endogenous
NsiI site present in the gene. Inside primers used in the
PCR mutagenesis were 1) 5
-ACTTCTGCTTCT
TCTGCTTGGTTC-3
, and 2) 5
-GAACCAAGCAGA
AGAAGCAGAAGT-3
. In a first step,
two separate PCR reactions were carried out 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 following PCR step using outside primer 1/outside primer 2. PCR reactions were performed on a Perkin-Elmer DNA thermal cycler with conditions consisting 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 (Stratagene). The resulting DNA fragment containing the T315A mutation was ligated into the YEp51:P45051 expression plasmid according to Fig. 1.
Introduction of the mutation and maintenance of the authentic sequence
was corroborated by DNA sequencing using Sequenase 2 (Amersham Corp.). All restriction enzymes and T4DNA ligase were obtained from Promega, and the recommended conditions for use were applied.
Strains and Culture Conditions for Heterologous Expression
The plasmid YEp51:P45051(T315A) was transformed into
S. cerevisiae strain GRF18 (MAT leu2-3,2-112
his3-11, 3-15 canr). Yeast transformants were grown
at 28 °C, 250 rpm with a 250-ml culture in 500-ml flasks. The media
used consisted of Difco yeast nitrogen base without amino acids (1.34%
w/v) supplemented with 100 mg/l histidine and 2% (w/v) glucose as an
initial carbon source. Heterologous expression was induced when the
glucose was exhausted at a cell density of approximately
108 cells/ml. The culture was left a further 4 h
before galactose was added to a concentration of 3% (w/v). After a
20-h induction, cells were harvested by centrifugation, resuspended in
buffer containing 0.4 M sorbitol, 50 mM
Tris-HCl, pH 7.4, and broken using a Braun disintegrator (Braun GmbH,
Mesungen, Germany) with four bursts of 30 s together with cooling
from liquid carbon monoxide. Cell debris was removed by centrifugation
at 1500 × g for 10 min using a bench centrifuge and
enzyme preparation was at 4 °C. The resulting supernatant was
centrifuged twice at 10,000 × g for 20 min to remove
mitochondria and then at 100,000 × g for 90 min to
yield the microsomal pellet. This was resuspended using a
Potter-Elvehjeim glass homogenizer at about 10 mg protein/ml in the
same buffer described above. P450 concentration by reduced carbon
monoxide difference spectroscopy was measured as described previously
(13), and protein concentrations were measured as indicated previously (14).
200
nmol of heterologously expressed C. albicans wild type and
mutant P45051 were solubilized in 100 mM potassium
phosphate buffer, pH 7.4, containing 2% (w/v) sodium cholate for
1 h at 4 °C, respectively. Solubilized P450 was recovered after
a 1-h centrifugation step at 100,000 × g and diluted
with a 20% (v/v) glycerol solution to 25 mM potassium
phosphate, 0.8% (w/v) sodium cholate. The supernatant was applied to
an amino-octyl-Sepharose column equilibrated with a 10 mM
potassium phosphate buffer, pH 7.4, containing 0.8% (w/v) sodium
cholate. The column was washed with 100 ml of 10 mM
potassium phosphate buffer, pH 7.4, containing 0.8% (w/v) sodium
cholate, by a second wash with a similar buffer containing 1.2% (w/v)
sodium cholate, and a third wash with 100 ml of 100 mM
potassium phosphate buffer containing 0.5% (w/v) sodium cholate.
P45051 was eluted from the column with 100 mM potassium
phosphate buffer, pH 7.4, containing 0.5% (w/v) sodium cholate and
0.3% (v/v) Tween 20. P45051-containing fractions were pooled and
dialyzed overnight against 2 liters of 10 mM potassium phosphate buffer, pH 6.8, containing 0.3% (w/v) sodium cholate. The
sample was then loaded onto a hydroxylapatite column equilibrated with
10 mM potassium phosphate buffer, pH 6.8. The column was washed with 10 mM potassium phosphate buffer, pH 6.8, and
eluted using a 10-150 mM potassium phosphate gradient.
P45051-containing fractions (assessed by reduced carbon monoxide
difference spectroscopy) were pooled and concentrated using an Amicon
Centricon 10 microconcentrator. Enzyme purity was assessed by
SDS-polyacrylamide gel electrophoresis and specific content. Purified
enzymes were stored at 80 °C until use.
A solution of NADP+ (2 mg), glucose
6-phosphate (5 mg) and glucose-6-phosphate dehydrogenase (3 units) in
100 mM potassium phosphate buffer containing 0.1 mM EDTA, 1 mM glutathione, and 20% (v/v)
glycerol (0.5 ml, pH 7.4) was incubated at 30 °C for 20 min. To this
was added microsomal protein (approximately 10 mg), and the volume was
made up to 1 ml with the above buffer. Following the addition of the
32-tritiated substrate (52 µg, 1.62 µCi in 10 µl of
dimethylformamide; see Fig. 3), 0.1-ml aliquots were removed (at
intervals of 0, 5, 10, 30, and 60 min) and added to a mixture of
dichloromethane (0.5 ml) and water (0.5 ml). The mixtures were
immediately shaken and then centrifuged. The organic layer was
discarded, further dichloromethane (2 × 0.5 ml) was added, and
the above procedure was repeated. To the resulting aqueous phase was
added charcoal, and the suspension was shaken, left at 4 °C for
1 h, and finally centrifuged to remove the charcoal. The
radioactivity of the aqueous phase was measured by liquid scintillation
counting. These reactions were compared with turnover of purified
reconstituted enzyme using equivalent substrate and P450 concentrations
(method described below).
Determination of Sterol 14
Each reaction mixture contained purified P45051 and P45051(T315A), respectively (0.2 nmol), and 1 unit of rabbit NADPH cytochrome P450 reductase in a total volume of 50 µl. To this, 50 µg of dilauroylphosphatidylcholine was added, and the reaction volume was adjusted to 950 µl with 100 mM potassium phosphate buffer, pH 7.4. 32-Tritiated substrate was added, and the mixture was sonicated until a white suspension formed. NADPH was added at a concentration of 23 mM to the mixture to start the reaction. For determination of enzyme kinetics, varying concentrations of substrate were used. Extraction and assessment of radioactivity was carried out as described above.
Inhibition of Sterol 14Sterol
14-demethylase activity of the purified preparations of P45051 and
P45051(T315A) was assayed as described above. Azole antifungal,
ketoconazole, and fluconazole (Fig. 2), were added to
the reaction mixtures, respectively, from 1000-fold stock
solutions.
Inhibition of Growth
Minimum inhibitory concentrations were estimated following the inoculation of 5 × 103 cells/ml in minimal medium containing 100 µg/ml histidine and varying doses of ketoconazole and fluconazole, respectively. Incubation was at 30 °C, 150 rpm in 60-ml sterile containers (Sterilin) using 2 ml of culture. Growth was determined by cell counts and colony-forming units/ml.
Difference SpectroscopyMicrosomal samples (4 × 107 M) of P45051 and P45051(T315A),
respectively, were placed in both sample and reference cuvettes (1-cm
path length) of a Philips PU8800 spectrophotometer. Azole antifungals,
ketoconazole, and fluconazole, dissolved in Me2SO were
added directly to the sample cuvette; the contents were mixed, and
after 1 min the spectrum between 500 and 350 nm was recorded. By adding
several increments of test substance, 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 added azole antifungal. The
maximum concentration of Me2SO used (1% (v/v)) caused no
change in the spectrum over the region scanned.
EPR spectra were recorded with an updated Bruker ER 200D-SRC spectrometer equipped with an Oxford Instruments ESR900 liquid helium cooling system, an NMR gaussmeter, and a microwave frequency counter. Spectra were recorded at 5 K with a microwave power of 0.5 milliwatts at 9.6 GHz using 1.0 millitesla field modulation at 100 kHz.
Previously (12), we have reported high level
expression of C. albicans sterol 14-demethylase in
S. cerevisiae, with P45051 levels exceeding 1.5 µmol/liter
of culture, which is higher than any other expression of heterologous
P450 in the literature. The expression system had already included
modifying the coding sequence at 263 due to the presence of CTG, which
encodes leucine in S. cerevisiae but serine in C. albicans (15). Introduction of TCT allowed the authentic amino
acid (serine) to be included when expressed in S. cerevisiae. Further mutagenesis was undertaken to change
Thr315 to Ala, involving a single base substitution of ACT
GCT, which is outlined in Fig. 1. S. cerevisiae strain
GRF18 transformed with P45051(T315A) expressed levels of P450
comparable with wild type enzyme with up to 2.5 nmol of P450/mg of
microsomal protein produced after expression from the GAL10 promoter of
YEp51 when determined by the optical absorption spectrum of the carbon
monoxide-bound form of reduced P450. The absorption maximum of the
CO-bound form of wild type P45051 was located at 448 nm. However, the
CO-bound form of P45051(T315A) had the Soret absorption maximum at 445 nm (Fig. 3). It was noted that no peaks at 420 nm were
obtained for either P45051 or P45051(T315A) (P420 is an inactive
derivative of P450), indicating stable production of both proteins.
Table I summarizes the results of each stage of the purification procedure starting from the microsomal fractions of P45051. The methods employed permitted the purification of both P45051 and P45051(T315A) with a yield of about 50% and to a specific content of approximately 17 nmol/mg microsomal protein, consistent with a purified hemoprotein of the predicted Mr. Upon SDS-polyacrylamide gel electrophoresis, pure preparations gave single protein bands confirming homogeneity with an apparent monomeric Mr = 55,000 (Fig. 4).
|
EPR Spectra of P45051(T315A)
Fig. 5 shows the
EPR spectrum of microsomal P45051(T315A) together with the spectrum of
a control sample not expressing C. albicans P45051. The
clearest features are at g values 2.43, 2.27, and 1.92 in the spectrum
from P45051(T315A); these are very similar to those reported for low
spin ferric iron in various cytochrome P450s and in particular are
essentially identical to the g values of 2.45, 2.27, and 1.92 from the
purified lanosterol 14-demethylating enzyme isolated from wild type
S. cerevisiae (16). These features were also present in the
spectrum for C. albicans P45051 (data not shown). Both
spectra also show peaks in the range 100-300 millitesla that are
probably from ferric contaminants as well as a derivative signal at
g = 2.0 which is presumably from a sum of free radical and 3Fe/S
centers. Since EPR is a sensitive probe of the electronic structure of
paramagnetic centers, the low spin ferric EPR spectra of Fig. 5 show
that the mutation T315A has no major effect on the heme
environment.
Sterol 14
P45051(T315A) showed a marked reduction in its ability to metabolize the alcohol derivative substrate compared with the wild type P45051 in both the purified and microsomal form reflected in a change in Km and Vmax The specific activity of the wild type P45051 for the release of formic acid from the 3H-labeled 32-oxo-derivative was 41 pmol/nmol of P450/min for microsomal protein compared with 125.2 pmol/nmol of P450/min for a reconstituted assay containing purified C. albicans P45051 and rabbit liver NADPH cytochrome P450 reductase. The difference in activity between microsomal and pure enzyme may reflect a limitation in available reductase in the microsomal fractions. As expected, the demethylation activity was below the base line of detectability in microsomes from the host strain harboring the parent vector YEp51 (data not shown).
Fig. 6 shows a double-reciprocal plot corresponding to
the metabolism of [32-3H]3-hydroxylanost-7-en-32-ol by
both purified P45051 and P45051(T315A). Wild type P45051 was shown to
have a Km of 29.4 µM and a maximal
enzymatic rate (Vmax) of 147 pmol of substrate
converted/min/nmol of P45051. In contrast, the affinity of
P45051(T315A) in binding sterol was reduced, as demonstrated by an
increase in the Km to 62.5 µM.
Additionally, the Vmax of P45051(T315A) was
reduced to 57% with 83.3 pmol of substrate converted/min/nmol of
P45051(T315A) (Table II).
|
S. cerevisiae transformants containing P45051 and P45051(T315A) showed no difference in their minimum inhibitory concentrations to ketoconazole and fluconazole following growth on yeast minimal medium containing 2% (w/v) glucose (Table III). Minimum inhibitory concentration values of 5 and 20 µM for ketoconazole and fluconazole, respectively, were obtained for transformants containing both P45051 and P45051(T315A) as well as for the host S. cerevisiae strain containing the empty expression cassette. However, minimum inhibitory concentration values determined following growth on galactose, resulting in induction of P450, revealed an increase in resistance of the yeast to the azole antifungals in both yeast strains expressing P45051 and P45051(T315A) when compared with the control host (Table III). A respective 4- and 5-fold increase in resistance to ketoconazole and fluconazole was observed for the strain expressing wild type P45051 in contrast to a 20-fold increase in resistance to both azole antifungals that was observed following expression of P45051(T315A). No difference was observed in the P450 content following the expression of wild type P45051 and P45051(T315A) during growth on galactose, with specific contents of approximately 1.2 nmol of P450/mg of microsomal protein being observed following expression of both proteins. No detectable P450 was observed following growth of the host strain on glucose.
|
Azole
antifungals caused characteristic changes in the oxidized forms of both
P45051 and P45051(T315A). The chemical interaction giving rise to the
type II change was the displacement of the native sixth ligand of the
heme iron by a nitrogen atom in the added azole antifungal and
resulting in a spectral peak (420-427 nm) and a corresponding trough
(390-410 nm) (17). The intensity of the resulting difference spectrum
has been characterized previously for wild type P45051 and found to be
proportional to the amount of the azole-bound form of the cytochrome
(18). Both ketoconazole and fluconazole caused the type II spectral
change in both P45051 and P45051(T315A), but the positions of the peaks
were altered in the mutant compared with the wild type. The spectral
peak was located at 425 nm for P45051(T315A) compared with 424 nm for
wild type P45051; the trough was located at 409 nm for P45051(T315A) compared with 412 nm for wild type P45051. Alteration in the positions of the peaks and troughs following azole antifungal binding in P45051(T315A) indicates an alteration in the conformational environment around the P450 heme. The apparent affinities of both ketoconazole and
fluconazole for P45051(T315A) were different when compared with the
wild type P45051. Ketoconazole and fluconazole were found to bind
stoichiometrically with wild type P45051, whereas both compounds only
formed one-to-one complexes with P45051(T315A) at an approximately
3-fold excess of azole antifungal over P450 (Fig. 7,
a and b). The results of the spectrophotometric
titration were consistent with those of the inhibition experiments
(Table IV), confirming that the difference in the
inhibitory effect resulted from the difference in the affinities of
both ketoconazole and fluconazole for P45051(T315A) compared with
P45051.
|
The
catalytic activity of purified P45051 and P45051(T315A), respectively,
was assayed by measuring the release of [3H]formic acid
plus [3H]water during the conversion of
[32-3H]3-hydroxylanost-7-en-32-ol to its C-14
demethylated product, 4,4-dimethyl-5
-cholesta-7,14-dien-3
-ol.
Fig. 8a shows the results of an experiment in
which 0.2 nmol of purified P45051 and P45051(T315A), respectively, were
inhibited with increasing amounts of ketoconazole. For P45051, the
inhibition of enzymatic activity was dependent on azole concentration,
and total inhibition of enzyme activity occurred at a concentration
equimolar to that of P45051. However, for purified P45051(T315A) a
4-fold excess concentration of ketoconazole was observed to inhibit
sterol 14
-demethylase activity. Essentially the same results were
obtained with fluconazole (Fig. 8b); total inhibition of
wild type P45051 activity occurred when equimolar amounts of
fluconazole were added, whereas for P45051(T315A) 4-fold excess amounts
of azole were required for total inhibition. The results are summarized
in Table IV, where similar differences in Ki were
observed between P45051 and P45051(T315A), the latter showing reduced
affinity for the azole antifungals.
The investigation of the three-dimensional structure of P450s remains of importance due to their roles in the metabolism of foreign compounds (carcinogens, drugs, and pollutants) as well as housekeeping roles in endogenous biosynthesis. Of the latter, considerable focus is now placed on the CYP51 family, which was cloned and sequenced first in S. cerevisiae (1) and later in other fungi (e.g. C. albicans; Ref. 19), rats (20), humans (2), and, from unpublished evidence, in plants also.2 The ancestor of these enzymes may have represented one of the earliest functions of P450.
In the sequences of known sterol 14-demethylase genes, residues
corresponding to Thr315 are conserved. Table
V shows the alignment of various sequences. This residue
is contained in the I-helix of P450 when modeling from the P450
structures solved for soluble bacterial enzymes (for review, see Ref.
21). Molecular modeling of P45051 of C. albicans predicted
that this residue could form a hydrogen bond to the 3-OH of sterol,
helping to locate the sterol in the active site (11). Our mutagenesis
and activity experiments support the validity of this model, since
reduced activity and affinity were observed for the mutant enzyme.
Gross conformational change would likely give rise to P420, the
inactive form of the enzyme, and a drastically altered active site
would be unlikely to retain the ability to undertake the highly
specific stereo- and regiospecific monoxygenase activity toward the
sterol 14
-methyl group. The absence of any perturbation of the EPR
spectra of mutant compared to the wild type protein also supports this
conclusion.
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Leaky P45051 mutants have been identified as being azole-resistant in other laboratory studies on U. maydis (9) and prompted further study of the mutant enzyme. Alteration of the Thr315 residue to alanine also subtly disturbed the hemoprotein as indicated by the change in the Soret maximum to 445 nm from 448 nm. This change in conformation is presumably responsible for the change in azole affinity observed in biochemical and microbiological assays of inhibition. In other studies, the stoichiometric binding of ketoconazole and fluconazole to purified P45051 of C. albicans has been established (18), but the T315A protein exhibited reduced affinity. This resistant mutation should be anticipated to occur in natural populations exposed to azole antifungals, since it results from a single base change. For AIDS patients, a limited number of fluconazole-resistant Candida species have been evaluated so far. Two resistant isolates were shown to have reduced accumulation of drug within cells (5), which explained their phenotype, rather than changes in the target site or of other sterol biosynthesis suppressor mutants as characterized for S. cerevisiae (7). Examination of resistant strains has also indicated increased efflux of drug as a cause of resistance in a limited number of further clinical strains, although comprehensive examination of other possible mechanisms of resistance was not included (6). An observation that has been made in a clinical setting is the raising of minimum inhibitory concentrations in increments during the development of resistance (22), and it seems likely that in some situations alterations in the active site will play a role. For other fungi, alteration in P45051 sensitivity has been correlated with resistance in isolates from AIDS patients of Cryptococcus neoformans (23) and of Aspergillus fumigatus3 rather than the other possible causes of resistance. This is the first paper to demonstrate a molecular mechanism altering the azole affinity of the P45051 enzyme to make it more resistant.