dihydroorotate dehydrogenase from the opportunisticpathogenic yeast Candida albicans Elke Zameitat1, Zoran Gojkovic´2,*, Wolfgang Knecht2,†, Jure Pisˇkur2,‡and Monika Lo¨ffler1 1 Institu
Trang 1dihydroorotate dehydrogenase from the opportunistic
pathogenic yeast Candida albicans
Elke Zameitat1, Zoran Gojkovic´2,*, Wolfgang Knecht2,†, Jure Pisˇkur2,‡and Monika Lo¨ffler1
1 Institute for Physiological Chemistry, Philipps-University, Marburg, Germany
2 BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
Many fungi, including certain yeasts, have been known
for decades as human pathogens Candida albicans
rep-resents the major group of yeast species identified in
clinical isolates This opportunistic pathogen causes
both trivial infections in normal people and serious
infections in immuno-compromised patients, especially
HIV-infected individuals [1] Yeast infections represent
a severe problem for clinicians, as a limited number of
antifungal agents are available In addition, these
organisms are becoming resistant to current classes of antifungal agents, particularly the azoles [2] Expres-sion of efflux pumps that reduce drug accumulation, and mutation or overexpression of antifungal target proteins are strategies that may be used by the patho-gens [3] The clinical consequences of antifungal resistance can be seen in treatment failures in patients and in changes in the prevalences of Candida spe-cies [4]
Keywords
antimycotics; Candida albicans;
dihydroorotate dehydrogenase; pyrimidines;
redoxal
Correspondence
E Zameitat or M Lo¨ffler, Institute for
Physiological Chemistry, Philipps-University,
Karl-von-Frisch-Str 1, D-35033 Marburg,
Germany
Fax: +49 6421 2865116
Tel: +49 6421 2865022
E-mail: zameitat@gmx.de;
loeffler@staff.uni-marburg.de
Present address
*AstraZeneca R&D Mo¨lndal, SE-431 83
Mo¨lndal, Sweden
†ZGene A ⁄ S, Anker Engelundsvej 1,
Build-ing 301, 2800 Lyngby, Denmark
‡Department of Cell and Organism Biology,
Lund University, So¨lvegatan 35, SE-223 62
Lund, Sweden
(Received 17 March 2006, revised 16 May
2006, accepted 18 May 2006)
doi:10.1111/j.1742-4658.2006.05327.x
Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents In this study we investigated C albicans dihydroorotate dehydroge-nase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron accep-tor and oxygen as the final electron accepaccep-tor Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C albicans,
recombinant-ly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity An inhibitor screening with 28 selected com-pounds was performed Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced
C albicans DHODH activity This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C albicans
Abbreviations
DHO, L -dihydroorotate; DHODH, dihydroorotate dehydrogenase; FeCy, potassium hexacyanoferrate(III); Q0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone; Q 6 , ubiquinone 30; Q 10 , ubiquinone 50; Q D , decylubiquinone.
Trang 2Whereas the pyrimidine metabolism of
Saccharomy-ces cerevisiae has received considerable attention, that
of C albicans has been addressed only indirectly For
example, 5-fluorocytosine possesses antifungal activity
in C albicans but no antineoplastic activity, as does
5-fluorouracil in humans [5] Expression of the salvage
enzymes cytosine deaminase and uracil
phosphoribo-syltransferase in C albicans makes pyrimidine salvage
different from that in mammals, because mammals can
only take up pyrimidine nucleosides for recycling [6]
As a prerequisite for development of antipyrimidine
agents that can enter cells through the salvage
path-way, permeases for pyrimidines and purines have been
well studied in yeast species [7]
The enzymes of the de novo pyrimidine synthesis
pathway have been shown to be drug targets, or
potential drug targets, in eukaryotes [8,9] This
biosyn-thetic pathway results in the formation of UMP and
consists of six enzymatic activities found in all
organ-isms [10,11] In most eukaryotes studied so far, five of
the corresponding enzymes are located in the cytosol,
whereas the fourth enzymatic reaction catalyzed by
dihydroorotate dehydrogenase takes place at the inner
mitochondrial membrane [10,12] The reaction
mech-anism of dihydroorotate dehydrogenase (DHODH, EC
1.3.99.11) (Fig 1) includes the stereospecific oxidation
of (S)-5,6-dihydroorotate to orotate with reduction of
flavin [13,14], and the transfer of electrons to
ubiqui-none, which is part of the respiratory chain Because
of this connection, pyrimidine formation requires a
sufficient concentration of oxygen in the cells Whereas
Schizosaccharomyces pombe possesses a mitochondrial
membrane-bound enzyme (classified as family 2
DHODH), S cerevisiae has a cytosolic DHODH
(clas-sified as family 1 DHODH), the activity of which is
independent of ubiquinone and the presence of oxygen
[15–17] This feature promotes growth of this yeast
under anaerobic conditions Saccharomyces kluyveri, a species relatively closely related to S cerevisiae, is the only yeast known to date that contains both enzyme forms [16,17] Even though S cerevisiae is a close rel-ative of Candida species, and is often used as a model pathogen, its DHODH of family 1 is unsuitable as a prototype for the search for enzyme inhibitors in other yeasts
We subcloned a gene coding for C albicans DHODH (accession number AY230865), overexpressed and purified the recombinant enzyme, and compared it with the DHODH from humans and other yeasts [17,18] This work evaluates C albicans DHODH as a target for the development of highly specific
antimycot-ic drugs against this widespread pathogen
Results
Genetic code and overexpression
In C albicans the standard leucine CUG codon is translated as serine [19] We found two CUG codons
in the DHODH ORF and changed them to UCG (L11S and L78S) by site-directed PCR mutagenesis for gene expression in a bacterial system By sequence alignment, we identified C albicans DHODH as a family 2 enzyme (Fig 2) In this class of enzyme, a catalytic serine residue corresponds to the active-site cysteine in family 1 A typical bipartite N-terminal sequence was identified in the sequence consisting of a targeting sequence that, analogously to the rat and human enzyme [12], promotes import into mitochon-dria and a hydrophobic transmembrane domain neces-sary for the correct insertion into the inner mitochondrial membrane Expression vectors were constructed to produce full-length CaDHODH and an N-terminally truncated mutant (DNCaDHODH), lack-ing the putative bipartite mitochondrial targetlack-ing motif and transmembrane domain
After purification of the full-length and truncated CaDHODH by affinity chromatography, SDS⁄ PAGE (Fig 3) showed that the purified enzymes were of the expected molecular mass of 48 kDa for the full-length enzyme and 42 kDa for the truncated enzyme The yield of recombinant proteins purified from 1 L E coli BL21 cultures was different for the full-length and truncated enzyme when cultured under similar condi-tions: 0.5 mg CaDHODH and 1.2 mg DNCaDHODH Compared with other mitochondrial yeast DHODH, the protein abundancies were in the same range: Sch pombe DHODH, 0.4 and 1.8 mg; S kluyveri DHODH, 1.4 and 1.8 mg (unpublished data) For the truncated and full-length human DHODH, the yields
Fig 1 Scheme of dihydroorotate dehydrogenase (DHODH) reaction
with chemical formulae Electron transfer from dihydroorotate to
FMN and further on to quinone.
Trang 3of purified proteins were approximately 10 times
higher [20] Obviously, truncated forms of the yeast
DHODH were expressed more efficiently than the
full-size enzymes It was not possible to increase the yield
of full-length DHODH by changing expression
condi-tions (temperature, oxygen supply, induction point and
period of expression) or using more or less Triton
X-100 as nonionic detergent through the purification
protocol (data not shown)
The Western blot in Fig 3, performed with human
DHODH antibodies, showed cross-reactivity with the
DHODH protein from C albicans Cross-reactivity was also observed with recombinant DHODH from Arabidopsis thaliana(unpublished data)
The flavin⁄ protein ratio (mol ⁄ mol) as estimated from fluorimetric cofactor analyses of the two recom-binant enzymes was in the range 0.2–0.3 mol flavin per mol protein
Kinetic characterization Activity measurements of CaDHODH and DNCaD-HODH in various buffers revealed maximum activity
at pH 8.0–8.5 From the characteristic bell-shaped activity profile, two pKa values could be calculated:
pKa1, 6.7 ± 0.05 for both enzymes; pKa2, 9.5 ± 0.1 for CaDHODH and 9.9 ± 0.15 for DNCaDHODH
We compared the activity of CaDHODH and DNCaDHODH with a variety of native and two artifi-cial electron acceptors CaDHODH and DNCaD-HODH could use the artificial acceptors potassium hexacyanoferrate(III) (FeCy) and 2,6-dichloroindophe-nol FeCy was the best electron acceptor Studies with different quinone acceptors [2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0), ubiquinone 30 (Q6), ubiquinone
50 (Q10), decylubiquinone (QD)] indicated a better acceptance of the ubiquinone derivative Q6 than Q10, which is the ubiquinone of most higher eukaryotes (Table 1) Fumarate and NAD were inadequate elect-ron acceptors for CaDHODH and DNCaDHODH
A
B
Fig 2 Dihydroorotate dehydrogenase (DHODH) amino-acid sequences (A) Alignment of the N-termini of the recombinantly expressed
C albicans DHODH and human DHODH ( CLUSTAL W version 1.8) Amino-acid residues that are identical in the human and C albicans enzyme are highlighted in black L11S and L78S mutations are shown in red Approximate positions of the domain that direct mitochondrial import and the hydrophobic, putative transmembrane domain are indicated In addition, the membrane-association motif forming a hydrophobic tun-nel for the electron acceptor in DHODH is indicated Numbers refer to amino-acid residues of the C albicans protein (B) Alignment of the catalytic centre of the recombinantly expressed C albicans DHODH and amino-acid sequences of different DHODH family 2 enzymes ( CLUSTAL W version 1.8) The highly conserved serine residue is marked in green.
Fig 3 Recombinant C albicans dihydroorotate dehydrogenase
(DHODH) (A) SDS ⁄ PAGE Lanes: M, molecular mass marker; 1,
CaDHODH; 2, DNCaDHODH 2 lg protein per lane (B) Western
blot Lanes: M, molecular mass marker; 1, CaDHODH; 2,
DNCaD-HODH 1 lg protein per lane.
Trang 4However, the presence of atmospheric oxygen seemed to
promote very low DHODH activity, suggesting that
molecular oxygen may be used as a poor electron
accep-tor The specific activity of the enzymes using QD and
2,6-dichloroindophenol as acceptor was 6 UÆmg)1
Km values for 2,6-dichloroindophenol, dihydroorotate
(DHO), and QDfor CaDHODH and DNCaDHODH,
respectively, were similar, as were kcat values for both
enzyme forms (Table 2)
Inhibition of the recombinant DHODH
Specific inhibitors for yeast DHODH have not yet
been described We studied the recombinant enzymes
from C albicans for their susceptibility to various
compounds, which have already been proven to be
inhibitors of human DHODH or DHODH from other
species or compounds implicated in interfering with
electron transport in mitochondria or pyrimidine
meta-bolism [18,21–23] Only the dianisidine derivative
red-oxal (0.5 mm) exhibited an inhibitory effect of more than 50% on CaDHODH and DNCaDHODH com-pared with the noninhibited reaction As compounds such as redoxal may have redox activity, we tested it
as a putative direct electron acceptor for the C albi-cans DHODH Redoxal (up to 100 lm) did not pro-mote oxidation of dihydroorotate to orotate (data not shown) Also, 1 mm brequinar reduced the activity by more than 50% of the full-length enzyme (Table 3)
IC50 values as a practical reflection of the relative effects of different substances on enzyme activity under comparable assay and laboratory conditions were obtained from dose–response curves The IC50 for redoxal was 106 ± 12 lm (CaDHODH) and
102 ± 12 lm (DNCaDHODH), respectively; that for brequinar was 439 ± 83 lm (CaDHODH)
Discussion
The availability and characterization of recombinant DHODH from C albicans in this work permitted the first screening of compounds as putative enzyme inhib-itors, with the rationale to interfere with the pyrimid-ine nucleotide pools of this pathogen
All DHODH proteins of family 2 must be translo-cated from the cytosol to the inner membrane of mito-chondria The proteins are directed by targeting sequences, which usually consist of various numbers of amino acids at the N-terminus [24] Although no con-sensus sequence has been identified, the pre-sequences have a high content of basic, hydrophobic and hydrox-ylated amino acids, and a length of about 10–80 amino acids Generally, the pre-sequence is cleaved on import, as it is not necessary for protein function [25] The length of the targeting sequence in the C albicans DHODH (37 amino acids) would suggest that there should be a cleavable site However, in silico studies (‘PeptideCutter’, http://www.expasy.org/tools) could not identify cleavage sites of known proteases Mam-malian DHODHs have a shorter targeting sequence of
Table 1 Alternative electron-accepting substrates for recombinant
C albicans dihydroorotate dehydrogenase (DHODH) Activities are
expressed relative to that with FeCy as the electron acceptor, and
mean ± SEM from three determinations is given as a percentage.
All reaction mixtures contained molecular oxygen at atmospheric
pressure (equivalent to about 230 l M ) and 1 m M DHO DCIP,
2,6-dichloroindophenol.
Electron acceptor
Relative velocity (%)
DCIP + QD (1 m M +0.1 m M ) 40.8 ± 1.7 50.6 ± 3.1
Table 2 Kinetic constants of the purified full-length and truncated C albicans dihydroorotate dehydrogenase (DHODH) All measurements were performed in triplicate For Kmand Vmax, the best fit (± asymptotic SEM) of the Michaelis–Menten equation to all data is given The
k cat values were calculated using the equation V max ¼ k cat [E], where [E] is the total enzyme concentration and is based on one active site ⁄ monomer U is the enzyme activity as lmol substrateÆmin)1.
DHODH
V max
(UÆmg)1)
K ma
(l M DHO)
K mb
(l M QD)
K mc
(l M DCIP)
k cat
(s)1)
k cat ⁄ K ma
( M DHO)1Æs)1)
k cat ⁄ K mb
( M Q 1
D Æs)1)
k cat ⁄ K mc
( M DCIP)1Æs)1)
5.0 · 10 4
1.7 · 10 3
a The concentration of DHO was varied (0–1.0 m M ) at fixed concentrations of 100 l M QDand 60 l M 2,6-dichloroindophenol (DCIP) as elec-tron acceptors b The concentration of QDwas varied (0–0.2 m M ) at a fixed DHO concentration of 1 m M c The concentration of DCIP was varied (0–0.2 m M ) at a fixed DHO concentration of 1 m M
Trang 5only 11–13 amino acids, which was found not to be
cleaved off after import into the inner mitochondrial
membrane [12] The targeting sequences of yeast
DHODH possess up to 5 times more amino acids [17];
therefore, proteolytic processing may be possible The
length of the targeting sequence seems to influence the
recombinant expression rate All yeast DHODHs have
40 amino acids in contrast with 10 amino acids in
the human DHODH [17] A similar observation was
made with the A thaliana DHODH, which has a
tar-geting sequence of 57 amino acids The expression rate
of the truncated plant DHODH was higher than that
of the full-size protein [26]
At the N-terminus, the adjoining hydrophobic region, which was identified as a transmembrane domain of 17 amino acids in rat DHODH [12], can be presumed to be a membrane-spanning a-helix Here,
we were able to predict a transmembrane domain of
16 amino acids with ‘ProtScale’ (http://www.expasy org/tools) in the C albicans DHODH amino-acid sequence (Fig 2)
As the recombinant CaDHODH and DNCaD-HODH had the same kinetic parameters, the trunca-tion seemed not to influence the enzyme activity of yeast DHODH However, the specific activity of
C albicansDHODH was considerably lower than that obtained with recombinant mammalian DHODH preparations, which were determined using the same assay (e.g human enzyme, 100–150 UÆmg)1) [20] In comparison with human species (Km¼ 6–15 lm for DHO and Km¼ 9–14 lm for QD), the Km values for
C albicansDHODH were 10-fold higher On the other hand, C albicans DHODH was very similar to other yeast DHODHs with regard to its kinetic properties Higher Kmvalues for QDwere described for full-length
S kluyveri and Sch pombe DHODH [17] compared with the truncated forms
Two a-helices after the hydrophobic domain at the N-terminus were predicted by structural alignment using ‘Swiss-PdbViewer 3.7’ comparing the structures
of human (RCSB PDB-ID, 1D3G) and E coli (RCSB PDB-ID, 1F78) with the amino-acid sequence of
C albicans DHODH (data not shown) They are sim-ilar to those of human DHODH and are thought to
be essential for membrane association and for facilita-ting the contact between the ubiquinone from the inner membrane and the active site of DHODH [23,27] Although there are some differences in processing and association in the organization of the fungal and mam-malian respiratory chain complexes, the assembly ensures the transfer of electrons from different sources
to oxygen by the respiratory chain complexes and the coupling of proton uptake from the matrix compart-ment [28]
The nature of the quinone in C albicans is not known In this study, recombinant C albicans DHODH was shown to use several native and two artificial electron acceptors, FeCy and 2,6-dichloroin-dophenol Q6, which has been described as a physiolo-gical electron acceptor in the respiratory chain of
S cerevisiae [29], was found to be superior to all the other quinones studied here Fumarate and NAD+, the physiological acceptors for DHODH of family 1A and 1B, respectively, were not acceptors for C albicans DHODH This provides functional evidence, addi-tional to its sequence similarity and catalytic-site
Table 3 Activity of recombinant C albicans dihydroorotate
dehy-drogenase (DHODH) in the presence of putative inhibitors Relative
velocities determined in chromogen reduction assays with 1 m M
DHO, 0.1 m M QDand 0.1 m M 2,6-dichloroindophenol (DCIP) as final
electron acceptor are given Values are mean ± SEM from three
determinations The activity of each enzyme without inhibitor was
set as 100% If not otherwise stated the concentration of the
com-pound was 1 m M TTFA, 2-thenoyltrifluoroacetone.
Compound
Relative velocity (%)
Control with dimethyl sulfoxide 100 100
Antimycin A (0.5 m M ) 53 ± 3 56 ± 3 (1 m M )
Licochalcone A (0.5 m M ) 100 ± 25 100 ± 5
4-Trifluoromethylaniline 91 ± 15 100 ± 20
Tournaire acid 3 (2.5 m M ) 98 ± 2 107 ± 5
Trang 6features, that C.albicans DHODH belongs to the
DHODH family 2 enzymes (Fig 2)
The respiratory chain complexes of fungi have been
shown to be inhibited by standard agents, e.g
rote-none, myxothiazol, antimycin A, and CN–, extensively
used to assay animal mitochondria [30] In contrast
with this high conservation of sensitivity, drugs that
have been shown to suppress DHODH activity both
in vitro and in vivo were found here not to interfere
with the DHODH of C albicans, e.g A77-1726,
ato-vaquone, and licochalcone A Some of these drugs
(Table 3) are in clinical use today: the antirheumatic
drug leflunomide⁄ A771726 (AravaTM) [31], the
antima-larial drug atovaquone (MalaroneTM) [22], and the
anticoccidial toltrazuril (BaycoxTM) [21] The
develop-ment of effective compounds against Plasmodium
falci-parum and Pneumocystis carinii took advantage of
species-specific differences between DHODH from
family 2 By structure–activity relationship studies,
some of these drugs have been shown to interfere with
the ubiquinone-binding site of mammal DHODH
[23,32] but not with that of E coli [27] Detailed
kin-etic investigations of the bisubstrate reaction catalyzed
by full-length rat DHODH revealed a noncompetitive
type of inhibition by brequinar with respect to the
co-substrate QD [33] LicA was described as a potent
inhibitor of E coli DHODH, but it affected neither
DHODH-1A and 1B from Lactococcus lactis (M
Han-sen, University of Copenhagen, personal
communica-tion) nor human DHODH (unpublished data)
Structural alignment using Swiss-PdbViewer 3.7 to
compare the structures of human and E coli DHODH
[23,27] and the amino-acid sequence of C albicans
DHODH showed considerable differences between the
inhibitor-binding sites (data not shown) Mainly,
hydrophobic interactions, which are important for the
binding of A771726 and brequinar, were reduced In
the structural alignment, we found hydrophobic amino
acids, which are important for inhibitor binding,
replaced with smaller or larger residues This may
explain the difference in binding of these drugs by the
fungal and animal DHODH and again highlights
DHODH as a very species-specific target for potential
intervention and drug discovery
In this study, considerable interference was observed
in the oxidation of DHO with QD by redoxal The
IC50value of 100 lm is higher for the fungal enzyme
than for the human (IC50¼ 368 nm) and rat (IC50¼
214 nm) enzyme [34] Interestingly, the distinct
species-related efficacy of inhibition of the human and rodent
enzyme observed with isoxazol, cinchoninic acid and
naphthoquinone derivatives seemed to be less obvious
with redoxal It was concluded that the binding of
o-dianisidines may be divergent from that of the other classes [34] As redoxal was superior to all the other compounds tested here in inhibiting fungi DHODH, it can be considered an attractive lead for the synthesis
of molecules with higher activity The high-resolution X-ray crystallographic structure of C albicans DHODH in complex with an o-dianisidine derivative will be necessary to understand the mode of binding and interference with enzyme catalysis
As the inactivation of any enzyme involved in a metabolic chain will render the whole chain inoperat-ive, the inactivation of any of the six proteins involved in pyrimidine de novo synthesis should result
in the same profound effect on the pyrimidine nuc-leotide pools in C albicans However, in mammalian cell lines, the development of drug resistance was observed with other agents and other enzymes of the
de novo pathway to a much greater degree than with DHODH [35] Therefore, it is reasonable to assume that the overexpression and proper location of an integral membrane protein would happen to a limited extent only Thus DHODH rather than a cytosolic enzyme of pyrimidine biosynthesis should be the preferential target for drug development The availab-ility of recombinant DHODH should expedite discov-ery of more potent agents for growth control strategies in C albicans, and permit the screening of
a large number of compounds, the examination of structure–activity relationships of inhibitors, and determination of the 3D structure of enzyme–inhib-itor complexes
Experimental procedures
Reagents Unless otherwise stated, the following chemicals were from Roche Diagnostics (Mannheim, Germany), Serva (Heidel-berg, Germany), Merck (Darmstadt, Germany) or Sigma (Sigma-Aldrich, Taufkirchen, Germany) at the purest grade available: anhydrotetracycline (Acros Organics, Geel, Bel-gium), DHO, dimethyl sulfoxide, QD, Q0, Q6, Q10, FeCy, fumarate, NAD, 2,6-dichloroindophenol
The inhibitors studied were: 2-hydroxyethylidene-cyano-acetic acid 4-trifluoromethyl anilide (A77-1726; Sanoif-Aventis, Frankfurt, Germany); trans-2-[4-(chlorophenyl)-cyclohexyl]-3-hydroxy-1,4-naphthoquinone (atovaquone, 566C80; Wellcome Foundation, Dartford, UK); 6-fluoro-2-(2¢-fluoro-1,1¢-biphenyl-4yl)-3-methyl-4-quinoline carboxy-lic acid (brequinar sodium, NSC 368390; DuPont Pharma GmbH, Bad Homburg, Germany); licochalcone A [36]; acetylsalicylic acid; alloxan; antimycin A; 3,4 dihydroxy-benzoic acid; 3,5 dihydroxydihydroxy-benzoic acid; 5-fluorouracil;
Trang 75-fluoroorotate; 5-fluorocytosine;
2-methyl-1,4-naphthoqui-none; menadione; salicylic acid; salicylhydroxamic acid
(Sigma); amytal (Serva); ciprofloxacin; toltrazuril (Bayer
AG, Leverkusen, Germany); clindamycin; carboxin; ectosin
(Fluka, Buchs, Switzerland); redoxal (NCI 73735) [35];
di-chloroallyllawson (NIH Drug Synthesis and Chemistry
Branch, Development Therapeutics Program, Division of
Cancer Treatment, Bethseda, MD, USA); lawson (Aldrich);
polyporic acid (Langner, University of Halle, Germany);
4-trifluoromethylaniline (Chemos GmbH, Regenstrauf,
Germany); tournaire acid 3 [37]
Oligonucleotides
ZGCaURA1–5¢, ATGTTTCGTCCAAGTATCAAAT
TC
ZGCaURA1–3¢, TCACTTATCATCAGAGCC
Ca-forlong2, ATGTTTCGTCCAAGTATCAAATTC
AAACAGTCGACTTTGTCC
CaKDHODH-mutfor1, CACAGATGCAGAGTCGG
GACATAAGTTGGGGGTT
CaKDHODH-mutrev1, CCAACTTATGTCCCGACT
CTGCATCTGTGAAAGT
CaDHODH-rev, CCGGAATTCCTTATCATCAGAG
CCAATTAT
AAGTATCAAATTCAAACAGTCG
CAGCAATCCATGAATATGTTTTGTGC
AGAGCCAATTATTTGCTCCCATG
Expression plasmids
The C albicans URA1 gene (accession number AY230865)
was subcloned with the oligonucleotides ZGCaURA1–5¢
and ZGCaURA1–3¢ The 1335-bp ORF was then amplified
from the URA1 PCR fragment with primers Ca-forlong2
and CaDHODH-rev Mutations were inserted by PCR with
primers Ca-forlong2 and CaKDHODH-mutrev1 for a first
fragment and with primers CaKDHODH-mutfor1 and
CaDHODH-rev for a second fragment The overlapping
PCR fragments were then used as templates for PCR with
primers Ca-forlong2 and CaDHODH-rev For subcloning
of the DHODH ORF the restriction sides for BamHI⁄
EcoRI were created with primers Ca-BamHI-for and
CaDHODH-rev3 for full-length C albicans DHODH The
resulting PCR fragment was cut by BamHI⁄ EcoRI and
sub-sequently ligated into pGEX-6P-3, cut by BamHI⁄ EcoRI
The resulting plasmid was named pGEX-6P-3-CaDHODH,
and the recombinant expressed enzyme is referred to as
CaDHODH A 55-amino acid N-terminal truncated form
of C albicans DHODH was constructed using
CaK-Bam-HI-for and CaDHODH-rev, cut by BamHI⁄ EcoRI and
subsequently ligated into corresponding sites of
pGEX-6P-3 The resulting plasmid was named pGEX-6P-3-DNCaD-HODH, and the recombinant expressed enzyme is referred to
as DNCaDHODH
Protein expression and purification All recombinant DHODHs were expressed as fusion pro-teins containing an N-terminal glutathione S-transferase (GST) tag The proteins were expressed in the E coli strain BL21 for 24 h at 18C after induction (A600¼ 0.5–0.6) with 1 mm isopropyl b-d-thiogalactoside in Luria–Bertani broth⁄ ampicillin (100 lgÆmL)1) medium plus 0.1 mm FMN For purification of the recombinant proteins, the cells were harvested at 4000 g for 15 min, resuspended in binding buf-fer (140 mm NaCl, 2.7 mm KCl, 0.1 mm FMN, 10 mm
Na2HPO4, 1.8 mm KH2PO4, 1% Triton X-100, pH 7.3), and disrupted by sonification After centrifugation for
60 min at 15 000 g, the supernatant was applied to a 1-mL GSTrapTM FF column (Amersham Biosciences Europe, Freiburg, Germany) The column was washed with 10 vol binding buffer and 10 vol pre-scission buffer (50 mm Tris⁄ HCl, 150 mm NaCl, 1 mm EDTA, 1 mm dithiothrei-tol, 1% Triton X-100, pH 7) The recombinant proteins were cut by pre-scission protease (Amersham Biosciences)
at 4C overnight The recombinant proteins without GST tag were eluted with 5 vol pre-scission buffer The exchange to buffer C [50 mm Tris⁄ HCl, 150 mm KCl, 10% (v⁄ v) glycerol, 0.1% (v ⁄ v) Triton X-100, pH 8] was per-formed using a PD-10 column (Amersham Biosciences) Protein determination and SDS⁄ PAGE were performed as described previously [17] For fluorimetric determination of flavin, 0.5–0.7 lgÆmL)1 protein was denatured by heating
up to 100C for 10 min After being allowed to cool, the solution was centrifuged and protected from light until measurement using a spectrofluorimeter (SFM 25, Bio-Tek Instruments, Bad Friedrichshall, Germany) at excita-tion⁄ emission wavelengths of 465 ⁄ 518 nm, with FMN as standard marker (0–100 lm)
Immunological methods Before immunodetection, recombinant C albicans DHODH from SDS⁄ PAGE was transferred on to Immobilon P (Milli-pore, Schwalbach, Germany) by semidry blotting (1.5 h at 0.8 mAÆcm)2; SDS⁄ polyacrylamide gel) After being blocked with 5% nonfat dried milk in 10 mm sodium phosphate buf-fer, pH 7.5, containing 150 mm NaCl, the membrane was exposed to affinity-purified rabbit antibodies to human DHODH (diluted 1 : 15000) [38] As secondary antibodies, goat anti-rabbit horseradish peroxidase-conjugated IgG (Sigma), diluted 1 : 10 000, were used Bound antibodies were detected with an ECL detection kit (Amersham Bio-sciences)
Trang 8Biochemical analysis of DHODH
The assay to determine enzyme activity and kinetic
parame-ters was performed in 50 mm Tris⁄ HCl, 150 mm KCl, 10%
(v⁄ v) glycerol, 0.1% (v ⁄ v) Triton X-100, pH 8 [18] At 30 C,
the oxidation of the substrate DHO with the quinone
cosub-strate was coupled to the reduction of the chromogen
2,6-di-chloroindophenol The Km of DHO was determined by
varying the concentration of DHO (1–1000 lm) at a fixed
concentration (200 lm) of QD The Kmof QDwas determined
by varying the concentration of QD(0.1–200 lm) at a fixed
concentration (1 mm) of DHO The additional Kmvalue for
2,6-dichloroindophenol (0.01–200 lm) was determined using
the same assay but without QD Kinetic data were evaluated
under initial velocity conditions [33]; the Michaelis–Menten
equation v¼ V[S] ⁄ (Km+ [S]) was fitted to all data
The pH-dependence of initial velocities was measured at
saturating substrate concentrations (1 mm DHO, 0.1 mm
QD) in different buffer systems (Mes⁄ HCl, Hepes ⁄ HCl,
Tris⁄ HCl) covering the pH range 5–9, using the chromogen
reduction assay with 2,6-dichloroindophenol as final
elec-tron acceptor Overlapping pH ranges were measured in
two buffer systems to exclude salt effects The equation
v¼ V ⁄ [(10–pH⁄ 10–pKa1) + (10–pKa2⁄ 10–pH) +1] was fitted
to the data
Various natural and artificial electron acceptors were
compared in the optimal Tris⁄ HCl buffer system at
pH 8.0 Reduction of the electron acceptors was measured at
the indicated wavelength: 2,6-dichloroindophenol (600 nm,
e¼ 18800 m)1Æcm)1), FeCy (420 nm, e¼ 1020 m)1Æcm)1),
NAD+ (340 nm, 6200 m)1Æcm)1) In an alternative
assay, UV absorption of the product orotate was
monit-ored in the presence of the electron acceptor fumarate
(280 nm, e¼ 7500 m)1Æcm)1) or oxygen only (280 nm, e¼
7500 m)1Æcm)1), and at the appropriate isosbestic
wave-length, with QD(300 nm, e¼ 2950 m)1Æcm-1), Q10(300 nm,
e¼ 2950 m)1Æcm)1), Q6 (293 nm, e¼ 4700 m)1Æcm)1), Q0
(287 nm, e¼ 5680 m)1Æcm)1), respectively
To determine the inhibitory potency of 28 different
com-pounds, the chromogen reduction assay was used with the
putative inhibitor up to a concentration of 1 mm as
des-cribed above Stock solutions of all inhibitors were prepared
freshly in Tris⁄ HCl buffer, pH 8.0, or in dimethyl sulfoxide
The appropriate controls were run in buffer or in the
pres-ence of dimethyl sulfoxide; 2% dimethyl sulfoxide in the
assays was found not to interfere with the DHODH activity
All measurements were performed in triplicate Percentage
of inhibition was related to controls (100% activity)
To determine the IC50 values for redoxal and breqinar,
the initial velocity of the DHODH reaction was measured
at saturating substrate concentrations of DHO (1 mm) and
QD (0.1 mm) with various concentrations of the putative
inhibitors (redoxal, 1 lm)1 mm; brequinar, 1 lm)8 mm)
The equation v¼ V ⁄ {1 + [I] ⁄ IC50}, where [I] is the
inhib-itor concentration, was fitted to the initial velocities to find
the drug concentration causing 50% inhibition of the enzyme activity (IC50value)
Acknowledgements
This study was supported by the Deutsche Fors-chungsgemeinschaft, Marburger Graduiertenkolleg
‘Protein Function at the Atomic Level’ to ML and by the Danish Research Council to JP We thank Maria-Bettina Kowalski and Ute Beck for technical assist-ance
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