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J. Biol. Chem., Vol. 280, Issue 12, 11295-11302, March 25, 2005

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The Functional Basis of Mycophenolic Acid Resistance in Candida albicans IMP Dehydrogenase^*

Gerwald A. Köhler¶, Xin Gong||, Stefan Bentink, Stephanie Theiss, Gina M. Pagani**, Nina Agabian, and Lizbeth Hedstrom||

From the ^||Department of Biochemistry and the ^**Graduate Program in Biophysics and Structural Biology, Brandeis University, Waltham, Massachusetts 02454-9110, the Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany, and the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143

Received for publication, August 26, 2004 , and in revised form, December 30, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to the same site as a mobile flap. In contrast, mizoribine monophosphate, the active metabolite of mizoribine, induces the closed conformation. Here, we report the characterization of IMPDH from wild-type and MPA-resistant strains of C. albicans. The wild-type enzyme displays significant differences from human IMPDHs, suggesting that selective inhibitors that could be novel antifungal agents may be developed. IMPDH from the MPA-resistant strain contains a single substitution (A251T) that is far from the MPA-binding site. The A251T variant was 4-fold less sensitive to MPA as expected. This substitution did not affect the k_cat value, but did decrease the K_m values for both substrates, so the mutant enzyme is more catalytically efficient as measured by the value of k_cat/K_m. These simple criteria suggest that the A251T variant would be the evolutionarily superior enzyme. However, the A251T substitution caused the enzyme to be 40-fold more sensitive to mizoribine monophosphate. This result suggests that A251T stabilizes the closed conformation, and this hypothesis is supported by further inhibitor analysis. Likewise, the MPA-resistant strain was more sensitive to mizoribine in cell culture. These observations illustrate the evolutionary challenge posed by the gauntlet of chemical warfare at the microbial level.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Candida albicans is an important fungal pathogen of immunocompromised patients (1, 2). Amphotericin B, 5-fluorocytosine, azoles, and echinocandins are currently used to treat candidiasis, although toxicity, limited availability, and the emergence of resistance create an urgent need for new drugs (3, 4). C. albicans is sensitive to mycophenolic acid (MPA)¹ and mizoribine when cultured in vitro (5, 6). Both of these compounds inhibit IMP dehydrogenase (IMPDH), so this enzyme presents a novel target for antifungal chemotherapy. In addition, the IMPDH gene IMH3 is a useful genetic marker for C. albicans because multiple copies confer MPA resistance (7). MPA^R, an IMH3 allele encoding an MPA-resistant IMPDH, confers drug resistance with a single gene copy (8–10). A second MPA-resistant allele of C. albicans IMPDH that carries mutations I47V, S102A, and G482D has been reported, although the functional effects of these mutations on enzyme activity have not been characterized (11, 12).

IMPDH catalyzes the rate-limiting step in guanine nucleotide metabolism: the oxidation of IMP to XMP with concomitant reduction of NAD⁺ (Fig. 1). IMPDH is a target for the immunosuppressive drugs mycophenolate mofetil (CellCept®) and mizoribine; the active metabolites of these drugs are MPA and mizoribine monophosphate, respectively (Fig. 1) (13, 14). The mechanism of the IMPDH reaction is complex, with important consequences for inhibitor selectivity (Fig. 1). IMPDH undergoes a large conformational change in mid-catalytic cycle that converts the enzyme from a dehydrogenase to a hydrolase (15). In the first half of the reaction, substrates bind, and hydride transfer occurs to form the covalent E·XMP* intermediate. NADH departs, and the mobile flap folds into the NADH site. This conformational change positions the conserved Arg-Tyr dyad to activate water, and E-XMP* is hydrolyzed to XMP (16). MPA traps E-XMP* by competing with the flap for the NADH site (18, 19). In contrast, mizoribine monophosphate (MZP) binds to the IMP site and induces the flap to close (15). Thus, the equilibrium between open and closed conformations controls drug sensitivity: the open conformation favors MPA binding, whereas the closed conformation favors MZP binding.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Mechanism of the IMPDH reaction and structure of inhibitors. A, chemical mechanism of the IMPDH reaction. XMP is an anion at pH 8, although the location of the negative charge is not well established. B, structures of inhibitors. C, substrates add randomly, and IMP is oxidized to form E-XMP* and NADH. The enzyme is in an open conformation in the ternary complexes, and the flap is disordered. NADH releases, and the flap folds into the NADH site, positioning the Arg-Tyr dyad to activate water. MPA binds to E·XMP* and blocks the conformational change, preventing hydrolysis. MZP binds to the IMP site and induces the closed conformation. Thus, the closed conformation blocks MPA inhibition, but promotes MZP inhibition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—IMP, ADP, NADH, MPA, mizoribine, and Tris were purchased from Sigma. NAD⁺ was purchased from Roche Applied Science. Dithiothreitol was purchased from Research Organics, Inc. Glycerin, EDTA, and KCl were purchased from Fisher. Tiazofurin was obtained from NCI. MZP was a gift from Drs. S. Shuto and A. Matsuda (Hokkaido University, Sapporo, Japan).

Strains and Growth Conditions—The C. albicans Ura^– auxotrophic strain CAI4 was kindly provided by Dr. William A. Fonzi (Department of Microbiology and Immunology, Georgetown University Medicine Center, Washington, D. C.). C. albicans cells were propagated in yeast extract/peptone/dextrose at 30 °C. The C. albicans strains used in this study are listed in Table I. Escherichia coli strain DH5 was used for cloning and plasmid isolation. E. coli strain H712, which lacks endogenous IMPDH, was obtained from the E. coli Genetic Stock Center of Yale University (New Haven, CT). Bacteria were grown at 37 °C in LB medium or M9CA medium with suitable supplements.

Generation of IMH3 Mutants—A heterozygous imh3/IMH3 mutant was generated using the URA3 flipper method (20) in C. albicans strain CAI4. For this purpose, plasmid pI4F was constructed by amplification of a 2.7-kb DNA fragment containing the IMH3 gene plus the flanking regions using the IMH3RXF/IDHBXR primer pair, followed by insertion of the EcoRI/BamHI-cut amplicon into pBluescript. Subsequently, the SalI and NotI restriction sites of pI4F were removed. The resulting plasmid was used as template for divergent PCR with primers KOIMHS (5'-CAGGGACGACTGGTCGACNRN₂RN₃RNCCCGGGAGAAATGGGAATGGGTAG-3') and NIMHKO (5'-TAATTGCGGCCGCTGCAGNWN₃WN₂SNGCATGCCCAAGGTGTCTCTGGTTC-3'), which introduced new SalI and NotI sites (underlined), respectively. (Degenerate regions of the primers were used to tag strains for a different study.) After a NotI/SalI restriction digest, the amplicon was ligated to the NotI/XhoI URA3 flipper cassette of pSFU1 (20), resulting in pSFI-MH3. The gel-purified ApaI/SacI fragment of pSFIMH3 containing the URA3 flipper with the 5'- and 3'-flanking regions of IMH3 was used for targeted IMH3 gene disruption in strain CAI4. C. albicans strain CAI4 was transformed by electroporation as described previously (7). Transformants were selected on uracil-deficient synthetic dextrose medium (yeast nitrogen base, complete supplement mixture without uracil, 2% glucose, and 2% agar) and subsequently tested for correct insertion of the disruption cassette by Southern analysis of genomic DNA (data not shown).

For this study, we used the heterozygous Ura⁺ imh3/IMH3 mutant Cl1M7 to generate mutants with a single copy of MPA^R substituted for IMH3. This was achieved by introducing the MPA^R mutation into strain Cl1M7 via transformation with a PCR fragment amplified from pEC352 using primers IMHSF (5'-GCTCCGTCGACTAGATGTTTATGATAC-3') and IDHCLAR (5'-GCATGGCATCGATGGAAC-3'). MPA-resistant Ura⁺ transformants were selected on uracil-deficient synthetic dextrose medium plus 10 µg/ml MPA, and the MPA^R mutation was verified by restriction digest (G-to-A transition in MPA^R eliminates an Fnu4HI site) and direct sequencing of PCR products. Two transformants (MPAR1 and MPAR2) were chosen for further testing.

Susceptibility Testing—The MPA and mizoribine resistance of C. albicans strains was measured quantitatively in a microtiter plate assay as described previously (7). Growth of the strains in synthetic medium in the presence of serial dilutions of the inhibiting substances was determined by A₅₆₀ measurements after 48 h using a microplate reader. Experiments were performed in triplicate.

Recombinant Expression of C. albicans IMPDH and MPA^R—Both the IMH3 gene (7) and MPA^R contain an intron at amino acid 150, which would prevent bacterial expression. Therefore, we isolated poly(A)⁺ RNA from strains CAI4 and EC3 using an Oligotex mRNA mini kit (Qiagen Inc.). Reverse transcription was carried out with Superscript II (Invitrogen) and primer DTNOT (5'-AATTCGCGGCCGCT₁₇-3'). The cDNAs were amplified using the primer pair CA5CDS (5'-CGGCGGAATTCATGGTGTTTGAAACTTCAAAAG-3') and CACDS3 (5'-TGGAACTGCAGTTAGTTGTGTAATCTCTTTTC-3'), which span the start and stop codons of the IMPDH genes, respectively. The PCR products were cut with EcoRI and PstI using the restriction sites introduced by the PCR primers and cloned into pUC18, resulting in pUCCDS4 (IMH3) and pUCCDS9 (MPA^R). These inserts were subsequently amplified using primers 5TGCDS (5'-P_i-TGGTGTTTGAAACTTCAAAAG-3') and CDS3HIND (5'-TGGTCAAGCTTAGTTGTGTAATCTCTTTTCATA-3') to introduce a HindIII restriction site for cloning into pTACTAC (21). pTACTAC was digested with NdeI, and the resulting ends were filled in with the Klenow fragment of DNA polymerase I and further digested with HindIII. The blunt end ligation at the NdeI site regenerated the ATG start codon of IMH3 and MPA^R, and the 3'-ligation regenerated a TAA stop codon. The ligation products were transformed into E. coli strain H712, a guanine auxotroph harboring a guaB^– mutation. Cells were plated on modified M9CA minimal medium (10 g/liter M9 minimal salts (Invitrogen), 17 mM NaCl, 0.1 mM CaCl₂, 1 mM MgSO₄, 1 g/liter casamino acids, 0.79 g/liter complete supplement mix, 1 g/liter thiamine, 2 g/liter glucose, 1 mM isopropyl 1-thio--D-galactopyranoside, and 50 mg/liter ampicillin) to isolate transformants correctly expressing the IMH3 or MPA^R plasmid and complementing the guaB^– mutation. For further studies, we used plasmids pTAC1_5 and pTAC4_13, which express wild-type IMH3 and MPA^R IMPDHs, respectively. The sequences of both plasmid inserts were determined and proved free of errors.

Protein Purification—E. coli H712 cells harboring either the pTAC1_5 or pTAC4_13 vector were cultured in LB medium containing 100 µg/ml ampicillin and 1 mM isopropyl 1-thio--D-galactopyranoside to express wild-type and A251T IMPDHs, respectively. The cells were harvested by centrifugation, and the pellet was resuspended in buffer A (50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, and 10% glycerol). All of the following manipulations were performed at 4 °C. The cells were disrupted by sonication. Debris was removed by centrifugation, and the supernatant was applied to a Cibacron blue-agarose column (Sigma) previously equilibrated with buffer A. IMPDH was eluted in a linear gradient of 0–1 M KCl in buffer A. Fractions containing IMPDH activity were pooled, concentrated by ultrafiltration, and dialyzed overnight against buffer A containing 0.5 mM IMP. IMPDH was further purified on an HQ anion exchange column using a BioCAD Sprint perfusion chromatography system (Applied Biosystems, Framingham, MA). Enzyme was eluted in a linear gradient of 0–1 M NaCl in 50 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol. The fractions containing IMPDH were pooled, dialyzed overnight against buffer A containing 0.5 mM IMP at 4 °C, and then applied to an HS cation exchange column. IMPDH was eluted in a linear gradient of 0–1 M NaCl in 50 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol. The protein concentration was measured using a NanoOrange® protein quantitation kit (Molecular Probes, Inc.) with bovine serum albumin as a standard. This protocol produced 12 mg of purified protein from 1 liter of cell culture.

Enzyme Kinetics—Standard IMPDH assays contained 100 mM KCl, 3 mM EDTA, 1 mM dithiothreitol, and 50 mM Tris (pH 8.0) (assay buffer). The production of NADH was monitored spectrophotometrically at 340 nm ( = 6.22 mM^–1 cm^–1) using a Hitachi U-2000 spectrophotometer. The concentrations of IMP and NAD⁺ were varied for K_m determinations. Initial velocity data were fit to the Michaelis-Menten equation (Equation 1) and the uncompetitive substrate inhibition equation (Equation 2) using SigmaPlot software (SPSS Inc.),

(Eq. 1)

(Eq. 2)

where v is the initial velocity, k_cat is the turnover number, K_m is the Michaelis constant of IMP or NAD⁺, and K_ii is the substrate inhibition constant. Steady-state parameters with respect to NAD⁺ were derived by first determining the apparent k_cat values for the initial velocity versus IMP plots (Equation 1 for wild-type IMPDH and Equation 2 for A251T IMPDH) and replotting these values against the NAD⁺ concentration (Equation 2 for wild-type IMPDH and Equation 1 for A251T IMPDH). Similarly, the K_m value for IMP was derived by first determining the apparent k_cat values for the initial velocity versus NAD⁺ concentration plots using Equation 2 for wild-type IMPDH and Equation 1 for the mutant enzyme and replotting these values against the IMP concentration (Equation 1 for wild-type IMPDH and Equation 2 for A251T IMPDH). The k_cat values determined by this method were similar to those determined above.

Inhibitor Kinetics—The K_i values for MPA inhibition were determined in experiments containing constant IMP concentrations (600 µM for wild-type IMPDH and 200 µM for A251T IMPDH) and varied NAD⁺ concentrations. Initial velocity data were fit to the uncompetitive tight-binding inhibition equation (Equation 3) and the noncompetitive inhibition equation (Equation 4) using SigmaPlot software,

(Eq. 3)

(Eq. 4)

where v is the initial velocity; V_max is the maximum velocity; S is substrate; K_m is the Michaelis constant for the substrate; K_is and K_ii are the slope and intercept inhibition constants, respectively; I is inhibitor; and E is enzyme. The best fits were determined by the relative fit error.

MZP is a slow tight-binding inhibitor under standard assay conditions. The K_i value was determined from the steady-state inhibition data, and the k_on and k_off values were determined from progress curves, both analyzed using Dynafit (22). The analysis presumed that MZP is a competitive inhibitor with respect to IMP, as has been observed for IMPDH from other organisms.

Multiple inhibition experiments with tiazofurin and ADP were performed with constant concentrations of IMP (600 µM for wild-type IMPDH and 200 µM for A251T IMPDH) and NAD⁺ (250 µM for wild-type IMPDH and 4000 µM for A251T IMPDH) at 25 °C in assay buffer. Data were fit to an equation describing multiple inhibition (Equation 5),

(Eq. 5)

where I and J are inhibitors; v₀ is the initial velocity in the absence of inhibitors; is the interaction constant; and K_i and K_j are the inhibition constants for I and J, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Isolation of MPA-resistant Strains of C. albicans—Strain CAI4 is sensitive to MPA, with an MIC₅₀ of 0.25 µg/ml. Electrocompetent C. albicans strain CAI4 was cultured in the presence of sub-lethal concentrations of MPA (3–5 µg/ml) as described under "Experimental Procedures." After prolonged incubation, several colonies appeared; cells were streaked onto agar plates with an increased concentration of MPA (10 µg/ml); and two strains were isolated (EC3 and EC5). The MIC₅₀ values for both strains EC3 and EC5 as determined by microdilution assays were 1.7 µg/ml MPA (Supplemental Fig. S1).

Identification of the MPA^R Mutation—Assuming that a mutant IMH3 allele confers MPA resistance, we amplified the entire coding regions of the IMH3 genes plus the flanking regions of both strains EC3 and EC5. Transformation of strain CAI4 with these amplification products yielded MPA-resistant clones, confirming that drug resistance results from mutation(s) in IMH3. Isolation and sequencing of the IMH3 genes from these strains revealed several differences from the published nucleic acid sequence of the IMH3 gene from strain SS (7). With one exception, all sequence differences within the coding region were silent. The G-to-A transition at nucleic acid 751 of the IMH3 coding sequence creates an Ala-to-Thr substitution at position 251 of the protein sequence. The mutant IMH3 gene also lacks the 200-bp solo phi element in the 3'-untranslated region, a long terminal repeat of a putative transposable element of C. albicans. phi was recently described by Goodwin and Poulter (23). We found that phi is present in one IMH3 allele of strains SC5314 and CAI4 (data not shown). Because neither SC5314 nor CAI4 shows MPA resistance similar to that shown by EC3 or EC5, we concluded that the phi element is not involved in MPA resistance. A single copy of the IMH3 gene carrying the A251T mutation could confer MPA resistance to C. albicans. This allele was subsequently developed as a dominant selection marker gene in Candida (8, 10, 24). However, the mechanistic basis of the drug resistance of MPA^R was unclear because Ala²⁵¹, although well conserved among IMPDHs from different sources, is not located in the MPA-binding site.

Characterization of Wild-type and A251T IMPDHs—Both the wild-type and MPA^R alleles of C. albicans IMPDH were expressed in E. coli by isopropyl 1-thio--D-galactopyranoside induction using vector pTACTAC. Both constructs complement the guanine auxotrophy of E. coli strain H712, indicating that active IMPDHs were produced, and both enzymes were purified to >95% homogeneity as described under "Experimental Procedures" (Supplemental Fig. S2).

The wild-type enzyme displayed strong NAD⁺ substrate inhibition, which is generally attributed to formation of a nonproductive E-XMP*·NAD complex. Initial velocity data collected at saturating IMP concentrations suggested that the K_m value for NAD⁺ is greater than the K_ii value, which complicates determination of steady-state parameters. This observation was confirmed when both substrates were varied (Figs. 2 and 3). Therefore, the k_cat and K_m values should not be considered well determined. Nevertheless, it is apparent that the kinetic parameters for C. albicans IMPDH are significantly different from those for human IMPDH, which is promising for the development of specific inhibitors. The parameters for human IMPDH type II are shown in Table II for reference; the parameters for human IMPDH type I are very similar to those for human IMPDH type II (25, 26).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Steady-state kinetics of C. albicans IMPDH: velocity versus IMP. A, wild-type IMPDH; B, A251T IMPDH. All assays contained 50 mM Tris-Cl (pH 8.0), 100 mM KCl, 1 mM dithiothreitol, and 3 mM EDTA at 25 °C. For the wild-type enzyme, the velocity versus IMP data were fit to the Michaelis-Menten equation (Equation 1) at each NAD concentration. Surprisingly, the A251T enzyme exhibited IMP substrate inhibition. The data were fit to the equation describing uncompetitive substrate inhibition (Equation 2).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Steady-state kinetics of C. albicans IMPDH: Vmax versus NAD. A, wild-type IMPDH; B, A251T IMPDH. IMP concentrations were varied in the presence of fixed NAD concentrations. For the wild-type enzyme, the Vmax values determined in Fig. 2 were plotted against the NAD concentration and fit to the equation describing uncompetitive substrate inhibition (Equation 2) to determine the values of Km for NAD and Vmax at saturating substrate concentrations. For the A251T enzyme, the Vmax values versus NAD data could be fit to the Michaelis-Menten equation (Equation 1) to determine the values of Km for NAD and Vmax at saturating substrate concentrations.

Inhibition of C. albicans IMPDHs—We determined the sensitivity of both C. albicans enzymes to well known IMPDH inhibitors. MPA is an uncompetitive inhibitor of IMPDHs from other sources (27); it binds selectively to the E·XMP* intermediate in the nicotinamide end of the dinucleotide site (after NADH departs) (Fig. 1) (18, 19). As expected, MPA is also an uncompetitive inhibitor of wild-type C. albicans IMPDH (Fig. 4 and Table III). The K_ii value is very similar to that of human IMPDH type II, which suggests that the drug-binding sites are also very similar in the fungal and human enzymes. MPA is also an uncompetitive inhibitor of A251T IMPDH. The K_ii value is 4-fold higher than that for the wild-type enzyme, comparable with the increase in MIC₅₀ observed in the growth of the MPA^R strains. These results confirm that drug resistance results from the substitution of Ala²⁵¹ with Thr.

View larger version (19K): [in this window] [in a new window]   FIG. 4. MPA inhibition of C. albicans IMPDH. A, wild-type IMPDH; B, A251T IMPDH. The Ki values for MPA inhibition were determined in experiments containing constant IMP concentrations (600 µM for the wild-type enzyme and 200 µM for the mutant enzyme) and varied NAD+ concentrations. Initial velocity data were best fit to the uncompetitive tight-binding inhibition equation (Equation 3).

In contrast to MPA and tiazofurin, ADP binds to the adenosine end of the dinucleotide site. As observed with other IMPDHs, ADP is a competitive inhibitor versus NAD⁺ of C. albicans IMPDH (27). The K_is value is 4-fold greater than that for the human enzyme. This difference probably reflects differences in the structure of the adenosine end of the dinucleotide site. The A251T mutation had a modest effect on ADP inhibition, decreasing the K_is value by a factor of 2.

We also investigated the MZP inhibition of the C. albicans enzymes. Like other IMPDHs, MZP is a slow tight-binding competitive inhibitor versus IMP of wild-type C. albicans IMPDH (Fig. 5 and Table III). MZP is a more potent inhibitor of the fungal enzyme, with the K_is value decreased by a factor of 4 relative to that for the human IMPDH (Table III) (26). Surprisingly, the A251T mutation increased the potency of MZP, with the K_is value decreased by a factor of 40 relative to that for the wild-type enzyme (Fig. 5 and Table III). In Tritrichomonas foetus IMPDH, MZP induces the closed conformation, where the flap has folded into the dinucleotide site (15). Therefore, one possible explanation for the increase in MZP affinity is that the equilibrium between the open and closed conformations has shifted to favor the closed conformation. Because the flap competes with MPA, such a shift in the conformational equilibrium could also explain MPA resistance.

View larger version (26K): [in this window] [in a new window]   FIG. 5. MZP inhibition of C. albicans IMPDH. A, wild-type IMPDH; B, A251T IMPDH. Conditions were as follow: IMP at 600 µM for the wild-type enzyme and 200 µM for the mutant enzyme, NAD+ at 250 µM for the wild-type enzyme and 4000 µM for the mutant enzyme, and 5.5 nM wild-type enzyme and 2.4 nM mutant enzyme. The data were fit to a mechanism describing slow-binding competitive inhibition using Dynafit (22).

View larger version (18K): [in this window] [in a new window]   FIG. 6. Tiazofurin/ADP inhibition of C. albicans IMPDH. A, wild-type IMPDH; B, A251T IMPDH. Multiple inhibition experiments with tiazofurin and ADP were performed with constant concentrations of IMP (600 µM for the wild-type enzyme and 200 µM for the mutant enzyme) and NAD+ (250 µM for the wild-type enzyme and 4000 µM for the mutant enzyme). The data were fit to Equation 5 to determine the value of the interaction constant .

View larger version (17K): [in this window] [in a new window]   FIG. 7. Mizoribine sensitivity of C. albicans strains. Shown is the mizoribine susceptibility of C. albicans strains as determined by microdilution assays. MIC50 values for mizoribine (MZR) are shown for the heterozygous imh3/IMH3 strain Cl1M7 and two imh3/MPAR strains, MPAR1 and MPAR2. MIC50 determinations were carried out in two independent experiments in triplicate. Error bars are the positive S.D.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Resistance to MPA can arise from a change in the topology of the MPA-binding site (which is also the nicotinamide subsite of the NAD⁺-binding site) or from a shift in the conformational equilibrium to favor the closed conformation (Fig. 1). These two mechanisms are not mutually exclusive and can be difficult to distinguish. Nevertheless, we believe the A251T mutation acts primarily by shifting the equilibrium to the closed conformation. First, the mutation had similar effects on both MPA and tiazofurin inhibition, increasing the K_ii values by factors of 3–4. These inhibitors have very different structures (Fig. 1), so it seems unlikely that a structural change would have similar effects on their potency. In contrast, changes in the open/closed conformational equilibrium should have similar effects on all inhibitors that bind to the open conformation. Second, the affinity of MZP increased 40-fold; because MZP induces the closed conformation, this increase in affinity can also be explained by a shift in the equilibrium to favor the closed conformation. Finally, the interaction between tiazofurin and ADP was more synergistic in the A251T enzyme as measured by the 2-fold decrease in the interaction constant . This increase in synergy also indicates that the mutation favors the closed conformation (16).

Most importantly, these data are remarkably self-consistent. The 4-fold increase in the K_ii value for MPA indicates that the fraction of E_open decreases 4-fold. Similarly, the 40-fold decrease in the K_is value for MZP indicates that E_closed increases 40-fold. Because the total enzyme must always be 1, then E_open + E_closed = 0.25 E_open + 40 E_closed. For the wild-type enzyme, E_open 0.98 and E_closed 0.02; for the A251T enzyme, E_open 0.2 and E_closed 0.8. If one assumes that tiazofurin and ADP bind independently to the open conformation, then the value is also an estimate of E_open (16). Both of these assumptions have been verified in experiments with IMPDH from Tritrichomonas foetus (16). Although not well determined, the values are consistent with the above estimates ( = E_open = 0.7 and 0.3 for the wild-type and A251T enzymes, respectively).

Unfortunately, at present, the structure of C. albicans IMPDH has not been solved, and the sequence of the flap is too different from that of the T. foetus enzyme to allow reliable modeling of the closed conformation. Therefore, we can only speculate about how the A251T mutation stabilizes the closed conformation. Ala²⁵¹ corresponds to Gly²³⁷ in T. foetus IMPDH (see Supplemental Fig. S5 for alignment) (Fig. 8). Gly²³⁷ is on the same segment as Arg²⁴¹, which interacts with NAD in the open conformation and with the flap in the closed conformation. With the exception of the side chain of Arg²⁴¹, this segment has the same structure in both the open and closed conformations. It is possible that a substitution at Gly²³⁷ could propagate to Arg²⁴¹, changing the equilibrium between the open and closed conformations. In addition, Gly²³⁷ is within 5.0 Å of Asp²⁶¹, which also interacts with NAD⁺ in the open conformation and with the flap in the closed conformation. Position 237 can easily accommodate an Ala residue, but Thr creates a potential steric conflict with the -carbon of Asp²⁶¹ (Fig. 8). Perhaps the repositioning of Asp²⁶¹ in response to the Thr substitution alters the equilibrium between the open and closed conformations. Interestingly, the IMPDH encoded by IMD2 in Saccharomyces cerevisiae also harbors a Ser residue at the equivalent position (Ser²⁵³, S. cerevisiae numbering). The IMD2 gene is the only member of the IMPDH gene family in S. cerevisiae that confers resistance to MPA (note that IMD1 is a pseudogene) (32, 33). Like the wild-type IMH3 gene in C. albicans, the IMPDHs encoded by S. cerevisiae IMD3 and IMD4 contain Ala at this position. Whether Ser²⁵³ in S. cerevisiae Imd2p is indeed responsible for the MPA resistance of this enzyme remains to be elucidated.

View larger version (34K): [in this window] [in a new window]   FIG. 8. Structural consequences of the A251T substitution. Ala251 corresponds to Gly237 in T. foetus IMPDH. This residue is near the NAD site and would therefore influence the equilibrium between the open and closed conformations. The open conformation is shown in orange, and -methylene thiazole-4-carboxamide adenine dinucleotide (TAD) is shown in blue (from the E·IMP·-methylene thiazole-4-carboxamide adenine dinucleotide complex of T. foetus IMPDH; Protein Data Bank code 1LRT [PDB] ). The closed conformation is shown in green (from the E·MZP complex of T. foetus IMPDH; Protein Data Bank code 1PVN [PDB] ). A Thr substitution at position 237 is shown in red.

More antifungal drugs are urgently needed, and our results suggest that IMPDH may be a new antifungal target. C. albicans is highly sensitive to IMPDH inhibitors, which suggests that guanine nucleotides are primarily supplied by de novo biosynthetic pathways. Although MPA resistance can be overcome in vitro by guanine supplementation, guanine levels in blood are too low to support growth, and C. albicans lacks the ability to salvage xanthine.³ Although MZP is a more potent inhibitor of C. albicans IMPDH compared with the human enzyme, it is not sufficiently selective to be useful in chemotherapy. Nevertheless, MZP (as well as ADP) demonstrates that selective inhibition is possible. Furthermore, alignment of the human and C. albicans sequences indicates that the adenosine subsites are very different. It may be possible to exploit the interactions of the adenosine subsite to develop fungus-specific IMPDH inhibitors.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grants GM54403 (to L. H.) and R01 AI33317 (to N. A.) and by Bundesministerium für Bildung und Forschung Grant O1 K18906-0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY864854 [GenBank] .

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S5.

^¶ Recipient of a Stipendienprogramm Infektionsforschung grant from the Bundesministerium für Bildung und Forschung.

To whom correspondence should be addressed: Dept. of Biochemistry, Brandeis University, MS 009, 415 South St., Waltham, MA 02454-9110. Tel.: 781-736-2333; Fax: 781-736-2349; E-mail: hedstrom{at}brandeis.edu.

¹ The abbreviations used are: MPA, mycophenolic acid; IMPDH, IMP dehydrogenase; MZP, mizoribine monophosphate; MIC₅₀, minimum concentration needed to inhibit cell growth by 50%.

² S. Bentink and G. A. Köhler, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. William A. Fonzi for strain CAI4, Dr. Joachim Morschhäuser for pSFU1, and Dr. Jane Koehler and the members of her laboratory for providing blood agar plates.

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