Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans

Abstract

Early genetic evidence suggested that A. nidulans possesses at least one uracil transporter. A gene, named furD, was recently identified by reverse genetics and in silico approaches and we confirm here that it encodes a high-affinity, high-capacity, uracil transporter. In this work, we study the regulation of expression of FurD and develop a kinetic model describing transporter-substrate interactions. The furD gene is not expressed in resting conidiospores, is transcriptionally activated and reaches a peak during the isotropic growth phase of conidiospore germination, and stays at a basic low level in mycelium. Transcriptional expression is correlated to uracil transport activity. Expression in a strain blocked in uracil biosynthesis (pyrG^-) is moderately increased and extended to later stages of germination. The presence of excess uracil in the medium leads to down-regulation of furD expression and FurD activity. A detailed kinetic analysis using a number of pyrimidine and purine analogues showed that FurD is able to recognize with high-affinity uracil (K_m 0.45 µM), thymine (K_i 3.3 µM) and several 5-substituted analogues of uracil, and with moderate affinity uric acid and xanthine (K_i 94–99 µM). Kinetic evidence supports a model in which the positions N1-H, =O2, N3-H, =O4, as well as planarity play a central role for the substrate binding. This model, which rationalizes the unique specificity of FurD for uracil, is compared to and found to be very similar to analogous models for protozoan uracil transporters.

• 5-fluorouracil
• permease
• specificity
• pyrimidine
• analogues

Introduction

Uracil transport is a ubiquitous cellular activity present in free-living prokaryotes and eukaryotes and also in differentiated cells of multicellular organisms. Genetic and biochemical evidence for transporter-mediated, specific uracil transport came from studies in bacteria and fungi nearly three decades ago (De Koning & Diallinas, [2000]). In most cases, uracil transport mutants have been identified as mutants resistant to the highly cytotoxic analogue 5-fluorouracil (Palmer et al. [1975], Jund et al. [1977], Andersen et al. [1995]). Such mutants led to the identification and cloning of the first genes encoding uracil permeases in Saccharomyces cerevisiae (fur4; Chevallier [1982]) and Escherichia coli (uraA; Andersen et al. [1995]). Fur4p and UraA belong to two protein families (De Koning & Diallinas, [2000]; http://www.tcdb.org/). Fur4p is the prototype of the Nucleobase/Cation Symporter 1 family (NCS1), conserved in prokaryotes and fungi, while UraA is a member of the nearly ubiquitous Nucleobase-Ascorbate Transporter (NAT) family, also known as NCS2. The NCS1 family consists of over two hundred currently sequenced proteins derived from Gram-negative and Gram-positive bacteria, archaea, yeast, fungi and plants (http://www.tcdb.org/). Among recognized substrates of NCS1 are purine cytosine uracil, allantoin, uridine or thiamine, and at least some NCS1 members function as substrate: H⁺ symporters. The NAT/NCS2 members are subdivided into three groups specific for uracil (bacteria), uric acid/xanthine (bacteria, fungi and plants) or ascorbate/Na⁺ (mammals) (De Koning & Diallinas, [2000]). The two families are probably distantly related. A third, plant-specific and evolutionary distinct allantoin transporter family, known as UPS, includes members which may also transport uracil and 5-fluorouracil (Schmidt et al. [2004]). Highly specific uracil transporters have been kinetically characterized in Trypanosoma brucei and Leishmania major (De Koning & Jarvis [1998], Papageorgiou et al. [2005], De Koning et al. [2005]) and in several mammalian tissues (Kraupp & Marz [1995], De Koning & Diallinas [2000], Cabrita et al. [2002]), but the corresponding genes remain unknown.

The S. cerevisie Fur4p uracil permease has become a paradigm of transporter regulation at a post-translational level and in particular at the level of protein trafficking and endocytosis. Work from Haguenauer-Tsapis and her colleagues has shown that the availability of uracil causes a decrease in the level of uracil permease by a double-lock mechanism, involving a moderate decrease of the FUR4 mRNA stability and a very rapid and efficient degradation of the existing permease (Galan et al. [1994], Seron et al. [1999]). Fur4p degradation involves phosphorylation in a PEST-like sequence located within the cytoplasmic N terminus, followed by efficient Rsp5p-dependent ubiquitination through lys63 that signals endocytosis and further vacuolar degradation (Marchal et al. [1998], Galan & Haguenauer-Tsapis [1997], Marchal et al. [2000]). Direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes (Seron et al. [1999]).

Fur4p-like uracil transporters have also been identified in Schizosaccharomyces pombe (de Montigny et al. [1998]) and, recently, we have deleted and characterized all the seven putative transporters belonging to this family in Aspergillus nidulans (Z. Hamari, S. Amillis, C. Drevet, A. Apostolaki, G. Diallinas and C. Scazzocchio, unpublished work). In this work we study the regulation of expression of a Fur4p-similar A. nidulans uracil transporter, called FurD, during germination and in response to uracil availability, and describe a model on how this transporter binds uracil. This model rationalizes the extreme specificity and affinity of FurD for uracil. Finally, FurD-uracil interactions are also compared to similar substrate interaction models of protozoan uracil transporters.

Materials and methods

Strains and media

Standard complete and minimal media for A. nidulans were used (Cove [1966]; http://www.fgsc.net/). In minimal media the sole nitrogen source, unless otherwise stated, was urea. Chemicals and most purine and pyrimidine analogues were purchased from Sigma (St. Louis, MO). Several pyrimidine analogues were gifts from Dr H de Koning. The A. nidulans strains used in uptake studies had the following genotype: ΔfurD::riboB yA2 riboB2, riboB2::riboB yA2, ΔfurD::riboB pyrG89 pyroA4 biA1 riboB2, pyrG89 pantoB100 pabaA1. ΔfurD::riboB is a total deletion of the furD open reading frame using the Double-Joint PCR approach (Yu et al. [2004]) and the riboB selection marker. riboB2::riboB is a homologous integration of the wild-type riboB gene in the endogenous riboB2 allele. pyrG89, pyroA4, riboB2, pantoB100, biA1 are uracil, pyridoxine, riboflavin, pantothenate and biotin auxotrophic mutations respectively, while yA2 is a colour mutation. Except pyrG89, these mutations do not affect the expression or function of FurD.

Northern blot analysis and standard nucleic acid manipulations

Northern blot analysis was performed using the glyoxal method (Sambrook et al. [1989]). Total RNA was isolated from resting conidiospores, germlings or mycelia by a standard protocol using the TRIzol Reagent of Invitrogen, (CA, USA), after initial manual breakage of Aspergillus material in dry ice (Tazebay et al., [1997]). In northerns, equal RNA loading was estimated by optical density measurements (260/280 nm) and controlled by estimating the amount of rRNA, as described in Sambrook et al. [1989]. Relative transcript levels were estimated by calculating the ratio of furD to rRNA messages by densitometry. [³²P]-dCTP labelled DNA molecules used as furD or rRNA specific probes were prepared using a random hexanucleotide-primer kit following the supplier's instructions (Promega).

Radiolabelled pyrimidine uptake measurements

[³H]-uracil uptake was assayed in germinating conidiospores at 37°C (Amillis et al. [2004]; Cecchetto et al. [2004]). Background uptake was calculated in the presence of 1000-fold excess of non-radiolabelled substrate and subtracted before estimation of kinetic parameters. The H⁺-uncoupler carbonylcyanide chlorophenylhydrazone (CCCP) and the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD) were added at final concentrations of 30 µM and 100 µM respectively, for 10 min before initiating the uptake assay. Cross competition assays were performed in the simultaneous presence of [³H]-uracil (0.2 µM) and various concentrations of non-radiolabelled putative competitors that had been pre-equilibrated in the assay mixture for 5 min. All transport assays were carried out in at least three independent experiments, with three replicates for each concentration or time point. Standard deviation was < 20%. Radiolabelled [³H]-5,6-uracil (52 Ci/mmol) was purchased from Amersham Biosciences.

Kinetic analysis

Data were fitted to the appropriate equations with the use of the Prism 3 software package (http://www.graphpad.com/) to obtain K_m, K_i and IC₅₀ values. IC₅₀ values for compounds inhibiting the uptake of radiolabelled permeates were determined from full dose-response curves with a minimum of eight points spread over the relevant range. K_i values were calculated from the Cheng & Prusoff ([1973]) equation K_i=IC₅₀/[1+(L/K_m)], in which L is the permeant concentration. It should be noted that the K_i value is an affinity constant implying binding to the transporter but does not indicate whether the ligand is being transported across the membrane. Free Gibbs energy ΔG° was calculated from ΔG°= − RTln(K_i), where R is the ideal gas constant and T the absolute temperature (°K).

Table I.  Substrate-binding specificity profile of FurD. Results are averages of at least three independent experiments with three replicates for each concentration point. Standard deviation was < 20%.

Substrates	Ki ( µM)	ΔG° (kJ/mol)
Uracil	0.45	−37.7
Xanthine	94.0	−23.9
Hypoxanthine	>1500	> − 16.8
Uric acid	99.0	−23.8
Guanine	>1500	> − 16.8
Adenine	>1500	> − 16.8
Thymine	3.3	−32.6
Cytosine	>1500	> − 16.8
1-methyl-uracil	3.7	−32.2
3-methyl-uracil	53.2	−25.4
2-thio-uracil	8.4	−30.1
4(3H)-pyrimidone	175.0	−22.3
4-thio-uracil	13.8	−28.9
5-fluoro-uracil	0.46	−37.6
5-chloro-uracil	0.45	−37.7
5-amino-uracil	0.86	−36.0
6-aza-uracil	1.9	−34.0
6-methyl-uracil	15.2	−28.6
Isocytosine	127	−22.2
5,6-dimethyl-uracil	12.0	−29.2
4,6-dihydroxy-pyrimidine	1354	17.0
5,6-dihydrouracil	19.0	−28.0
Glutarimide	1077	−17.8
Uridine	>1500	> − 16.8
Thymidine	>1500	> − 16.8

Results and discussion

FurD is a high-affinity, high-capacity, uracil/H⁺ symporter

Early genetic screens for 5-fluorouracil resistant mutants of A. nidulans suggested the existence of at least one major uracil specific transporter, corresponding to the product of the fulF gene (Palmer et al. [1975]). However this gene was never cloned and no uptake studies have confirmed this suggestion. fulF mutants are no longer available.

In the annotated sequence of the A. nidulans genome there are 6 genes similar to the Fur4p uracil transporter of S. cerevisiae (http://www.membranetransport.org/). However, we detected an extra gene, ‘split’ at the edges of two contigs (Contigs 1.158 + 1.209). Thus, the seven fur-like genes are: AN0660.2 (FurA), AN4152.2 (FurB), AN3352.2 (FurC), [sequence 6103-6752 from contig 1.209 + sequence 283357 to 281825 from contig 1.158] (FurD), AN8416.2 (FurE), AN9326.2 (FurF), AN7955.2 (FurG). These seven fur-like genes were systematically knocked-out by the Double-Joint PCR method (Yu et al. [2004]) and analyzed by growth tests and uptake assays (not shown). Only the furD deletion showed a phenotype compatible with lack of uracil transport (full resistance to 5-fluorouracil and lack of radiolabelled uracil uptake). The detailed description of furD cloning and knock-out will be published in a more specialized fungal journal, as part of a systematic work to characterize A. nidulans nucleobase/nucleoside transporters.

Here, we have characterized kinetically FurD-mediated uracil transport. Uptake studies were performed at 37°C, using conidiospores geminated in the absence of uracil from the medium, at a stage prior to germ tube emergence. At this stage, a number of A. nidulans transporters studied are significantly expressed (Tazebay et al. [1997], Amillis et al. [2004]). Figure 1A displays the time course of uracil uptake in furD⁺ and ΔfurD strains, showing that the uptake rate for uracil in germinating condiospores is exclusively FurD-mediated. Uptake reaches a steady-state at 2 min and is linear for at least 90 sec. This chronological profile is typical of all A. nidulans transporters studied up to now and very similar to most transporters from S. cerevisiae. Kinetic analyses of FurD performed at 30 sec and 1 min have given identical K_m and V_m values. Results from 1 min assays were used for subsequent work due to higher V values and reduced standard errors.

Figure 1.  Biochemical identification of FurD. (A) Time course of uptake of 0.2 µM [³H]-uracil in a wild-type (wt) and a ΔfurD strain. (B) FurD-mediated [³H]-uracil transport kinetics. (C) Energetic and pH dependence of uracil uptake. For details see text and Materials and methods. Results represent mean values from three independent determinations with bars of standard deviations shown (<15%).

Uracil uptake showed hyperbolic kinetics in relation to substrate concentration, as expected from the kinetic characteristics of a single transporter (Figure 1B). The apparent K_m and V_m for uracil were calculated to be 0.45±0.3 µM and 0.5 pmoles/min 10⁷ conidiospores, respectively. Although the K_m characterizes this transporter, V_m depends on the absolute quantity of transporter inserted into the membrane. The latter depends on growth conditions but is also under developmental control, as shown later. Thus this parameter is contingent to the exact conditions of the experiment. Inhibition experiments with the H⁺-uncoupler carbonylcyanide chlorophenylhydrazone (CCCP) and the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD) suggest that, as most fungal transporters, FurD functions as H⁺ symporter (Figure 1C). This is further supported by a pH effect on FurD activity. Maximal FurD activity was obtained at apH range between the 6.8 and 10 (Figure 1C).

FurD should not be the only transporter taking-up uracil in A. nidulans. ΔfurD pyrG^- strains are rescued by high concentrations of uracil added in the growth medium. The alternative explanation, that simple diffusion of uracil is the basis of this observation is probably not valid, as the very low level of radiolabelled uracil uptake in a ΔfurD strains is completely inhibited by excess ‘cold’ uracil (results not shown). Thus, apart from FurD, there must be at least one ‘cryptic’, very low-affinity, uracil transporter. Such very low-affinity, low-capacity, transporters cannot be analyzed kinetically due to technical reasons associated with the radiolabelled purines/pyrimidines available commercially. One possibility is that the ‘missing’ transporter might be the recently identified general nucleoside transporter, CntA, as is the case in S. cerevisiae, where the uridine transporter Fui1p can also transport, albeit very inefficiently, uracil (Zhang et al. [2006]).

FurD activity appears early during germination and responds to uracil pools

FurD-mediated uracil transport activity was measured by direct uptake experiments using radiobelled uracil. Uptake experiments were performed in furD⁺ and ΔfurD strains, at various stages during germination, at 37°C. Germination media contained no uracil. Results are shown in Figure 2A. In the furD⁺ strain, resting and 1 h germinating conidiospores showed no uracil uptake. FurD-mediated uracil uptake was first detected after 2 and dramatically increased to a maximum after 3 h of germination. After this peak, uracil uptake quickly dropped to a basal level (4–5 h) and approached a steady-state in young mycelium (6–8 h). No significant uracil uptake was detected at any stage in the ΔfurD strain. A similar uracil uptake profile was obtained with several A. nidulans strains (not shown).

Figure 2.  Expression of purine transporters during conidiospore germination. FurD activity was measured by estimating initial uptake rates of [³H]-uracil in a wt and a pyrG89 strain. (A) [³H]-uracil uptake rates in resting (0 h) and germinating (1–7 h) conidiospores in MM supplemented with urea as sole N source. (B) [³H]-uracil uptake rates in resting (0 h) and germinating (1–7 h) conidiospores in MM supplemented with urea as sole N source and in the presence of uracil. Uptake measurements represent the averages of at least three independent experiments with standard deviations of <20%. For technical details, see Materials and methods. (C) Northern blot analyses of furD mRNA steady-state levels from resting and germinating (0–6 h) conidiospores of a wt and a pyrG89 strain grown in MM supplemented with urea as sole N source and in the presence of uracil. Loading of RNA was estimated by hybridization with an rRNA-specific probe.

We performed the same analysis in the presence of 100 µM and 1 mM concentrations of uracil in the germination medium. Radiolabelled uracil uptake was measured after thorough washing of samples to exclude the possibility that excess ‘cold’ uracil remains in the uptake assays. Results (shown in Figure 2B) showed that the presence of excess uracil in the growth medium down-regulates FurD-mediated uracil transport. In order to further investigate the response of the FurD regulation to uracil or UMP cellular pools, we performed the same uptake analysis using furD⁺pyrG^−- and ▵furD pyrG^−- strains, geminating in the absence of exogenous uracil (shown in Figure 2A). pyrG^−- (orotidine-5'-phosphate decarboxylase) mutants are blocked in UMP biosynthesis (Palmer et al. [1975], Oakley et al. [1987]) and can only grow when exogenous uracil is supplied at concentrations > 2.5 mM. No uracil uptake was detected throughout germination and mycelium development in the ΔfurD pyrG^−- strain. The furD⁺pyrG^−- mutant showed increased and prolonged FurD activity, showing significantly higher uracil uptake in mycelium than a ΔfurD pyrG⁺ strain.

Expression of FurD activity reflects de novo transcription and a post-transcriptional effect

We examined whether regulation of FurD activity directly reflects de novo transcription of the furD gene. furD mRNA steady state levels were detected by Northern blot analysis using total RNA isolated from furD⁺ or furD⁺pyrG^−- strains, as previously described for the uptake experiments. Results are summarized in Figure 2C. In both furD⁺ and furD⁺pyrG⁻ strains, de novo transcription of furD mRNA was evident. The timing of mRNA steady state level appearance is practically identical to that of uracil transport activities. In a furD⁺ wild-type strain, preloading with excess uracil down-regulates furD mRNA steady state levels. This is evident after 4 h of germination (2-fold reduction as estimated by densitometry, not shown) but mostly after 5 h of germination (4.5-fold reduction). In contrast, in a furD⁺pyrG^−- strain, which should suffer from depleted uracil pools, furD mRNA basal levels in mycelia (6 h) are 2.4-higher than in a furD⁺ wild-type strain. Compared to FurD transporter activity (Figure 2A), changes in mRNA steady levels, despite following a similar pattern depending on uracil pools, are less prominent. These results suggest that FurD transport activity reflects both de novo transcription and post-transcriptional regulation.

The early transcriptional activation during the isotropic growth phase of germination is common in most A. nidulans transporters examined so far. The kinetics of mRNA accumulation are also typical of transporter genes. In that sense, furD expression does not differ from that of other transporters involved in nitrogen or carbon source accumulation. Uracil cannot be catabolized in A. nidulans. The presence of exogenous excess uracil or alteration in cellular pools due to block in uracil biosynthesis affected moderately furD mRNA levels. Accumulation of uracil down-regulates furD mRNA levels, while depleted pools have the opposite effect. However, these moderate alterations in mRNA steady state levels cannot be the only mechanism accounting for the more prominent changes in FurD activity observed. In S. cerevisiae, uracil availability causes both a decrease in FUR4 mRNA stability and a very rapid degradation of the existing permease (Galan et al. [1996], Seron et al. [1999]). We predict that in A. nidulans, FurD might also be degraded in response to excess uracil, through ubiquitination and routing to the vacuole (see introduction). In fact, in A. nidulans, a similar vacuolar degradation behavior has been shown for purine and amino acid transporters in response to the presence of ammonium (Valdez-Taubas et al. [2004]; Pantazopoulou et al. [2006]). It seems that fungi impose this rapid vacuolar degradation mechanism for most transmembrane proteins either as a negative feedback mechanism (e.g., excess substrate) or when a sudden change in physiological conditions (e.g., a preferred metabolite) obligates a change in the metabolic needs of the cell. The extremely high-affinity for uracil and the effect of uracil pools on its expression, suggest that FurD, similar to Fur4p in yeast, serves a scavenging role as a back-up to uracil biosynthesis.

Specificity of FurD and modelling of substrate recognition

FurD-mediated transport of [³H]-uracil was assessed in the presence of pyrimidines, purines and analogues as described in Materials and methods. Competition of radiolabelled uracil uptake by nucleobases and analogues was competitive and thus the criteria for using of the Cheng & Prusoff equation to determine K_i value were met (see Materials and methods). By calculating the Gibbs free energy involved in the interactions between the transporter and substrates (see Materials and methods), a model for permeant binding could be constructed (Table I), as we have previously described for other bacterial, fungal and protozoan transporters (Papageorgiou et al. [2005], Goudela et al. [2005], Goudela et al. [2006]).

FurD had virtually no affinity for cytosine, purines or pyrimidine nucleosides, displaying a high degree of specificity for uracil transport. The affinity for uracil (K_m 0.45 µM) is at least 15–30 fold higher than that exhibited by any other A. nidulans high-affinity transporter studied, such as those specific for purines, amino acids, nitrate or ammonium. Interestingly, despite the fact that thymine is not taken-up by A. nidulans (Z. Hamari, S. Amillis, C. Drevet, A. Apostolaki, G. Diallinas and C. Scazzocchio, unpublished work), FurD was shown to have a relatively high-affinity for thymine binding (K_i 3.3 µM). To our knowledge, thymine recognition by Fur4p or UraA has not been shown. FurD also recognizes with moderate affinities (∼100 µM) uric acid and xanthine, two purines which possess pyrimidine rings identical to uracil. Thus, FurD might be responsible for the very leaky growth observed when ΔuapA ΔuapC strains, lacking the two established uric acid/xanthine transporters, grow on these purines as sole nitrogen sources. This assumption is further supported by the observation that ΔuapA ΔuapC leaky growth on uric acid or xanthine is reduced when excess uracil is included in the medium (G. Diallinas, unpublished). A ΔfurA ΔuapA ΔuapC strain would clarify these issues.

FurD also recognizes with relatively high-affinity several uracil analogues. Analogues with substitutions at position 5 (5-fluorouracil, 5-chlorouracil, 5-aminouracil) displayed a very high affinity for FurD, similar to that of uracil. 5-fluorouracil recognition and transport by uracil carriers has also been observed in bacteria, yeast, plants and mammals (De Koning & Diallinas, [2000]). Thymine (5-methyluracil) had also a relatively high affinity, albeit 8-fold reduced compared to uracil (δ(ΔG°) 5 kJ/mol). This suggests that the H at position 5 is not involved in interactions with FurD, and that bulky replacements of this H atom have moderate steric effects on binding. Non-bulky substitution at position 6 (6-azauracil) similarly led to minor reduction (3.7 kJ/mol) of FurD binding affinity, suggesting that the higher energy loss in the binding of 6-methyluracil (9.1 kJ/mol) should be due to steric hindrance.

2-Thiouracil displayed a 19-fold reduced affinity for FurD, compared to uracil. This indicates that FurD forms a hydrogen bond of 7.6 kJ/mol with the keto group at position 2, as the predominant form of 2-thiouracil is as thione (=S) rather than as thiol (-SH) (Brown [1994]) and the thione group is much less capable of forming an H-bond than the keto group (Donohue [1969]). If the thione group does engage in any H-bonding with the FurD binding pocket, this would lead to an underestimation of the ΔG° for the keto groups. However, the energy of any S2 or S4 H-bond would be expected to be an order of magnitude less than from the corresponding keto group (Pitha & Scheit [1975]). The keto group at position 4 was likewise involved in the substrate binding of FurD, most probably as a hydrogen bond acceptor. This follows directly from the loss in Gibbs free energy, δ(ΔG°), between 4-thiouracil and uracil (8.8 kJ/mol), indicating a similar apparent bond with = O2. The non-detectable affinity for cytosine strongly suggests involvement of N3-H in another strong hydrogen bond. Cytosine differs from uracil in position 4, where =O4 is replaced by an amino group, and in the state of protonation of N3. The δ(ΔG°) between cytosine and uracil is ∼20 kJ/mol. Considering that the loss from position 4 is 8.8 kJ/mol, the contribution of the H in N3 of uracil should be at least 11.2 kJ/mol. This prediction conforms nicely to the loss of binding energy comparing 3-methyluracil and uracil, which was calculated to be 12.3 kJ/mol. Thus, the estimated contributions from = O2,=O4 and N3 sum up to 7.6 + 8.8 + 12.3 = 28.7 kJ/mol.

One additional weak interaction between uracil and FurD appears to involve N1-H. This follows from the Σ(ΔG°) between 1-methyluracil and uracil which is 5.5. kJ/mol. If N1-H was not involved in any interaction, the δ(ΔG°) between 4-(3H)-pyrimidone and uracil would only be 7.6 kJ/mol, the loss from = O2. The observed δ(ΔG°) was however 15.4 kJ/mol. This suggests that N1-H in uracil contributes 7.8 kJ/mol, close to the 5.5 kJ/mol δ(ΔG°) estimated from comparing 1-methyluracil and uracil. This further confirms that the loss in 3-methyluracil was not simply due to steric hindrance. In addition, the δ(ΔG°) estimated from comparing isocytosine and uracil was 15.5 kJ/mol, a little higher from what should be expected (13.1 kJ/mol) for the loss of interactions from positions 1 and 2. Thus, including contribution from N1-H, the σ(ΔG°) comes to 28.7 + 5.5 = 34.2 kJ/mol. The observed ΔG° for uracil was 37.7 kJ/mol. Two assumptions can be made for the ‘missing’ 3.5 kJ/mol. This minor energetic difference is either the product of underestimation of contributions made by the thione groups when using 2- or 4-thiouracil to assign the energy from H bonds of positions 2 and 4, or derives from stacking of Π-orbitals of the pyrimidine ring with the respective orbitals of an aromatic residue in the FurD binding pocket. The analogue 5, 6-dihydrouracil, lacking a conjugated Π-system in the pyrimidine ring, displayed a 42-fold lower affinity than uracil and an estimated δ(ΔG°) of 9.7 kJ/mol for FurD. Because bulky substitutions at positions 5 and 6 had no or minor effects on binding affinities, energy loss in the interaction of 5, 6-dihydrouracil should not derive from steric effects. However, the loss of 9.7 kJ/mol exceeds significantly what was expected from all previous calculations concerning contributions of other positions. Another, much more probable assumption is that the loss of 9.7 kJ/mol derives from the loss of planarity, by 0.136 A°, in (Chem3D Ultra, CambridgeSoft) 5, 6-dihydrouracil. The importance of planarity conferred by a conjugated Π-system, is further established by the loss of 19.9 kJ/mol in glutarimide. This uracil analogue is non planar by 0.20 A° (Chem3D Ultra, CambridgeSoft). A model of FurD-uracil interactions is proposed below (Figure 3).

The interactions proposed for FurD binding of the pyrimidine ring explain the moderate affinities for xanthine and uric acid, and for other purines. The pyrimidine ring of xanthine and uric acid is identical to uracil, thus the loss of 13.8–13.9 kJ/mol should be attributed to steric effects caused by the presence of the imidazol ring. In contrast, adenine, hypoxanthine and guanine do not bind at all FurD probably due to both steric hindrance from the imidazol ring and lack of specific hydrogen donors and acceptors in the pyrimidine ring.

Conclusion

Using several pyrimidine analogues we modelled FurD-substrate interactions. We are aware however of the fact that most changes to the pyrimidine ring will affect size, bond angles, bond length, planarity, charge distribution in the Π-orbitals etc. (Brown [1994]), makes quantitative modelling of the transporter-substrate interactions speculative. The model presented in Figure 3 must therefore be regarded as such. According to the model proposed =O2, =O4, N1-H and N3-H contribute, via H bonds, in contacts with FurD. Furthermore, the aromaticity and planarity of the pyrimidine ring are necessary criteria for efficient binding, and thus contribute to the extremely high affinity and specificity with which FurD binds uracil. Comparing this model to analogous models for protozoan uracil transporters, we could detect impressive similarities, shown in Figure 3. Protozoan genomes do not include FurD homologues and thus these similarities might reflect a case of convergent evolution.

Figure 3.  Comparative speculative models for possible interactions of FurD or LmU1 with their substrates. ΔG°_obs is the ΔG° calculated directly from the K_i of the relevant substrate. Σ(ΔG°) is the sum of the differences in ΔG° values obtained from binary comparisons of the relevant substrate and selected analogues. Estimated apparent ΔG° values are in kJ/mol. R stands for an amino acid in the transporter interacting, most probably via H-bonds, with a specific purine position. Shaded areas depict positions of interactions with R. The LmU1 interactions were reproduced from Papageorgiou et al. ([2005]).

Modeling uracil recognition by protozoan or fungal transporters has an obvious medical importance. Uracil analogues are, or can be developed to be, powerful drugs against infections and cancer. 5-fluororuracil is commonly used as an anticancer agent (Kraupp & Marz [1995]). Kinetic analysis has already shown that protozoan and fungal purine transporters are sufficiently different from mammalian transporters in the selective uptake of therapeutic agents (de Koning et al. [2005]). Knowledge on the modes of uracil recognition by the host and target cell transporters should constitute a critical step in developing targeted pharmacological therapies.

This paper was first published online on iFirst on 10 January 2007.

Acknowledgements

We thank H. P. de Koning for providing several pyrimidine analogues and for his continuous constructive criticism on aspects related to modelling transporter-substrate interactions. Work in the laboratory of G.D. was supported by The University of Athens (ELKE). Work at Orsay by the CNRS, the Université Paris-Sud and Institut Universitaire de France. Z. H. was supported by Marie Curie ERG (MERG-CT-2005-517914).