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

Figures & data

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

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%).

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.

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]).

Papageorgiou IG, Yakob L, Al Salabi MI, Diallinas G, Soteriadou KP, de Koning HP. Identification of the first pyrimidine nucleobase transporter in Leishmania: similarities with the Trypanosoma brucei U1 transporter and antileishmanial activity of uracil analogues. Parasitology 2005; 130: 275–283

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