In fungi, uptake of salvageable purines is carried out by members of two 
evolutionarily distinct protein families, the Purine-Related Transporters 
(PRT/NCS1) and the AzgA-like Transporters. We carried out a comparative kinetic 
analysis of two prototypes of these transporter families. The first was Fcy21p, 
a herein characterized protein of Candida albicans, and the second was AzgA, a 
transporter of Aspergillus nidulans. Our results showed that: (i) AzgA and 
Fcy21p are equally efficient high-affinity, high-capacity, purine transporters, 
(ii) Fcy21p, but not AzgA, is an efficient cytosine and 5-fluorocytosine 
transporter, interacting with =O2 and C4-NH2 of the pyrimidine ring, (iii) the 
major interactions of AzgA and Fcy21p with the purine ring are similar, but not 
identical, involving in all cases positions 6 and 7, and for some substrates, 
positions 1 and 9 as well, and (iv) in AzgA, bulky groups at position N3 have a 
detrimental steric effect on substrate binding, while similar substitutions at 
C2 or N9 are fully or partially tolerated. In contrast, in Fcy21p, C2 and N9 
bulky substitutions abolish substrate binding, while similar substitutions in N3 
are fully tolerated. These results suggest that all fungal purine transporters 
might have evolved from a single ancestral protein, and show that fungal 
transporters use different substrate interactions compared to the analogous 
protozoan or mammalian proteins. Finally, results are also discussed in respect 
of the possibility of using fungal purine transporters as specific gateways for 
the development of targeted antifungal pharmacological therapies.