Allopurinol and xanthine use different translocation mechanisms and trajectories in the fungal UapA transporter

In Aspergillus nidulans UapA is a H⁺-driven transporter specific for xanthine, uric acid and several analogues. Here, genetic and physiological evidence is provided showing that allopurinol is a high-affinity, low-capacity, substrate for UapA. Surprisingly however, transport kinetic measurements showed that, uniquely among all recognized UapA substrates, allopurinol is transported by apparent facilitated diffusion and exhibits a paradoxical effect on the transport of physiological substrates. Specifically, excess xanthine or other UapA substrates inhibit allopurinol uptake, as expected, but the presence of excess allopurinol results in a concentration-dependent enhancement of xanthine binding and transport. Flexible docking approaches failed to detect allopurinol binding in the major UapA substrate binding site, which was recently identified by mutational analysis and substrate docking using all other UapA substrates. These results and genetic evidence suggest that the allopurinol translocation pathway is distinct from, but probably overlapping with, that of physiological UapA substrates. Furthermore, although the stimulating effect of allopurinol on xanthine transport could, in principle, be rationalized by a cryptic allopurinol-specific allosteric site, evidence was obtained supporting that accelerated influx of xanthine is triggered through exchange with cytoplasmically accumulated allopurinol. Our results are in line with recently accumulating evidence revealing atypical and complex mechanisms underlying transport systems.