Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes

UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidul ans, is a member of a large family of nucleobase-ascorbate transporters con served in all domains of life. We have investigated structure-function rela tionships in UapA, by studying chimeric transporters and missense mutations , and showed that specific polar or charged an-Lino acid residues (E412, E4 14, Q449, N450, T457) on either side of an amphipathic alpha -helical trans membrane segment (TMS10) are critical for purine binding and transport. Her e, the mutant Q449E, having no uric acid-xanthine transport activity at 25 degreesC, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine . All seven revertants were found to carry the mutation F569S within the la st transmembrane segment (TMS14) of UapA. Further kinetic analysis of a sel ected suppressor showed that UapA-Q449E/F569S transports with high affinity (K-M values of 4-10 muM) xanthine, hypoxanthine and uracil. Uptake competi tion experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thio guanine, adenosine or ascorbic acid. A strain carrying mutation F569S by it self conserves high-capacity, high-affinity (K-M values of 1.5-15 muM), tra nsport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compo unds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10 -L10) are critical for the uptake specificity and transport kinetics of Uap A. (C) 2001 Academic Press.