title: Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii.
abstract: The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K(m)=0.91+/-0.19 microM) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K(m) values of 105 and 134 microM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine (K(m) values 0.28-1.5 microM) as well as cytidine (K(i)=32 microM). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3' and 5' hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably pi-pi-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.
authors.affiliation: Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, UK. h.de-koning@bio.gla.ac.uk
authors.forename: Harry P
authors.initials: HP
authors.lastname: De Koning
authors.forename: Mohammed I
authors.initials: MI
authors.lastname: Al-Salabi
authors.forename: Adrian M
authors.initials: AM
authors.lastname: Cohen
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Wastling
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D006790:Host-Parasite Interactions; D019271:Hypoxanthine / Q000378:metabolism; D007288:Inosine / Q000378:metabolism; D033702:Nucleobase Transport Proteins / Q000378:metabolism*; D033703:Nucleoside Transport Proteins / Q000378:metabolism*; D009705:Nucleosides / Q000378:metabolism; D011684:Purine Nucleosides / Q000378:metabolism; D013379:Substrate Specificity; D014122:Toxoplasma / Q000378:metabolism* / Q000502:physiology
chemical_list: D033702:Nucleobase Transport Proteins; D033703:Nucleoside Transport Proteins; D009705:Nucleosides; D011684:Purine Nucleosides; D019271:Hypoxanthine; D007288:Inosine; D000241:Adenosine