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J. Biol. Chem., Vol. 283, Issue 9, 5380-5388, February 29, 2008

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Adenosine Kinase Mediates High Affinity Adenosine Salvage in Trypanosoma brucei^*

Munender Vodnala, Artur Fijolek, Reza Rofougaran, Marc Mosimann, Pascal Mäser, and Anders Hofer¹

From the Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden and the Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavage-dependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K_m = 0.04–0.08 µM depending on [phosphate]) and by being negatively regulated by adenosine (K_i = 8–14 µM). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.

Received for publication, July 9, 2007 , and in revised form, December 20, 2007.

^* This work was supported by grants from the Swedish Research Council, the Magnus Bergvall's Foundation, the Åke Wiberg's Foundation, the Kempe's Foundation, and the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 46-90-7867840; Fax: 46-90-7869795; E-mail: anders.hofer{at}medchem.umu.se.

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