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Identification and Characterization of a Unique Adenosine Kinase from Mycobacterium tuberculosis

Southern Research Institute, Birmingham, Alabama 35205,¹ Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294²

Received 7 May 2003/ Accepted 15 August 2003

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (K_m = 0.8 ± 0.08 µM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 µM. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with K_m values of 79 and 960 µM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (K_m = 1100 ± 140 µM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg²⁺, and activity was stimulated by potassium, as reflected by a decrease in the K_m and an increase in V_max for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.

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