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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.sub.m]= 0.8[+ or -]0.08[micro]M) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 [micro]M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with [K.sub.m] values of 79 and 960 [micro]M, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor ([K.sub.m]=1100[+ or -]140[micro]M; 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 [Mg.sup.2+], and activity was stimulated by potassium, as reflected by a decrease in the [K.sub.m] and an increase in [V.sub.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.

Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3121, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a serious health problem throughout the world, and new drugs are needed to help control this disease. We have identified several purine nucleoside analogs that exhibit selective activity against Mycobacterium tuberculosis. The lead compound in this series is 2-methyl-adenosine (methyl-Ado), which is active against proliferating and nonproliferating bacteria due to its ability to inhibit protein synthesis. Methyl-Ado is activated by adenosine kinase that is expressed in M. tuberculosis cells. The primary intracellular metabolite is 2-methyl-AMP, although some methyl-ATP was also produced in the cells. Adenosine kinase has been purified from M. tuberculosis cells and its biochemical activity has been characterized and compared to that of the human homolog. The gene for adenosine kinase has been determined to be Rv2202c, which had been putatively identified as a sugar kinase. Because very little is known about purine metabolism in M. tuberculosis, we have initiated studies to characterize the enzymes that are involved in salvage of purine nucleosides. We believe that enhanced knowledge of the characteristics of the enzymes involved in purine salvage in M. tuberculosis should aid in the rational design of more potent purine analogs that can selectively inhibit M. tuberculosis replication. Compounds in this class should be active against strains of M. tuberculosis that are resistant to current agents used to treat this disease and may also target latent disease.