adenosine-kinase and cordycepin

Adenosine kinase (

Topics: Adenosine Kinase; Deoxyadenosines; Mutation; S-Adenosylmethionine; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Cordycepin is an efficient component of Cordyceps spp, a traditional Chinese medicine widely used for healthcare in China, and has been recently acted as a strong anticancer agent for clinic. However, whether and how it may play a role in combating tuberculosis, caused by Mycobacterium tuberculosis, remains unknown. Here we report that cordycepin can kill Mycobacterium by hijacking the bacterial adenosine kinase (AdoK), a purine salvage enzyme responsible for the phosphorylation of adenosine (Ado) to adenosine monophosphate (AMP). We show that cordycepin is a poor AdoK substrate but it competitively inhibits the catalytic activity of AdoK for adenosine phosphorylation. Cordycepin does not affect the activity of the human adenosine kinase (hAdoK), whereas hAdoK phosphorylates cordycepin to produce a new monophosphate derivative. Co-use of cordycepin and deoxycoformycin, an inhibitor of adenosine deaminase (ADD), more efficiently kills M. bovis and M. tuberculosis. The add-deleted mycobacterium is more sensitive to cordycepin. This study characterized cordycepin as a new mycobactericidal compound and also uncovered a potential anti-mycobacterial mechanism.

Topics: Adenosine Kinase; Antitubercular Agents; Chromatography, High Pressure Liquid; Chromatography, Liquid; Deoxyadenosines; Dose-Response Relationship, Drug; Microbial Sensitivity Tests; Molecular Structure; Mutation; Mycobacterium tuberculosis; Polymorphism, Single Nucleotide; Tandem Mass Spectrometry

Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic- and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3'-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.

Topics: Adenosine; Adenosine Kinase; Animals; Blotting, Northern; Blotting, Southern; Blotting, Western; Deoxyadenosines; Fluorescent Antibody Technique; Gene Silencing; Genetic Complementation Test; Mutation; Phylogeny; Protozoan Proteins; RNA Interference; Saccharomyces cerevisiae; Trypanocidal Agents; Trypanosoma brucei brucei

The adenosine analogue cordycepin (3'-deoxyadenosine) inhibits growth and causes aberrant cell morphology in the fission yeast, Schizosaccharomyces pombe. Exogenously added thiamine, the pyrimidine moiety of the thiamine molecule, and adenine alleviate its growth-disturbing effect. At concentrations that do not inhibit growth, the drug reduces mating and sporulation and causes a decrease in the mRNA level of gene ste11 and the ste11-dependent gene, mei2. The mating- and sporulation-inhibiting effect of cordycepin is overcome by adenine. A mutant disrupted for the ado1 gene encoding adenosine kinase exhibits a cordycepin-resistant and methionine-sensitive phenotype, excretes adenosine into the medium and mates and sporulates poorly in the presence of adenine. A S. pombe mutant containing a frameshift mutation at the beginning of the carboxy-terminal half of gene ufd1 (the Saccharomyces cerevisiae UFD1 homologue) is cordycepin-resistant and sterile. Strains disrupted for the ufd1 gene only form microcolonies.

Topics: Adenosine Kinase; Antifungal Agents; Deoxyadenosines; Drug Resistance, Fungal; Mutation; Phenotype; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Sequence Homology; Spores; Vesicular Transport Proteins

The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases. This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB. The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated. Adenosine (Km 3 microM) is its primary substrate. In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM). Other kinetic properties of the recombinant enzyme have also been determined.

Topics: Adenosine; Adenosine Kinase; Blotting, Western; Chromatography, Affinity; Deoxyadenosines; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Kinetics; Recombinant Proteins; Saccharomyces cerevisiae

Sequencing of the Saccharomyces cerevisiae genome revealed an open reading frame (YJR105w) encoding a putative protein highly similar to adenosine kinases from other species. Disruption of this gene (renamed ADO1) affected utilization of S-adenosyl methionine (AdoMet) as a purine source and resulted in a severe reduction of adenosine kinase activity in crude extracts. Furthermore, knock-out of ADO1 led to adenosine excretion in the medium and resistance to the toxic adenosine analogue cordycepin. From these data we conclude that ADO1 encodes yeast adenosine kinase. We also show that ADO1 does not play a major role in adenine utilization in yeast and we propose that the physiological role of adenosine kinase in S. cerevisiae could primarily be to recycle adenosine produced by the methyl cycle.

Topics: Adenosine; Adenosine Kinase; Amino Acid Sequence; Deoxyadenosines; Drug Resistance; Genes, Fungal; Molecular Sequence Data; Mutation; Phenotype; S-Adenosylmethionine; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Terminology as Topic

A cordycepin-resistant mutant strain of Saccharomyces cerevisiae (CD-R2) was found to be deficient in adenosine kinase. This mutant accumulated S-adenosylhomocysteine during growth in the presence of exogenous adenosine and it grew in a pseudohyphal manner in the presence of this nucleotide.

Topics: Adenosine; Adenosine Kinase; Biotransformation; Cell Division; Deoxyadenosines; Drug Resistance, Microbial; Mutation; S-Adenosylhomocysteine; Saccharomyces cerevisiae; Thiamine