Plasmodium falciparum purine nucleoside phosphorylase as a model in the search for new inhibitors by high throughput screening.

Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (PfPNP) blocks the purine salvage pathway in vitro and in vivo. In this study, PfPNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. PfPNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant PfPNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: KM 17 µM, kcat 1.2 s-1, VMax 2.2 U/mg and kcat/KM 7 × 10-4; for MESG they were: KM 131 µM, kcat 2.4 s-1, VMax 4.4 U/mg and kcat/KM 1.8 × 10-4. The thermodynamic parameters for the substrate Phosphate were: ΔG - 5.8 cal mol-1, ΔH - 6.5 cal mol-1 and ΔS - 2.25 cal mol-1/degree. The ITC results demonstrated that the binding of phosphate to free PfPNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with PfPNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant PfPNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by PfPNP and a model using PfPNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria.

Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis.

5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.

S-Adenosylmethionine regulates connexins sub-types expressed by hepatocytes.

Intercellular communication via GAP Junctions plays an important role in tissue homeostasis, apoptosis, carcinogenesis, cell proliferation and differentiation. Hepatocyte connexins (Cx) 26 and 32 levels are decreased during the de-differentiation process of primary hepatocytes in culture, a situation that is also characterized by a decrease in S-Adenosylmethionine (SAMe) levels. In this current study, we show that SAMe supplementation in cultured hepatocytes every 12h, leads to an up-regulation of Cx26 and 32 mRNA and protein levels and blocks culture-induced Cx43 expression, although it failed to increase Cx26 and 32 membrane localization and GAP junction intracellular communication. SAMe reduced nuclear ß-catenin accumulation, which is known to stimulate the TCF/LEF-dependent gene transcription of Cx43. Moreover SAMe-induced reduction in Cx43 and ß-catenin was prevented by the proteasome inhibitor MG132, and was not mediated by GSK3 activity. SAMe, and its metabolite 5'-methylthioadenosine (MTA) increased Cx26 mRNA in a process partially mediated by Adenosine A(2A) receptors but independent of PKA. Finally livers from MAT1A knockout mice, characterized by low hepatic SAMe levels, express higher Cx43 and lower Cx26 and 32 protein levels than control mice. These results suggest that SAMe maintains a characteristic expression pattern of the different Cxs in hepatocytes by differentially regulating their levels.

Functional analysis of methylthioribose kinase genes in plants.

Through a biochemical and a genetic approach, we have identified several plant genes encoding methylthioribose (MTR) kinase, an enzyme involved in recycling of methionine through the methylthioadenosine (MTA) cycle. OsMTK1, an MTR kinase from rice (Oryza sativa), is 48.6 kD in size and shows cooperative kinetics with a V(max) of 4.9 pmol/min and a K0.5 of 16.8 microm. MTR kinase genes are the first genes to be identified from the MTA cycle in plants. Insertional mutagenesis of the unique AtMTK gene in Arabidopsis (Arabidopsis thaliana) resulted in an inability of plants to grow on MTA as a supplemental sulfur source. MTK knock-out plants were not impaired in growth under standard conditions, indicating that the MTA cycle is a nonessential metabolic pathway in Arabidopsis when sulfur levels are replete. In rice, OsMTK genes were strongly up-regulated in shoots and roots when plants were exposed to sulfur starvation. Gene expression was largely unaffected by lack of nitrogen or iron in the nutrient solution, indicating that OsMTK regulation was linked specifically to sulfur metabolism.

Conversion of 5'-methylthioadenosine into S-adenosylmethionine by yeast cells.

5'-Methylthio[U-14C]adenosine was used as a culture supplement for Candida utilis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The (U-14C)-labeled adenine of 5'-methylthio[U-14C]adenosine did not contribute to the labeling of the amino acid components of the sulfonium compound.