Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

The results of several decades of studying the catalytic mechanism of Escherichia colt purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared, The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme

Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule

In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium’s high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Assisted phytoextraction of trace metals: Cu, Cd, Zn, Pb

Wydział BiologiiFitoekstrakcja wykorzystuje naturalne lub wspomagane zdolności roślin do oczyszczania ziemi z pierwiastków śladowych. Dla optymalizacji tej metody ważne jest wyselekcjonowanie roślin wydajnie akumulujących metale, o wysokiej produkcji suchej biomasy. Celem wykonanych badań było określenie, w jaki sposób obecność jonów metali (Cu, Zn, Cd, Pb) w mieszankach dwuskładnikowych wpływa na pobieranie i translokację metali, oraz czy uprawa współrzędna, inokulacja bakteriami wspomagającymi wzrost roślin, suplementacja chelatorami i konsorcjum mikroorganizmów może podwyższyć wydajność fitoekstrakcji. Badania przeprowadzono na gorczycy sarepskiej Brassica juncea, uprawianej samodzielnie lub współrzędnie z kukurydzą Zea mays oraz lucerną Medicago sativa. Wykazano, że obecność jonów metali w mieszankach podwójnych może mieć znaczny wpływ na pobieranie oraz translokację metali. Najważniejsze zaobserwowane interakcje to synergia akumulacji Zn i Pb oraz Zn i Cd, i konkurencja pomiędzy Zn i Cu. Wykazano również, że prowadzenie uprawy współrzędnej, inokulacja ryzobakteriami oraz suplementacja chelatorami i konsorcjum mikroorganizmów może podwyższyć wydajność fitoekstrakcji metali. W najbardziej wydajnym wariancie - uprawie współrzędnej gorczycy i lucerny inokulowanych Burkholderia phytofirmans PsJNT - uzyskano średni wzrost plonu metali o 95% dla Zn, 90% dla Cd, i 160% dla Pb.Phytoextraction uses natural or stimulated ability of plants to purify the soil contaminated with trace elements. To optimize this method, it is important to select plants efficiently accumulating metals, and with high production of dry biomass. The aim of the research was to determine how the presence of metal ions (Cu, Zn, Cd, Pb) in binary mixtures affects the uptake and translocation of metals, and whether intercropping, inoculation with bacteria promoting plant growth, supplementation of chelators and a consortium of microorganisms can increase the efficiency of phytoextraction. The study was conducted on Indian mustard Brassica juncea, grown alone or co-planted with maize Zea mays and alfalfa Medicago sativa. Results have shown that the presence of metal ions in the binary mixtures may have a significant impact on the uptake and translocation of metals. The most important observed interactions are: synergy in accumulation of Zn and Pb, and Zn and Cd, and competition between Zn and Cu. It was also shown that co-planting, inoculation with rhizobacteria and supplementation with chelators and a consortium of microorganisms can increase efficiency of metal phytoextraction. An increase in metal yield by 95% for Zn, 90% for Cd and 160% for Pb was achieved in the most efficient variant: co-planting of Indian mustard with alfalfa, and with PGPR inoculation

Trace metals membrane transporters in plants

Metale śladowe obecne w roztworze glebowym można podzielić na dwie grupy: metale konieczne do prawidłowego funkcjonowania komórek roślinnych (m.in. Fe, Cu, Mn, Co, Zn, Ni) oraz metale niekonieczne (np. Cd, Hg, Pb, Cr). Obecne w roślinach białka transportujące niezbędne metale z gleby do korzenia i dalej do pędu charakteryzują się szeroką specyficznością, stanowiąc drogę wnikania także dla metali niekoniecznych. Utrzymywanie homeostazy poprzez kontrolowanie pobierania, transportu, wypompowywania i sekwestracji tych pierwiastków jest kluczowe dla prawidłowego działania organizmów. Praca ta została poświęcona krótkiej charakterystyce zidentyfikowanych do tej pory roślinnych transporterów błonowych, należących do następujących rodzin białek: ZIP, YSL, NRAMP, P1B-ATP-az, transporterów ABC, COPT, CAX, CDF i IREG.Trace metals present in soil can be divided into two groups: essential metals indispensable for proper functioning of plant cells (e.g. Fe, Cu, Mn, Co, Zn, Ni), and non-essential metals (e.g. Cd, Hg, Pb, Cr). Transporters for essential trace metals in plants usually have a wide specificity and can equally transport non-essential metals. Maintaining homeostasis by controlling the uptake, transport, removal and sequestration of metals is crucial for organisms. This work is dedicated to a short characterization of identified plant membrane transporters, belonging to several protein families: ZIP, YSL, NRAMP, P1B-ATP-ases, ABC transporters, COPT, CAX, CDF and IREG

I'm 2.8% Neanderthal - The Beginning of Genetic Exhibitionism?

International audienceDirect-to-consumer genetic testing is gaining popularity. How- ever, the sensitive nature of personal genomic sequencing re- sults might not be fully understood by the general public. In this paper we study the examples of disclosure of this sen- sitive information on social networks. We found that Twit- ter users often post their results publicly. We observed that information on ethnic background is much more frequently released than other information, for example relating to dis- ease risk. This data could be of potential value to entities such as insurance companies.We found that about 24% of the analyzed tweets that men- tioned ethnicity results also contained percentage data. In cases of users disclosing more details of their ethnic back- ground, we found about 96% of these profiles also included identifying information and consequently can be attributed to individuals.As a result, external entities such as insurance companies can gain an insight in the genetic test results and in the end the users could be subject to genetic discrimination