Ligand chirality effects on the dynamics of human 3-phosphoglycerate kinase: comparison between D- and L-nucleotides

l-nucleoside analogues are now largely used as antiviral drugs for the treatment of viral infections like HBV, HCV and HIV. However, in order to be fully active, they need to be phosphorylated by cellular or viral kinases. Human 3-phosphogycerate kinase (hPGK) was shown to catalyze the last step of activation of l-enantiomers and thus constitutes an attractive target for theoretical predictions of its phosphorylation efficiency. Molecular dynamics simulations were carried out with four different nucleotides (d-/l-ADP and d-/l-CDP) in complex with hPGK and 1,3-bisphospho-d-glycerate (bPG). The binding affinities of CDPs (both enantiomers) for hPGK were found very weak while d- and l-ADP were better substrates. Interestingly, the binding affinity of the bPG substrate was found to be lower in presence of d-ADP than l-ADP which indicates a potential antagonistic effect on one substrate to the other. A detailed analysis of the simulations unravels important dynamic conditions for efficient phosphorylation. Indeed, as previously described for the natural substrate, the hinge bending motion of the domains upon substrates binding should be more correlated and directional. Interestingly, the unforeseen finding was the larger dynamics freedoms observed for the substrates that was favored by the protein atoms flexibility around the nucleobase binding site.     

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.     

An Acidophilic Bacterial-Archaeal-Fungal Ecosystem Linked to Formation of Ferruginous Crusts and Stalactites

The presence of specialized microbial associations between populations of chemoautotrophic bacteria and archaea with ascomycetous fungi was observed inside stalactite-shaped mineral formations in a highly acidic cave environment. Metagenomic, chemical and electron microscopy analyses were used to investigate the relevance of these microbial ecosystems in the formation of stalactites. Ferric hydroxide produced by acidophilic bacteria and archaea was shown to be deposited onto fungal hyphae, resulting in complex mineralized stalactite-shaped structures. Thus, both archaeal-bacterial and fungal members of the ecosystem were shown to play an active role in the formation of stalactites.     

FRACTIONATION OF GLYCOPROTEINS ASSOCIATED TO ADULT RAT BRAIN MYELIN FRACTIONS

Abstract— The chloroform-methanol insoluble residue of adult rat brain myelin fractions (My-CMI) contains only 20% of protein but all myelin-associated glycoproteins (Z anetta et al ., 1977a). After solubilization in sodium dodecyl sulphate, these glycoproteins were separated by sequential affinity chromatography on 4 immobilized lectins. Ten fractions (9 of which contained only glycoproteins) were obtained. Glycoproteins added up to at least 25% of My-CMI proteins. Many minor glycoproteins were detected in the different fractions. However most of them appeared not to be intrinsic to myelin. On the contrary a major glycoprotein electrophoretic band, component A, appeared to be intrinsic to myelin although its presence also on oligodendrocyte plasma membrane cannot be excluded. Component A was tentatively identified with the'major myelin associated glycoprotein'described by QUARLES (1972, 1973 a, b ). It accounted for less than 0.4% of proteins and 8% of glycoproteins of myelin fractions and consisted of at least two'glycopolypeptides'which, both, bind to concanavalin A and to the Ulex europeus lectin. The other major glycoprotein, component B, did not bind to any of the lectins used and, thus, must have N -acetyl neuraminic acid as only terminal sugar. The separation of myelin-associated glycoproteins according to their affinity for lectins allowed a tentative identification of the lectin binding sites of myelin sheath.     

Biocatalytic asymmetric hydroxylation of hydrocarbons by free and immobilized Bacillus megaterium cells

Screening of soil bacteria with allylbenzene resulted in a Bacillus megaterium strain, which hydroxylates simple hydrocarbons in high enantiomeric excess (ee up to 99%). Benzylic and nonbenzylic hydroxylation products were obtained, without the usually observed high preference for the benzylic position. The immobilization of the B. megaterium cells in alginate gel effectively improved the stability of the cells and increased the amounts of products formed, without loss of enantioselectivity. The product ratio ( vs. β hydroxylation) was shifted towards benzylic hydroxylation, which suggests that at least two hydroxylating enzymes with distinct regioselectivity are involved. Comparison to free-cell fermentations in small- and large-scale bioreactors (up to 2000 ml) showed that the use of immobilized cells is advantageous, as they are easier to handle and yield higher amounts of oxidation products.