A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis.
The presence of industrially produced chemicals in water is often not monitored while their passive transport and accumulation can cause serious damage in living cells. Molecular dynamics simulations make an...
A simplified, fast, and effective production method has been developed for the synthesis of manganese ferrite (MnFe2O4) magnetic nanoparticles (MNPs). In addition to the wide applicability of MnFe2O4 MNPs, this work also reports their application in DNA isolation for the first time. An ultrasonic-cavitation-assisted combustion method was applied in the synthesis of MnFe2O4 MNPs at different furnace temperatures (573 K, 623 K, 673 K, and 773 K) to optimize the particles’ properties. It was shown that MnFe2O4 nanoparticles synthesized at 573 K consist of a spinel phase only with adequate size and zeta potential distributions and superparamagnetic properties. It was also demonstrated that superparamagnetic manganese ferrite nanoparticles bind DNA in buffer with a high NaCl concentration (2.5 M), and the DNA desorbs from the MNPs by decreasing the NaCl concentration of the elution buffer. This resulted in a DNA yield comparable to that of commercial DNA extraction products. Both the DNA concentration measurements and electrophoresis confirmed that a high amount of isolated bacterial plasmid DNA (pDNA) with adequate purity can be extracted with MnFe2O4 (573 K) nanoparticles by applying the DNA extraction method proposed in this article.
In this article, we describe the antimicrobial properties of a new composite based on anodic aluminium oxide (AAO) membranes containing propyl-copper-phosphonate units arranged at a predetermined density inside the AAO channels. The samples were prepared with four concentrations of copper ions and tested as antimicrobial drug on four different strains of Escherichia coli (K12, R2, R3 and R4). For comparison, the same strains were tested with three types of antibiotics using the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests. Moreover, DNA was isolated from the analysed bacteria which was additionally digested with formamidopyrimidine-DNA glycosylase (Fpg) protein from the group of repair glycosases. These enzymes are markers of modified oxidised bases in nucleic acids produced during oxidative stress in cells. Preliminary cellular studies, MIC and MBC tests and digestion with Fpg protein after modification of bacterial DNA suggest that these compounds may have greater potential as antibacterial agents than antibiotics such as ciprofloxacin, bleomycin and cloxacillin. The described composites are highly specific for the analysed model Escherichia coli strains and may be used in the future as new substitutes for commonly used antibiotics in clinical and nosocomial infections in the progressing pandemic era. The results show much stronger antibacterial properties of the functionalised membranes on the action of bacterial membranes in comparison to the antibiotics in the Fpg digestion experiment. This is most likely due to the strong induction of oxidative stress in the cell through the breakdown of the analysed bacterial DNA. We have also observed that the intermolecular distances between the functional units play an important role for the antimicrobial properties of the used material. Hence, we utilised the idea of the 2D solvent to tailor them.
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