Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra( N -methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes . To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes . The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes . These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1, 3.2.2.2, 3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases Rih and catalyzes the cleavage of ribonucleosides to nitrogenous bases and ribose. RihC is one of the enzymes that are synthesized by lactobacilli in response to the presence of Klebsiella. To characterize this protein from Limosilactobacillus reuteri LR1, we cloned and expressed it. The activity of the enzyme was studied towards a wide range of substrates, including ribonucleosides, deoxyribonucleosides as well as an arabinoside. It was shown that the enzyme is active only with ribonucleosides and arabinoside, with the best substrate being uridine. The thermal stability of this enzyme was studied, and its crystal structure was obtained, which demonstrated the tetrameric architecture of the enzyme and allowed to shed light on a correlation between its structure and enzymatic activity. Comprehensive comparisons of all known RihC structures, both existing crystal structures and computed model structures from various species, were made, allowing for the identification of structural motifs important for enzyme functioning.

Prosthetic joint infections (PJIs) remain a significant challenge, occurring in 1% to 2% of joint arthroplasties and potentially leading to a 20% to 30% mortality rate within 5 years. The primary pathogens responsible for PJIs include Staphylococcus aureus, coagulase-negative staphylococci, and Gram-negative bacteria, typically treated with intravenous antibiotic drugs. However, this conventional approach fails to effectively eradicate biofilms or the microbial burden in affected tissues. As a result, innovative strategies are being explored to enhance the efficacy of infection prevention through the development of antibacterial-coated implants. These coatings are required to demonstrate broad-spectrum antimicrobial activity, minimal local and systemic toxicity, favorable cost-effectiveness, and support for bone healing. In the present review, the analysis of various methodologies for developing antibacterial coatings was performed, emphasizing studies that conducted in vivo tests to advance potential clinical applications. A diversity of techniques employed for the development of coatings incorporating antimicrobial agents highlights promising avenues for reducing infection-related surgical failures.

Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD⁺ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments — the differences in maxima of the melting curves (T _m) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T _m decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH — Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.

The effect of medium pH and ion strength on the thermal stability of formate dehydrogenases (EC 1.2.1.2, FDH) from model plant Arabidopsis thaliana (AthFDH) and soybean Glycine max(SoyFDH) are...

The analysis of a formate dehydrogenase (FDH) structure in the apo form and in a complex with nicotinamide (NAD⁺) and azide ion has shown a high probability of efficient fluorescence quenching during the formation of such a triple complex. The excitation and fluorescence spectra indicated that the enzyme fluorescence is determined by tryptophan residues. The dependence of FDH fluorescence quenching on the NAD⁺ and azide concentrations was studied. The obtained data were used to determine the concentration of active sites and the catalytic rate constant of recombinant FDH from Glycine max.