RudS: bacterial desulfidase responsible for tRNA 4-thiouridine de-modification.

In this study, we present an extensive analysis of a widespread group of bacterial tRNA de-modifying enzymes, dubbed RudS, which consist of a TudS desulfidase fused to a Domain of Unknown Function 1722 (DUF1722). RudS enzymes exhibit specific de-modification activity towards the 4-thiouridine modification (s4U) in tRNA molecules, as indicated by our experimental findings. The heterologous overexpression of RudS genes in Escherichia coli significantly reduces the tRNA 4-thiouridine content and diminishes UVA-induced growth delay, indicating the enzyme's role in regulating photosensitive tRNA s4U modification. Through a combination of protein modeling, docking studies, and molecular dynamics simulations, we have identified amino acid residues involved in catalysis and tRNA binding. Experimental validation through targeted mutagenesis confirms the TudS domain as the catalytic core of RudS, with the DUF1722 domain facilitating tRNA binding in the anticodon region. Our results suggest that RudS tRNA modification eraser proteins may play a role in regulating tRNA during prokaryotic stress responses.

Insights into the Alcyoneusvirus Adsorption Complex.

The structures of the Caudovirales phage tails are key factors in determining the host specificity of these viruses. However, because of the enormous structural diversity, the molecular anatomy of the host recognition apparatus has been elucidated in only a number of phages. Klebsiella viruses vB_KleM_RaK2 (RaK2) and phiK64-1, which form a new genus Alcyoneusvirus according to the ICTV, have perhaps one of the most structurally sophisticated adsorption complexes of all tailed viruses described to date. Here, to gain insight into the early steps of the alcyoneusvirus infection process, the adsorption apparatus of bacteriophage RaK2 is studied in silico and in vitro. We experimentally demonstrate that ten proteins, gp098 and gp526-gp534, previously designated as putative structural/tail fiber proteins (TFPs), are present in the adsorption complex of RaK2. We show that two of these proteins, gp098 and gp531, are essential for attaching to Klebsiella pneumoniae KV-3 cells: gp531 is an active depolymerase that recognizes and degrades the capsule of this particular host, while gp098 is a secondary receptor-binding protein that requires the coordinated action of gp531. Finally, we demonstrate that RaK2 long tail fibers consist of nine TFPs, seven of which are depolymerases, and propose a model for their assembly.

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N4-acylcytosine derivatives.

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N4-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (kcat/Km up to 2.8 × 106 M-1 s-1). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N4-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases.

Consecutive Marcus Electron and Proton Transfer in Heme Peroxidase Compound II-Catalysed Oxidation Revealed by Arrhenius Plots.

Electron and proton transfer reactions in enzymes are enigmatic and have attracted a great deal of theoretical, experimental, and practical attention. The oxidoreductases provide model systems for testing theoretical predictions, applying experimental techniques to gain insight into catalytic mechanisms, and creating industrially important bio(electro)conversion processes. Most previous and ongoing research on enzymatic electron transfer has exploited a theoretically and practically sound but limited approach that uses a series of structurally similar ("homologous") substrates, measures reaction rate constants and Gibbs free energies of reactions, and analyses trends predicted by electron transfer theory. This approach, proposed half a century ago, is based on a hitherto unproved hypothesis that pre-exponential factors of rate constants are similar for homologous substrates. Here, we propose a novel approach to investigating electron and proton transfer catalysed by oxidoreductases. We demonstrate the validity of this new approach for elucidating the kinetics of oxidation of "non-homologous" substrates catalysed by compound II of Coprinopsis cinerea and Armoracia rusticana peroxidases. This study - using the Marcus theory - demonstrates that reactions are not only limited by electron transfer, but a proton is transferred after the electron transfer event and thus both events control the reaction rate of peroxidase-catalysed oxidation of substrates.

New 1-(3-nitrophenyl)-5,6-dihydro-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepines: synthesis and computational study.

Triazole derivatives constitute an important group of heterocyclic compounds have have been the subject of extensive study in the recent past. These compounds have shown a wide range of biological and pharmacological activities. In this work, new fused tricyclic 1-(3-nitrophenyl)-5,6-dihydro-4H-[1,2,4]triazolo[4,3-a][1,5]-benzodiazepines have been synthesized by the thermal cyclization of N'-(2,3-dihydro-1H-1,5-benzodiazepin-4-yl)-3-nitrobenzohydrazides. After screening ethanol, toluene and 1-butanol as solvents, butanol-1 was found to be the best choice for the cyclization reaction in order to obtain the highest yields of tricyclic derivatives. The chemical structures of the synthesized compounds were elucidated by the analysis of their IR, 1H- and 13C-NMR spectral data. For tentative rationalization of the reaction processes, the global and local reactivity indices of certain compounds, taking part in the reaction pathway, were assessed by means of quantum mechanical calculations using the conceptual density functional theory (DFT) approach. This work could be useful for the synthesis of new heterocyclic compounds bearing a fused triazole ring.

The study of NADPH-dependent flavoenzyme-catalyzed reduction of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans).

The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs) towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and two-electron (hydride) transferring NAD(P)H: quinone oxidoreductase (NQO1) was examined in this work. Since the =N+ (→O)O- moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs) whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs) of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity indices were assessed by quantum mechanical methods. In P-450R-catalyzed reactions, both BFXs and NACs showed the same reactivity dependence on their electron-accepting potency which might be consistent with an "outer sphere" electron transfer mechanism. In NQO1-catalyzed two-electron (hydride) transferring reactions, BFXs acted as more efficient substrates than NACs, and the reduction efficacy of BFXs by NQO1 was in general higher than by single-electron transferring P-450R. In NQO1-catalyzed reactions, QSARs obtained showed that the reduction efficacy of BFXs, as well as that of NACs, was determined by their electron-accepting potency and could be influenced by their binding mode in the active center of NQO1 and by their global softness as their electronic characteristic. The reductive conversion of benzofuroxan by both flavoenzymes yielded the same reduction product of benzofuroxan, 2,3-diaminophenazine, with the formation of o-benzoquinone dioxime as a putative primary reductive intermediate, which undergoes a further reduction process. Overall, the data obtained show that by contrast to NACs, the flavoenzyme-catalyzed reduction of BFXs is unlikely to initiate their redox-cycling, which may argue for a minor role of the redox-cycling-type action in the cytotoxicity of BFXs.