T. A. Seregina

Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli

Significance Hydrogen sulfide (H2S) is a highly toxic gas that interferes with cellular respiration; however, at low physiological amounts, it plays an important role in cell signaling. Remarkably, in bacteria, endogenously produced H2S has been recently recognized as a general protective molecule, which renders multiple bacterial species highly resistant to oxidative stress and various classes of antibiotics. The mechanism of this phenomenon remains poorly understood. In this paper, we use Escherichia coli as a model system to elucidate its major enzymatic source of H2S and establish the principle biochemical pathways that account for H2S-mediated protection against reactive oxygen species. Understanding those mechanisms has far-reaching implications in preventing bacterial resistance and designing effective antimicrobial therapies. Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli. Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ∆mstA cells hypersensitive to H2O2. Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (Ptet-mstA) renders ∆fur cells fully resistant to H2O2. Furthermore, the endogenous level of H2S is reduced in ∆fur or ∆sodA ∆sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.

Construction of a butyrate-producing E. coli strain without the use of heterologous genes

Multistage construction of an E. coli strain containing no foreign genes which is capable of producing butyrate has been carried out. At the first stage, deletions in gene fadR encoding a protein repressor of an operon for fatty acid degradation and gene aceF responsible for the synthesis of pyruvate dehydrogenase were introduced in the strain MG1655 genome. Then, a mutant obtained from the above strain by induced mutagenesis and capable of growth on ethanol as a sole carbon source under aerobic conditions was selected. It was shown that growth of the mutant on ethanol is provided by two mutations. One of them (a substitution: 257G → A) is located in the regulatory region of gene adhE that controls the synthesis of alcohol-dehydrogenase; the other, containing a substitution Glu568 → Lys, affects the structural portion of the gene. As a result of the consequent mutagenesis of the obtained strain and selection on indicating media, variants capable of growing on butyrate and butanol as sole carbon sources and putatively bearing mutations in gene atoC (encoding transcriptional activator of atoDAB operon) were selected. At the last stage of the work, gene atoB, encoding the synthesis of the thiolase II enzyme, was placed under the control of a constitutive promoter Ptet, and the functional allele of gene aceF was introduced. The resulting E. coli strain (ΔfadR, adhE, atoC, Ptet-atoB) accumulates 800 mg/l of butyrate upon growth on glucose-containing medium under semi-anaerobic (oxygen limited) conditions. Introduction of an additional deletion in gene ldhA encoding lactate dehydrogenase in the strain genome leads to a further growth of a butyrate production up to 1.3 g/l.

Crystallization and preliminary X-ray study of Vibrio cholerae uridine phosphorylase in complex with 6-methyluracil

Uridine phosphorylase catalyzes the phosphorolysis of ribonucleosides, with the nitrogenous base and ribose 1-phosphate as products. Additionally, it catalyzes the reverse reaction of the synthesis of ribonucleosides from ribose 1-phosphate and a nitrogenous base. However, the enzyme does not catalyze the synthesis of nucleosides when the substrate is a nitrogenous base substituted at the 6-position, such as 6-methyluracil (6-MU). In order to explain this fact, it is essential to investigate the three-dimensional structure of the complex of 6-MU with uridine phosphorylase. 6-MU is a pharmaceutical agent that improves tissue nutrition and enhances cell regeneration by normalization of nucleotide exchange in humans. 6-MU is used for the treatment of diseases of the gastrointestinal tract, including infectious diseases. Here, procedures to obtain the uridine phosphorylase from the pathogenic bacterium Vibrio cholerae (VchUPh), purification of this enzyme, crystallization of the complex of VchUPh with 6-MU, and X-ray data collection and preliminary X-ray analysis of the VchUPh-6-MU complex at atomic resolution are reported.

Isolation and phenotypic characteristics of the Escherichia coli butanol-tolerant mutants

Resistance to butanol is a key factor affecting microbial ability to produce economically profitable amounts of butanol. In this study, an Escherichia coli strain capable of growth in the presence of 1.5% butanol was isolated. The mutant MG1655 ButR was characterized by increased resistance to ethanol, isopropanol, and bivalent ions but exhibited supersensitivity to osmotic shock. Compared to the wild type strain, the butanol-tolerant mutant was more sensitive to antibiotics inhibiting protein synthesis but was more resistant to membrane-penetrating antibiotics, such as surfactin. Increased content of unsaturated fatty acids was found in the membranes of butanol-tolerant mutants. It was revealed that overexpression of the genes encoding cold-shock proteins decreased butanol tolerance of both mutant and the wild-type strain. It was concluded that butanol tolerance was associated with multiple rearrangements of the cell genetic system, rather than with single mutations.

Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine.

A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh-thymidine complex (of dimensions ∼200-350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µl VchUPh (15 mg ml(-1)), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl(2).6H2O in 0.1 M Tris-HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a=91.80, b=95.91, c=91.89 Å, β=119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da(-1); the corresponding solvent content was 43.74%.

Uridine phosphorylase in biomedical, structural, and functional aspects: A review

The activation of xenobiotics often causes malignant tumor cells to resist chemotherapeutic treatment. Uridine phosphorylase is the key enzyme of pyrimidine metabolism and catalyzes the reversible phosphorylation of uridine with the formation of uracil and ribose-1-phosphate. High-selectivity anticancer agents based on uridine phosphorylase inhibitors are promising for treating both oncological and infection diseases. New medicinal preparations can be predicted and rationally developed only on the basis of detailed biomedical, structural, and functional knowledge about the biomacromolecular target enzyme-drug complex.

Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2′-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation

Butanol as a regulatory factor of ompC gene expression in E. coli cells

The influence of butanol on the expression of ompC gene encoding synthesis of OmpC porin in the MG1655 strain of E. coli and butanol-tolerant mutant ButR was studied. It was shown that in the case of wild bacteria, the addition of butanol to the growth medium results in an increased level of ompC transcription. However, OmpC porin is not detected in the membrane fraction of cells. ButR mutant exhibits a higher level of ompC gene expression. A direct correlation is observed between the level of OmpC porin expression and its content in the membrane fraction of ButR mutant cells. In the regulatory region of the ompC gene of the ButR mutant, three nucleotide substitutions located in the binding sites of OmpR and CpxR activator proteins were identified. It was shown that mutations in the regulatory region of the ompC gene in the ButR mutant are responsible for the decreased level of OmpC porin expression under normal growth conditions. However, these mutations lead to an increased level of OmpC porin synthesis in the presence of butanol. These data suggest an additional mechanism of ompC gene regulation with the participation of butanol as a positive transcription effector.

Structure of the homodimer of uridine phosphorylase from Salmonella typhimurium in the native state at 1.9 Å resolution

Uridine phosphorylase (UPh) belongs to pyrimidine nucleoside phosphorylases. This enzyme catalyzes cleavage of the C-N glycoside bond in uridine to form uracil and ribose-1’-phosphate. Uridine phosphorylase supplies cells with nucleotide precursors by catalyzing the phosphorolysis of purine and pyrimidine nucleosides. This is an alternative to de novo nucleotide synthesis. The three-dimensional structure of native uridine phosphorylase from Salmonella typhimurium (StUPh) in a new crystal form was solved and refined at 1.90 Å resolution (Rst = 20.37%; Rfree = 24.69%; the rmsd of bond lengths and bond angles are 0.009 Å and 1.223°, respectively). A homodimer containing two asynchronously functioning active sites was demonstrated to be the minimum structural unit necessary for function of the hexameric StUPh molecule (L33L2). Each active site is formed by amino acid residues of both subunits.