Dealkylation of Macromolecules by Eukaryotic α-Ketoglutarate-Dependent Dioxygenases from the AlkB-like Family.

Alkylating modifications induced by either exogenous chemical agents or endogenous metabolites are some of the main types of damage to DNA, RNA, and proteins in the cell. Although research in recent decades has been almost entirely devoted to the repair of alkyl and in particular methyl DNA damage, more and more data lately suggest that the methylation of RNA bases plays an equally important role in normal functioning and in the development of diseases. Among the most prominent participants in the repair of methylation-induced DNA and RNA damage are human homologs of Escherichia coli AlkB, nonheme Fe(II)/α-ketoglutarate-dependent dioxygenases ABH1-8, and FTO. Moreover, some of these enzymes have been found to act on several protein targets. In this review, we present up-to-date data on specific features of protein structure, substrate specificity, known roles in the organism, and consequences of disfunction of each of the nine human homologs of AlkB. Special attention is given to reports about the effects of natural single-nucleotide polymorphisms on the activity of these enzymes and to potential consequences for carriers of such natural variants.

An Insight into the Mechanism of DNA Cleavage by DNA Endonuclease from the Hyperthermophilic Archaeon Pyrococcus furiosus.

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes.

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5' to an AP-site; can recognize and process some damaged nucleosides; and possess 3'-phosphodiesterase, 3'-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions' concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3'-5' exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

Cation-Dependent Self-assembly of Vanadium Polyoxoniobates.

Reaction of Na7H[Nb6O19]·15H2O with NaVO3·2H2O at 220 °C in the presence of NaHCO3 gives new bicapped α-Keggin vanadododecaniobate [VNb12O40{NbO(CO3)}2]13-, isolated and structurally characterized as Na9H4[VNb12O40{NbO(CO3)}2]·37H2O (1). According to 51V NMR and ESI-MS data, this anion equilibrates in solution with [VNb12O40]15- and oligomeric species that result from dissociation of the {NbO(CO3)}+ fragments. In the presence of potassium, the same reaction gives [VxNb24O76]n- (x = 4, n = 12 (2a); x = 3, n = 17 (2b)). The anions with x = 3 and 4 cocrystallize together, but exist as separate entities both in solid and in solution according to 51V MAS NMR and ESI-MS data.

Bioremediation of Polycyclic Aromatic Hydrocarbons by Means of Bacteria and Bacterial Enzymes.

Polycyclic aromatic hydrocarbons (PAHs) are widespread, persistent, and toxic environmental pollutants. Many anthropogenic and some natural factors contribute to the spread and accumulation of PAHs in aquatic and soil systems. The effective and environmentally friendly remediation of these chemical compounds is an important and challenging problem that has kept scientists busy over the last few decades. This review briefly summarizes data on the main sources of PAHs, their toxicity to living organisms, and physical and chemical approaches to the remediation of PAHs. The basic idea behind existing approaches to the bioremediation of PAHs is outlined with an emphasis on a detailed description of the use of bacterial strains as individual isolates, consortia, or cell-free enzymatic agents.

The Role of DNMT Methyltransferases and TET Dioxygenases in the Maintenance of the DNA Methylation Level.

This review deals with the functional characteristics and biological roles of enzymes participating in DNA methylation and demethylation as key factors in epigenetic regulation of gene expression. The set of enzymes that carry out such processes in human cells is limited to representatives of two families, namely DNMT (DNA methyltransferases) and TET (DNA dioxygenases). The review presents detailed information known today about each functionally important member of these families and describes the catalytic activity and roles in the mammalian body while also providing examples of dysregulation of the expression and/or activity of these enzymes in conjunction with the development of some human disorders, including cancers, neurodegenerative diseases, and developmental pathologies. By combining the up-to-date information on the dysfunction of various enzymes that control the DNA "methylome" in the human body, we hope not only to draw attention to the importance of the maintenance of a required DNA methylation level (ensuring epigenetic regulation of gene expression and normal functioning of the entire body) but also to help identify new targets for directed control over the activity of the enzymes that implement the balance between processes of DNA methylation and demethylation.

Individual Contributions of Amido Acid Residues Tyr122, Ile168, and Asp173 to the Activity and Substrate Specificity of Human DNA Dioxygenase ABH2.

Human Fe(II)/α-ketoglutarate-dependent dioxygenase ABH2 plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases, e.g., N1-methyladenine (m1A), N3-methylcytosine (m3C), and some etheno derivatives. Moreover, ABH2 is capable of a less efficient oxidation of an epigenetic DNA mark called 5-methylcytosine (m5C), which typically is a specific target of DNA dioxygenases from the TET family. In this study, to elucidate the mechanism of the substrate specificity of ABH2, we investigated the role of several active-site amino acid residues. Functional mapping of the lesion-binding pocket was performed through the analysis of the functions of Tyr122, Ile168, and Asp173 in the damaged base recognition mechanism. Interactions of wild-type ABH2, or its mutants Y122A, I168A, or D173A, with damaged DNA containing the methylated base m1A or m3C or the epigenetic marker m5C were analyzed by molecular dynamics simulations and kinetic assays. Comparative analysis of the enzymes revealed an effect of the substitutions on DNA binding and on catalytic activity. Obtained data clearly demonstrate the effect of the tested amino acid residues on the catalytic activity of the enzymes rather than the DNA-binding ability. Taken together, these data shed light on the molecular and kinetic consequences of the substitution of active-site residues for the mechanism of the substrate recognition.

The Enigma of Substrate Recognition and Catalytic Efficiency of APE1-Like Enzymes.

Despite significant achievements in the elucidation of the nature of protein-DNA contacts that control the specificity of nucleotide incision repair (NIR) by apurinic/apyrimidinic (AP) endonucleases, the question on how a given nucleotide is accommodated by the active site of the enzyme remains unanswered. Therefore, the main purpose of our study was to compare kinetics of conformational changes of three homologous APE1-like endonucleases (insect Drosophila melanogaster Rrp1, amphibian Xenopus laevis xAPE1, and fish Danio rerio zAPE1) during their interaction with various damaged DNA substrates, i.e., DNA containing an F-site (an uncleavable by DNA-glycosylases analog of an AP-site), 1,N 6-ethenoadenosine (εA), 5,6-dihydrouridine (DHU), uridine (U), or the α-anomer of adenosine (αA). Pre-steady-state analysis of fluorescence time courses obtained for the interaction of the APE1-like enzymes with DNA substrates containing various lesions allowed us to outline a model of substrate recognition by this class of enzymes. It was found that the differences in rates of DNA substrates' binding do not lead to significant differences in the cleavage efficiency of DNA containing a damaged base. The results suggest that the formation of enzyme-substrate complexes is not the key factor that limits enzyme turnover; the mechanisms of damage recognition and cleavage efficacy are related to fine conformational tuning inside the active site.

The impact of single-nucleotide polymorphisms of human apurinic/apyrimidinic endonuclease 1 on specific DNA binding and catalysis.

Human apurinic/apyrimidinic (AP) endonuclease APE1 is a crucial enzyme of the base excision repair (BER) pathway, which is in charge of recognition and initiation of removal of AP-sites in DNA. It is known that some single-nucleotide polymorphism (SNP) variants of APE1 have a reduced activity as compared to wild-type APE1. It has been hypothesized that genetic variation in APE1 might be responsible for an increased risk of some types of cancer. In the present work, analysis of SNPs of the APE1 gene was performed to select the set of variants having substitutions of amino acid residues on the surface of the enzyme globule and in the DNA-binding site, thereby affecting protein-protein interactions or the catalytic reaction, respectively. For seven APE1 variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S), conformational dynamics and catalytic activities were examined. The conformational changes in the molecules of APE1 variants and in a DNA substrate were recorded as fluorescence changes of Trp and 2-aminopurine residues, respectively, using the stopped-flow technique. The results made it possible to determine the kinetic mechanism underlying the interactions of the APE1 variants with DNA substrates, to calculate the rate constants of the elementary stages, and to identify the stages of the process affected by mutation.