Crystal structure of a DNA catalyst.

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.

Developing Wound Dressings Using 2-deoxy-D-Ribose to Induce Angiogenesis as a Backdoor Route for Stimulating the Production of Vascular Endothelial Growth Factor.

2-deoxy-D-Ribose (2dDR) was first identified in 1930 in the structure of DNA and discovered as a degradation product of it later when the enzyme thymidine phosphorylase breaks down thymidine into thymine. In 2017, our research group explored the development of wound dressings based on the delivery of this sugar to induce angiogenesis in chronic wounds. In this review, we will survey the small volume of conflicting literature on this and related sugars, some of which are reported to be anti-angiogenic. We review the evidence of 2dDR having the ability to stimulate a range of pro-angiogenic activities in vitro and in a chick pro-angiogenic bioassay and to stimulate new blood vessel formation and wound healing in normal and diabetic rat models. The biological actions of 2dDR were found to be 80 to 100% as effective as VEGF in addition to upregulating the production of VEGF. We then demonstrated the uptake and delivery of the sugar from a range of experimental and commercial dressings. In conclusion, its pro-angiogenic properties combined with its improved stability on storage compared to VEGF, its low cost, and ease of incorporation into a range of established wound dressings make 2dDR an attractive alternative to VEGF for wound dressing development.

Enzyme promiscuity shapes adaptation to novel growth substrates.

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.

Antioxidant and Antimutagenic Activities of Different Fractions from the Leaves of Rhododendron arboreum Sm. and Their GC-MS Profiling.

In this era of urbanization and environmental pollution, antioxidants and antimutagens derived from plants are promising safeguards for human health. In the current investigation, we analyzed the antioxidant and antimutagenic effects of the hexane, chloroform, and ethyl acetate fractions of Rhododendron arboreum Sm. leaves and determined their chemical composition. The different fractions inhibited lipid peroxidation, repressed the production of nitric oxide radicals, and prevented deoxyribose degradation. The antimutagenic activity of the leaf fractions was analyzed against 4-nitro-O-phenylenediamine, sodium azide and 2-aminofluorene mutagens in two test strains (TA-98 and TA-100) of Salmonella typhimurium. The experiment was conducted using pre- and co-incubation modes. The best results were obtained in the pre-incubation mode, and against indirect acting mutagen. The presence of a number of bioactive constituents was confirmed in the different fractions by GC-MS analysis. The study reveals the strong antioxidant and antimutagenic activity of R. arboreum leaves. We propose that those activities of R. arboreum might correspond to the combined effect of the phytochemicals identified by GC-MS analysis. To the best of our knowledge, this is the first report on the antimutagenic activity of R. arboreum leaves.

Enzymatic transhalogenation of dendritic RGD peptide constructs with the fluorinase.

The substrate scope of fluorinase enzyme mediated transhalogenation reactions is extended. Substrate tolerance allows a peptide cargo to be tethered to a 5'-chloro-5'-deoxynucleoside substrate for transhalogenation by the enzyme to a 5'-fluoro-5'-deoxynucleoside. The reaction is successfully extended from that previously reported for a monomeric cyclic peptide (cRGD) to cargoes of dendritic scaffolds carrying two and four cyclic peptide motifs. The RGD peptide sequence is known to bind upregulated αVß3 integrin motifs on the surface of cancer cells and it is demonstrated that the fluorinated products have a higher affinity to αVß3 integrin than their monomeric counterparts. Extending the strategy to radiolabelling of the peptide cargoes by tagging the peptides with [(18)F]fluoride was only moderately successful due to the poor water solubility of these higher order peptide scaffolds although the strategy holds promise for peptide constructs with improved solubility.

Effect of Selected Plant Phenolics on Fe2+-EDTA-H2O2 System Mediated Deoxyribose Oxidation: Molecular Structure-Derived Relationships of Anti- and Pro-Oxidant Actions.

In the presence of transition metal ions and peroxides, polyphenols, well-known dietary antioxidants, can act as pro-oxidants. We investigated the effect of 13 polyphenols and their metabolites on oxidative degradation of deoxyribose by an •OH generating Fenton system (Fe2+-ethylenediaminetetraacetic acid (EDTA)-H2O2). The relationship between phenolics pro-oxidant/anti-oxidant effects and their molecular structure was analyzed using multivariate analysis with multiple linear regression and a backward stepwise technique. Four phenolics revealed a significant inhibitory effect on OH-induced deoxyribose degradation, ranging from 54.4% ± 28.6% (3,4-dihydroxycinnamic acid) to 38.5% ± 10.4% (catechin) (n = 6), correlating with the number of -OH substitutions (r = 0.58). Seven phenolics augmented the oxidative degradation of deoxyribose with the highest enhancement at 95.0% ± 21.3% (quercetin) and 60.6% ± 12.2% (phloridzin). The pro-oxidant effect correlated (p < 0.05) with the number of -OH groups (r = 0.59), and aliphatic substitutes (r = -0.22) and weakly correlated with the occurrence of a catechol structure within the compound molecule (r = 0.17). Selective dietary supplementation with phenolics exhibiting pro-oxidant activity may increase the possibility of systemic oxidative stress in patients treated with medications containing chelating properties or those with high plasma concentrations of H2O2 and non-transferrin bound iron.

DNA polymerase λ inactivation by oxidized abasic sites.

Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase ß (Pol ß) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol ß and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol ß for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol ß, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.

Evaluation of in vivo T cell kinetics: use of heavy isotope labelling in type 1 diabetes.

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.

A hydroxyl-scavenging assay based on DNA damage in vitro.

Previous deoxyribose degradation method lacks biological relevance, specificity, and even reliability. In this study, a new hydroxyl radical-scavenging assay based on DNA damage is described. 2-Thiobarbituric acid-reactive substance (TBARS, λmax = 530 nm) was chosen as the biomarker of hydroxyl-mediated DNA damage. On the basis of systematic investigations into various factors affecting A530 nm and solvent interference, the experimental procedure was developed. The successful measurement of 30 selected antioxidants demonstrated that the proposed DNA damage method is reliable, simple, specific, and biologically relevant. It is suitable for all types of antioxidants in vitro.

Glycation of glutamate cysteine ligase by 2-deoxy-d-ribose and its potential impact on chemoresistance in glioblastoma.

The antioxidant glutathione (GSH) plays a critical role in maintaining intracellular redox homeostasis but in tumors the GSH biosynthetic pathway is often dysregulated, contributing to tumor resistance to radiation and chemotherapy. Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction in GSH synthesis, and enzyme function is controlled by GSH feedback inhibition or by transcriptional upregulation of the catalytic (GCLC) and modifier (GCLM) subunits. However, it has recently been reported that the activity of GCLC and the formation of GCL can be modified by reactive aldehyde products derived from lipid peroxidation. Due to the susceptibility of GCLC to posttranslational modifications by reactive aldehydes, we examined the potential for 2-deoxy-D-ribose (2dDR) to glycate GCLC and regulate enzyme activity and GCL formation. 2dDR was found to directly modify both GCLC and GCLM in vitro, resulting in a significant inhibition of GCLC and GCL enzyme activity without altering substrate affinity or feedback inhibition. 2dDR-mediated glycation also inhibited GCL subunit heterodimerization and formation of the GCL holoenzyme complex while not causing dissociation of pre-formed holoenzyme. This PTM could be of particular importance in glioblastoma (GBM) where intratumoral necrosis provides an abundance of thymidine, which can be metabolized by thymidine phosphorylase (TP) to form 2dDR. TP is expressed at high levels in human GBM tumors and shRNA knockdown of TP in U87 GBM cells results in a significant increase in cellular GCL enzymatic activity.

One-pot approach to functional nucleosides possessing a fluorescent group using nucleobase-exchange reaction by thymidine phosphorylase.

Herein, we describe ß-selective coupling between a modified uracil and a deoxyribose to produce functionalized nucleosides catalyzed by thymidine phosphorylase derived from Escherichia coli. This enzyme mediates nucleobase-exchange reactions to convert unnatural nucleosides possessing a large functional group such as a fluorescent molecule, coumarin or pyrene, linked via an alkyl chain at the C5 position of uracil. 5-(Coumarin-7-oxyhex-5-yn)uracil (C4U) displayed 57.2% conversion at 40% DMSO concentration in 1.0 mM phosphate buffer pH 6.8 to transfer thymidine to an unnatural nucleoside with C4U as the base. In the case of using 5-(pyren-1-methyloxyhex-5-yn)uracil (P4U) as the substrate, TP also could catalyse the reaction to generate a product with a very large functional group at 50% DMSO concentration (21.6% conversion). We carried out docking simulations using MF myPrest for the modified uracil bound to the active site of TP. The uracil moiety of the substrate binds to the active site of TP, with the fluorescent moiety linked to the C5 position of the nucleobase located outside the surface of the enzyme. As a consequence, the bulky fluorescent moiety binding to uracil has little influence on the coupling reaction.

Definition of the intermediates and mechanism of the anticancer drug bleomycin using nuclear resonance vibrational spectroscopy and related methods.

Bleomycin (BLM) is a glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage. The last detectable intermediate prior to DNA cleavage is a low spin Fe(III) peroxy level species, termed activated bleomycin (ABLM). DNA strand scission is initiated through the abstraction of the C-4' hydrogen atom of the deoxyribose sugar unit. Nuclear resonance vibrational spectroscopy (NRVS) aided by extended X-ray absorption fine structure spectroscopy and density functional theory (DFT) calculations are applied to define the natures of Fe(III)BLM and ABLM as (BLM)Fe(III)─OH and (BLM)Fe(III)(η(1)─OOH) species, respectively. The NRVS spectra of Fe(III)BLM and ABLM are strikingly different because in ABLM the δFe─O─O bending mode mixes with, and energetically splits, the doubly degenerate, intense O─Fe─N(ax) transaxial bends. DFT calculations of the reaction of ABLM with DNA, based on the species defined by the NRVS data, show that the direct H-atom abstraction by ABLM is thermodynamically favored over other proposed reaction pathways.

The dgt gene of Escherichia coli facilitates thymine utilization in thymine-requiring strains.

The Escherichia coli dGTP triphosphohydrolase (dGTPase) encoded by the dgt gene catalyses the hydrolysis of dGTP to deoxyguanosine and triphosphate. The recent discovery of a mutator effect associated with deletion of dgt indicated participation of the triphosphohydrolase in preventing mutagenesis. Here, we have investigated the possible involvement of dgt in facilitating thymine utilization through its ability to provide intracellular deoxyguanosine, which is readily converted by the DeoD phosphorylase to deoxyribose-1-phosphate, the critical intermediate that enables uptake and utilization of thymine. Indeed, we observed that the minimal amount of thymine required for growth of thymine-requiring (thyA) strains decreased with increased expression level of the dgt gene. As expected, this dgt-mediated effect was dependent on the DeoD purine nucleoside phosphorylase. We also observed that thyA strains experience growth difficulties upon nutritional shift-up and that the dgt gene facilitates adaptation to the new growth conditions. Blockage of the alternative yjjG (dUMP phosphatase) pathway for deoxyribose-1-phosphate generation greatly exacerbated the severity of thymine starvation in enriched media, and under these conditions the dgt pathway becomes crucial in protecting the cells against thymineless death. Overall, our results suggest that the dgt-dependent pathway for deoxyribose-1-phosphate generation may operate under various cell conditions to provide deoxyribosyl donors.

Cutting edge: C1q binds deoxyribose and heparan sulfate through neighboring sites of its recognition domain.

C1q, the recognition subunit of the C1 complex of complement, is an archetypal pattern recognition molecule with the striking ability to sense a wide variety of targets, including a number of altered self-motifs. The recognition properties of its globular domain were further deciphered by means of x-ray crystallography using deoxy-D-ribose and heparan sulfate as ligands. Highly specific recognition of deoxy-D-ribose, involving interactions with Arg C98, Arg C111, and Asn C113, was observed at 1.2 A resolution. Heparin-derived tetrasaccharide interacted more loosely through Lys C129, Tyr C155, and Trp C190. These data together with previous findings define a unique binding area exhibiting both polyanion and deoxy-D-ribose recognition properties, located on the inner face of C1q. DNA and heparin compete for C1q binding but are poor C1 activators compared with immune complexes. How the location of this binding area in C1q may regulate the level of C1 activation is discussed.

Effects of an aqueous extract of Orbignya phalerata Mart on locomotor activity and motor coordination in mice and as antioxidant in vitro.

The antioxidant activities of aqueous extract (AE) of Orbignya phalerata were assessed in vitro as well as its effect on locomotor activity and motor coordination in mice. AE does not possesses a strong antioxidant potential according to the scavenging assays; it also did not present scavenger activity in vitro. Following oral administration, AE (1, 2 and 3 g/kg) did not significantly change the motor activity of animals when compared with the control group, up to 24 h after administration and did not alter the remaining time of the animals on the Rota-rod apparatus. Further studies currently in progress will enable us to understand the mechanisms of action of the aqueous extract of Orbignya phalerata widely used in Brazilian flok medicine.

(±)-Catechin-A Mass-Spectrometry-Based Exploration Coordination Complex Formation with FeII and FeIII.

Catechin is an extensively investigated plant flavan-3-ol with a beneficial impact on human health that is often associated with antioxidant activities and iron coordination complex formation. The aim of this study was to explore these properties with FeII and FeIII using a combination of nanoelectrospray-mass spectrometry, differential pulse voltammetry, site-specific deoxyribose degradation assay, FeII autoxidation assay, and brine shrimp mortality assay. Catechin primarily favored coordination complex formation with Fe ions of the stoichiometry catechin:Fe in the ratio of 1:1 or 2:1. In the detected Fe-catechin coordination complexes, FeII prevailed. Differential pulse voltammetry, the site-specific deoxyribose degradation, and FeII autoxidation assays proved that coordination complex formation affected catechin's antioxidant effects. In situ formed Fe-catechin coordination complexes showed no toxic activities in the brine shrimp mortality assay. In summary, catechin has properties for the possible treatment of pathological processes associated with ageing and degeneration, such as Alzheimer's and Parkinson's diseases.

Fe binding properties of two soybean (Glycine max L.) LEA4 proteins associated with antioxidant activity.

Late embryogenesis abundant (LEA) group 4 (LEA4) proteins play an important role in the water stress tolerance of plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, the protective functions and mechanisms of LEA4 proteins are still not clear. In this study, the metal binding properties of two related soybean LEA4 proteins, GmPM1 and GmPM9, were tested using immobilized metal ion affinity chromatography (IMAC). The metal ions Fe(3+), Ni(2+), Cu(2+) and Zn(2+) were observed to bind these two proteins, while Ca(2+), Mg(2+) or Mn(2+) did not. Results from isothermal titration calorimetry (ITC) indicated that the binding affinity of GmPM1 for Fe(3+) was stronger than that of GmPM9. Hydroxyl radicals generated by the Fe(3+)/H(2)O(2) system were scavenged by both GmPM1 and GmPM9 in the absence or the presence of high ionic conditions (100 mM NaCl), although the scavenging activity of GmPM1 was significantly greater than that of GmPM9. These results suggest that GmPM1 and GmPM9 are metal-binding proteins which may function in reducing oxidative damage induced by abiotic stress in plants.

The sweetness of the DNA backbone drives Toll-like receptor 9.

The prevailing paradigm ascribes activation of Toll-like receptor 9 (TLR9) to the detection of CpG-motifs within pathogen derived DNA. However, new work ties natural phospho-diester (PD) DNA recognition by TLR9 to the detection of the DNA sugar backbone 2' deoxyribose. PD 2' deoxyribose homopolymers lacking DNA bases (abasic) are shown to act as TLR9 agonist while abasic phospho-thioate (PS) 2' deoxyribose functions as TLR9 antagonist. Alignment of bases to PD 2' deoxyribose enhanced its TLR9 agonistic function, while only CpG-motifs introduced to inhibitory PS 2' deoxyribose converted the antagonistic activity into powerful agonistic function. These new data thus restrict the CpG-motif dependency of TLR9 activation to the promising group of immunopharmacons that are based on PS modified synthetic DNA. They also show that natural PD DNA drives TLR9 activation sequence-independently as is the case for ds RNA recognizing TLR3 and ss RNA recognizing TLR7 and TLR8. Thus evolutionary pressure might have exiled nucleic acid recognizing TLRs such as TLR9 to endosomes in order to avoid activation by host (self) derived nucleic acids.

Antimicrobial peptides and self-DNA in autoimmune skin inflammation.

Toll-like receptor (TLR)-mediated detection of viral nucleic acids and production of type I interferons (IFNs) by plasmacytoid dendritic cells (pDCs) are key elements of antiviral defense. By contrast, inappropriate recognition of self-nucleic acids with induction of IFN responses in pDCs can lead to autoimmunity. In this review we describe how pDC responses to self-DNA are normally avoided and focus on our recent finding that in psoriasis, a common autoimmune disease of the skin, these barriers can be breached by the cationic antimicrobial peptide LL37. LL37 binds extracellular self-DNA fragments into aggregated particles that enter pDCs and trigger robust IFN responses by activating endosomal TLR9 as if they were viruses. We also describe the mechanisms that normally control production and activity of LL37 in human skin and propose that the persistent overexpression of LL37 in psoriasis leads to uncontrolled IFN responses that drive autoimmune skin inflammation.

Sugar metabolism, an additional virulence factor in enterobacteria.

Enterobacteria display a high level of flexibility in their fermentative metabolism. Biotyping assays have thus been developed to discriminate between clinical isolates. Each biotype uses one or more sugars more efficiently than the others. Recent studies show links between sugar metabolism and virulence in enterobacteria. In particular, mechanisms of carbohydrate utilization differ substantially between pathogenic and commensal E. coli strains. We are now starting to gain insight into the importance of this variability in metabolic function. Studies using various animal models of intestinal colonization showed that the presence of the fos and deoK loci involved in the metabolism of short-chain fructoligosaccharides and deoxyribose, respectively, help avian and human pathogenic E. coli to outcompete with the normal flora and colonize the intestine. Both PTS and non-PTS sugar transporters have been found to modulate virulence of extraintestinal pathogenic E. coli strains. The vpe, GimA, and aec35-37 loci contribute to bacterial virulence in vivo during experimental septicemia and urinary tract infection, meningitis, and colibacillosis, respectively. However, in most cases, the sugars metabolized, and the precise role of their utilization in the expression of bacterial virulence is still unknown. The massive development of powerful analytical methods over recent years will allow establishing the knowledge of the metabolic basis of bacterial pathogenesis that appears to be the next challenge in the field of infectious diseases.