Ribonucleotide Synthesis Research Articles

Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress.

Isolation, purification and characterization of 5'-phosphodiesterase from Aspergillus fumigatus.

5′-Phosphodiesterase (5′-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5′-guanosine monophosphate and 5′-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from Aspergillus fumigatus. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe3+, Cu2+, and Zn2+, but slightly improved by the presence of K+ and Na+. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5′-PDE active site. Furthermore, purified 5′-PDE could specifically catalyze the synthesis of ribonucleotides with a Vmax 0.71 mmol/mg·min and a KM of 13.60 mg/mL.

Scalable Synthesis of 2,2′-Anhydro-arabinofuranosyl Imidazoles

We report the efficient and scalable synthesis of 2,2′-anhydro-5-amino-1-β-arabinofuranosylimidazole-4-carboxamide and 2,2′-anhydro-5-amino-1-β-arabinofuranosylimidazole-4-carbonitrile from commercial arabino-adenosine. 2,2′-Anhydro-5-amino-1-β-arabinofuranosylimidazole-4-carboxamide is synthesised in only five steps with a single chromatographic purification. Additionally, we report a high-yielding, three-step conversion of 2,2′-anhydro-5-amino-1-β-arabinofuranosylimidazole-4-carboxamide into 2,2′-anhydro-5-amino-1-β-arabinofuranosylimidazole-4-carbonitrile. They are proposed key intermediates of the divergent prebiotic synthesis of ribonucleotides and this facile synthesis is anticipated to be instrumental in continued investigation of the origins of nucleotides.

The Surface UV Environment on Planets Orbiting M Dwarfs: Implications for Prebiotic Chemistry and the Need for Experimental Follow-up

Abstract Potentially habitable planets orbiting M dwarfs are of intense astrobiological interest because they are the only rocky worlds accessible to biosignature search over the next 10+ years because of a confluence of observational effects. Simultaneously, recent experimental and theoretical work suggests that UV light may have played a key role in the origin of life on Earth, especially the origin of RNA. Characterizing the UV environment on M-dwarf planets is important for understanding whether life as we know it could emerge on such worlds. In this work, we couple radiative transfer models to observed M-dwarf spectra to determine the UV environment on prebiotic Earth-analog planets orbiting M dwarfs. We calculate dose rates to quantify the impact of different host stars on prebiotically important photoprocesses. We find that M-dwarf planets have access to 100–1000 times less bioactive UV fluence than the young Earth. It is unclear whether UV-sensitive prebiotic chemistry that may have been important to abiogenesis, such as the only known prebiotically plausible pathways for pyrimidine ribonucleotide synthesis, could function on M-dwarf planets. This uncertainty affects objects like the recently discovered habitable-zone planets orbiting Proxima Centauri, TRAPPIST-1, and LHS 1140. Laboratory studies of the sensitivity of putative prebiotic pathways to irradiation level are required to resolve this uncertainty. If steady-state M-dwarf UV output is insufficient to power these pathways, transient elevated UV irradiation due to flares may suffice; laboratory studies can constrain this possibility as well.

Prebiotic selection and assembly of proteinogenic amino acids and natural nucleotides from complex mixtures

2-aminothiazole — a hybrid of prebiotic amino acid and nucleotide precursors — sequentially accumulates and purifies glycolaldehyde and glyceraldehyde from complex mixtures in the order required for ribonucleotide synthesis, dynamically resolves glyceraldehyde from its ketose-isomer dihydroxyacetone, and provides the first strategy to select natural amino acids from abiotic aldehydes and ketones.

Quantitation of Teriflunomide in Human Serum/Plasma Across a 40,000-Fold Concentration Range by LC/MS/MS.

Leflunomide is a prodrug used primarily for treatment of rheumatoid arthritis. The active metabolite, teriflunomide (A77 1726), inhibits the enzyme dihydroorotate dehydrogenase and thereby reduces the synthesis of pyrimidine ribonucleotides. Teriflunomide is also administered directly and finds use in treating multiple sclerosis. Therapeutic concentrations are generally in the tens of μg/mL serum or plasma and, due to adverse effects and the time required to reach steady state, therapeutic drug monitoring is beneficial. The drug is also a potential teratogen. A method was developed and validated to quantify the drug teriflunomide over a 40,000-fold concentration range of 5 ng/mL to 200 μg/mL in serum or plasma. This is accomplished by dividing the quantitative range into two separate but overlapping regions; a high curve and a low curve range. Samples are evaluated first against the high curve after a 100-fold dilution of the sample extract. Samples falling below the upper curve region are evaluated again without dilution and quantified, if possible, against the low curve calibration standards. Appropriate choice of a concentration for the deuterated internal standard (D4-teriflunomide) allows for a single, identical, extraction procedure to be performed for both curve regions but with the dilution performed for high curve samples. The method is rugged and reliable with good accuracy and precision statistics.

Influence of the UV Environment on the Synthesis of Prebiotic Molecules

Ultraviolet radiation is common to most planetary environments and could play a key role in the chemistry of molecules relevant to abiogenesis (prebiotic chemistry). In this work, we explore the impact of UV light on prebiotic chemistry that might occur in liquid water on the surface of a planet with an atmosphere. We consider effects including atmospheric absorption, attenuation by water, and stellar variability to constrain the UV input as a function of wavelength. We conclude that the UV environment would be characterized by broadband input, and wavelengths below 204 nm and 168 nm would be shielded out by atmospheric CO2 and water, respectively. We compare this broadband prebiotic UV input to the narrowband UV sources (e.g., mercury lamps) often used in laboratory studies of prebiotic chemistry and explore the implications for the conclusions drawn from these experiments. We consider as case studies the ribonucleotide synthesis pathway of Powner et al. (2009) and the sugar synthesis pathway of Ritson and Sutherland (2012). Irradiation by narrowband UV light from a mercury lamp formed an integral component of these studies; we quantitatively explore the impact of more realistic UV input on the conclusions that can be drawn from these experiments. Finally, we explore the constraints solar UV input places on the buildup of prebiotically important feedstock gasses like CH4 and HCN. Our results demonstrate the importance of characterizing the wavelength dependence (action spectra) of prebiotic synthesis pathways to determine how pathways derived under laboratory irradiation conditions will function under planetary prebiotic conditions.

Aldehydes and sugars from evolved precometary ice analogs: Importance of ices in astrochemical and prebiotic evolution

Evolved interstellar ices observed in dense protostellar molecular clouds may arguably be considered as part of precometary materials that will later fall on primitive telluric planets, bringing a wealth of complex organic compounds. In our laboratory, experiments reproducing the photo/thermochemical evolution of these ices are routinely performed. Following previous amino acid identifications in the resulting room temperature organic residues, we have searched for a different family of molecules of potential prebiotic interest. Using multidimensional gas chromatography coupled to time-of-flight mass spectrometry, we have detected 10 aldehydes, including the sugar-related glycolaldehyde and glyceraldehyde--two species considered as key prebiotic intermediates in the first steps toward the synthesis of ribonucleotides in a planetary environment. The presence of ammonia in water and methanol ice mixtures appears essential for the recovery of these aldehydes in the refractory organic residue at room temperature, although these products are free of nitrogen. We finally point out the importance of detecting aldehydes and sugars in extraterrestrial environments, in the gas phase of hot molecular clouds, and, more importantly, in comets and in primitive meteorites that have most probably seeded the Earth with organic material as early as 4.2 billion years ago.

Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons

Abstract We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D2). Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996) J. Am. Chem. Soc. 118, 7929–7940). Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL) RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL.

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein.

Synthesis of aldehydic ribonucleotide and amino acid precursors by photoredox chemistry.

Synthesis of aldehydic ribonucleotide and amino acid precursors by photoredox chemistry.

Abstract 3681: Aspirin acetylates glucose 6 phosphate dehydrogenase and inhibits its activity in colon cancer cells.

Abstract Epidemiological studies have demonstrated a significant correlation between regular aspirin use and reduced colon cancer incidence and mortality. Although a consensus is emerging on the view that aspirin has significant anti-cancer properties, it is not yet clear which pathways or molecular targets might be involved. We hypothesized that aspirin's anti-cancer effect may occur through acetylation of a panel of protein targets, including novel candidates with modulated functional activity, as a result of aspirin-mediated acetylation. In the present study, using anti-acetyl lysine antibody, we determined aspirin-mediated protein acetylation profiles in three different colon cancer cell lines namely HCT-116, HT-29 and GC3/c1, by treating cells with aspirin at different concentration, and analyzing them in Western Blot. We observed that in all cell lines tested, aspirin induced acetylation of multiple proteins in a concentration dependent fashion spanning a molecular weight range from 20 to 200 kDa. Aspirin's acetylation targets included the tumor suppressor protein p53, and glucose 6 phosphate dehydrogenase (G6PD), an enzyme in the pentose phosphate pathway. Mass spectrometry analysis of the recombinant G6PD, treated with aspirin in vitro, revealed acetylation of up to 14 distinct lysine residues, including lysine 235 at its active site. In vitro G6PD assay in lysates isolated from the cancer cell lines showed that aspirin caused a 70% decrease in the G6PD enzyme activity in HCT116 cells; however, the reduction in the enzyme activity was less in HT-29/GC3/c1 cells (up to 30%). Inhibition of G6PD activity leading to a reduction in ribonucleotide synthesis and cell proliferation may represent an important mechanism by which aspirin exerts its anticancer effects. Citation Format: Guoqiang Ai, Fred K. Hagen, Jayarama B. Gunaje. Aspirin acetylates glucose 6 phosphate dehydrogenase and inhibits its activity in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3681. doi:10.1158/1538-7445.AM2013-3681

Structural Phylogenomics Reveals Gradual Evolutionary Replacement of Abiotic Chemistries by Protein Enzymes in Purine Metabolism

The origin of metabolism has been linked to abiotic chemistries that existed in our planet at the beginning of life. While plausible chemical pathways have been proposed, including the synthesis of nucleobases, ribose and ribonucleotides, the cooption of these reactions by modern enzymes remains shrouded in mystery. Here we study the emergence of purine metabolism. The ages of protein domains derived from a census of fold family structure in hundreds of genomes were mapped onto enzymes in metabolic diagrams. We find that the origin of the nucleotide interconversion pathway benefited most parsimoniously from the prebiotic formation of adenine nucleosides. In turn, pathways of nucleotide biosynthesis, catabolism and salvage originated ∼300 million years later by concerted enzymatic recruitments and gradual replacement of abiotic chemistries. Remarkably, this process led to the emergence of the fully enzymatic biosynthetic pathway ∼3 billion years ago, concurrently with the appearance of a functional ribosome. The simultaneous appearance of purine biosynthesis and the ribosome probably fulfilled the expanding matter-energy and processing needs of genomic information.

A BH3 masked H-phosphonate for coupling in oligonucleotide synthesis

A method of masking 3′-H-phosphonate group for the solution-phase synthesis of ribonucleotide by H-phosphonate approach was described. The phosphonic acid group was masked by bis-(2-cyanoethyl) boranophosphate during the reactions. After a successive demasking treatment by triethylamine, trityl cation, and triethylamine, the triethylammonium 3′-H-phosphonate nucleotide can be obtained efficiently ready for the coupling cycle in synthesis of oligonucleotide.

Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase

Bacillus anthracis is the causative agent of anthrax, which is associated with a high mortality rate. Like several medically important bacteria, B. anthracis lacks glutathione but encodes many genes annotated as thioredoxins, thioredoxin reductases, and glutaredoxin-like proteins. We have cloned, expressed, and characterized three potential thioredoxins, two potential thioredoxin reductases, and three glutaredoxin-like proteins. Of these, thioredoxin 1 (Trx1) and NrdH reduced insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and the manganese-containing type Ib ribonucleotide reductase (RNR) from B. anthracis in the presence of NADPH and thioredoxin reductase 1 (TR1), whereas thioredoxin 2 (Trx2) could only reduce DTNB. Potential TR2 was verified as an FAD-containing protein reducible by dithiothreitol but not by NAD(P)H. The recently discovered monothiol bacillithiol did not work as a reductant for RNR, either directly or via any of the redoxins. The catalytic efficiency of Trx1 was 3 and 20 times higher than that of Trx2 and NrdH, respectively, as substrates for TR1. Additionally, the catalytic efficiency of Trx1 as an electron donor for RNR was 7-fold higher than that of NrdH. In extracts of B. anthracis, Trx1 was responsible for almost all of the disulfide reductase activity, whereas Western blots showed that the level of Trx1 was 15 and 60 times higher than that of Trx2 and NrdH, respectively. Our findings demonstrate that the most important general disulfide reductase system in B. anthracis is TR1/Trx1 and that Trx1 is the physiologically relevant electron donor for RNR. This information may provide a basis for the development of novel antimicrobial therapies targeting this severe pathogen.

Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets

Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. Invitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis

Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes.Analysis showed that in hemoplasmas genes involved in carbohydrate metabolism are limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source. The majority of the pentose phosphate pathway enzymes that catalyze the de novo synthesis of ribonucleotides were absent, as were cell division protein FtsZ and chaperonins GroEL/ES. Uncharacterized protein paralogs containing putative surface expression motifs, comprised 62% of M. haemofelis and 19% of Mycoplasma suis genome coverage respectively, the majority of which were present in a small number of unstructured islands. Limited mass spectrometry and immunoblot data matched a number of characterized proteins and uncharacterized paralogs, confirming their expression and immunogenicity in vivo.These data have allowed further characterization of these important pathogens, including their limited metabolic capabilities, which may contribute to their uncultivatable status. A number of immunogenic proteins, and a potential mechanism for host immune system evasion, have been identified.

A Missing Prebiotic Link: Discovery of a Plausible Synthesis of Pyrimidine Nucleotides

Back to the future: The synthesis of pyrimidine ribonucleotides under plausibly prebiotic conditions is revisited with a new outcome.

Mechanism of biological effect of the delta-sleep-inducing peptide includes activation of deoxyribonucleotide synthesis

To find out the mechanism of oncoprotective effects of the delta-sleep-inducing peptide, we have studied the dynamics of ribonucleotide reductase activity, which is a vital enzyme in the DNA biosynthesis, using electron paramagnetic resonance (EPR), and a concentration of low molecular mass iron complexes in the murine spleen after intraperitoneal injection of the peptide and the antitumor preparation methylnitrosourea. The data obtained give evidence on the different effects on the ribonucleotide synthesis system by the substances under study. The peptide induced significant activation of the enzyme as a consequence of activation of processes of protein synthesis. Methylnitrosourea injection leads to a decrease in the concentration of the ribonucleotide reductase active form due to destruction of the enzyme active center. The prospects of the study are discussed.

Forming phosphates

Support for RNA as the origin of life is provided by the synthesis of pyrimidine ribonucleotides under plausible prebiotic conditions