Thermodynamic reaction control of nucleoside phosphorolysis

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.DFG, 390540038, EXC 2008: UniSysCatTU Berlin, Open-Access-Mittel - 201

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.DFG, 390540038, EXC 2008: UniSysCatDFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

The two α-dox genes of Nicotiana attenuata: overlapping but distinct functions in development and stress responses

<p>Abstract</p> <p>Background</p> <p>Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In <it>Nicotiana attenuata</it>, herbivory strongly induces the expression of an <it>α-dox1 </it>gene. To determine its role, we silenced its expression using <it>Agrobacterium</it>-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second <it>α-dox </it>isoform. This led us to identify the corresponding <it>α-dox2 </it>gene in <it>N. attenuata </it>and examine the regulation of both <it>α-dox </it>genes as well as the consequences of their silencing in plant development and anti-herbivore defense.</p> <p>Results</p> <p>The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both <it>α-dox </it>genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for <it>α-dox1</it>, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product <it>in planta </it>(2-HOT) and showed no signs of oxidative stress. In contrast to <it>α-dox1</it>, the expression of <it>α-dox2 </it>gene is not induced by wounding or elicitors in the oral secretions of <it>Manduca sexta</it>. Instead, <it>α-dox2 </it>is expressed in roots and flowers which lack <it>α-dox1 </it>expression, but both genes are equally regulated during leaf maturation. We transiently silenced <it>α-dox </it>gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of <it>α-dox1 </it>or <it>α-dox2 </it>had no effects on plant growth, the co-suppression of both <it>α-dox </it>genes decreased plant growth. Plants transiently silenced for both <it>α-dox </it>genes had increased constitutive levels of JA and ABA but silencing <it>α-dox1 </it>alone resulted in lower <it>M. sexta</it>-induced levels of JA, 2-HOT and ABA.</p> <p>Conclusions</p> <p>Thus, both <it>α-dox </it>isoforms function in the development of <it>N. attenuata</it>. In leaf maturation, the two <it>α-dox </it>genes have overlapping functions, but only <it>α-dox2 </it>is involved in root and flower development and only <it>α-dox1 </it>functions in anti-herbivore defense.</p

A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases

Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.DFG, 390540038, EXC 2008: UniSysCa

The hazards of genotype imputation in chromosomal regions under selection: A case study using the Lactase gene region

Although imputation of missing SNP results has been widely used in genetic studies, claims about the quality and usefulness of imputation have outnumbered the few studies that have questioned its limitations. But it is becoming clear that these limitations are real—for example, disease association signals can be missed in regions of LD breakdown. Here, as a case study, using the chromosomal region of the well-known lactase gene, LCT, we address the issue of imputation in the context of variants that have become frequent in a limited number of modern population groups only recently, due to selection. We study SNPs in a 500 bp region covering the enhancer of LCT, and compare imputed genotypes with directly genotyped data. We examine the haplotype pairs of all individuals with discrepant and missing genotypes. We highlight the nonrandom nature of the allelic errors and show that most incorrect imputations and missing data result from long haplotypes that are evolutionarily closely related to those carrying the derived alleles, while some relate to rare and recombinant haplotypes. We conclude that bias of incorrectly imputed and missing genotypes can decrease the accuracy of imputed results substantially