Nucleoside Phosphorylases make N7-xanthosine.

Modern, highly evolved nucleoside-processing enzymes are known to exhibit perfect regioselectivity over the glycosylation of purine nucleobases at N9. We herein report an exception to this paradigm. Wild-type nucleoside phosphorylases also furnish N7-xanthosine, a "non-native" ribosylation regioisomer of xanthosine. This unusual nucleoside possesses several atypical physicochemical properties such as redshifted absorption spectra, a high equilibrium constant of phosphorolysis and low acidity. Ultimately, the biosynthesis of this previously unknown natural product illustrates how even highly evolved, essential enzymes from primary metabolism are imperfect catalysts.

Thermal segment microwell plate control for automated liquid handling setups.

Automated high-throughput liquid handling operations in biolabs necessitate miniaturised and automatised equipment for effective space utilisation and system integration. This paper presents a thermal segment microwell plate control unit designed for enhanced microwell-based experimentation in liquid handling setups. The development of this device stems from the need to move towards geometry standardization and system integration of automated lab equipment. It incorporates features based on Smart Sensor and Sensor 4.0 concepts. An enzymatic activity assay is implemented with the developed device on a liquid handling station, allowing fast characterisation via a high-throughput approach. The device outperforms other comparable devices in certain metrics based on automated liquid handling requirements and addresses the needs of future biolabs in automation, especially in high-throughput screening.

Purine nucleoside antibiotics: recent synthetic advances harnessing chemistry and biology.

Covering: 2019 to 2023Nucleoside analogues represent one of the most important classes of small molecule pharmaceuticals and their therapeutic development is successfully established within oncology and for the treatment of viral infections. However, there are currently no nucleoside analogues in clinical use for the management of bacterial infections. Despite this, a significant number of clinically recognised nucleoside analogues are known to possess some antibiotic activity, thereby establishing a potential source for new therapeutic discovery in this area. Furthermore, given the rise in antibiotic resistance, the discovery of new clinical candidates remains an urgent global priority and natural product-derived nucleoside analogues may also present a rich source of discovery space for new modalities. This Highlight, covering work published from 2019 to 2023, presents a current perspective surrounding the synthesis of natural purine nucleoside antibiotics. By amalgamating recent efforts from synthetic chemistry with advances in biosynthetic understanding and the use of recombinant enzymes, prospects towards different structural classes of purines are detailed.

Bioprocess development to produce a hyperthermostable S-methyl-5'-thioadenosine phosphorylase in Escherichia coli.

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5'-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1-90.1 g L-1 were observed together with an overproduction of ApMTAP in a 1.9%-3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg-1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases.

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T. maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T. maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

Generation of a Perfusable 3D Lung Cancer Model by Digital Light Processing.

Lung cancer still has one of the highest morbidity and mortality rates among all types of cancer. Its incidence continues to increase, especially in developing countries. Although the medical field has witnessed the development of targeted therapies, new treatment options need to be developed urgently. For the discovery of new drugs, human cancer models are required to study drug efficiency in a relevant setting. Here, we report the generation of a non-small cell lung cancer model with a perfusion system. The bioprinted model was produced by digital light processing (DLP). This technique has the advantage of including simulated human blood vessels, and its simple assembly and maintenance allow for easy testing of drug candidates. In a proof-of-concept study, we applied gemcitabine and determined the IC50 values in the 3D models and 2D monolayer cultures and compared the response of the model under static and dynamic cultivation by perfusion. As the drug must penetrate the hydrogel to reach the cells, the IC50 value was three orders of magnitude higher for bioprinted constructs than for 2D cell cultures. Compared to static cultivation, the viability of cells in the bioprinted 3D model was significantly increased by approximately 60% in the perfusion system. Dynamic cultivation also enhanced the cytotoxicity of the tested drug, and the drug-mediated apoptosis was increased with a fourfold higher fraction of cells with a signal for the apoptosis marker caspase-3 and a sixfold higher fraction of cells positive for PARP-1. Altogether, this easily reproducible cancer model can be used for initial testing of the cytotoxicity of new anticancer substances. For subsequent in-depth characterization of candidate drugs, further improvements will be necessary, such as the generation of a multi-cell type lung cancer model and the lining of vascular structures with endothelial cells.

Production of Modified Nucleosides in a Continuous Enzyme Membrane Reactor.

Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2'-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2'-deoxyribonucleoside and 6-chloro-2-fluoro-2'-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2'-deoxyadenosine, 2,6-dichloropurine-2'-deoxyribonucleoside, and 6-chloro-2-fluoro-2'-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.

Biased Borate Esterification during Nucleoside Phosphorylase-Catalyzed Reactions: Apparent Equilibrium Shifts and Kinetic Implications.

Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have evolved into a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an additional means for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides and ribosyl phosphates with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase.

Optimization of Inclusion Body Formation and Purification in Multi-well Plates.

Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5'-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.

Industrial potential of the enzymatic synthesis of nucleoside analogs: existing challenges and perspectives.

Nucleoside phosphorylases have progressed from an enzymatic curiosity to a viable synthetic tool. However, despite the recent advances in nucleoside phosphorylase-catalyzed nucleoside synthesis, the widespread application of these enzymes in industrial processes is still lacking. We attribute this gap to three key challenges, which are outlined in this short review. To address these persistent obstacles, we believe that biocatalytic nucleoside synthesis needs to embrace interdisciplinary partnerships with the fields of organic chemistry, process engineering, and flow chemistry.

A Comprehensive IT Infrastructure for an Enzymatic Product Development in a Digitalized Biotechnological Laboratory.

Typical product development in biotechnological laboratories is a distributed and versatile process. Today's biotechnological laboratory devices are usually equipped with multiple sensors and a variety of interfaces. The existing software for biotechnological research and development is often specialized on specific tasks and thus generates task-specific information. Scientific personnel is confronted with an abundance of information from a variety of sources. Hence a comprehensive software backbone that structures the developmental process and maintains data from various sources is missing. Thus, it is not possible to maintain data access, documentation, reporting, availability, and proper data exchange. This chapter envisions a comprehensive digital infrastructure handling the data throughout an enzymatic product development process in a laboratory. The platform integrates a variety of software products, databases, and devices to make all product development life cycle (PDLC) data available and accessible to the scientific staff.

Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases.

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0-500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 12 modified) substrates. The screening results correlated well with literature data, and additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

Thermostable adenosine 5'-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies.

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5'-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2-6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius.

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

Optimized Biocatalytic Synthesis of 2-Selenopyrimidine Nucleosides by Transglycosylation*.

Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2-selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pH 9 under hypoxic conditions to improve the solubility and stability of the 2-Se-nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2-Se-pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40-48 % conversion to the target nucleoside. Scale-up of the optimized conditions provided four Se-containing nucleosides in 6-40 % isolated yield, which compares favorably to established chemical routes.

The Peculiar Case of the Hyper-thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus*.

The poor solubility of many nucleosides and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes that can withstand these conditions. Herein, we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space (up to 80 % (v/v) nonaqueous medium), and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 106 for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.

Kinetic Analysis of the Hydrolysis of Pentose-1-phosphates through Apparent Nucleoside Phosphorolysis Equilibrium Shifts*.

Herein, we report an addition to the toolbox for the monitoring and quantification of the hydrolytic decay of pentose-1-phosphates, which are known to be elusive and difficult to quantify. This communication describes how apparent equilibrium shifts of a nucleoside phosphorolysis reaction can be employed to calculate hydrolytic loss of pentose-1-phosphates based on the measurement of post-hydrolysis equilibrium concentrations of a nucleoside and a nucleobase. To demonstrate this approach, we assessed the stability of the relatively stable ribose-1-phosphate at 98 °C and found half-lives of 1.8-11.7 h depending on the medium pH. This approach can be extended to other sugar phosphates and related reaction systems to quantify the stability of UV-inactive and hard-to-detect reaction products and intermediates.