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.

Quality Data from Messy Spectra: How Isometric Points Increase Information Content in Highly Overlapping Spectra.

Spectroscopic techniques are immensely useful for obtaining information about chemical transformations while they are happening. However, such data are often messy, and it is challenging to extract reliable information from them without careful calibrations or internal standards. This short introductory review discusses how isometric points (points in a spectrum where the signal intensity remains constant throughout the progress of a chemical transformation) can be used to derive high-quality data from messy spectra. Such analyses are helpful in a variety of (bio-)chemical settings, as selected case studies demonstrate.

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.

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.

G-type Halohydrin Dehalogenases Catalyze Ring Opening Reactions of Cyclic Epoxides with Diverse Anionic Nucleophiles.

Halohydrin dehalogenases are promiscuous biocatalysts, which enable asymmetric ring opening reactions of epoxides with various anionic nucleophiles. However, despite the increasing interest in such asymmetric transformations, the substrate scope of G-type halohydrin dehalogenases toward cyclic epoxides has remained largely unexplored, even though this subfamily is the only one known to display activity with these sterically demanding substrates. Herein, we report on the exploration of the substrate scope of the two G-type halohydrin dehalogenases HheG and HheG2 and a newly identified, more thermostable member of the family, HheG3, with a variety of sterically demanding cyclic epoxides and anionic nucleophiles. This work shows that, in addition to azide and cyanide, these enzymes facilitate ring-opening reactions with cyanate, thiocyanate, formate, and nitrite, significantly expanding the known repertoire of accessible transformations.

Chemo-enzymatic synthesis of natural products and their analogs.

Enzymes continue to gain recognition as valuable tools in synthetic chemistry as they enable transformations, which elude conventional organochemical approaches. As such, the progressing expansion of the biocatalytic arsenal has introduced unprecedented opportunities for new synthetic strategies and retrosynthetic disconnections. As a result, enzymes have found a solid foothold in modern natural product synthesis for applications ranging from the generation of early chiral synthons to endgame transformations, convergent synthesis, and cascade reactions for the rapid construction of molecular complexity. As a primer to the state-of-the-art concerning strategic uses of enzymes in natural product synthesis and the underlying concepts, this review highlights selected recent literature examples, which make a strong case for the admission of enzymatic methodologies into the standard repertoire for complex small-molecule synthesis.

Alternative Assay Reagents for UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module.

This report advises against the use of 5-iodoridine or 5-ethynyluridine as alternative assay reagents in the PUB module, primarily due to their lack of an isosbestic point of phosphorolysis under moderately alkaline conditions.

The effects of positive end-expiratory pressure on cardiac function: a comparative echocardiography-conductance catheter study.

BACKGROUND: Echocardiographic parameters of diastolic function depend on cardiac loading conditions, which are altered by positive pressure ventilation. The direct effects of positive end-expiratory pressure (PEEP) on cardiac diastolic function are unknown. METHODS: Twenty-five patients without apparent diastolic dysfunction undergoing coronary angiography were ventilated noninvasively at PEEPs of 0, 5, and 10 cmH2O (in randomized order). Echocardiographic diastolic assessment and pressure-volume-loop analysis from conductance catheters were compared. The time constant for pressure decay (τ) was modeled with exponential decay. End-diastolic and end-systolic pressure volume relationships (EDPVRs and ESPVRs, respectively) from temporary caval occlusion were analyzed with generalized linear mixed-effects and linear mixed models. Transmural pressures were calculated using esophageal balloons. RESULTS: τ values for intracavitary cardiac pressure increased with the PEEP (n = 25; no PEEP, 44 ± 5 ms; 5 cmH2O PEEP, 46 ± 6 ms; 10 cmH2O PEEP, 45 ± 6 ms; p < 0.001). This increase disappeared when corrected for transmural pressure and diastole length. The transmural EDPVR was unaffected by PEEP. The ESPVR increased slightly with PEEP. Echocardiographic mitral inflow parameters and tissue Doppler values decreased with PEEP [peak E wave (n = 25): no PEEP, 0.76 ± 0.13 m/s; 5 cmH2O PEEP, 0.74 ± 0.14 m/s; 10 cmH2O PEEP, 0.68 ± 0.13 m/s; p = 0.016; peak A wave (n = 24): no PEEP, 0.74 ± 0.12 m/s; 5 cmH2O PEEP, 0.7 ± 0.11 m/s; 10 cmH2O PEEP, 0.67 ± 0.15 m/s; p = 0.014; E' septal (n = 24): no PEEP, 0.085 ± 0.016 m/s; 5 cmH2O PEEP, 0.08 ± 0.013 m/s; 10 cmH2O PEEP, 0.075 ± 0.012 m/s; p = 0.002]. CONCLUSIONS: PEEP does not affect active diastolic relaxation or passive ventricular filling properties. Dynamic echocardiographic filling parameters may reflect changing loading conditions rather than intrinsic diastolic function. PEEP may have slight positive inotropic effects. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02267291 , registered 17. October 2014.

Coloring Chemistry-How Mindful Color Choices Improve Chemical Communication.

Color is a central element to scientific communication, but its use comes with the responsibility to ensure universally accessible and accurate data presentation. This short Viewpoint Article aims to sensitize the chemical community to the importance of mindful color choices in scientific illustrations.

UV-Spectroscopic Detection of (Pyro-)Phosphate with the PUB Module.

Despite the prevalence of ortho- and pyrophosphate in biochemistry, operationally simple and versatile high-throughput methodologies for their quantification are lacking. We herein introduce PUB, a module for phosphate detection by continuous UV-spectroscopic monitoring of 5-bromouridine phosphorolysis. The PUB module uses cheaply available, bench-stable reagents and can be employed for continuous and discontinuous reaction monitoring in biochemical assays to detect (pyro-)phosphate concentrations spanning almost 4 orders of magnitude, as demonstrated with representative use cases.

Two-Phase Biocatalysis in Microfluidic Droplets.

This Perspective discusses the literature related to two-phase biocatalysis in microfluidic droplets. Enzymes used as catalysts in biocatalysis are generally less stable in organic media than in their native aqueous environments; however, chemical and pharmaceutical compounds are often insoluble in water. The use of aqueous/organic two-phase media provides a solution to this problem and has therefore become standard practice for multiple biotransformations. In batch, two-phase biocatalysis is limited by mass transport, a limitation that can be overcome with the use of microfluidic systems. Although, two-phase biocatalysis in laminar flow systems has been extensively studied, microfluidic droplets have been primarily used for enzyme screening. In this Perspective, we summarize the limited published work on two-phase biocatalysis in microfluidic droplets and discuss the limitations, challenges, and future perspectives of this technology.

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.

Estimating cardiac output based on gas exchange during veno-arterial extracorporeal membrane oxygenation in a simulation study using paediatric oxygenators.

Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) therapy is a rescue strategy for severe cardiopulmonary failure. The estimation of cardiac output during VA-ECMO is challenging. A lung circuit ([Formula: see text]Lung) and an ECMO circuit ([Formula: see text]ECMO) with oxygenators for CO2 removal ([Formula: see text]CO2) and O2 uptake ([Formula: see text]O2) simulated the setting of VA-ECMO with varying ventilation/perfusion ([Formula: see text]/[Formula: see text]) ratios and shunt. A metabolic chamber with a CO2/N2 blend simulated [Formula: see text]CO2 and [Formula: see text]O2. [Formula: see text] Lung was estimated with a modified Fick principle: [Formula: see text]Lung = [Formula: see text]ECMO × ([Formula: see text] CO2 or [Formula: see text]O2Lung)/([Formula: see text]CO2 or [Formula: see text]O2ECMO). A normalization procedure corrected [Formula: see text]CO2 values for a [Formula: see text]/[Formula: see text] of 1. Method agreement was evaluated by Bland-Altman analysis. Calculated [Formula: see text]Lung using gaseous [Formula: see text]CO2 and [Formula: see text]O2 correlated well with measured [Formula: see text]Lung with a bias of 103 ml/min [- 268 to 185] ml/min; Limits of Agreement: - 306 ml/min [- 241 to - 877 ml/min] to 512 ml/min [447 to 610 ml/min], r2 0.85 [0.79-0.88]). Blood measurements of [Formula: see text]CO2 showed an increased bias (- 260 ml/min [- 1503 to 982] ml/min), clinically not applicable. Shunt and [Formula: see text]/[Formula: see text] mismatch decreased the agreement of methods significantly. This in-vitro simulation shows that [Formula: see text]CO2 and [Formula: see text]O2 in steady-state conditions allow for clinically applicable calculations of [Formula: see text]Lung during VA-ECMO therapy.

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.

Corrigendum: Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.

[This corrects the article DOI: 10.3389/fbioe.2020.00854.].

Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.

Nucleoside-5'-triphosphates (NTPs) and their analogs are building blocks of DNA and are important compounds in both pharmaceutical and molecular biology applications. Currently, commercially available base or sugar modified NTPs are mainly synthesized chemically. Since the chemical production of NTPs is time-consuming and generally inefficient, alternative approaches are under development. Here we present a simple, efficient and generalizable enzymatic synthesis method for the conversion of nucleosides to NTPs. Our one-pot method is modular, applicable to a wide range of natural and modified nucleotide products and accesses NTPs directly from cheap nucleoside precursors. Nucleoside kinases, nucleoside monophosphate (NMP) kinases and a nucleoside diphosphate (NDP) kinase were applied as biocatalysts. Enzymes with different substrate specificities were combined to produce derivatives of adenosine and cytidine triphosphate with conversions of 4 to 26%. The implementation of a (deoxy)ATP recycling system resulted in a significant increase in the conversion to all NTP products, furnishing 4 different NTPs in quantitative conversion. Natural (deoxy)NTPs were synthesized with 60 to >99% conversion and sugar- and base-modified NTPs were produced with 69 to >99% and 27 to 75% conversion, respectively. The presented method is suitable for the efficient synthesis of a wide range of natural and modified NTPs in a sustainable one-pot process.

Spectral Unmixing-Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases.

The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC-like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.