Editorial

3 pages, 208 KiB

Open AccessEditorial

Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides

by Jesús Fernández-Lucas

Biomolecules 2021, 11(8), 1147; https://doi.org/10.3390/biom11081147 - 03 Aug 2021

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Nucleic acid derivatives are involved in cell growth and replication, but they are also particularly important as building blocks for RNA and DNA synthesis [...] Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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12 pages, 2294 KiB

Open AccessEditor’s ChoiceArticle

Enzymatic and Chemical Syntheses of Vacor Analogs of Nicotinamide Riboside, NMN and NAD

by Lars Jansen Sverkeli, Faisal Hayat, Marie E. Migaud and Mathias Ziegler

Biomolecules 2021, 11(7), 1044; https://doi.org/10.3390/biom11071044 - 16 Jul 2021

Cited by 11 | Viewed by 3675

Abstract

It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide [...] Read more.

It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide (VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor, vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by nicotinamide phosphoribosyltransferase (NamPT). Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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16 pages, 5028 KiB

Open AccessArticle

Green Production of Cladribine by Using Immobilized 2′-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

by Cintia Wanda Rivero, Natalia Soledad García, Jesús Fernández-Lucas, Lorena Betancor, Gustavo Pablo Romanelli and Jorge Abel Trelles

Biomolecules 2021, 11(5), 657; https://doi.org/10.3390/biom11050657 - 29 Apr 2021

Cited by 7 | Viewed by 2447

Abstract

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2′-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was [...] Read more.

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2′-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiG_{PEI 25000}-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5–9) and temperature (30–60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiG_{PEI 25000}-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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13 pages, 1936 KiB

Open AccessArticle

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

by Martin Becker, Patrick Nikel, Jennifer N. Andexer, Stephan Lütz and Katrin Rosenthal

Biomolecules 2021, 11(4), 590; https://doi.org/10.3390/biom11040590 - 16 Apr 2021

Cited by 20 | Viewed by 3731

Abstract

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics [...] Read more.

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2′3′-cGAMP) formation was successfully developed. The 2′3′-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2′3′-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2′3′-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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22 pages, 2960 KiB

Open AccessArticle

Multi-Enzymatic Cascades in the Synthesis of Modified Nucleosides: Comparison of the Thermophilic and Mesophilic Pathways

by Ilja V. Fateev, Maria A. Kostromina, Yuliya A. Abramchik, Barbara Z. Eletskaya, Olga O. Mikheeva, Dmitry D. Lukoshin, Evgeniy A. Zayats, Maria Ya. Berzina, Elena V. Dorofeeva, Alexander S. Paramonov, Alexey L. Kayushin, Irina D. Konstantinova and Roman S. Esipov

Biomolecules 2021, 11(4), 586; https://doi.org/10.3390/biom11040586 - 16 Apr 2021

Cited by 9 | Viewed by 4199

Abstract

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of [...] Read more.

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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17 pages, 3378 KiB

Open AccessArticle

Radical Dehalogenation and Purine Nucleoside Phosphorylase E. coli: How Does an Admixture of 2′,3′-Anhydroinosine Hinder 2-fluoro-cordycepin Synthesis

by Alexey L. Kayushin, Julia A. Tokunova, Ilja V. Fateev, Alexandra O. Arnautova, Maria Ya. Berzina, Alexander S. Paramonov, Olga I. Lutonina, Elena V. Dorofeeva, Konstantin V. Antonov, Roman S. Esipov, Igor A. Mikhailopulo, Anatoly I. Miroshnikov and Irina D. Konstantinova

Biomolecules 2021, 11(4), 539; https://doi.org/10.3390/biom11040539 - 07 Apr 2021

Cited by 2 | Viewed by 2046

Abstract

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3′-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure [...] Read more.

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3′-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2′,3′-anhydroinosine, a byproduct in the preparation of 3-′deoxyinosine. Moreover, 2′,3′-anhydroinosine forms during radical dehalogenation of 9-(2′,5′-di-O-acetyl-3′-bromo- -3′-deoxyxylofuranosyl)hypoxanthine, a precursor of 3′-deoxyinosine in chemical synthesis. The products of 2′,3′-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2′,3′-anhydroinosine hydrolysis was investigated. The reactions were performed in D₂O instead of H₂O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2′,3′-anhydroinosine hydrolysis in D₂O is fully determined for the first time. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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16 pages, 4237 KiB

Open AccessArticle

A Novel One-Pot Enzyme Cascade for the Biosynthesis of Cladribine Triphosphate

by Julia Frisch, Tin Maršić and Christoph Loderer

Biomolecules 2021, 11(3), 346; https://doi.org/10.3390/biom11030346 - 25 Feb 2021

Cited by 12 | Viewed by 2741

Abstract

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade [...] Read more.

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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11 pages, 4586 KiB

Open AccessFeature PaperArticle

Molecular Basis of NDT-Mediated Activation of Nucleoside-Based Prodrugs and Application in Suicide Gene Therapy

by Javier Acosta, Elena Pérez, Pedro A. Sánchez-Murcia, Cristina Fillat and Jesús Fernández-Lucas

Biomolecules 2021, 11(1), 120; https://doi.org/10.3390/biom11010120 - 18 Jan 2021

Cited by 6 | Viewed by 3037

Abstract

Herein we report the first proof for the application of type II 2′-deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of LdNDT over the nucleoside-based prodrugs 2′-deoxy-5-fluorouridine [...] Read more.

Herein we report the first proof for the application of type II 2′-deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of LdNDT over the nucleoside-based prodrugs 2′-deoxy-5-fluorouridine (dFUrd), 2′-deoxy-2-fluoroadenosine (dFAdo), and 2′-deoxy-6-methylpurine riboside (d6MetPRib). Such activity was significantly increased (up to 30-fold) in the presence of an acceptor nucleobase. To shed light on the strong nucleobase dependence for enzymatic activity, different molecular dynamics simulations were carried out. Finally, as a proof of concept, we tested the LdNDT/dFAdo system in human cervical cancer (HeLa) cells. Interestingly, LdNDT/dFAdo showed a pronounced reduction in cellular viability with inhibitory concentrations in the low micromolar range. These results open up future opportunities for the clinical implementation of nucleoside 2′-deoxyribosyltransferases (NDTs) in cancer treatment. Full article

(This article belongs to the Special Issue Biotechnological and Biomedical Applications of Enzymes Involved in the Synthesis of Nucleosides and Nucleotides)

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