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2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases

Crespo, N.; Sánchez Murcia, Pedro Alejandro; Gago, Federico; Cejudo Sanches, J.; Galmes, M.A.; Fernandez Lucas, Jesus; Mancheño, José Miguel (Applied Microbiology and Biotechnology, 2017)

Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly ...
Characterization Of An Atypical, Thermostable, Organic Solvent- And Acid-Tolerant 2′-Deoxyribosyltransferase From Chroococcidiopsis Thermalis

Del Arco, Jon; Sánchez Murcia, Pedro Alejandro; Mancheño José Miguel, José Miguel; Gago, Federico; Fernandez Lucas, Jesus (Applied Microbiology And Biotechnology, 2018-05-23)

In our search for thermophilic and acid-tolerant nucleoside 2′-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical ...
Enzymatic Synthesis Of Therapeutic Nucleosides Using A Highly Versatile Purine Nucleoside 2’-Deoxyribosyltransferase From Trypanosoma Brucei

Pérez, Elena; Sánchez Murcia, Pedro Alejandro; Jordaan, Justin; Blanco, María Dolores; Mancheño, José Miguel; Gago, Federico; Fernandez Lucas, Jesus (ChemCatChem, 2018-07-18)

The use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo-, regio- and stereoselectivity as well as milder reaction conditions. Herein, the ...
N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs

Acosta, Javier; Del Arco, Jon; Pisabarro, Victor; Gago, Federico; Fernández-Lucas, Jesús (Frontiers in Bioengineering and Biotechnology, 2020-06-20)

Nucleoside-2′-deoxyribosyl-transferases (NDTs) catalyze a transglycosylation reaction consisting of the exchange of the 2′-deoxyribose moiety between a purine and/or pyrimidine nucleoside and a purine and/or pyrimidine ...
Reaction mechanism of nucleoside 2’-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate

Del Arco, Jon; Perona Requena, Almudena; González, Leticia; Fernández Lucas, Jesús; Gago, Federico; Sánchez-Murcia, Pedro Alejandro (Universidad de la Costa, 2019)

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2′-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape of the ...
Structure‐guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2' deoxyribosyltransferase

Del Arco, Jon; Mills, Alberto; Gago, Federico; Fernández Lucas, Jesús (Universidad de la Costa, 2019-07-02)

The use of nucleoside 2’‐deoxyribosyl‐transferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2’‐deoxyribonucleosides. We now show that a ...

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2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases
...
Crespo, N. | 2017

Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2'-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8-10) across a broad range of temperatures (30-70 °C) and ionic strengths (0-500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2'-fluoro-2'-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2'-fluoro-2'-deoxyguanosine, ara G, and ara H by a 2'-deoxyribosyltransferase is reported.

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Characterization Of An Atypical, Thermostable, Organic Solvent- And Acid-Tolerant 2′-Deoxyribosyltransferase From Chroococcidiopsis Thermalis
...
Del Arco, Jon | 2018-05-23

In our search for thermophilic and acid-tolerant nucleoside 2′-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50–100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2′-deoxynucleosides but is also proficient using cytosine and 2′-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT’s substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80–100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.

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Enzymatic Synthesis Of Therapeutic Nucleosides Using A Highly Versatile Purine Nucleoside 2’-Deoxyribosyltransferase From Trypanosoma Brucei
...
Pérez, Elena | 2018-07-18

The use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo-, regio- and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2’-deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50–70 °C), pH (4–7) and ionic strength (0–500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8–10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure-guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8-fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde-activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for >25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.

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N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs
...
Acosta, Javier | 2020-06-20

Nucleoside-2′-deoxyribosyl-transferases (NDTs) catalyze a transglycosylation reaction consisting of the exchange of the 2′-deoxyribose moiety between a purine and/or pyrimidine nucleoside and a purine and/or pyrimidine base. Because NDTs are highly specific for 2′-deoxyribonucleosides they generally display poor activity on modified C2′ and C3′ nucleosides and this limitation hampers their applicability as biocatalysts for the synthesis of modified nucleosides. We now report the production and purification of a novel NDT from Archaeoglobus veneficus that is endowed with native ribosyltransferase activity and hence it is more properly classified as an N-ribosyltransferase (AvNRT). Biophysical and biochemical characterization revealed that AvNRT is a homotetramer that displays maximum activity at 80°C and pH 6 and shows remarkably high stability at high temperatures (60–80°C). In addition, the activity of AvNRT was found to increase up to 2-fold in 4 M NaCl aqueous solution and to be retained in the presence of several water-miscible organic solvents. For completeness, and as a proof of concept for possible industrial applications, this thermophilic and halotolerant biocatalyst was successfully employed in the synthesis of different purine ribonucleoside analogs.

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Reaction mechanism of nucleoside 2’-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate
...
Del Arco, Jon | 2019

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2′-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an SN1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C–N bond cleavage and C–N bond formation.

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Structure‐guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2' deoxyribosyltransferase
...
Del Arco, Jon | 2019-07-02

The use of nucleoside 2’‐deoxyribosyl‐transferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2’‐deoxyribonucleosides. We now show that a highly versatile purine nucleoside 2’‐deoxyribosyltransferase from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates, most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved ~3‐fold by introducing a single amino acid replacement at position 5 following a structure‐guided approach. Biophysical and biochemical characterization revealed that the TbPDTY5F variant is a homodimer that displays maximum activity at 50 oC and pH 6.5 and shows a remarkably high melting temperature of 69 oC. Substrate specificity studies demonstrated that 6‐oxopurine ribonucleosides are the best donors (inosine > guanosine >> adenosine) whereas no significant preferences exist between 6‐aminopurines and 6‐oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7‐deaza purines. TbPDTY5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.

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Browsing by Author "Gago, Federico"

2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases ﻿

Characterization Of An Atypical, Thermostable, Organic Solvent- And Acid-Tolerant 2′-Deoxyribosyltransferase From Chroococcidiopsis Thermalis ﻿

Enzymatic Synthesis Of Therapeutic Nucleosides Using A Highly Versatile Purine Nucleoside 2’-Deoxyribosyltransferase From Trypanosoma Brucei ﻿

N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs ﻿

Reaction mechanism of nucleoside 2’-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate ﻿

Structure‐guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2' deoxyribosyltransferase ﻿

Envíos recientes

2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases

Characterization Of An Atypical, Thermostable, Organic Solvent- And Acid-Tolerant 2′-Deoxyribosyltransferase From Chroococcidiopsis Thermalis

Enzymatic Synthesis Of Therapeutic Nucleosides Using A Highly Versatile Purine Nucleoside 2’-Deoxyribosyltransferase From Trypanosoma Brucei

N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs

Reaction mechanism of nucleoside 2’-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate

Structure‐guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2' deoxyribosyltransferase

2′-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases

Characterization Of An Atypical, Thermostable, Organic Solvent- And Acid-Tolerant 2′-Deoxyribosyltransferase From Chroococcidiopsis Thermalis

Enzymatic Synthesis Of Therapeutic Nucleosides Using A Highly Versatile Purine Nucleoside 2’-Deoxyribosyltransferase From Trypanosoma Brucei

N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs

Reaction mechanism of nucleoside 2’-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate

Structure‐guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2' deoxyribosyltransferase