Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold signif...

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Autores: Hormigo Cisneros, Daniel, Arco Arrieta, Jon del, Acosta Bueno, Javier, Fürst, Maximilian, J. L., Fernández Lucas, Jesús
Tipo de documento: artigo
Data de publicação:2024
País:España
Recursos:Universidad Europea (UEM)
Repositório:ABACUS. Repositorio de Producción Científica
Idioma:inglês
OAI Identifier:oai:abacus.universidadeuropea.com:11268/13616
Acesso em linha:http://hdl.handle.net/11268/13616
Access Level:Acceso aberto
Palavra-chave:Biotecnología
Goal 9: Build resilient infrastructure, promote sustainable industrialization and foster innovation
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spelling Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside AnalogsHormigo Cisneros, DanielArco Arrieta, Jon delAcosta Bueno, JavierFürst, Maximilian, J. L.Fernández Lucas, JesúsBiotecnologíaGoal 9: Build resilient infrastructure, promote sustainable industrialization and foster innovationNucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 ◦C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 ºC over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.20252025-02-0120242024-01-0120242024-01-01journal articlehttp://purl.org/coar/resource_type/c_6501info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/11268/13616reponame:ABACUS. Repositorio de Producción Científicainstname:Universidad Europea (UEM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:abacus.universidadeuropea.com:11268/136162026-06-11T12:41:27Z
dc.title.none.fl_str_mv Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
title Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
spellingShingle Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
Hormigo Cisneros, Daniel
Biotecnología
Goal 9: Build resilient infrastructure, promote sustainable industrialization and foster innovation
title_short Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
title_full Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
title_fullStr Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
title_full_unstemmed Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
title_sort Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs
dc.creator.none.fl_str_mv Hormigo Cisneros, Daniel
Arco Arrieta, Jon del
Acosta Bueno, Javier
Fürst, Maximilian, J. L.
Fernández Lucas, Jesús
author Hormigo Cisneros, Daniel
author_facet Hormigo Cisneros, Daniel
Arco Arrieta, Jon del
Acosta Bueno, Javier
Fürst, Maximilian, J. L.
Fernández Lucas, Jesús
author_role author
author2 Arco Arrieta, Jon del
Acosta Bueno, Javier
Fürst, Maximilian, J. L.
Fernández Lucas, Jesús
author2_role author
author
author
author
dc.contributor.none.fl_str_mv
dc.subject.none.fl_str_mv Biotecnología
Goal 9: Build resilient infrastructure, promote sustainable industrialization and foster innovation
topic Biotecnología
Goal 9: Build resilient infrastructure, promote sustainable industrialization and foster innovation
description Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 ◦C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 ºC over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.
publishDate 2024
dc.date.none.fl_str_mv 2024
2024-01-01
2024
2024-01-01
2025
2025-02-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/11268/13616
url http://hdl.handle.net/11268/13616
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution-NonCommercial-NoDerivatives 4.0 Internacional
http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution-NonCommercial-NoDerivatives 4.0 Internacional
http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:ABACUS. Repositorio de Producción Científica
instname:Universidad Europea (UEM)
instname_str Universidad Europea (UEM)
reponame_str ABACUS. Repositorio de Producción Científica
collection ABACUS. Repositorio de Producción Científica
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.812429

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