One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry

Biocatalysis reproduce nature’s synthetic strategies in order to synthesize different organic compounds. Natural metabolic pathways usually involve complex networks to support cellular growth and survival. In this regard, multi-enzymatic systems are valuable tools for the production of a wide variet...

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Autores:
Acosta, Javier
Del Arco, Jon
Martínez Pascual, Sara
Clemente Suárez, Vicente Javier
Fernandez Lucas, Jesus
Tipo de recurso:
Article of journal
Fecha de publicación:
2018
Institución:
Corporación Universidad de la Costa
Repositorio:
REDICUC - Repositorio CUC
Idioma:
eng
OAI Identifier:
oai:repositorio.cuc.edu.co:11323/1211
Acceso en línea:
https://hdl.handle.net/11323/1211
https://repositorio.cuc.edu.co/
Palabra clave:
2 0 -deoxyribosyltransferase
phosphoribosyltransferases
cascade reactions
purine nucleoside analogues
dietary nucleotides
Rights
openAccess
License
Atribución – No comercial – Compartir igual
id RCUC2_99af05ee53dfd4b79ad001e0bc5843c8
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repository_id_str
dc.title.eng.fl_str_mv One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
title One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
spellingShingle One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
2 0 -deoxyribosyltransferase
phosphoribosyltransferases
cascade reactions
purine nucleoside analogues
dietary nucleotides
title_short One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
title_full One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
title_fullStr One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
title_full_unstemmed One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
title_sort One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry
dc.creator.fl_str_mv Acosta, Javier
Del Arco, Jon
Martínez Pascual, Sara
Clemente Suárez, Vicente Javier
Fernandez Lucas, Jesus
dc.contributor.author.spa.fl_str_mv Acosta, Javier
Del Arco, Jon
Martínez Pascual, Sara
Clemente Suárez, Vicente Javier
Fernandez Lucas, Jesus
dc.subject.eng.fl_str_mv 2 0 -deoxyribosyltransferase
phosphoribosyltransferases
cascade reactions
purine nucleoside analogues
dietary nucleotides
topic 2 0 -deoxyribosyltransferase
phosphoribosyltransferases
cascade reactions
purine nucleoside analogues
dietary nucleotides
description Biocatalysis reproduce nature’s synthetic strategies in order to synthesize different organic compounds. Natural metabolic pathways usually involve complex networks to support cellular growth and survival. In this regard, multi-enzymatic systems are valuable tools for the production of a wide variety of organic compounds. Methods: The production of different purine nucleosides and nucleoside-50 -monophosphates has been performed for first time, catalyzed by the sequential action of 2 0 -deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) and hypoxanthine-guanine-xanthine phosphoribosyltransferase from Thermus themophilus HB8 (TtHGXPRT). Results: The biochemical characterization of LdNDT reveals that the enzyme is active and stable in a broad range of pH, temperature, and ionic strength. Substrate specificity studies showed a high promiscuity in the recognition of purine analogues. Finally, the enzymatic production of different purine derivatives was performed to evaluate the efficiency of multi-enzymatic system LdNDT/TtHGXPRT. Conclusions: The production of different therapeutic purine nucleosides was efficiently catalyzed by LdNDT/TtHGXPRT. In addition, the resulting by-products were converted to IMP and GMP. Taking all of these features, this bioprocess entails an efficient, sustainable, and economical alternative to chemical synthetic methods.
publishDate 2018
dc.date.accessioned.none.fl_str_mv 2018-11-17T13:38:54Z
dc.date.available.none.fl_str_mv 2018-11-17T13:38:54Z
dc.date.issued.none.fl_str_mv 2018
dc.type.spa.fl_str_mv Artículo de revista
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dc.identifier.issn.spa.fl_str_mv 2073-4344
dc.identifier.uri.spa.fl_str_mv https://hdl.handle.net/11323/1211
dc.identifier.instname.spa.fl_str_mv Corporación Universidad de la Costa
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dc.language.iso.none.fl_str_mv eng
language eng
dc.relation.references.spa.fl_str_mv 1. Parker, W.B. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem. Rev. 2009, 109, 2880–2893. [CrossRef] [PubMed] 2. De Clercq, E. Recent highlights in the development of new antiviral drugs. Curr. Opin. Microbiol. 2005, 8, 552–560. [CrossRef] [PubMed] 3. Behrens, M.; Meyerhof, W.; Hellfritsch, C.; Hofmann, T. Sweet and umami taste: Natural products, their chemosensory targets, and beyond. Angew. Chem. Int. Ed. 2011, 50, 2220–2242. [CrossRef] [PubMed] 4. Mikhailopulo, I.A. Biotechnology of nucleic acid constituents—State of the art and perspectives. Curr. Org. Chem. 2007, 11, 317–335. [CrossRef] 5. Fresco-Taboada, A.; de la Mata, I.; Arroyo, M.; Fernández-Lucas, J. New insights on nucleoside 2 0 -deoxyribosyltransferases: A versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs. Appl. Microbiol. Biotechnol. 2013, 97, 3773–3785. [CrossRef] [PubMed] 6. Lapponi, M.J.; Rivero, C.W.; Zinni, M.A.; Britos, C.N.; Trelles, J.A. New developments in nucleoside analogues biosynthesis: A review. J. Mol. Catal. B Enzym. 2016, 133, 218–233. [CrossRef] 7. Del Arco, J.; Fernández-Lucas, J. Purine and Pyrimidine Phosphoribosytransferases: A versatile tool for enzymatic synthesis of nucleoside-50 -monophosphates. Curr. Pharm. Des. 2017, 23. [CrossRef] 8. Lewkowicz, E.S.; Iribarren, A.M. Nucleoside phosphorylases. Curr. Org. Chem. 2006, 10, 1197–1215. [CrossRef] 9. Del Arco, J.; Acosta, J.; Pereira, H.M.; Perona, A.; Lokanath, N.K.; Kunishima, N.; Fernández-Lucas, J. Enzymatic production of non-natural nucleoside-50 -monophosphates by a novel thermostable uracil phosphoribosyltransferase. ChemCatChem 2017. [CrossRef] 10. Del Arco, J.; Martinez, M.; Donday, M.; Clemente-Suarez, V.J.; Fernández-Lucas, J. Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8. Biocatal. Biotransfor. 2017, 1–8. [CrossRef] 11. Del Arco, J.; Cejudo-Sanches, J.; Esteban, I.; Clemente-Suarez, V.J.; Hormigo-Cisneros, D.; Perona, A.; Fernández-Lucas, J. Enzymatic production of dietary nucleotides from low-soluble purine bases by an efficient, thermostable and alkali-tolerant biocatalyst. Food Chem. 2017, 237, 605–611. [CrossRef] [PubMed] 12. Serra, I.; Conti, S.; Piškur, J.; Clausen, A.R.; Munch-Petersen, B.; Terreni, M.; Ubiali, D. Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides. Adv. Synth. Catal. 2014, 356, 563–570. [CrossRef] 13. Fernández-Lucas, J. Multienzymatic synthesis of nucleic acid derivatives: A general perspective. Appl. Microbiol. Biotechnol. 2015, 99, 4615–4627. [CrossRef] [PubMed] 14. Zou, Z.; Ding, Q.; Ou, L.; Yan, B. Efficient production of deoxynucleoside-50 -monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system. Appl. Microbiol. Biotechnol. 2013, 97, 9389–9395. [CrossRef] [PubMed] 15. Mori, H.; Iida, A.; Fujio, T.; Teshiba, S. A novel process of inosine 50 -monophosphate production using overexpressed guanosine/inosine kinase. Appl. Microbiol. Biotechnol. 1997, 48, 693–698. [CrossRef] [PubMed] 16. Zhou, X.; Szeker, K.; Janocha, B.; Böhme, T.; Albrecht, D.; Mikhailopulo, I.A.; Neubauer, P. Recombinant purine nucleoside phosphorylases from thermophiles: Preparation, properties and activity towards purine and pyrimidine nucleosides. FEBS J. 2013, 280, 1475–1490. [CrossRef] [PubMed] 17. Iglesias, L.E.; Lewkowicz, E.S.; Medici, R.; Bianchi, P.; Iribarren, A.M. Biocatalytic approaches applied to the synthesis of nucleoside prodrugs. Biotechnol. Adv. 2015, 33, 412–434. [CrossRef] [PubMed] 18. Fernández-Lucas, J.; Acebal, C.; Sinisterra, J.V.; Arroyo, M.; de la Mata, I. Lactobacillus reuteri 2’-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides. Appl. Environ. Microbiol. 2010, 76, 1462–1470. [CrossRef] [PubMed] 19. Crespo, N.; Sánchez-Murcia, P.A.; Gago, F.; Cejudo-Sanches, J.; Galmes, M.A.; Fernández-Lucas, J.; Mancheño, J.M. 20 -Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases. Appl. Microbiol. Biotechnol. 2017, 101, 7187–7200. [CrossRef] [PubMed] 20. Fresco-Taboada, A.; Serra, I.; Arroyo, M.; Fernández-Lucas, J.; de la Mata, I.; Terreni, M. Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine. Catal. Today 2016, 259, 197–204. [CrossRef] 21. De Clercq, E. Highlights in the discovery of antiviral drugs: A personal retrospective. J. Med. Chem. 2010, 53, 1438–1450. [CrossRef] [PubMed] 22. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym. Microb. Technol. 2007, 40, 1451–1463. [CrossRef] 23. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [CrossRef] [PubMed] 24. Gill, S.C.; Von Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182, 319–326. [CrossRef]
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spelling Acosta, JavierDel Arco, JonMartínez Pascual, SaraClemente Suárez, Vicente JavierFernandez Lucas, Jesus2018-11-17T13:38:54Z2018-11-17T13:38:54Z20182073-4344https://hdl.handle.net/11323/1211Corporación Universidad de la CostaREDICUC - Repositorio CUChttps://repositorio.cuc.edu.co/Biocatalysis reproduce nature’s synthetic strategies in order to synthesize different organic compounds. Natural metabolic pathways usually involve complex networks to support cellular growth and survival. In this regard, multi-enzymatic systems are valuable tools for the production of a wide variety of organic compounds. Methods: The production of different purine nucleosides and nucleoside-50 -monophosphates has been performed for first time, catalyzed by the sequential action of 2 0 -deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) and hypoxanthine-guanine-xanthine phosphoribosyltransferase from Thermus themophilus HB8 (TtHGXPRT). Results: The biochemical characterization of LdNDT reveals that the enzyme is active and stable in a broad range of pH, temperature, and ionic strength. Substrate specificity studies showed a high promiscuity in the recognition of purine analogues. Finally, the enzymatic production of different purine derivatives was performed to evaluate the efficiency of multi-enzymatic system LdNDT/TtHGXPRT. Conclusions: The production of different therapeutic purine nucleosides was efficiently catalyzed by LdNDT/TtHGXPRT. In addition, the resulting by-products were converted to IMP and GMP. Taking all of these features, this bioprocess entails an efficient, sustainable, and economical alternative to chemical synthetic methods.Acosta, Javier-db7704d1-7f5b-4e1e-840f-6bb5e26da600-0Del Arco, Jon-c5ed68af-857c-4b28-99a7-33a4254ed926-0Martínez Pascual, Sara-30107093-0d62-402f-be21-5cdce39381a4-0Clemente Suárez, Vicente Javier-0000-0002-2397-2801-600Fernandez Lucas, Jesus-3f36c351-7522-42ea-8605-cd7e804a6387-0engCatalystsAtribución – No comercial – Compartir igualinfo:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf22 0 -deoxyribosyltransferasephosphoribosyltransferasescascade reactionspurine nucleoside analoguesdietary nucleotidesOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industryArtículo de revistahttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/acceptedVersion1. Parker, W.B. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem. Rev. 2009, 109, 2880–2893. [CrossRef] [PubMed] 2. De Clercq, E. Recent highlights in the development of new antiviral drugs. Curr. Opin. Microbiol. 2005, 8, 552–560. [CrossRef] [PubMed] 3. Behrens, M.; Meyerhof, W.; Hellfritsch, C.; Hofmann, T. Sweet and umami taste: Natural products, their chemosensory targets, and beyond. Angew. Chem. Int. Ed. 2011, 50, 2220–2242. [CrossRef] [PubMed] 4. Mikhailopulo, I.A. Biotechnology of nucleic acid constituents—State of the art and perspectives. Curr. Org. Chem. 2007, 11, 317–335. [CrossRef] 5. Fresco-Taboada, A.; de la Mata, I.; Arroyo, M.; Fernández-Lucas, J. New insights on nucleoside 2 0 -deoxyribosyltransferases: A versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs. Appl. Microbiol. Biotechnol. 2013, 97, 3773–3785. [CrossRef] [PubMed] 6. Lapponi, M.J.; Rivero, C.W.; Zinni, M.A.; Britos, C.N.; Trelles, J.A. New developments in nucleoside analogues biosynthesis: A review. J. Mol. Catal. B Enzym. 2016, 133, 218–233. [CrossRef] 7. Del Arco, J.; Fernández-Lucas, J. Purine and Pyrimidine Phosphoribosytransferases: A versatile tool for enzymatic synthesis of nucleoside-50 -monophosphates. Curr. Pharm. Des. 2017, 23. [CrossRef] 8. Lewkowicz, E.S.; Iribarren, A.M. Nucleoside phosphorylases. Curr. Org. Chem. 2006, 10, 1197–1215. [CrossRef] 9. Del Arco, J.; Acosta, J.; Pereira, H.M.; Perona, A.; Lokanath, N.K.; Kunishima, N.; Fernández-Lucas, J. Enzymatic production of non-natural nucleoside-50 -monophosphates by a novel thermostable uracil phosphoribosyltransferase. ChemCatChem 2017. [CrossRef] 10. Del Arco, J.; Martinez, M.; Donday, M.; Clemente-Suarez, V.J.; Fernández-Lucas, J. Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8. Biocatal. Biotransfor. 2017, 1–8. [CrossRef] 11. Del Arco, J.; Cejudo-Sanches, J.; Esteban, I.; Clemente-Suarez, V.J.; Hormigo-Cisneros, D.; Perona, A.; Fernández-Lucas, J. Enzymatic production of dietary nucleotides from low-soluble purine bases by an efficient, thermostable and alkali-tolerant biocatalyst. Food Chem. 2017, 237, 605–611. [CrossRef] [PubMed] 12. Serra, I.; Conti, S.; Piškur, J.; Clausen, A.R.; Munch-Petersen, B.; Terreni, M.; Ubiali, D. Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides. Adv. Synth. Catal. 2014, 356, 563–570. [CrossRef] 13. Fernández-Lucas, J. Multienzymatic synthesis of nucleic acid derivatives: A general perspective. Appl. Microbiol. Biotechnol. 2015, 99, 4615–4627. [CrossRef] [PubMed] 14. Zou, Z.; Ding, Q.; Ou, L.; Yan, B. Efficient production of deoxynucleoside-50 -monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system. Appl. Microbiol. Biotechnol. 2013, 97, 9389–9395. [CrossRef] [PubMed] 15. Mori, H.; Iida, A.; Fujio, T.; Teshiba, S. A novel process of inosine 50 -monophosphate production using overexpressed guanosine/inosine kinase. Appl. Microbiol. Biotechnol. 1997, 48, 693–698. [CrossRef] [PubMed] 16. Zhou, X.; Szeker, K.; Janocha, B.; Böhme, T.; Albrecht, D.; Mikhailopulo, I.A.; Neubauer, P. Recombinant purine nucleoside phosphorylases from thermophiles: Preparation, properties and activity towards purine and pyrimidine nucleosides. FEBS J. 2013, 280, 1475–1490. [CrossRef] [PubMed] 17. Iglesias, L.E.; Lewkowicz, E.S.; Medici, R.; Bianchi, P.; Iribarren, A.M. Biocatalytic approaches applied to the synthesis of nucleoside prodrugs. Biotechnol. Adv. 2015, 33, 412–434. [CrossRef] [PubMed] 18. Fernández-Lucas, J.; Acebal, C.; Sinisterra, J.V.; Arroyo, M.; de la Mata, I. Lactobacillus reuteri 2’-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides. Appl. Environ. Microbiol. 2010, 76, 1462–1470. [CrossRef] [PubMed] 19. Crespo, N.; Sánchez-Murcia, P.A.; Gago, F.; Cejudo-Sanches, J.; Galmes, M.A.; Fernández-Lucas, J.; Mancheño, J.M. 20 -Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases. Appl. Microbiol. Biotechnol. 2017, 101, 7187–7200. [CrossRef] [PubMed] 20. Fresco-Taboada, A.; Serra, I.; Arroyo, M.; Fernández-Lucas, J.; de la Mata, I.; Terreni, M. Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine. Catal. Today 2016, 259, 197–204. [CrossRef] 21. De Clercq, E. Highlights in the discovery of antiviral drugs: A personal retrospective. J. Med. Chem. 2010, 53, 1438–1450. [CrossRef] [PubMed] 22. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym. Microb. Technol. 2007, 40, 1451–1463. [CrossRef] 23. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. [CrossRef] [PubMed] 24. Gill, S.C.; Von Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182, 319–326. [CrossRef]PublicationORIGINALOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry.pdfOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry.pdfapplication/pdf1109365https://repositorio.cuc.edu.co/bitstreams/360db415-6903-4aef-9f75-b2ac3ae7f6ba/download7567640f9169fdbb501b0c6d021b2ca5MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://repositorio.cuc.edu.co/bitstreams/7c15618a-0ccc-48f1-82c2-943a8098cd7a/download8a4605be74aa9ea9d79846c1fba20a33MD52THUMBNAILOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry.pdf.jpgOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry.pdf.jpgimage/jpeg67272https://repositorio.cuc.edu.co/bitstreams/a26a554f-e37e-41a7-b939-b8e1bffcc3fa/download301f516d1bf536395c6ac44509335adeMD54TEXTOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry.pdf.txtOne-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry.pdf.txttext/plain44749https://repositorio.cuc.edu.co/bitstreams/3d6bd68d-0939-4605-8245-8e12443b5d01/downloadad3443da466cb0dbb2cdbe4307f43186MD5511323/1211oai:repositorio.cuc.edu.co:11323/12112024-09-17 10:58:52.263open.accesshttps://repositorio.cuc.edu.coRepositorio de la Universidad de la Costa CUCrepdigital@cuc.edu.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