Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the firs...

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Authors: Acosta, Javier, Del Arco, Jon, Pozo, María Luisa del, Herrera-Tapias, Beliña, Clemente-Suárez, Vicente J., Berenguer, José, Hidalgo, Aurelio, Fernández-Lucas, Jesús
Format: article
Status:Published version
Publication Date:2020
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/236845
Online Access:http://hdl.handle.net/10261/236845
Access Level:Open access
Keyword:Enzymatic synthesis
Nucleotides
Phosphoribosyltransferase
nucleoside-50-monophosphate kinase
Dual domain protein
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spelling Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and TriphosphatesAcosta, JavierDel Arco, JonPozo, María Luisa delHerrera-Tapias, BeliñaClemente-Suárez, Vicente J.Berenguer, JoséHidalgo, AurelioFernández-Lucas, JesúsEnzymatic synthesisNucleotidesPhosphoribosyltransferasenucleoside-50-monophosphate kinaseDual domain proteinIn our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg and Co as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5′-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.European University of Madrid to JF-L. A grant from the Spanish Ministry of Science and Innovation BIO2016-77031-RUniversidad Europea de MadridMinisterio de Ciencia e Innovación (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2021202120202021info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/236845reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.3389/fbioe.2020.00677Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2368452026-05-22T06:33:51Z
dc.title.none.fl_str_mv Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
title Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
spellingShingle Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
Acosta, Javier
Enzymatic synthesis
Nucleotides
Phosphoribosyltransferase
nucleoside-50-monophosphate kinase
Dual domain protein
title_short Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
title_full Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
title_fullStr Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
title_full_unstemmed Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
title_sort Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
dc.creator.none.fl_str_mv Acosta, Javier
Del Arco, Jon
Pozo, María Luisa del
Herrera-Tapias, Beliña
Clemente-Suárez, Vicente J.
Berenguer, José
Hidalgo, Aurelio
Fernández-Lucas, Jesús
author Acosta, Javier
author_facet Acosta, Javier
Del Arco, Jon
Pozo, María Luisa del
Herrera-Tapias, Beliña
Clemente-Suárez, Vicente J.
Berenguer, José
Hidalgo, Aurelio
Fernández-Lucas, Jesús
author_role author
author2 Del Arco, Jon
Pozo, María Luisa del
Herrera-Tapias, Beliña
Clemente-Suárez, Vicente J.
Berenguer, José
Hidalgo, Aurelio
Fernández-Lucas, Jesús
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universidad Europea de Madrid
Ministerio de Ciencia e Innovación (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Enzymatic synthesis
Nucleotides
Phosphoribosyltransferase
nucleoside-50-monophosphate kinase
Dual domain protein
topic Enzymatic synthesis
Nucleotides
Phosphoribosyltransferase
nucleoside-50-monophosphate kinase
Dual domain protein
description In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg and Co as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5′-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.
publishDate 2020
dc.date.none.fl_str_mv 2020
2021
2021
2021
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/236845
url http://hdl.handle.net/10261/236845
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.3389/fbioe.2020.00677
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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