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...

Full description

Autores:: Acosta, Javier
Del Arco, Jon
Del Pozo, Maria Luisa
Herrera, Beliña
Clemente-Suárez, Vicente Javier
Berenguer, José
Hidalgo, Aurelio
Fernández-Lucas, Jesús

Tipo de recurso:: Article of journal

Fecha de publicación:: 2020

Institución:: Corporación Universidad de la Costa

Repositorio:: REDICUC - Repositorio CUC

Idioma:: eng

id	RCUC2_4b14444eefa4542e8b4020096f53fc19
oai_identifier_str	oai:repositorio.cuc.edu.co:11323/6477
network_acronym_str	RCUC2
network_name_str	REDICUC - Repositorio CUC
repository_id_str
dc.title.spa.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 Enzymatic synthesis Nucleotides Phosphoribosyltransferase Nucleoside-5cpsdummy′-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.fl_str_mv	Acosta, Javier Del Arco, Jon Del Pozo, Maria Luisa Herrera, Beliña Clemente-Suárez, Vicente Javier Berenguer, José Hidalgo, Aurelio Fernández-Lucas, Jesús
dc.contributor.author.spa.fl_str_mv	Acosta, Javier Del Arco, Jon Del Pozo, Maria Luisa Herrera, Beliña Clemente-Suárez, Vicente Javier Berenguer, José Hidalgo, Aurelio Fernández-Lucas, Jesús
dc.subject.spa.fl_str_mv	Enzymatic synthesis Nucleotides Phosphoribosyltransferase Nucleoside-5cpsdummy′-monophosphate kinase Dual domain protein
topic	Enzymatic synthesis Nucleotides Phosphoribosyltransferase Nucleoside-5cpsdummy′-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 Mg2+ and Co2+ 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.accessioned.none.fl_str_mv	2020-07-07T19:25:59Z
dc.date.available.none.fl_str_mv	2020-07-07T19:25:59Z
dc.date.issued.none.fl_str_mv	2020-06-24
dc.type.spa.fl_str_mv	Artículo de revista
dc.type.coar.fl_str_mv	http://purl.org/coar/resource_type/c_2df8fbb1
dc.type.coar.spa.fl_str_mv	http://purl.org/coar/resource_type/c_6501
dc.type.content.spa.fl_str_mv	Text
dc.type.driver.spa.fl_str_mv	info:eu-repo/semantics/article
dc.type.redcol.spa.fl_str_mv	http://purl.org/redcol/resource_type/ART
dc.type.version.spa.fl_str_mv	info:eu-repo/semantics/acceptedVersion
format	http://purl.org/coar/resource_type/c_6501
status_str	acceptedVersion
dc.identifier.issn.spa.fl_str_mv	2296-4185
dc.identifier.uri.spa.fl_str_mv	https://hdl.handle.net/11323/6477
dc.identifier.doi.spa.fl_str_mv	https://doi.org/10.3389/fbioe.2020.00677
dc.identifier.instname.spa.fl_str_mv	Corporación Universidad de la Costa
dc.identifier.reponame.spa.fl_str_mv	REDICUC - Repositorio CUC
dc.identifier.repourl.spa.fl_str_mv	https://repositorio.cuc.edu.co/
identifier_str_mv	2296-4185 Corporación Universidad de la Costa REDICUC - Repositorio CUC
url	https://hdl.handle.net/11323/6477 https://doi.org/10.3389/fbioe.2020.00677 https://repositorio.cuc.edu.co/
dc.language.iso.none.fl_str_mv	eng
language	eng
dc.relation.references.spa.fl_str_mv	Acosta, J., Del Arco, J., Martinez-Pascual, S., Clemente-Suárez, V., and Fernández-Lucas, J. (2018). One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry. Catalysts 8:9. doi: 10.3390/catal8010009 Ådén, J., Weise, C. F., Brännström, K., Olofsson, A., and Wolf-Watz, M. (2013). Structural topology and activation of an initial adenylate kinase–substrate complex. Biochemistry 52, 1055–1061. doi: 10.1021/bi301460k Ali, L. Z., and Sloan, D. L. (1986). Activation of hypoxanthine/guanine phosphoribosyltransferase from yeast by divalent zinc and nickel ions. J. Inorg. Biochem. 28, 407–415. doi: 10.1016/0162-0134(86)80026-5 Argos, P. (1990). An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J. Mol. Biol. 211, 943–958. doi: 10.1016/0022-2836(90)90085-Z Brown, P. H., and Schuck, P. (2006). Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys. J. 90, 4651–4661. doi: 10.1529/biophysj.106.081372 Case, D., Betz, R. M., Cerutti, D. S., Cheatham, T., Darden, T., Duke, R., et al. (2016). AMBER 2016. San Francisco: University of California. Chen, X., Zaro, J., and Shen, W. C. (2013). “Fusion protein linkers: effects on production, bioactivity, and pharmacokinetics,” in Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges, ed. S. R. Schmidt (Hoboken, NJ: John Wiley & Sons), 57–73. doi: 10.1016/j.addr.2012.09.039 Davlieva, M., and Shamoo, Y. (2010). Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability. Proteins 78, 357–364. doi: 10.1002/prot.22549 Del Arco, J., Cejudo-Sanches, J., Esteban, I., Clemente-Suárez, V. J., Hormigo, D., Perona, A., et al. (2017). Enzymatic production of dietary nucleotides from low-soluble purine bases by an efficient, thermostable and alkali-tolerant biocatalyst. Food Chem. 237, 605–611. doi: 10.1016/j.foodchem.2017.05.136 Del Arco, J., and Fernández-Lucas, J. (2017). Purine and pyrimidine phosphoribosyltransferases: a versatile tool for enzymatic synthesis of nucleoside-5’-monophosphates. Curr. Pharm. Des. 23, 6898–6912. doi: 10.2174/1381612823666171017165707 Del Arco, J., and Fernández-Lucas, J. (2018). Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives. Appl. Microbiol. Biotechnol. 102, 7805–7820. doi: 10.1007/s00253-018-9242-8 Del Arco, J., Acosta, J., Pereira, H. M., Perona, A., Lokanath, N. K., Kunishima, N., et al. (2018a). Enzymatic production of non-natural nucleoside-5’-monophosphates by a Thermostable uracil phosphoribosyltransferase. Chemcatchem 10, 439–448. doi: 10.1002/cctc.201701223 Del Arco, J., Martinez, M., Donday, M., Clemente-Suarez, V. J., and Fernández-Lucas, J. (2018b). Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8. Biocatal. Biotransform. 36, 216–223. doi: 10.1080/10242422.2017.1313837 Del Arco, J., Martínez-Pascual, S., Clemente-Suárez, V. J., Corral, O. J., Jordaan, J., Hormigo, D., et al. (2018c). One-pot, one-step production of dietary nucleotides by magnetic biocatalysts. Catalysts 8:184. doi: 10.3390/catal8050184 Del Arco, J., Sánchez-Murcia, P. A., Mancheño, J. M., Gago, F., and Fernández-Lucas, J. (2018d). Characterization of an atypical, thermostable, organic solvent-and acid-tolerant 2’-deoxyribosyltransferase from Chroococcidiopsis thermalis. Appl. Microbiol. Biotechnol. 102, 6947–6957. doi: 10.1007/s00253-018-9134-y Del Arco, J., Mills, A., Gago, F., and Fernández-Lucas, J. (2019a). Structure-guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2’-deoxyribosyltransferase. Chembiochem 20, 2996–3000. doi: 10.1002/cbic.201900397 Del Arco, J., Pérez, E., Naitow, H., Matsuura, Y., Kunishima, N., and Fernández-Lucas, J. (2019b). Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5’-monophospate analogues. Bioresour. Technol. 276, 244–252. doi: 10.1016/j.biortech.2018.12.120 Delano, W. L. (2002). The PyMOL Molecular Graphics System. San Carlos, CA: De Lano Scientific. Ding, Q., and Ou, L. (2017). NTP regeneration and its application in the biosynthesis of nucleotides and their derivatives. Curr. Pharm. Des. 23, 6936–6947. doi: 10.2174/1381612823666171024155247 Ebina, T., Toh, H., and Kuroda, Y. (2011). DROP: an SVM domain linker predictor trained with optimal features selected by random forest. Bioinformatics 27, 487–494. doi: 10.1093/bioinformatics/btq700 el Kouni, M. H. (2003). Potential chemotherapeutic targets in the purine metabolism of parasites. Pharmacol. Ther. 99, 283–309. doi: 10.1016/S0163-7258(03)00071-8 Fernández-Lucas, J. (2015). Multienzymatic synthesis of nucleic acid derivatives: a general perspective. Appl. Microbiol. Biotechnol. 99, 4615–4627. doi: 10.1007/s00253-015-6642-x Fernández-Lucas, J., Acebal, C., Sinisterra, J. V., Arroyo, M., and de la Mata, I. (2010). Lactobacillus reuteri 2’-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides. Appl. Environ. Microbiol. 76, 1462–1470. doi: 10.1128/aem.01685-09 Fernández-Lucas, J., Fresco-Taboada, A., de la Mata, I., and Arroyo, M. (2012). One-step enzymatic synthesis of nucleosides from low water-soluble purine bases in non-conventional media. Bioresour. Technol. 115, 63–69. doi: 10.1016/j.biortech.2011.11.127 Formoso, E., Limongelli, V., and Parrinello, M. (2015). Energetics and structural characterization of the large-scale functional motion of adenylate kinase. Sci. Rep. 5:8425. doi: 10.1038/srep08425 Fresco-Taboada, A., de la Mata, I., Arroyo, M., and Fernández-Lucas, J. (2013). New insights on nucleoside 2’-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs. Appl. Microbiol. Biotechnol. 97, 3773–3785. doi: 10.1007/s00253-013-4816-y George, R. A., and Heringa, J. (2002). An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. Des. Sel. 15, 871–879. doi: 10.1093/protein/15.11.871 Hochstadt, J. (1978). Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase from enteric bacteria. Methods Enzymol. 51, 549–558. doi: 10.1016/S0076-6879(78)51077-X Kamel, S., Yehia, H., Neubauer, P., and Wagner, A. (2019). “Enzymatic synthesis of nucleoside analogues by nucleoside phosphorylases,” in Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives, ed. M. J. Fernández-Lucas (Weinheim: Wiley-VCH), 1–28. doi: 10.1002/9783527812103.ch1 Kanagawa, M., Baba, S., Ebihara, A., Shinkai, A., Hirotsu, K., Mega, R., et al. (2010). Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 893–898. doi: 10.1107/S1744309110023079 Lapponi, M. J., Rivero, C. W., Zinni, M. A., Britos, C. N., and Trelles, J. A. (2016). New developments in nucleoside analogues biosynthesis: a review. J. Mol. Catal. B Enzym. 133, 218–233. doi: 10.1016/j.molcatb.2016.08.015 Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992). “Computer aided interpretation of analytical sedimentation data for proteins,” in Analytical Ultracentrifugation In Biochemistry And Polymer Science, eds S. E. Harding, J. C. Horton, and A. J. Rowe (Cambridge: Royal Society of Chemistry), 90–125. Lewkowicz, E. S., and Iribarren, A. M. (2017). Whole cell biocatalysts for the preparation of nucleosides and their derivatives. Curr. Pharm. Design. 23, 6851–6878. doi: 10.2174/1381612823666171011101133 Mbewe, B., Chibale, K., and McIntosh, D. B. (2007). Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized reactive red 120. Protein Expr. Purif. 52, 153–158. doi: 10.1016/j.pep.2006.09.014 Mikhailopulo, I. A. (2007). Biotechnology of nucleic acid constituents-State of the art and perspectives. Curr. Org. Chem. 11, 317–335. doi: 10.2174/138527207780059330 Minton, A. P. (1997). Alternative strategies for the characterization of associations in multicomponent solutions via measurement of sedimentation equilibrium. Prog. Colloid Polym. Sci. 107, 11–19. doi: 10.1007/BFb0118010 Montero, C., and Llorente, P. (1991). Artemia purine phosphoribosyltransferases. Purification and characterization. Biochem. J. 275, 327–334. doi: 10.1042/bj2750327 Motulsky, H., and Christopoulos, A. (2019). Fitting Models to Biological Data using Linear and Nonlinear Regression. A Practical Guide to Curve Fitting. New York, NY: Oxford University Press. Mukhopadhyay, A., Kladova, A. V., Bursakov, S. A., Gavel, O. Y., Calvete, J. J., Shnyrov, V. L., et al. (2010). Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria. J. Biol. Inorg. Chem. 16, 51–61. doi: 10.1007/s00775-010-0700-8 Munagala, N. R., Chin, M. S., and Wang, C. C. (1998). Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. Biochemistry 37, 4045–4051. doi: 10.1021/bi972515h Niesen, F. H., Berglund, H., and Vedadi, M. (2007). The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221. doi: 10.1038/nprot.2007.321 Panayiotou, C., Solaroli, N., and Karlsson, A. (2014). The many isoforms of human adenylate kinases. Int. J. Biochem. Cell. B 49, 75–83. doi: 10.1016/j.biocel.2014.01.014 Pérez, E., Sánchez-Murcia, P. A., Jordaan, J., Blanco, M. D., Mancheño, J. M., Gago, F., et al. (2018). Enzymatic synthesis of therapeutic nucleosides using a highly versatile purine nucleoside 2’-deoxyribosyltransferase from Trypanosoma brucei. Chemcatchem 10, 4406–4416. doi: 10.1002/cctc.201800775 Raman, J., Sumathy, K., Anand, R. P., and Balaram, H. (2004). A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity. Arch. Biochem. Biophys. 427, 116–122. doi: 10.1016/j.abb.2004.04.014 Reddy Chichili, V. P., Kumar, V., and Sivaraman, J. (2013). Linkers in the structural biology of protein–protein interactions. Protein Sci. 22, 153–167. doi: 10.1002/pro.2206 Roe, D. R., and Cheatham, T. E. I. I. I. (2013). PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095. doi: 10.1021/ct400341p Serra, I., Ubiali, D., Piškur, J., Munch-Petersen, B., Bavaro, T., and Terreni, M. (2017). Immobilization of deoxyadenosine kinase from Dictyostelium discoideum (DddAK) and its application in the 5’-phosphorylation of arabinosyladenine and arabinosyl-2-fluoroadenine. Chem. Select 2, 5403–5408. doi: 10.1002/slct.201700558 Sinha, S. C., and Smith, J. L. (2001). The PRT protein family. Curr. Opin. Struct. Biol. 11, 733–739. doi: 10.1016/S0959-440X(01)00274-3 Ubiali, D., and Speranza, G. (2019). “Enzymatic phosphorylation of nucleosides,” in Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives, in Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives, eds J. Fernández-Lucas and M. J. Camarasa (Weinheim: Wiley), 29–42. doi: 10.1002/9783527812103.ch2 Vajda, S., Yueh, C., Beglov, D., Bohnuud, T., Mottarella, S. E., Xia, B., et al. (2017). New additions to the ClusPro server motivated by CAPRI. Proteins 85, 435–444. doi: 10.1002/prot.25219 Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., et al. (2018). SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303. doi: 10.1093/nar/gky427 Wenck, M. A., Medrano, F. J., Eakin, A. E., and Craig, S. P. (2004). Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi. BBA Proteins Proteom. 1700, 11–18. doi: 10.1016/j.bbapap.2004.03.009 Whitford, P. C., Gosavi, S., and Onuchic, J. N. (2007). Conformational transitions in adenylate kinase. J. Biol. Chem. 283, 2042–2048. doi: 10.1074/jbc.m707632200 Yoshikawa, M., Kato, T., and Takenishi, T. (1967). A novel method for phosphorylation of nucleosides to 5’-nucleotides. Tetrahed. Lett. 8, 5065–5068. doi: 10.1016/S0040-4039(01)89915-9 Yoshikawa, M., Kato, T., and Takenishi, T. (1969). Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 42, 3505–3508. doi: 10.1246/bcsj.42.3505 Zeller, F., and Zacharias, M. (2015). Substrate binding specifically modulates domain arrangements in adenylate kinase. Biophys. J. 109, 1978–1985. doi: 10.1016/j.bpj.2015.08.049 Zhou, X., Hu, J., Zhang, C., Zhang, G., and Zhang, Y. (2019). Assembling multidomain protein structures through analogous global structural alignments. PNAS 116, 15930–15938. doi: 10.1073/pnas.1905068116
dc.rights.spa.fl_str_mv	CC0 1.0 Universal
dc.rights.uri.spa.fl_str_mv	http://creativecommons.org/publicdomain/zero/1.0/
dc.rights.accessrights.spa.fl_str_mv	info:eu-repo/semantics/openAccess
dc.rights.coar.spa.fl_str_mv	http://purl.org/coar/access_right/c_abf2
rights_invalid_str_mv	CC0 1.0 Universal http://creativecommons.org/publicdomain/zero/1.0/ http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv	openAccess
dc.publisher.spa.fl_str_mv	Frontiers in Bioengineering and Biotechnology
institution	Corporación Universidad de la Costa
bitstream.url.fl_str_mv	https://repositorio.cuc.edu.co/bitstream/11323/6477/1/Hypoxanthine-Guanine%20Phosphoribosyltransferase%20adenylate%20Kinase%20From%20Zobellia%20galactanivorans.pdf https://repositorio.cuc.edu.co/bitstream/11323/6477/2/license_rdf https://repositorio.cuc.edu.co/bitstream/11323/6477/3/license.txt https://repositorio.cuc.edu.co/bitstream/11323/6477/4/Hypoxanthine-Guanine%20Phosphoribosyltransferase%20adenylate%20Kinase%20From%20Zobellia%20galactanivorans.pdf.jpg https://repositorio.cuc.edu.co/bitstream/11323/6477/5/Hypoxanthine-Guanine%20Phosphoribosyltransferase%20adenylate%20Kinase%20From%20Zobellia%20galactanivorans.pdf.txt
bitstream.checksum.fl_str_mv	6db1950fe119cd51afc342dca818b21e 42fd4ad1e89814f5e4a476b409eb708c e30e9215131d99561d40d6b0abbe9bad df335f2add7998316b0158ebec6fef02 c335ebbd3c17c45cbe9193bc6a067127
bitstream.checksumAlgorithm.fl_str_mv	MD5 MD5 MD5 MD5 MD5
repository.name.fl_str_mv	Repositorio Universidad de La Costa
repository.mail.fl_str_mv	bdigital@metabiblioteca.com
_version_	1808400008235450368
spelling	Acosta, Javier7698a1f34892df8735789cd6b651f8dbDel Arco, Jon923cae3a1d238f02847617e53bdc6387Del Pozo, Maria Luisae65055cc9e17382c5632d494ab00e973Herrera, Beliñabebcc6a7545fa460c7426fbec5b9a5b3Clemente-Suárez, Vicente Javieraa432216f3fbd15b4192bba77ed7f848Berenguer, José9af418f40115a56362d9b45f5440cb68Hidalgo, Aurelio4cc2ce2709a7706275a8e97402e7cb50Fernández-Lucas, Jesús203a2ac57497988acae8aa40b528a49c2020-07-07T19:25:59Z2020-07-07T19:25:59Z2020-06-242296-4185https://hdl.handle.net/11323/6477https://doi.org/10.3389/fbioe.2020.00677Corporación Universidad de la CostaREDICUC - Repositorio CUChttps://repositorio.cuc.edu.co/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 Mg2+ and Co2+ 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.engFrontiers in Bioengineering and BiotechnologyCC0 1.0 Universalhttp://creativecommons.org/publicdomain/zero/1.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Enzymatic synthesisNucleotidesPhosphoribosyltransferaseNucleoside-5cpsdummy′-monophosphate kinaseDual domain proteinHypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and TriphosphatesArtículo 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/acceptedVersionAcosta, J., Del Arco, J., Martinez-Pascual, S., Clemente-Suárez, V., and Fernández-Lucas, J. (2018). One-pot multi-enzymatic production of purine derivatives with application in pharmaceutical and food industry. Catalysts 8:9. doi: 10.3390/catal8010009Ådén, J., Weise, C. F., Brännström, K., Olofsson, A., and Wolf-Watz, M. (2013). Structural topology and activation of an initial adenylate kinase–substrate complex. Biochemistry 52, 1055–1061. doi: 10.1021/bi301460kAli, L. Z., and Sloan, D. L. (1986). Activation of hypoxanthine/guanine phosphoribosyltransferase from yeast by divalent zinc and nickel ions. J. Inorg. Biochem. 28, 407–415. doi: 10.1016/0162-0134(86)80026-5Argos, P. (1990). An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J. Mol. Biol. 211, 943–958. doi: 10.1016/0022-2836(90)90085-ZBrown, P. H., and Schuck, P. (2006). Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys. J. 90, 4651–4661. doi: 10.1529/biophysj.106.081372Case, D., Betz, R. M., Cerutti, D. S., Cheatham, T., Darden, T., Duke, R., et al. (2016). AMBER 2016. San Francisco: University of California.Chen, X., Zaro, J., and Shen, W. C. (2013). “Fusion protein linkers: effects on production, bioactivity, and pharmacokinetics,” in Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges, ed. S. R. Schmidt (Hoboken, NJ: John Wiley & Sons), 57–73. doi: 10.1016/j.addr.2012.09.039Davlieva, M., and Shamoo, Y. (2010). Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability. Proteins 78, 357–364. doi: 10.1002/prot.22549Del Arco, J., Cejudo-Sanches, J., Esteban, I., Clemente-Suárez, V. J., Hormigo, D., Perona, A., et al. (2017). Enzymatic production of dietary nucleotides from low-soluble purine bases by an efficient, thermostable and alkali-tolerant biocatalyst. Food Chem. 237, 605–611. doi: 10.1016/j.foodchem.2017.05.136Del Arco, J., and Fernández-Lucas, J. (2017). Purine and pyrimidine phosphoribosyltransferases: a versatile tool for enzymatic synthesis of nucleoside-5’-monophosphates. Curr. Pharm. Des. 23, 6898–6912. doi: 10.2174/1381612823666171017165707Del Arco, J., and Fernández-Lucas, J. (2018). Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives. Appl. Microbiol. Biotechnol. 102, 7805–7820. doi: 10.1007/s00253-018-9242-8Del Arco, J., Acosta, J., Pereira, H. M., Perona, A., Lokanath, N. K., Kunishima, N., et al. (2018a). Enzymatic production of non-natural nucleoside-5’-monophosphates by a Thermostable uracil phosphoribosyltransferase. Chemcatchem 10, 439–448. doi: 10.1002/cctc.201701223Del Arco, J., Martinez, M., Donday, M., Clemente-Suarez, V. J., and Fernández-Lucas, J. (2018b). Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8. Biocatal. Biotransform. 36, 216–223. doi: 10.1080/10242422.2017.1313837Del Arco, J., Martínez-Pascual, S., Clemente-Suárez, V. J., Corral, O. J., Jordaan, J., Hormigo, D., et al. (2018c). One-pot, one-step production of dietary nucleotides by magnetic biocatalysts. Catalysts 8:184. doi: 10.3390/catal8050184Del Arco, J., Sánchez-Murcia, P. A., Mancheño, J. M., Gago, F., and Fernández-Lucas, J. (2018d). Characterization of an atypical, thermostable, organic solvent-and acid-tolerant 2’-deoxyribosyltransferase from Chroococcidiopsis thermalis. Appl. Microbiol. Biotechnol. 102, 6947–6957. doi: 10.1007/s00253-018-9134-yDel Arco, J., Mills, A., Gago, F., and Fernández-Lucas, J. (2019a). Structure-guided tuning of a selectivity switch towards ribonucleosides in Trypanosoma brucei purine nucleoside 2’-deoxyribosyltransferase. Chembiochem 20, 2996–3000. doi: 10.1002/cbic.201900397Del Arco, J., Pérez, E., Naitow, H., Matsuura, Y., Kunishima, N., and Fernández-Lucas, J. (2019b). Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5’-monophospate analogues. Bioresour. Technol. 276, 244–252. doi: 10.1016/j.biortech.2018.12.120Delano, W. L. (2002). The PyMOL Molecular Graphics System. San Carlos, CA: De Lano Scientific.Ding, Q., and Ou, L. (2017). NTP regeneration and its application in the biosynthesis of nucleotides and their derivatives. Curr. Pharm. Des. 23, 6936–6947. doi: 10.2174/1381612823666171024155247Ebina, T., Toh, H., and Kuroda, Y. (2011). DROP: an SVM domain linker predictor trained with optimal features selected by random forest. Bioinformatics 27, 487–494. doi: 10.1093/bioinformatics/btq700el Kouni, M. H. (2003). Potential chemotherapeutic targets in the purine metabolism of parasites. Pharmacol. Ther. 99, 283–309. doi: 10.1016/S0163-7258(03)00071-8Fernández-Lucas, J. (2015). Multienzymatic synthesis of nucleic acid derivatives: a general perspective. Appl. Microbiol. Biotechnol. 99, 4615–4627. doi: 10.1007/s00253-015-6642-xFernández-Lucas, J., Acebal, C., Sinisterra, J. V., Arroyo, M., and de la Mata, I. (2010). Lactobacillus reuteri 2’-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides. Appl. Environ. Microbiol. 76, 1462–1470. doi: 10.1128/aem.01685-09Fernández-Lucas, J., Fresco-Taboada, A., de la Mata, I., and Arroyo, M. (2012). One-step enzymatic synthesis of nucleosides from low water-soluble purine bases in non-conventional media. Bioresour. Technol. 115, 63–69. doi: 10.1016/j.biortech.2011.11.127Formoso, E., Limongelli, V., and Parrinello, M. (2015). Energetics and structural characterization of the large-scale functional motion of adenylate kinase. Sci. Rep. 5:8425. doi: 10.1038/srep08425Fresco-Taboada, A., de la Mata, I., Arroyo, M., and Fernández-Lucas, J. (2013). New insights on nucleoside 2’-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs. Appl. Microbiol. Biotechnol. 97, 3773–3785. doi: 10.1007/s00253-013-4816-yGeorge, R. A., and Heringa, J. (2002). An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. Des. Sel. 15, 871–879. doi: 10.1093/protein/15.11.871Hochstadt, J. (1978). Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase from enteric bacteria. Methods Enzymol. 51, 549–558. doi: 10.1016/S0076-6879(78)51077-XKamel, S., Yehia, H., Neubauer, P., and Wagner, A. (2019). “Enzymatic synthesis of nucleoside analogues by nucleoside phosphorylases,” in Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives, ed. M. J. Fernández-Lucas (Weinheim: Wiley-VCH), 1–28. doi: 10.1002/9783527812103.ch1Kanagawa, M., Baba, S., Ebihara, A., Shinkai, A., Hirotsu, K., Mega, R., et al. (2010). Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 893–898. doi: 10.1107/S1744309110023079Lapponi, M. J., Rivero, C. W., Zinni, M. A., Britos, C. N., and Trelles, J. A. (2016). New developments in nucleoside analogues biosynthesis: a review. J. Mol. Catal. B Enzym. 133, 218–233. doi: 10.1016/j.molcatb.2016.08.015Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992). “Computer aided interpretation of analytical sedimentation data for proteins,” in Analytical Ultracentrifugation In Biochemistry And Polymer Science, eds S. E. Harding, J. C. Horton, and A. J. Rowe (Cambridge: Royal Society of Chemistry), 90–125.Lewkowicz, E. S., and Iribarren, A. M. (2017). Whole cell biocatalysts for the preparation of nucleosides and their derivatives. Curr. Pharm. Design. 23, 6851–6878. doi: 10.2174/1381612823666171011101133Mbewe, B., Chibale, K., and McIntosh, D. B. (2007). Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized reactive red 120. Protein Expr. Purif. 52, 153–158. doi: 10.1016/j.pep.2006.09.014Mikhailopulo, I. A. (2007). Biotechnology of nucleic acid constituents-State of the art and perspectives. Curr. Org. Chem. 11, 317–335. doi: 10.2174/138527207780059330Minton, A. P. (1997). Alternative strategies for the characterization of associations in multicomponent solutions via measurement of sedimentation equilibrium. Prog. Colloid Polym. Sci. 107, 11–19. doi: 10.1007/BFb0118010Montero, C., and Llorente, P. (1991). Artemia purine phosphoribosyltransferases. Purification and characterization. Biochem. J. 275, 327–334. doi: 10.1042/bj2750327Motulsky, H., and Christopoulos, A. (2019). Fitting Models to Biological Data using Linear and Nonlinear Regression. A Practical Guide to Curve Fitting. New York, NY: Oxford University Press.Mukhopadhyay, A., Kladova, A. V., Bursakov, S. A., Gavel, O. Y., Calvete, J. J., Shnyrov, V. L., et al. (2010). Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria. J. Biol. Inorg. Chem. 16, 51–61. doi: 10.1007/s00775-010-0700-8Munagala, N. R., Chin, M. S., and Wang, C. C. (1998). Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. Biochemistry 37, 4045–4051. doi: 10.1021/bi972515hNiesen, F. H., Berglund, H., and Vedadi, M. (2007). The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221. doi: 10.1038/nprot.2007.321Panayiotou, C., Solaroli, N., and Karlsson, A. (2014). The many isoforms of human adenylate kinases. Int. J. Biochem. Cell. B 49, 75–83. doi: 10.1016/j.biocel.2014.01.014Pérez, E., Sánchez-Murcia, P. A., Jordaan, J., Blanco, M. D., Mancheño, J. M., Gago, F., et al. (2018). Enzymatic synthesis of therapeutic nucleosides using a highly versatile purine nucleoside 2’-deoxyribosyltransferase from Trypanosoma brucei. Chemcatchem 10, 4406–4416. doi: 10.1002/cctc.201800775Raman, J., Sumathy, K., Anand, R. P., and Balaram, H. (2004). A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity. Arch. Biochem. Biophys. 427, 116–122. doi: 10.1016/j.abb.2004.04.014Reddy Chichili, V. P., Kumar, V., and Sivaraman, J. (2013). Linkers in the structural biology of protein–protein interactions. Protein Sci. 22, 153–167. doi: 10.1002/pro.2206Roe, D. R., and Cheatham, T. E. I. I. I. (2013). PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095. doi: 10.1021/ct400341pSerra, I., Ubiali, D., Piškur, J., Munch-Petersen, B., Bavaro, T., and Terreni, M. (2017). Immobilization of deoxyadenosine kinase from Dictyostelium discoideum (DddAK) and its application in the 5’-phosphorylation of arabinosyladenine and arabinosyl-2-fluoroadenine. Chem. Select 2, 5403–5408. doi: 10.1002/slct.201700558Sinha, S. C., and Smith, J. L. (2001). The PRT protein family. Curr. Opin. Struct. Biol. 11, 733–739. doi: 10.1016/S0959-440X(01)00274-3Ubiali, D., and Speranza, G. (2019). “Enzymatic phosphorylation of nucleosides,” in Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives, in Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives, eds J. Fernández-Lucas and M. J. Camarasa (Weinheim: Wiley), 29–42. doi: 10.1002/9783527812103.ch2Vajda, S., Yueh, C., Beglov, D., Bohnuud, T., Mottarella, S. E., Xia, B., et al. (2017). New additions to the ClusPro server motivated by CAPRI. Proteins 85, 435–444. doi: 10.1002/prot.25219Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., et al. (2018). SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303. doi: 10.1093/nar/gky427Wenck, M. A., Medrano, F. J., Eakin, A. E., and Craig, S. P. (2004). Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi. BBA Proteins Proteom. 1700, 11–18. doi: 10.1016/j.bbapap.2004.03.009Whitford, P. C., Gosavi, S., and Onuchic, J. N. (2007). Conformational transitions in adenylate kinase. J. Biol. Chem. 283, 2042–2048. doi: 10.1074/jbc.m707632200Yoshikawa, M., Kato, T., and Takenishi, T. (1967). A novel method for phosphorylation of nucleosides to 5’-nucleotides. Tetrahed. Lett. 8, 5065–5068. doi: 10.1016/S0040-4039(01)89915-9Yoshikawa, M., Kato, T., and Takenishi, T. (1969). Studies of phosphorylation. III. Selective phosphorylation of unprotected nucleosides. Bull. Chem. Soc. Jpn. 42, 3505–3508. doi: 10.1246/bcsj.42.3505Zeller, F., and Zacharias, M. (2015). Substrate binding specifically modulates domain arrangements in adenylate kinase. Biophys. J. 109, 1978–1985. doi: 10.1016/j.bpj.2015.08.049Zhou, X., Hu, J., Zhang, C., Zhang, G., and Zhang, Y. (2019). Assembling multidomain protein structures through analogous global structural alignments. PNAS 116, 15930–15938. doi: 10.1073/pnas.1905068116ORIGINALHypoxanthine-Guanine Phosphoribosyltransferase adenylate Kinase From Zobellia galactanivorans.pdfHypoxanthine-Guanine Phosphoribosyltransferase adenylate Kinase From Zobellia galactanivorans.pdfapplication/pdf4990041https://repositorio.cuc.edu.co/bitstream/11323/6477/1/Hypoxanthine-Guanine%20Phosphoribosyltransferase%20adenylate%20Kinase%20From%20Zobellia%20galactanivorans.pdf6db1950fe119cd51afc342dca818b21eMD51open accessCC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8701https://repositorio.cuc.edu.co/bitstream/11323/6477/2/license_rdf42fd4ad1e89814f5e4a476b409eb708cMD52open accessLICENSElicense.txtlicense.txttext/plain; charset=utf-83196https://repositorio.cuc.edu.co/bitstream/11323/6477/3/license.txte30e9215131d99561d40d6b0abbe9badMD53open accessTHUMBNAILHypoxanthine-Guanine Phosphoribosyltransferase adenylate Kinase From Zobellia galactanivorans.pdf.jpgHypoxanthine-Guanine Phosphoribosyltransferase adenylate Kinase From Zobellia galactanivorans.pdf.jpgimage/jpeg60731https://repositorio.cuc.edu.co/bitstream/11323/6477/4/Hypoxanthine-Guanine%20Phosphoribosyltransferase%20adenylate%20Kinase%20From%20Zobellia%20galactanivorans.pdf.jpgdf335f2add7998316b0158ebec6fef02MD54open accessTEXTHypoxanthine-Guanine Phosphoribosyltransferase adenylate Kinase From Zobellia galactanivorans.pdf.txtHypoxanthine-Guanine Phosphoribosyltransferase adenylate Kinase From Zobellia galactanivorans.pdf.txttext/plain60519https://repositorio.cuc.edu.co/bitstream/11323/6477/5/Hypoxanthine-Guanine%20Phosphoribosyltransferase%20adenylate%20Kinase%20From%20Zobellia%20galactanivorans.pdf.txtc335ebbd3c17c45cbe9193bc6a067127MD55open access11323/6477oai:repositorio.cuc.edu.co:11323/64772023-12-14 11:25:11.926CC0 1.0 Universal\|\|\|http://creativecommons.org/publicdomain/zero/1.0/open accessRepositorio Universidad de La Costabdigital@metabiblioteca.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

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

Publicaciones similares