Acosta, JavierDel Arco, JonDel Pozo, María LuisaHerrera-Tapias, BeliñaClemente-Suárez, Vicente JavierBerenguer, JoséHidalgo, AurelioFernández Lucas, Jesús2024-10-302024-10-302020Acosta J, Del Arco J, Del Pozo ML, Herrera-Tapias B, Clemente-Suárez VJ, Berenguer J, Hidalgo A and Fernández-Lucas J (2020) Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates. Front. Bioeng. Biotechnol. 8:677. doi: 10.3389/fbioe.2020.006772296-418510.3389/fbioe.2020.00677https://hdl.handle.net/20.500.14352/109789In 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.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal articlehttps://doi.org/10.3389/fbioe.2020.00677https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.00677/fullopen access577.113.3577.15577.260Enzymatic synthesisNucleotidesPhosphoribosyltransferaseNucleoside-5cpsdummy′-monophosphate kinaseDual domain proteinBioquímica (Biología)Biología molecular (Biología)Biotecnología2403 Bioquímica2415 Biología Molecular2302.09 Enzimología2302.23 Ácidos Nucleicos