Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates
Javier Acosta, Jon Del Arco, María Luisa del Pozo, Beliña Annery Herrera Tapias, Vicente Javier Clemente‐Suárez, José Berenguer, Aurélio Hidalgo, Jesús Fernández‐Lucas
Abstract
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 (AMK) from Zobellia galactanivorans (ZgHGPRT/AMK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMK. 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 ºC. Thermal denaturation experiments revealed that ZgHGPRT/AMK exhibits an apparent unfolding temperature (Tm) of 45 ºC and a retained activity of around 80 % when incubated at 40 ºC 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/AMK 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/AMK 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.