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

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

doi:10.3389/fbioe.2020.00677

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 (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.

Highlights

Purine nucleotides are involved in multitude of biochemical processes, but they are important as building blocks for RNA and DNA synthesis
After an in silico mining, we discovered an ORF that potentially encodes a putative hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/AMPK bifunctional protein annotated throughout the genome
According to the multiple sequence alignment of amino acid sequences of closest PRT homologs, ZgHGPRT/AMPK would belong to class I PRTs, which display a conserved 13-residue “fingerprint” region (PRPP binding-motif) in their amino acid sequence (Del Arco and Fernández-Lucas, 2017; Figure 4A)

Summary

Introduction

Purine nucleotides are involved in multitude of biochemical processes, but they are important as building blocks for RNA and DNA synthesis. Biosynthesis of purine nucleotides is performed through two different metabolic routes, de novo and salvage pathways. In the de novo pathway, purine nucleotides are synthesized from simple precursors like glycine, glutamine, or aspartate. Salvage pathway employs purine nucleobases to generate the corresponding nucleoside-5 -monophosphates (NMPs). This requirement for purines is satisfied by means of different endogenous and/or exogenous sources of preformed nitrogen bases (el Kouni, 2003; Del Arco and Fernández-Lucas, 2018). Both metabolic routes, de novo and salvage pathways, lead to inosine-5 -monophosphate (IMP) synthesis. IMP is converted to guanosine-5 -monophosphate (GMP) and adenosine-5 monophosphate (AMP), which are subsequently phosphorylated to get guanosine-5 -triphosphate (GTP) and adenosine-5 triphosphate (ATP), respectively

Methods

Results

Conclusion