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

Frontiers in Bioengineering and Biotechnology
Javier AcostaJesús 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 (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 analysi...Continue Reading

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Datasets Mentioned

BETA
CAZ96627
CAZ96936.1
CAZ95963.1
CAZ97836.1
CAZ95571.1

Methods Mentioned

BETA
phosphotransferase
protein folding

Software Mentioned

SEDFIT
GraphPad Prism
OPtimal features server ( DROP
BLASTP
Model
Domain Enhanced Modeling
Swiss
Domain linker pRediction
ClusPro
pmemd

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