Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8

Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvagepathway of living organisms. They are involved in the formation of C-N glycosidic bonds in pur-ine nucleosides-50-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl groupfrom 5-phospho-a-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence ofMg2þ. Herein, we report a simple and thermostable process for the one-pot, one-step synthesisof some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribo-syltransferase, APRT2, fromThermus thermophilusHB8. In this sense, the cloning, expression andpurification ofTtXPRT andTtAPRT2 is described for the first time. Both genes,xprtandaprt2were expressed as his-tagged enzymes inE. coliBL21(DE3) and purified by a heat-shock treat-ment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatog-raphy. Biochemical characterization revealedTtXPRT as a tetramer andTtAPRT2 as a dimer. Inaddition, both enzymes displayed a strong temperature dependence (relative activity>75% in atemperature range from 70 to 90C), but they also showed very different behaviour under theinfluence of pH. WhileTtXPRT is active in a pH range from 5 to 7,TtAPRT2 has a high depend-ence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally,substrate specificity studies