Comparative investigations on thermostable pyrimidine nucleoside phosphorylases from Geobacillus thermoglucosidasius and Thermus thermophilus

The recombinant expression and biocatalytic characterization of two thermostable pyrimidine nucleoside phosphorylases (PyNP), isolated from Geobacillus thermoglucosidasius (Gt) and Thermus thermophilus (Tt) is described. Both enzymes are highly thermostable (half life of GtPyNP is 1.6 h at 70 °C, half life of TtPyNP is >24 h at 80 °C). Kinetic parameters for the phosphorolysis of natural substrates were determined for GtPyNP at 60 °C (Km for uridine 2.3 mM, Km for thymidine 1.3 mM) and TtPyNP at 80 °C (Km for uridine 0.15 mM, Km for thymidine 0.43 mM). The kcat values for uridine are almost identical for both enzymes (ca. 277 s−1), while the kcat value for thymidine is about 8 times higher for TtPyNP than for GtPyNP (679 s−1 vs. 83 s−1).Both enzymes were tested towards the ability to catalyze the phosphorolytic cleavage of 2′-fluorosubstituted pyrimidine nucleosides – a prerequisite for the efficient synthesis of a number of relevant purine nucleoside analogues. GtPyNP showed poor activity towards 2′-deoxy-2′-fluorouridine (dUrd2′F; 0.4% substrate conversion after 30 min), and the phosphorolysis of the epimeric counterpart 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (dUrd2′F) could not be detected at all. By contrast, TtPyNP showed dramatically higher conversion rates (15.6% and 1.6% conversion in 30 min of both substrates, respectively). The amount of converted pyrimidine nucleosides increased significantly with time. After 17 h 65% of dUrd2′F and 46% of dUrd2′F was phosphorolytically cleaved.Our results demonstrate the potential of TtPyNP as a biocatalyst in transglycosylation reactions aiming at the production of 2′-fluorosubstituted purine nucleosides that are highly bioactive but hardly accessible by chemical methods.

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► Kinetic parameters and thermal characteristics of pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius and Thermus thermophilus are described.
► Both enzymes efficiently catalyze the phosphorolysis of natural pyrimidine nucleosides substrates at high temperatures (60 °C and 80 °C).
► Striking differences in the acceptance of 2′-fluorosubstituted nucleosides as substrates can be seen.
► In particular the T. thermophilus enzyme appears to be a promising biocatalyst for chemo-enzymatic syntheses of modified purine nucleosides.