The activity of PyNP-04 was determined by performing phosphorolysis reactions with the substrates uridine (a), thymidine (b) and the model substrate 5-iodouridine (c). Samples were analyzed by the UV/Vis spectroscopy-based assay described recently. Briefly, a 500 µL reaction mixture consisting of 1 mM nucleoside and 50 mM K2HPO4 in 50 mM buffer (glycine/NaOH pH 9 or MOPS/NaOH pH 7) was preheated to the desired temperature (60 °C or 80 °C). The reaction was started by the addition of the enzyme (typically 10 µL of enzyme stock solution prediluted in 2 mM potassium phosphate buffer pH 7). Final concentrations of PyNP-04 from 150 to 750 ng mL -1 were applied. At timely intervals, samples of 60 µL were taken and quenched in 450 µL 100 mM NaOH and 200 µL of the diluted sample was transferred to a UV/Vis-transparent 96well plate to record the UV absorption spectra from 250 to 350 nm in steps of 1 nm. Spectral analysis was carried out as described previously with software and reference spectra freely available online.