The Peculiar Case of the Hyperthermostable Pyrimidine Nucleoside Phosphorylase from Thermus Thermophilus

The poor solubility of many nucleoside and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes which withstand these conditions. Herein we report that the pyrimidine nucleoside phosphorylase from Thermus thermophilus is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space (up to 80% (v/v) non-aqueous medium) and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10⁶ for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleoside substrates at low concentrations, which is unprecedented among non-specific pyrimidine nucleoside phosphorylases.