Abstract
The poor solubility of many nucleosides 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 that can 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) nonaqueous 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 nucleobases at low concentrations, which is unprecedented among nonspecific pyrimidine nucleoside phosphorylases.
Highlights
Nucleoside phosphorylases are useful biocatalysts for the synthesis of pentose-1-phosphates and nucleoside analogs.[1,2,3,4,5,6,7,8,9,10,11] These enzymes catalyze the reversible phosphorolysis of nucleosides to the corresponding pentose1-phosphates and nucleobases (Scheme 1) and can be employed in the reverse reaction for glycosylation and transglycosylation reactions to furnish nucleosides of interest directly from free nucleobases
In the course of our work we were surprised to discover that TtPyNP is inhibited by nucleoside substrates even at low concentrations, which is atypical for pyrimidine nucleoside phosphorylases
Following heterologous expression of the enzyme in Escherichia coli and purification via heat treatment and affinity chromatography, we explored the working space of TtPyNP using the phosphorolysis of uridine (1a) as a model reaction (Figure 1A)
Summary
Nucleoside phosphorylases are useful biocatalysts for the synthesis of pentose-1-phosphates and nucleoside analogs.[1,2,3,4,5,6,7,8,9,10,11] These enzymes catalyze the reversible phosphorolysis of nucleosides to the corresponding pentose1-phosphates and nucleobases (Scheme 1) and can be employed in the reverse reaction for glycosylation and transglycosylation reactions to furnish nucleosides of interest directly from free nucleobases.
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