The Peculiar Case of the Hyper‐thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**

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 10 6 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.

Diversification of 4ʹ-Methylated Nucleosides by Nucleoside Phosphorylases

“…Similarly, no conversion was observed under reaction conditions outside of the working space of TtPyNP (pH 3 or pH 12, Figure 1A). [26] NMR analysis of a reaction mixture with TtPyNP and 1a corroborated the proposed reactivity and creation of the pentose-1-phosphate 3, as evident from the rise of an additional 1 H NMR signal at 5.57 ppm showing a strong H,P-HMQC signal (Figure 1B). Consistent with the native reactivity of PyNPs, inversion at the anomeric position was evident by this signal lacking NOE contacts to the 4ʹ-methyl group of 3, while the corresponding anomeric proton in 1a showed clear correlation to the methyl substituent.…”

“…Interestingly, the apparent Michalis-Menten constant KMʹ of the phosphorolysis of 1a (KMʹ = 3.37 mM) indicated that TtPyNP has a much lower affinity for 1a compared to natural nucleosides like uridine or thymidine (KM < 1 mM), [27] suggesting that productive binding of the modified substrate 1a might present a challenge due to the increased steric bulk. In addition to a lower affinity for 1a, TtPyNP also displayed a lower rate constant compared to uridine (0.59 vs 5.05 s -1 for 1 mM substrate at 60 °C and pH 9) [26] which showed a similar temperature-dependence as indicated by phosphorolysis experiments at different temperatures monitored by UV spectroscopy (Figure 1D). [29,30] Collectively, these results demonstrate that, unlike other nucleoside phosphorylases, TtPyNP selectively converts the 4ʹ-methylated nucleoside 1a to the corresponding sugar phosphate 3, albeit with a lower rate constant and substrate affinity compared to the native substrates.…”

“…Having established the activity of TtPyNP with 1a, we conducted kinetic experiments to provide further insights into this enzymatic transformation. Although TtPyNP is inhibited by pyrimidine nucleobases such as uracil (2a), [26] we could observe Michaelis-Menten behavior of the enzyme with 1a (Figure 1C). Interestingly, the apparent Michalis-Menten constant KMʹ of the phosphorolysis of 1a (KMʹ = 3.37 mM) indicated that TtPyNP has a much lower affinity for 1a compared to natural nucleosides like uridine or thymidine (KM < 1 mM), [27] suggesting that productive binding of the modified substrate 1a might present a challenge due to the increased steric bulk.…”

“…The data for uridine in D were taken from ref. 26. Please see the Supporting Information for details and the externally hosted supplementary information for raw data.…”

pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius

“…[29] While purine NPs have been researched intensively, [30][31][32][33][34][35][36] including their ratetemperature relationships, [10,37] comparably little is known about rate-temperature relationships of reactions catalyzed by the structurally distinct pyrimidine NPs. [38] Since these enzymes typically operate across a wide pH window with similar rate constants [39,40] and convert electronically diverse substrates, [38,39] we hypothesized that they would present a convenient model system to interrogate pH and deprotonation effects on the Eyring plots of nucleoside phosphorolysis, as an example of a simple nucleophilic substitution. Thus, we questioned if there exists an activation heat capacity change during the phosphorolysis reactions catalyzed by thermostable pyrimidine NPs and, if so, whether this effect shows any pH-and/or protonationdependence across the broad working space of these enzymes.…”