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 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.

Supplementary Information [2] and can, alternatively, be obtained from the Supplementary Information of previous publications. [6] The degree of conversion was determined directly from the spectra fit which considers the UV-active substrate and product in relation to one another. [4] For activity determination, only sampling points showing 3−10% conversion of the nucleoside substrate were considered. This lower bound was set due to the inherent inaccuracy of the UV-based method employed (roughly ±0.3 percentage points, due to the inherent error in spectral acquisition, as described in the original publication) [4] and the upper bound was applied as recommended by Cornish-Bowden [7] for equilibrium reactions. All datapoints outside this window were not included for calculation of activity and marked accordingly in the Supplementary Information. [2] Datapoints that displayed baseline shifts or other spectral anomalies were also excluded from consideration.
Background correction was performed as described recently. [5] Experimental spectra were fitted either across the entire spectrum or over the information-rich shoulder region of pyrimidine nucleosides/nucleobases, as appropriate for the analysis. All background corrections and the corresponding datafiles are detailed in the metadata files in the externally hosted supplementary information. [2] Enzymatic activity was determined by linear approximation of the conversion over time with a forced intercept at the origin. All raw data and the datapoints considered for calculation are freely available online with outliers and excluded datapoints clearly marked. [2] The observed rate constant was obtained by considering the degree of conversion (mol per second) per mol enzyme applied, using the molar extinction coefficient of TtPyNP of 26.930 cm -1 M -1 as predicted by Protparam [8] (i.e. the stock solution of 1 g L -1 had a concentration of 37.1 µM).
The activity of TtPyNP between pH 3 and 10 was determined using reaction mixtures of 1 mM 1a, 50 mM potassium phosphate and 8 µg mL -1 TtPyNP (2 µg mL -1 for pH 4−6) at 60 °C in a buffer mix consisting of 5 mM citrate, 10 mM MOPS and 20 mM glycine (all final concentration; adjusted to the respective pH value with NaOH and HCl solutions, not equated for ionic strength) in a final volume of 500 µL. Samples of 50 µL were withdrawn, quenched and analyzed after 1, 2 and 3 min as described above.
The activity of TtPyNP from 40−100 °C was determined using reaction mixtures of 1 mM 1a and 50 mM potassium phosphate in 50 mM glycine/NaOH buffer at pH 9 and the indicated temperature in a total volume of 500 µL. Depending on the temperature (and, therefore, on rate of phosphorolysis), 0.06−20 µg mL -1 TtPyNP were used (Table S1), to permit sampling of all reactions within the same time domain.
From all reactions, samples of 50 µL were withdrawn after 1, 2 and 3 min, quenched and analyzed as described above. The obtained data were fit to the Eyring equation [9] = ℎ exp − ‡ − ‡ The fit of the experimental data shown in Figure 1C yielded ∆ ‡ = 94.68 ± 3.06 kJ mol -1 and ‡ = 27.04 ± 1.08 J mol -1 K -1 (R 2 = 0.994). TtPyNP (0.12 µg mL -1 for 90 °C and 0.40 µg mL -1 for 80 °C) in 20 mM glycine/NaOH buffer pH 9 containing the indicated amount of cosolvent in a total volume of 500 µL were performed with samples taken after 1, 2 and 3 min. Quenching and data analysis was carried out as described above and data were fit as detailed in the metadata files available online. [2] The half-life of TtPyNP under different conditions was determined by incubation of TtPyNP (8.4−12.5 µg mL -1 ) in 20 mM potassium phosphate and 20 mM glycine/NaOH buffer pH 9 at the indicated temperatures in a total volume of 220 µL in a PCR tube (please note that the PCR tube was chosen intentionally for incubation since it ensures homogenous heating of the entire mixture without any sample cooling or evaporation). The tubes were incubated in a PCR cycler with lid heating for various timespans at a constant temperature as indicated. The lid temperature was set to 10 °C above the incubation temperature to prevent sample condensation at the lid. At certain timepoints, tubes were removed from the PCR cycler and immediately cooled on ice until activity determination. To assay for (residual) enzymatic activity, 150 µL of the incubated enzyme mixture were used to start a reaction consisting of 1 mM 1a, 20 mM potassium phosphate, 20 mM glycine/NaOH buffer pH 9 at 60 °C in a total volume of 500 µL (i.e. 150 µL enzyme mixture were added to 350 µL nucleoside mixture with both mixtures containing the same phosphate and buffer concentrations and a final total concentration of 1 mM 1a and 2.50−3.75 µg mL -1 TtPyNP). The assay was performed at 60 °C to ensure enzymatic activity during the activity assay without denaturation during the reaction. From these reactions, samples of 50 µL were withdrawn after 1, 2 and 4 min (or as indicated in the metadata file), quenched and analyzed as described above. Reactions containing organic solvent were performed analogously with the incubation mixture and the assay mixture containing the respective concentration of cosolvent. All data regarding residual activity were compared to the activity without any incubation (both aqueous and cosolvent incubations, i.e. since initial rate varied with cosolvent content, these data were referenced to the initial rate under exactly these conditions). To obtain the half-life of TtPyNP, activity data were fit to the first-order exponential decay function with definitions from above. Please see Table S2 for an overview of all incubated enzyme amounts and incubation times for each condition. All raw and transformed data can be obtained free of charge from an external online repository. [2] The predicted total turnovers pTTN were calculated as pTTN = , with , being the observed initial rate constant at the given temperature (as shown in Figures   1C, 2A and 2B), and definitions from above.
Please see the externally hosted Supporting Information for all raw data and fit results. [2]  The activity of TtPyNP with different pyrimidine substrates was determined using reaction mixtures of 1 mM nucleoside (1a−1i), 50 mM potassium phosphate and 0.25 µg mL -1 TtPyNP in 50 mM glycine/NaOH buffer at pH 9 and 80 °C in a total volume of 500 µL. Samples of 50 µL were withdrawn after 1, 2 and 3 min, quenched and analyzed as described above. Samples from reactions with 1a−1d were quenched in 100 mM NaOH and samples from reactions with 1e−1i were quenched in 200 mM NaOH. [4,5] Reference spectra for all nucleosides can be obtained from the externally hosted Supplementary Information [2] or from our previous publication about spectral unmixing. [5,6] 7  (Table S3), quenched and analyzed as described above. Supplementary items Figure S1. Activity of TtPyNP in dimethyl formamide (DMF). TtPyNP (0.12 µg mL -1 for 90 °C and 0.40 µg mL -1 for 80 °C) in 20 mM glycine/NaOH buffer pH 9

The activity of TtPyNP in reaction mixtures with DMF
containing the indicated amount of cosolvent in a total volume of 500 µL were performed with samples taken after 1, 2 and 3 min. Quenching and data analysis was carried out as described above and data were fit as detailed in the metadata files available online. [2] Figure S2. Kinetics of TtPyNP at 40 °C and pH 9. 9 The kinetics of TtPyNP at 40 °C and pH 9 were analyzed using reaction mixtures of 50 mM potassium phosphate and 20 or 50 µg mL -1 enzyme in 50 mM glycine/NaOH buffer pH 9 at 40 °C with the indicated nucleoside concentration in a total volume of 200 or 500 µL. Reactions were run for variable times (Table S4) and samples were withdrawn, quenched and analyzed as described above (no duplicates were performed).   (Table S5) and samples were withdrawn, quenched and analyzed as described above (no duplicates were performed). GtPyNP has an extinction coefficient of 21,890 cm -1 M -1 as predicted by Protparam, [8] thus the stock solution of 1.2 g L -1 had a concentration of 54.8 µM. The data were fit to the Michaelis-Menten equation (6) shown below. was used in all reactions for a temperature and substrate. Reactions were run for variable times and samples were withdrawn, quenched and analyzed as described above (Table S3). [2] Selected data were 19.95 ± 17.08 s -1 (R 2 = 0.676). However, it must be noted that the data in Figure S4 do not describe substrate inhibition, but instead a product inhibition which manifests itself in data resembling substrate inhibition.
with definitions from above. Figure S5. Non-linearity of product formation rate at higher substrate concentrations under quasisteady-state conditions. This figure shows selected raw data of the experiments shown in Figure S4.