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

Article abstract of DOI:10.1002/cbic.202000679

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.

Full text of DOI:10.1002/cbic.202000679

Combining Chemistry and Biology
Accepted Article
Title: The Peculiar Case of the Hyperthermostable Pyrimidine
Nucleoside Phosphorylase from Thermus thermophilus
Authors: Felix Kaspar, Peter Neubauer, and Anke Kurreck
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To be cited as: ChemBioChem 10.1002/cbic.202000679
Link to VoR: https://doi.org/10.1002/cbic.202000679
01/2020

10.1002/cbic.202000679
ChemBioChem
COMMUNICATION
The Peculiar Case of the Hyperthermostable Pyrimidine
Nucleoside Phosphorylase from Thermus thermophilus
F. Kaspar[a],[b],*, P. Neubauer[a] and A. Kurreck[a],[b],
*
[a]
F. Kaspar, Dr. P. Neubauer and Dr. A. Kurreck
Department of Biotechnology, Chair of Bioprocess Engineering
Technical University of Berlin
Ackerstraße 76, ACK24, D-13355, Germany
E-mail: anke.wagner@tu-berlin.de
F. Kaspar and Dr. A. Kurreck
[b]
BioNukleo GmbH
Ackerstraße 76, D-13355, Berlin, Germany
E-mail: felix.kaspar@web.de
Supporting information for this article is given via a link at the end of the document.
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 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 nucleobases at low concentrations, which is
reaction mixtures containing high concentrations of organic
solvents like DMSO and DMF.[15] Therefore, we hypothesized that
such
a hyperthermostable enzyme would withstand harsh
conditions such as near-boiling cosolvent-heavy media and
enable greatly increased substrate loading compared to
commonly performed nucleoside transglycosylations.[8,9,16] This
prompted us to investigate TtPyNP in more detail for applications
in nucleoside synthesis and probe the limits of its tolerance to
harsh conditions. In this communication, we report that TtPyNP is
active and stable over
temperature, pH and cosolvents and accepts
a
vast working space regarding
range of
a
substituted pyrimidine nucleosides. In the course of our work we
were surprised to discover that TtPyNP is inhibited by
nucleobases even at low concentrations, which is atypical for
pyrimidine nucleoside phosphorylases. Hence, TtPyNP is a
suboptimal candidate for industrial applications where high
substrate loading is a prerequisite.
unprecedented
phosphorylases.
among
non-specific
pyrimidine
nucleoside
Nucleoside phosphorylases are useful biocatalysts for the
synthesis of pentose-1-phosphates and nucleoside analogs.[1–11]
These enzymes catalyze the reversible phosphorolysis of
nucleosides to the corresponding pentose-1-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. The
current primary bottleneck for the efficient application of these
enzymes for synthetic purposes is the low water-solubility of many
nucleosides and nucleobases, restricting the substrate loading to
the low millimolar range.[12] Increased substrate solubility can be
achieved through the use of harsh reaction conditions (base, heat,
cosolvent) and, consequently, thermostable nucleoside
phosphorylases have attracted particular interest due to their
stability and activity under these conditions.[1,2,4] Recent work from
our group has demonstrated that some of these enzymes can be
employed reliably at temperatures of up to 70 °C and over a broad
pH range up to at least pH 9, which creates an exceptionally large
working space and allows adjustments of the reaction conditions
to suit the substrate(s).[7,13,14] Further benefits of these
thermostable enzymes, including their long shelf life and easy
purification via heat treatment of crude extracts, make these
catalysts attractive for various applications.
Scheme 1. Phosphorolysis of pyrimidine nucleosides catalyzed e.g. by
Thermus thermophilus pyrimidine nucleoside phosphorylase (TtPyNP, PDB
2dsj).
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).
Since this reaction is under tight thermodynamic control (K = 0.2
at 60 °C and pH 9),[7] we applied an excess of phosphate in these
experiments to drive the reaction in the phosphorolysis direction.
TtPyNP displayed phosphorolytic activity from pH 4 to 10 (Figure
1B), with a clear preference for acidic conditions as the observed
rate constants kobs differed by more than a factor of five between
pH 4 and 10. This observation is somewhat counterintuitive since
i) pentose-1-phosphates are prone to hydrolysis under acidic con-
The pyrimidine nucleoside phosphorylase from Thermus
thermophilus (TtPyNP, Scheme 1) has been reported to be active
at up to 100 °C in aqueous media and to display some activity in
1
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10.1002/cbic.202000679
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the working space of TtPyNP covers most of the pH and
temperature range accessible in water and, given the activity of
the enzyme, we anticipated that its overall performance would
only be limited by its stability under the applied reaction conditions.
Next, we turned our attention to the activity of TtPyNP in the
presence of organic cosolvents. Previous work with this enzyme
indicated that some activity is retained in mixtures containing 50%
(v/v) DMSO or DMF.[15] These two solvents fall into the short list
of common organic solvents that meet the prerequisites to be
considered as cosolvents for high-temperature biocatalytic
reactions involving nucleosides: i) a boiling point > 100 °C, ii)
water-miscibility[21] and iii) resistance to hydrolysis. These
prerequisites readily disqualify commonly used alcohols such as
methanol, ethanol or isopropanol,[22] as well as aromatics, ethers,
hydrocarbons and esters. Considering that DMF is toxic, highly
harmful to the environment[23] and rather detrimental to the activity
of TtPyNP (Figure S1), we selected DMSO and ethylene glycol as
promising cosolvents and interrogated TtPyNP’s activity at
different concentrations of these solvents, again employing the
phosphorolysis of 1a as a model reaction. TtPyNP displayed
activity at up to 60% (v/v) DMSO and 80% (v/v) ethylene glycol,
with more cosolvent being tolerated at 80 °C than at 90 °C (Figure
Figure 1. Working space of TtPyNP. The phosphorolysis of uridine (1a, A) was
employed to investigate the pH-dependence (B) and the temperature-
dependence (C) of the phosphorolytic activity as quantified by the observed rate
constant kobs. To assay across the pH space, reactions were performed with
1 mM 1a, 50 mM potassium phosphate, 2 or 8 µg mL-1 TtPyNP 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 at 25 °C, not equated for
ionic strength) in a final volume of 500 µL. To assay across the temperature
space, reactions were carried out with 1 mM 1a, 50 mM potassium phosphate
and 0.06−20 µg mL-1 TtPyNP in 50 mM glycine/NaOH buffer at pH 9 and the
indicated temperature. Data shown represent the average of two experiments.
Error bars are too small to see and were omitted for clarity. Fit results for C are
available from the Supporting Information. Please see the Supporting
Information for more experimental details and the externally hosted Supporting
Information for raw data and calculations.[20]
ditions,[2,17] ii) an intracellular pH in the range of 7 would suggest
a preference for neutral conditions and iii) other pyrimidine
nucleoside phosphorylases are inactive at pH values below 6.[18]
In accordance with previous reports,[13,15] TtPyNP was active at
temperatures of up to 100 °C (Figure 1C), with the temperature-
dependence of kobs (in contrast to previous reports)[15] following
the trends predicted by conventional transition state theory as
described by the Eyring equation.[19] These results indicated that
Figure 2. Phosphorolytic activity of TtPyNP in the presence of the organic
cosolvents dimethyl sulfoxide (DMSO, A) and ethylene glycol (B). Reactions
were run with 1 mM 1a, 20 mM potassium phosphate and 0.12 or 0.40 µg mL-1
TtPyNP in 20 mM glycine/NaOH buffer pH 9 containing the indicated amount of
cosolvent at the indicated temperature. Data shown represent the average of
two experiments. Error bars are too small to see and were omitted for clarity.
Please see the Supporting Information for more experimental details and the
externally hosted Supporting Information for raw data and calculations.[20]
2
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10.1002/cbic.202000679
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2A and B). Interestingly, moderate amounts of either cosolvent
(i.e. 20−40% (v/v)) promoted higher activities than observed in
purely aqueous solution, presumably due to decreased enzymatic
flexibility and a higher affinity for both substrates in a slightly less
polar environment.[24,25]
Having established the activity of TtPyNP at high
temperatures and with considerable amounts of cosolvent, we
were interested in the half-life and total turnovers of the enzyme
to evaluate if these harsh conditions were feasible for any
Next, we probed the substrate scope of this enzyme by
subjecting a series of substituted pyrimidine nucleosides to
phosphorolysis. Previous work had demonstrated that 1a, its 2’-
deoxy analogue 1b and thymidine (1d) are converted by TtPyNP
to obtain the corresponding sugar phosphate and
nucleobase.[13,15] Our results corroborate and extend these
reports by showing that substitutions in the 5-position at the
nucleobase, such as aliphatic residues or halogens, are generally
well tolerated without any loss of activity compared to the native
reactions that exceeded the short reaction times of activity assays. substrates 1a and 1d (Scheme 2). Comparable or slightly higher
To determine the half-life t1/2 and predicted total turnover number
(pTTN) of TtPyNP, we incubated the enzyme in buffered solution
with phosphate, determined its residual activity after various
incubation times and approximated the incubation time-
dependent decrease in activity as a first-order exponential decay.
Despite its remarkable activity at 100 °C and pH 9 in purely
aqueous solution (Figure 1B), the half-life of TtPyNP under these
conditions was only 2.2 min, corresponding to a pTTN of only
around 28,000 (Table 1, entry 1). At 90 °C and 80 °C, we found
half-lives of 100 min and more than 19 h, respectively, with the
pTTNs increasing accordingly to well above 106, despite the lower
turnover rates at these temperatures (Table 1, entries 2 and 3).
These experiments demonstrated that denaturation-driven loss of
activity proceeds rapidly near the boiling point of water. However,
slightly lower temperatures facilitated increased stability and
enzymatic activity, as reflected by the predicted total turnovers.
As transformations at 80−90 °C appeared feasible from a stability
standpoint, we then assessed the stability of TtPyNP in the
presence of DMSO and ethylene glycol, using the same
experimental set-up. Although both solvents permitted excellent
activity of TtPyNP at 90 °C, the half-life of the enzyme did not
surpass 30 min at 40% (v/v) of either solvent at this temperature,
reflected by pTTNs below 200,000 (Figure 3A and B).
Nonetheless, a modest decrease in temperature to 80 °C resulted
in much higher half-lives and pTTNs. For example, TtPyNP had a
half-life of 4.4 h at 80 °C and 60% (v/v) ethylene glycol,
corresponding to around 1,500,000 predicted total turnovers
(Figure 3B). Even DMSO was tolerated reasonably well, with
TtPyNP achieving a pTTN of 3,100,000 at 80 °C and 30% (v/v)
DMSO (Figure 3A). Taken together, these data demonstrate that
TtPyNP performs favorably at up to 80 °C in media with up to
around 50% (v/v) of DMSO or ethylene glycol.
levels of activity were obtained with 2’-deoxy nucleosides, which
agrees well with the broad substrate spectrum typically observed
for non-specific pyrimidine nucleoside phosphorylases.[7–9] Given
all the above results, TtPyNP indeed seemed useful as a catalyst
Table 1. Activity and stability of TtPyNP in aqueous solution.
Temperature
[°C]
ꢀꢁꢂꢃ
[b]
ꢄꢅ/ꢆ
pTTN [106][c]
[s-1][a]
Figure 3. Performance of TtPyNP assessed by the predicted total turnover
number (pTTN) of the substrate 1a in hot cosolvent-heavy reaction mixtures.
The half-life of TtPyNP was determined by incubating the enzyme at
concentrations of 8.4−12.5 µg mL-1 in 20 mM potassium phosphate and 20 mM
glycine/NaOH buffer pH 9 and the indicated concentrations of DMSO (A) or
ethylene glycol (B) at the indicated temperatures in a total volume of 220 µL in
a PCR tube and measuring the residual activity through phosphorolysis of the
model substrate 1a at timely intervals (assay conditions of 1 mM 1a, 20 mM
potassium phosphate and 2.5−3.75 µg mL-1 TtPyNP in 20 mM glycine buffer pH
9 at 60 °C). Fitting of the incubation time-dependent decrease in activity as a
first-order exponential decay yielded the half-life under the respective conditions,
which was used to calculate pTTN via the initial rate constant under these
conditions (Figure 2A and B). Please see the externally hosted Supporting
Information for all raw and calculated data as well as tabulated half-lives and
predicted total turnover numbers.[20]
100
90
215.6
117.1
45.7
2.2 min
100.2 min
19.5 h
0.028
1.296
80
15.139
[a] Determined as initial rate with 1a at pH 9 and the respective temperature as
shown in Figure 1C. [b] Determined from incubation experiments from the
incubation time-dependent decay of enzymatic activity (initial rate) at pH 9 and
the respective temperature (please see the externally hosted Supporting
Information for all raw and calculated data).[20] The activity assay was performed
at 60 °C to ensure enzyme stability during the assay. [c] Determined via
multiplication of ꢀꢁꢂꢃ and the mean catalyst lifetime ꢇ as detailed in the
Supporting Information.
3
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Scheme 2. Phosphorolysis of 5-substituted pyrimidine nucleosides by TtPyNP.
Reactions were performed with 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. Data shown represent the average of two experiments. Standard
deviations were all 5% or below and were omitted for clarity. Please see the
Supporting Information for more experimental details and the externally hosted
Supporting Information for raw data and calculations.[20]
for the (reversible) phosphorolysis of various pyrimidine
nucleosides as it displays a reasonably broad substrate scope
and remarkable stability under harsh conditions including high
temperature and cosolvent content.
However, despite TtPyNP’s excellent activity and stability,
its use as a biocatalyst in synthetic applications is limited due to
an apparent inhibition by nucleobases. We discovered this
phenomenon when we attempted to determine the Michaelis-
Menten constant KM for the substrate 1a using concentrations of
up to 50 mM (Figure S2). Previous work had reported KM values
in the range of 0.2−1.0 mM at 80 °C and pH 7 (for 1a and 1d in
phosphate buffer)[13,15] which compares well with other pyrimidine
nucleoside phosphorylases.[26–28] However, our data at 40 °C and
pH 9 suggested a strong inhibition behavior which did not appear
to resemble any Michaelis-Menten-type kinetics (Figure S2).
Follow-up experiments with 1a at 80 °C and pH 7 revealed that,
indeed, TtPyNP appeared to follow Michaelis-Menten-like kinetics
for nucleoside concentrations up to 1−2 mM (under saturating
phosphate concentrations for which typical Michaelis-Menten
behavior was observed; Figure S4). In contrast, the observed rate
constants of phosphorolysis at higher nucleoside concentrations
suggested a possible substrate inhibition by nucleosides with an
apparent inhibitory constant Ki within the same order of magnitude
as KM. However, the non-linearity of the conversion over time at
higher substrate concentrations under quasi-steady state
conditions (Figure S5) provided some evidence that TtPyNP
might instead be inhibited by one of the products. Indeed, non-
zero initial concentrations of the nucleobase 2a caused a drastic
decrease of the kobs of phosphorolysis, while the sugar phosphate
3 had no effect (Figure 4A). Due to this strong inhibition of TtPyNP
by 2a, we were unable to determine the Ki from Michaelis-Menten
plots of the phosphorolysis reaction. Therefore, we attempted to
approximate the Ki by measuring the KM for 2a in the reverse
reaction (glycosylation with 3). However, instead of saturation-
Figure 4. Inhibition of TtPyNP by substrates and products. TtPyNP is inhibited
in the phosphorolysis direction by increasing concentrations of the nucleobase
2a, but not by the sugar phosphate 3 (A). The reverse reaction is also inhibited
by increasing concentrations of 2a (B). Reactions in A were carried out with
1 mM 1a, 50 mM potassium phosphate and 0.33 mL-1 TtPyNP in 50 mM
MOPS/NaOH buffer pH 7 at 80 °C with the indicated concentration of either
product (2a or 3) in a total volume of 200 µL. Reactions in B were performed
with 2 mM 3 and 0.17 µg mL-1 TtPyNP in 50 mM MOPS/NaOH buffer pH 7 at
80 °C with the indicated concentration of 2a in a total volume of 200 µL. For
each condition, reactions were carried out in duplicates and all data points are
shown. Raw data and calculations are freely available from an external online
repository.[20]
type kinetics we only observed a significant decrease of the kobs
of glycosylation with increasing concentrations of 2a, indicating
that this nucleobase also inhibits its own glycosylation (Figure 4B).
4
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To exclude any of our assay conditions causing these effects, we
determined the KM for 1a of the corresponding enzyme from
Geobacillus thermoglucosidasius (GtPyNP) and observed classic
Michaelis-Menten behavior with no evidence of inhibition up to
50 mM 1a in agreement with the available literature (Figure
S3).[13] Interestingly, similar inhibition effects of TtPyNP were also
observed, to varying degrees, with the nucleosides 1d and 1f
(Figure S6), revealing that inhibition of this enzyme by
nucleobases is not limited to 2a. Together, these results present
evidence that TtPyNP is inhibited by nucleobases through a yet
unknown mechanism. The reason(s) for this apparent product
inhibition (or substrate inhibition, depending on the direction) are
unclear to date as TtPyNP shares high structural[29] and sequence
identity (see Figure 1 in [13]) to other thymidine and pyrimidine
nucleoside phosphorylases concerning the active site residues
and overall protein structure. Although substrate inhibition is
known for uridine phosphorylases,[28,30] this is, to the best of our
knowledge, the first reported example of a pyrimidine nucleoside
phosphorylase being competitively substrate/product-inhibited.
However, we doubt that this inhibition of TtPyNP holds any
physiological significance, as the intracellular concentrations of
nucleosides and their bases are typically in the low micromolar
range, which is more than two orders of magnitude lower than the
concentrations necessary to effect significant inhibition of TtPyNP.
In any case, this clearly makes TtPyNP a rather suboptimal
candidate for preparative purposes. In order to achieve
satisfactory product titers, substrate concentrations of at least
100 mM typically need to be applied.[12,31,32] Our characterization
revealed that TtPyNP is severely inhibited by nucleobases at
concentrations upwards of 0.5 mM, which limits its reactivity both
in the phosphorolysis and glycosylation direction and renders its
performance in potential industrial applications subpar.
with metadata and calculations are freely available from an
external online repository.[20] Likewise, reference spectra and
software for spectral unmixing are available from the same
repository[35,36] and described in previous works.[33,34]
Acknowledgements
The authors thank Kerstin Heinecke (TU Berlin) for proofreading
and critical comments.
Keywords: nucleoside • nucleoside phosphorylase •
thermostable • cosolvent • enzyme
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
H. Yehia, S. Kamel, K. Paulick, A. Wagner, P. Neubauer, Curr. Pharm.
Des. 2017, 23, 6913–6935.
S. Kamel, M. Weiß, H. F. T. Klare, I. A. Mikhailopulo, P. Neubauer, A.
Wagner, Mol. Catal. 2018, 458, 52–59.
M. Rabuffetti, T. Bavaro, R. Semproli, G. Cattaneo, M. Massone, F. C.
Morelli, G. Speranza, D. Ubiali, Catalysts 2019, 9, 355.
S. Kamel, H. Yehia, P. Neubauer, A. Wagner, in Enzym. Chem. Synth.
Nucleic Acid Deriv., 2019, 1–28.
R. T. Giessmann, N. Krausch, F. Kaspar, N. M. Cruz Bournazou, A.
Wagner, P. Neubauer, M. Gimpel, Processes 2019, 7, 380.
F. Kaspar, R. T. Giessmann, K. F. Hellendahl, P. Neubauer, A. Wagner,
M. Gimpel, ChemBioChem 2020, 21, 1428–1432.
F. Kaspar, R. T. Giessmann, P. Neubauer, A. Wagner, M. Gimpel, Adv.
Synth. Catal. 2020, 362, 867–876.
M. S. Drenichev, C. S. Alexeev, N. N. Kurochkin, S. N. Mikhailov, Adv.
Synth. Catal. 2018, 360, 305–312.
C. S. Alexeev, M. S. Drenichev, E. O. Dorinova, R. S. Esipov, I. V
Kulikova, S. N. Mikhailov, Biochim. Biophys. Acta - Proteins Proteomics
2020, 1868, 140292.
In conclusion, we characterized the hyperthermostable
pyrimidine nucleoside phosphorylase from Thermus thermophilus
and revealed its exceptional working space, broad substrate
scope as well as excellent stability and tolerance to organic
cosolvents. However, its inhibition by nucleobases even at low
concentrations discourages its use in synthetic applications.
These observations make TtPyNP an outlier among pyrimidine
nucleoside phosphorylases, displaying unmatched stability and a
rare example of substrate/product inhibition.
[10] X. Zhou, K. Szeker, B. Janocha, T. Böhme, D. Albrecht, I. A. Mikhailopulo,
P. Neubauer, FEBS J. 2013, 280, 1475–1490.
[11] I. Serra, T. Bavaro, D. A. Cecchini, S. Daly, A. M. Albertini, M. Terreni, D.
Ubiali, J. Mol. Catal. B Enzym. 2013, 95, 16–22.
[12] F. Kaspar, M. R. L. Stone, P. Neubauer, A. Kurreck, ChemRxiv 2020,
DOI 10.26434/chemrxiv.12753413.v1
[13] K. Szeker, X. Zhou, T. Schwab, A. Casanueva, D. Cowan, I. A.
Mikhailopulo, P. Neubauer, J. Mol. Catal. B Enzym. 2012, 84, 27–34.
[14] K. F. Hellendahl, F. Kaspar, X. Zhou, Z. Huang, P. Neubauer, A. Wagner,
2020, in preparation.
[15] M. Almendros, J. Berenguer, J.-V. Sinisterra, Appl. Environ. Microbiol.
2012, 78, 3128–3135.
Experimental Section
[16] H. Yehia, S. Westarp, V. Röhrs, F. Kaspar, T. R. Giessmann, F. T. H.
Klare, K. Paulick, P. Neubauer, J. Kurreck, A. Wagner, Molecules 2020,
25, 934.
Enzymatic reactions were prepared from stock solutions of
nucleoside, potassium phosphate and buffer and started via the
addition of enzyme. Reaction conditions were applied as stated in
the figure captions and the Supporting Information. Reaction
monitoring was performed as described previously.[7,33,34]
Typically, samples of 50 µL were withdrawn from reaction
mixtures containing 1 mM UV-active compounds and pipetted into
450 µL 100 mM aqueous NaOH to stop the reaction. To record
the UV spectra of these alkaline samples, 200 µL of the quenched
samples were transferred to a UV/Vis-transparent 96-well plate
(UV star, GreinerBioOne, Kremsmünster, Austria) and UV
absorption spectra were recorded from 250 to 350 nm in steps of
1 nm with a high-throughput plate reader (BioTek Instruments,
Winooski, USA). The obtained experimental UV spectra were
then deconvoluted via spectral unmixing using suitable reference
spectra of the nucleoside and nucleobase to derive the respective
degree of conversion. All raw data presented in this report, along
[17] R. T. Giessmann, F. Kaspar, P. Neubauer, A. Kurreck, M. Gimpel in
preparation,
preliminary
dataset
available
at
DOI
10.5281/zenodo.3572070.
[18] N. Hori, M. Watanabe, Y. Yamazaki, Y. Mikami, Agric. Biol. Chem. 1990,
54, 763–768.
[19] H. Eyring, J. Chem. Phys. 1935, 3, 107–115.
[20] F. Kaspar, 2020, DOI 10.5281/zenodo.4284188.
[21] Biphasic systems involving hydrophobic solvents are not an option as
nucleosides are virtually insoluble in all apolar solvents.
[22] Even at lower temperatures, small alcohols are unfavorable cosolvents
as they work excellently as quenching agents for nucleoside
phosphorylases.
[23] C. M. Alder, J. D. Hayler, R. K. Henderson, A. M. Redman, L. Shukla, L.
E. Shuster, H. F. Sneddon, Green Chem. 2016, 18, 3879–3890.
[24] H. J. Wiggers, J. Cheleski, A. Zottis, G. Oliva, A. D. Andricopulo, C. A.
Montanari, Anal. Biochem. 2007, 370, 107–114.
[25] S. Roy, B. Jana, B. Bagchi, J. Chem. Phys. 2012, 136, 115103.
5
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10.1002/cbic.202000679
ChemBioChem
COMMUNICATION
[26] D. F. Visser, F. Hennessy, J. Rashamuse, B. Pletschke, D. Brady, J. Mol.
Catal. B Enzym. 2011, 68, 279–285.
[27] P. P. Saunders, B. A. Wilson, G. F. Saunders, J. Biol. Chem. 1969, 244,
3691–3697.
[28] T. A. Krenitsky, Biochim. Biophys. Acta - Enzymol. 1976, 429, 352–358.
[29] K. Shimizu, N. Kunishima, 2006, DOI 10.2210/pdb2DSJ/pdb.
[30] R. G. Silva, V. L. Schramm, Biochemistry 2011, 50, 9158–9166.
[31] Y. Ni, D. Holtmann, F. Hollmann, ChemCatChem 2014, 6, 930–943.
[32] R. A. Sheldon, D. Brady, ChemSusChem 2019, 12, 2859–2881.
[33] F. Kaspar, R. T. Giessmann, N. Krausch, P. Neubauer, A. Wagner, M.
Gimpel, Methods Protoc. 2019, 2, 60.
[34] F. Kaspar, R. T. Giessmann, S. Westarp, K. F. Hellendahl, N. Krausch,
I. Thiele, M. C. Walczak, P. Neubauer, A. Wagner, ChemBioChem 2020,
21, 2604.
[35] R. T. Giessmann, N. Krausch, 2019, DOI 10.5281/zenodo.3243376.
[36] R. T. Giessmann, F. Kaspar, 2019, DOI 10.5281/zenodo.3333469.
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10.1002/cbic.202000679
ChemBioChem
COMMUNICATION
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What? The pyrimidine nucleoside phosphorylase from Thermus thermophilus is an outlier among its peers. The enzyme is extremely
stable and performs remarkably well in near-boiling cosolvent-heavy media, but an apparent inhibition by nucleobases renders its
performance in preparative applications rather subpar.
7
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