Enzymatic Production of Non-Natural Nucleoside-5′-Monophosphates by a Thermostable Uracil Phosphoribosyltransferase

Article abstract of DOI:10.1002/cctc.201701223

The use of enzymes as biocatalysts applied to synthesis of modified nucleoside-5′-monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo-, regio-, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate-binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several 5 and 6-substituted pyrimidines as substrates. These experimental results suggest TtUPRT could be a valuable biocatalyst for the synthesis of modified NMPs.

Full text of DOI:10.1002/cctc.201701223

Accepted Article
Title: Enzymatic production of non-natural nucleoside-5'-
monophosphates by a novel thermostable uracil
phosphoribosyltransferase
Authors: Jon Del Arco, Javier Acosta, Humberto M. Pereira, Almudena
Perona, Neratur K. Lokanath, Naoki Kunishima, and Jesús
Fernández-Lucas
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To be cited as: ChemCatChem 10.1002/cctc.201701223
Link to VoR: http://dx.doi.org/10.1002/cctc.201701223
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10.1002/cctc.201701223
ChemCatChem
FULL PAPER
Enzymatic
production
by
phosphoribosyltransferase
of
non-natural
novel
nucleoside-5'-
monophosphates
a
thermostable
uracil
Jon del Arco,[a] Javier Acosta,[a] Humberto M. Pereira,[b] Almudena Perona,[a] Neratur K. Lokanath,[c]
Naoki Kunishima, * [d] Jesús Fernández-Lucas * [a, e]
.
Abstract: The use of enzymes as biocatalysts applied to synthesis
of modified nucleoside-5'-monophosphates (NMPs) is an
interesting alternative to traditional multistep chemical methods
which offers several advantages, such as stereo, regio and
enantioselectivity, simple downstream processing, and mild reaction
conditions. Herein we report the recombinant expression,
production and purification of uracil phosphoribosyltransferase from
Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has
been determined by protein crystallography, and its substrate
specificity and biochemical characteristics have been analysed,
providing new structural insights into the substrate-binding mode.
Biochemical characterization of the recombinant protein indicates
that the enzyme is a homotetramer, with activity and stability across
a broad range of temperatures (50-80 °C), pH (5.5-9) and ionic
strength (0-500 mM NaCl). Surprisingly, TtUPRT is able to
recognize several 5 and 6-substituted pyrimidines as substrates.
These experimental results, suggest TtUPRT could be a valuable
biocatalyst for the synthesis of modified NMPs.
triphosphates (NTPs) has been the target of several chemical
and enzymatic synthetic Moreover, 2'-
efforts.[4-5]
deoxynucleoside-5'-monophosphates (dNMPs) are basic
precursors of corresponding 2'-deoxynucleoside-5'-
triphosphates (dNTPs), which are widely used in modern
molecular biology experiments. In this respect, the development
of new synthetic processes of NMPs and dNMPs is a main
focus area of pharmaceutical companies.
Chemical synthesis of NMPs is commonly performed by 5'-
phosphorylation of the precursor nucleosides. It often requires
the use of chemical reagents (phosphoryl chloride, POCl3, or
phosphorus pentoxide, P2O5), acidic conditions and organic
[6]
solvents which are expensive and environmentally harmful.
Indeed, chemical glycosylation of precursor nucleosides often
requires time-consuming multistep processes.[7] These
synthetic routes usually provide poor or moderate global yields
and low product purity, and are also associated with harsh
reaction conditions and waste disposal issues.
In this context, the enzymatic synthesis of NMPs shows many
advantages, such as one-pot reactions under mild conditions,
high stereo- and regio-selectivity, and an environmentally-
friendly technology. Due to this, many different enzymes have
been employed as valuable catalysts for mono or multi-
enzymatic synthesis of NMPs,[8-12] including nucleoside kinases
(NK),[13-15] acid phosphotransferases (AP/PTase),[16-17] 5'-
Introduction
Nucleosides are essential metabolites in numerous biochemical
processes. In this sense, nucleoside analogues have been
widely used in the treatment of several serious human illnesses
such as cancer or viral diseases, among others.[1-3]
.
phosphodiesterases[18] or phosphoribosyltransferases (PRT).[11-
The intracellular phosphorylation of nucleosides leads to
corresponding nucleotides. In this regard, the availability of
synthetic nucleotide analogues allows the study of structure-
12, 19]
Uracil phosphoribosyltransferase (EC 2.4.2.9, UPRT) catalyzes
the reversible transfer of the 5-phosphoribosyl group from 5-
phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to uracil N1 to
form uridine-5'-monophosphate, UMP, in the presence of Mg2+
(Scheme 1).[20-22] Since UMP is a common precursor of all
pyrimidine nucleotides, UPRT is an essential enzyme in the
salvage pathways for pyrimidine nucleotide synthesis.
activity relationships, kinetics, and other
metabolic
consequences of nucleoside administration. As a consequence,
the development of suitable and efficient methodologies for the
synthesis of nucleoside-5'-mono- (NMPs), -di (NDPs) and
[a]
J. del Arco, J. Acosta, Dr. A. Perona and Dr. J. Fernández- Lucas
Applied Biotechnology Group,
Universidad Europea de Madrid,
Urbanización El Bosque, Calle Tajo, s/n, 28670 Villaviciosa de
Odón (Madrid), Spain.
E-mail: jesus.fernandez2@universidadeuropea.es
Dr. HM Pereira
Instituto de Física de São Carlos, Universidade de São Paulo,
CP369, 13560-970, São Carlos, SP, Brazil.
Dr. Neratur K. Lokanath
Department of Studies in Physics, University of Mysore, Mysore
570 006, India
Dr. Naoki Kunishima
[b]
[c]
[d]
[e]
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148,
Japan. E-mail: kunisima@spring8.or.jp
Dr. J. Fernández- Lucas
Grupo de Investigación en Desarrollo Agroindustrial Sostenible,
Universidad de la Costa, CUC,
Calle 58 # 55 - 66. Barranquilla, Colombia
Scheme 1. Enzymatic synthesis of different pyrimidine nucleoside-5'-
monophosphates catalyzed by TtUPRT.
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10.1002/cctc.201701223
ChemCatChem
FULL PAPER
phosphoribosyltransferases were annotated throughout the
genome. Among them, two ORFs, ttha0783 and ttha1312 (uprt
gene), which are annotated as putative PyrR bifunctional protein
UPRT shows a remarkable preference for uracil as substrate,
and neither thymine nor cytosine can be recognized by any
UPRT up to date. However, several bacterial, protozoan and
plant UPRTs, such as uracil phosphoribosyltransferase from
Eschericha coli, Leishmania donovani and Arabidopsis thaliana,
have shown slight activity over different pyrimidine derivatives,
pyrimidine
phosphoribosyltransferase
operon
regulatory
and
protein/uracil
putative Uracil
a
phosphoribosyltransferase (TtUPRT), were subjected to
bioinformatics analyses to identify their physical and chemical
properties and possible structures.
23]
such as 4-thiouracil, 6-azauracil or 5-fluorouracil.[5,
Unfortunately, an extensive functional characterization of their
potential as biocatalysts has not been carried out to date.
Mesophilic enzymes do not usually exhibit high activity and
stability under aggressive reaction conditions, such us extreme
pH values, high temperatures, or the presence of organic
solvents, which are frequently used to circumvent the most
habitual problems to scale up bioprocesses from laboratory to
industry, e.g. viscosity of the medium and concentration of
substrates. The use of enzymes from thermophiles
(thermozymes) offers the possibility of using high temperature
reactions, resulting in a higher solubilization of substrates, a
diminution of medium-viscosity and an increase of substrate
diffusion coefficients, leading to higher overall reaction rates.
The present work aims to explore the potential of uracil
phosphoribosyltransferase from Thermus thermophilus HB8,
TtUPRT, as biocatalyst for the synthesis of natural and non-
natural NMPs. In this regard, we report here for the first time the
cloning of the uprt gene from Thermus thermophilus HB8, the
expression in Escherichia coli, purification of the recombinant
protein (TtUPRT), and characterization of the enzymatic activity,
specificity and oligomeric state of the protein. Furthermore, we
also report the complete crystal structure of TtUPRT, which
Figure 1. Multiple sequence alignment of uracil phosphoribosyltransferases
from Thermus thermophilus (TtUPRT), Sulfolobus sulfotaricus (SsUPRT),
Toxoplasma gondii (TgUPRT), Mycobacterium tuberculosis (MtUPRT),
Escherichia coli (EcUPRT) and Bacillus caldolyticus (BcUPRT). Amino acid
sequences were aligned by CLUSTALΩ. Amino acids for each polypeptide
sequence were independently numbered. The secondary structural elements
(dark grey cylinder for α helices and black arrows for β strands) for the
TtUPRT are indicated above the aligned amino acid sequences. Conserved
regions (I-IV) are encircled. Asterisk (*) indicates single, fully conserved
residues; colon (:) indicates conservation of strong groups; and period (.)
indicates conservation of weak groups.
exhibits
a canonical PRT fold shared widely with other
BLAST
analysis
of
amino
acid
sequence
structurally known UPRTs. Details of active site architecture
have also been determined. Our results reveal TtUPRT as a
homotetramer, active and stable over a broad temperature
range (50-80 °C), pH (5.5-9) and ionic strength (0-500 mM
sodium chloride). Specificity studies show that TtUPRT is able to
use uracil as base donor (as expected), but surprisingly thymine
is also slightly recognized by TtUPRT. More interestingly,
TtUPRT is able to recognize several 5 and 6-substituted
pyrimidines as substrates, this being the first time that the
synthesis of 5-bromouridine-5'-monophosphate, 5-BrUMP, 5-
chlorouridine-5'-monophosphate, 5-ClUMP, 5-iodouridine-5'-
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) reveals uprt encodes a
putative uracil phosphoribosyltransferase. The pairwise
aminoacid sequence alignment reveals TtUPRT displays high
sequence identity with uracil phosphoribosyltransferase from
Bacillus caldolyticus, BcUPRT, (62 %), Thermotoga maritima,
TmUPRT (59 %) and E. coli, EcUPRT (51 %). The identity
percentage with other UPRTs was below 42 %.
Multiple sequence alignment of predicted TtUPRT aminoacid
sequence with uracil phosphoribosyltransferases from other
thermophilic and mesophilic bacteria and protozoan
characterized so far, reveals that 5-phospho-α-D-ribosyl-1-
pyrophosphate and uracil binding sites are conserved in TtUPRT
sequence (Figure 1). All these characteristics suggest that this
monophosphate,
monophosphate,
5-IUMP,
5-OHMeUMP
5-hydroxymethyluridine-5'-
and 6-methyluridine-5'-
gene
codes
for
a
putative
intracellular
uracil
phosphoribosyltransferase. TtUPRT was predicted to contain
208 amino acid residues and have a relative molecular mass of
17.66 kDa (Protparam, http://web.expasy.org/protparam).
Moreover, multiple sequence alignment of TtUPRT with other
homologous UPRTs reveals a high similarity between different
UPRTs and clearly suggests that their three-dimensional
structures will be very similar, with a conserved core structure
encompassing the PRPP/PPi and pyrimidine nucleobase binding
sites and sharing similar substrate binding contacts. In this
regard, four typical short regions (I–IV) of the UPRTs family,
involved in substrate recognition, catalysis, and stability of the
monophosphate, 6-MeUMP, has been carried out by an UPRT
enzyme. All of these features indicate that TtUPRT could be an
interesting biocatalyst for the synthesis of different non-natural
uridine-5'-monophosphate analogues.
Results and Discussion
Bioinformatics analysis of uracil phosphoribosyltransferase
encoding gene of T. thermophilus HB8
protein, can easily be recognized in TtUPRT (Figure 2), a finding
24]
which supports a similar active site (Figure 3).[20-22,
All
described UPRTs contain a conserved PRPP binding domain
(region III), thirteen-residue PRPP-binding motif region, which
typically comprises four hydrophobic residues followed by two
The genomic information of T. thermophilus HB8 has been
analysed and published online (Genbank No. AP008226).
Several genes that potentially encode purine and pyrimidine
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10.1002/cctc.201701223
ChemCatChem
FULL PAPER
acidic residues, two hydrophobic residues, and four small
residues.[21, 25] In addition, several conserved residues in regions
I-II are involved in PRPP binding and stabilization. Near the end
C-terminal, bacterial UPRTs display a highly conserved motif
(region IV) which is critical in uracil-binding.
were reported as dimers. However, TgUPRT has also been
[30]
described as monomeric
or tetrameric (in presence of GTP
which affects the oligomerization state in solution).[31] It seems
that the oligomerization state of the PRTs could be essential in
catalysis and stabilization of the active conformation as well as
in thermal stability of the protein. [20, 32]
Overall structure and architecture of active site
We have determined the crystal structure of TtUPRT (PDB 1v9s)
at 2.1 Å resolution (Table 1). The asymmetric unit of TtUPRT
crystal contains four subunit chains A, B, C, and D with a
canonical PRT fold shared widely with other structurally known
UPRTs.[33] These four subunits comprise
a homotetramer
(Figure 2) which is commonly observed in other reported crystal
structures of UPRT from various microorganisms: UPRT from
Thermotoga maritima (TmUPRT; PDB 1o5o), UPRT from
Escherichia coli (EcUPRT; PDB 2ehj), UPRT from Burkholderia
pseudomallei (BpUPRT; PDB 3dmp), UPRT from Aquifex
aeolicus (AaUPRT; PDB 2e55), UPRT from Mycobacterium
tuberculosis (MtUPRT; PDB 5e38),[34] UPRT from Toxoplasma
31]
gondii (TgUPRT; PDB 1bd3/1upu/1jlr/1jls),[25,
UPRT from
Sulfolobus solfataricus (SsUPRT; PDB 1xtt/1xtu/1xtv).[20] In
addition, the two subunits A/D or B/C of 1v9s may represent a
protomeric homodimer as observed in the crystal structure of
UPRT from Bacillus caldolyticus (BcUPRT; PDB 1i5e).[21]
Although the four subunits in the TtUPRT homotetramer are
arranged in the 222 molecular symmetry, two of three pseudo-
two fold axes are almost broken, suggesting a structural
asymmetry in detail. Of the four subunits, A and D adopt an
open conformation in which the active-site pocket is exposed to
the solvent due to the disorder of the active-site loop
(Arg103−Val111); the root-mean-square deviation (r.m.s.d.)
value from the Cα superposition of the open subunits (197 Cα
atoms) was 0.303 Å. On the other hand, the subunits B and C
adopt a closed conformation in which the active site is covered
by the ordered active-site loop and is excluded from the solvent;
the r.m.s.d. value for the closed subunits (208 Cα atoms) was
0.234 Å. The Cα superposition from another combination of
subunits (197−199 Cα atoms) yielded the higher r.m.s.d. values
in the range of 0.417−0.728 Å.
Figure 2. Overall structure of UPRT from Thermus thermophilus HB8. A
homotetrameric TtUPRT molecule is shown as a ribbon model. Four subunits
are distinguished by different coloring and labeling; subunits in open and
closed conformations are shown with cool (green and blue) and warm (yellow
and red) colors, respectively. The N- and C-termini, the active-site loop, the
UMP binding site, and the effector binding site are labeled on subunit B; the
binding sites are delineated by gray circles. Invisible active-site loops in
subunits A and D are represented as dotted lines. This figure was prepared
with Discovery Studio (Accelrys Inc.).
Production and purification of TtUPRT
The
uprt
gene
which
from
codes
for
the
uracil
phosphoribosyltransferase
Thermus
thermophilus
The active-site loop (Arg103−Val111) seems to serve as a flap
to open/close the active-site pocket. Interestingly, clear residual
electron density compatible with UMP molecule was observed
only in the active-site pocket of the closed subunits (B and C),
although it was not modeled due to the lack of experimental
evidence for identification (Figure 3A). The density may be
derived from a co-purified UMP, because we did not add UMP in
the crystallization. This putative UMP is recognized by fourteen
well-conserved residues in the active-site pocket: the phosphate
moiety by Arg103, Ala134, Thr135, Gly136, Gly137, Ser138, the
ribose moiety by Arg103, Asp130, Met132, the uracil moiety by
Arg103, Met132, Ala134, Gly191, Tyr192, Ile193, Gly198,
Asp199, Ala200. Similar co-purified UMP was also reported in
SsUPRT (PDB 1xtv).[20] This observation means that only two of
four active-site pockets in the tetramer are occupied by UMP,
which is reminiscent of the half-of-the-sites reactivity observed in
the acyl-CoA thioesterase PaaI from Thermus thermophilus,[35]
for instance. Another residual electron density, probably for a co-
purified effector, was found in the protomeric dimer interface of
all four subunits, although we could not estimate the ligand from
the shape of the density (Figure 3B).
(TtUPRT), was cloned and overexpressed in Escherichia coli
BL21(DE3) as described in experimental section. The
recombinant N-terminal His6-tagged TtUPRT was purified by
two chromatographic steps. SDS-PAGE analysis of purified
enzyme shows only one protein band with an apparent
molecular mass of 24 kDa (Figure S1). This result agrees with
results from analytical ultracentrifugation analysis. Under the
conditions assayed, sedimentation velocity experiments
revealed TtUPRT as a single specie with an experimental
sedimentation coefficient of 5.22 S (s20,w = 5.27) compatible with
a tetrameric state (Mw = 98.250 kDa). This value is also
compatible with a single-species monomeric model of 24.562
kDa, similar to the molecular mass calculated from the amino
acid sequence (24.563 kDa).
Different oligomeric states are described for different bacterial,
archaeon and protozoan UPRTs. On the one hand, UPRT from
Gram-positive thermophile Bacillus caldolyticus, BcUPRT has
been described as a dimer,[21] whereas UPRTs from E. coli,
EcUPRT,
and
thermoacidophilic
archaeon
Sulfolobus
solfutaricus, SsUPRT, were described as
a
trimer[26] and
tetramer[27] respectively. On the other hand, UPRTs from several
protozoan parasites, such as Crithidia luciliae, ClUPRT,[28]
Giardia lambia, GlUPRT,[29] and Toxoplasma gondii, TgUPRT,[25]
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FULL PAPER
Table 1. Data collection and refinement statistics.
Data collection
Space group
P3221
Unit-cell parameters (Å)
Resolution range (Å)
Completeness (%)
<I/σ(I)>
a=116.15, c=157.64
30.0−2.1 (2.18−2.10)
99.7 (100.0)
15.5 (4.4)
a Rmerge (%)
5.1 (29.4)
Refinement
Resolution range (Å)
b Rcryst / Rfree (%)
30.0−2.1 (2.20−2.10)
23.8 (31.2) / 27.3 (35.0)
0.006
R.m.s.d. bond lengths (Å)
R.m.s.d. bond angles (º)
1.30
a Rmerge = ∑hkl ∑i |Ii(hkl) – <I(hkl)>| / ∑hkl ∑i Ii(hkl), where Ii(hkl) is the ith observation of reflection hkl and <I(hkl)> is the weighted average intensity for all
b
observations i of reflection hkl. Rcryst = ∑hkl ||Fobs| – |Fcalc|| / ∑hkl |Fobs|, where |Fobs| and |Fcalc| are the observed and calculated structure-factor amplitudes,
respectively. Rfree was calculated with 5% of the reflections chosen at random and omitted from refinement. Values in parentheses are for the outermost shell.
Figure 3. Residual electron densities for putative ligands found in the crystal structure. Stereo representations of crystal structure relevant to the ligand binding in
the UMP site of subunit B (a) and in the effector site of subunit C (b) are shown. Atoms are depicted as stick models with the atom-type coloring. Important
residues for ligand recognition are labeled; residues with asterisks denote those on the other subunit. The mFo−DFc electron density maps after simulated
annealing are overlaid with two different contour levels: 3.0 σ and 4.0 σ for cyan and orange maps, respectively. This figure was prepared with Discovery Studio
(Accelrys
Inc).
This putative effector site is similar to but distinct from those
reported: the CTP site in SsUPRT (PDB 1xtu)[20] and the GTP
site in TgUPRT (PDB 1jlr).[31] This putative effector is surrounded
by twelve moderately-conserved residues: His13, Lys14, Asp27,
extension by TmUPRT (r.m.s.d. of 3.22 Å for 199 Cα atoms and
a sequence identity of 54 %; PDB entry 1o5o) and BcUPRT
(r.m.s.d. of 1.35 Å for 199 Cα atoms and a sequence identity of
59 %; PDB 1i5e). According to these results, TgUPRT was
Glu30, Leu31, Glu34 from one subunit, and Lys70, Leu71, Val91, chosen as the best candidate for structural alignment (as it is
Pro92, His93, Ala94 from the other subunit. The invariant
arginine residue Arg78, which is reported to be important for an
intersubunit communication in SsUPRT (PDB 1xtv),[20] is located
in the vicinity of the active-site pocket and adopts the cis main-
chain conformation in all four subunits.
described below).
Substrate specificity
Table 2 summarizes the specific activities of TtUPRT using
uracil, thymine and cytosine as acceptors. TtUPRT has a strong
preference for uracil, but it also displays activity over thymine.
Notably, TtUPRT shows higher specific activities than most
other reported UPRTs under similar enzymatic assay conditions.
By way of example, for the biosynthesis of UMP, the specific
activity showed by TtUPRT (3.7 µmol min-1 mg-1) was 1.2, 1.82,
2.4, 6.2 and 26.4 times higher than activity values of UPRTs
In order to elucidate the architecture of the active site, the
structural alignment of TtUPRT (PDB 1v9s) was performed with
other UPRTs. Three-dimensional superposition with the
FATCAT[36] shows mutant Cys128Val TgUPRT as the closest
structural homologue (PDB 1bd3; r.m.s.d. of 2.41 Å for 199 Cα
atoms and a sequence identity of 35 %), followed in a similar
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10.1002/cctc.201701223
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from Giardia intestinalis, GiUPRT (0.6 µmol min-1 mg-1);[29]
Toxoplasma gondii, TgUPRT (2.03 µmol min-1 mg-1);[30]
Mycobacterium tuberculosis, MtUPRT (1.7 µmol min-1 mg-1);[24]
Crithidia luciliae, ClUPRT (0.6 µmol min-1 mg-1; adapted from)[28]
and Sulfolobus shibatae, SsUPRT, (0.14 µmol min-1 mg-1,
adapted from),[37] respectively. UPRTase activity of UPRT from
E. coli, EcUPRT, and Bacillus caldolyticus, BcUPRT, is higher
than specific activity showed by TtUPRT. However, neither
EcUPRT nor BcUPRT exhibit higher thermal stability than
TtUPRT, as described below.
Steady-state kinetics
Steady-state kinetic studies were carried out at variable
concentrations of uracil or PRPP, in order to determine KM, kcat
and kcat/KM values. The results are summarized in Table 3.
TtUPRT shows lower KM values for uracil than for PRPP. These
results agree with kinetic data described for other UPRTs, [21, 22,
24, 27, 29]. In this sense, it is reasonable to think that similar to
other UPRTs, catalysis could follow a sequential mechanism, in
which PRPP binding is followed by uracil, and PPi product is
released first, followed by UMP
Table 2. Reactivity of TtUPRT using different natural pyrimidine bases as
substrates
Table 3. Steady-state kinetic parameters for UMP formation reaction catalyzed
by TtUPRT.
Pyrimidine basea
Uracil
Specific activity (IU/mgenz)
3.70
n.d.
Variable uracila
Variable PRPPb
Cytosine
KM
(mM)
kcat
(s-1)
kcat/KM
(s-1 mM-1)
KM
(mM)
kcat
(s-1)
kcat/KM
(s-1 mM-
Thymine
0.80
1
)
aReaction conditions were 3 µg of enzyme in 40 µl at 60 °C for 10 min.
Substrate concentrations were 10 mM PRPP, 10 mM pyrimidine base, 12 mM
MgCl2 in 12 mM TrisHCl buffer, pH 8.0.
0.19±0.03
28.87±0.5
151.94
3.33±0.7
44.32±1.3
13.30
a Concentration range 0.12-17 mM at a fixed PRPP concentration of 10 mM.
b Concentration range 0.25-32 mM at a fixed Uracil concentration of 10 mM.
Biochemical characterization of TtUPRT
Experimental results reveal that the enzymatic activity of the
TtUPRT is strongly dependent on temperature, as expected for
proteins from hyperthermophilic organisms. The temperature
profile shows that TtUPRT is active (more than 75 %) across a
broad temperature range (50-80 °C), with maximum activity at
60 °C (Figure 4A). Interestingly, recombinant TtUPRT also
displays high activity (more than 70 %) in a pH range from 5.5 to
9, with maximum activity values at pH 7-8 (Figure 4B). In
addition, negligible loss of activity was observed in presence of
500 mM of sodium chloride, and a slight activity decrease was
observed when sodium chloride concentration was increased
(70 % activity remained at 1 M sodium chloride) (Figure 4C).
Biocatalyst stability
Some enzymes, especially in partially purified preparations, may
be remarkably unstable, losing a significant amount of activity
over the period of incubation. In this sense, it is necessary to
determine if the enzyme is active and stable under operational
conditions.The stability of a biocatalyst is defined as the time
during which it retains 50 % of its initial activity and is affected by
environmental conditions such as pH and temperature.
Storage of TtUPRT at 4 °C was analysed to ensure enzyme
stability, and TtUPRT retained its activity for more than 130 days
(85 % relative activity) (data not shown). Unfortunately, activity
decreased considerably (25 % of retained activity) when stored
at -20 °C or -80 °C.
In addition, the effect of temperature on enzyme stability was
evaluated by incubating TtUPRT in 20 mM Tris-HCl, pH 8.0 at
different temperatures (50-80 °C) during 2 hours (Figure 4D).
Experimental data shows that TtUPRT is stable in a temperature
range from 50 to 70 °C, whereas unfortunately TtUPRT
undergoes a significant loss of activity when stored at 80 °C for
incubation periods of more than 45 min, probably due to protein
suffering irreversible denaturation. These results agree with
thermostability of UPRT from extreme thermoacidophilic
archaebacterium Sulfolobus shibatae, which displays tolerated
incubation at 80 °C for at least one hour without detectable loss
of activity. In addition, both enzymes, TtUPRT and SsUPRT,
exhibit higher stability than other described UPRTs, such as
UPRT from Gram-positive thermophile Bacillus caldolyticus,
BcUPRT (60
%
of retained activity at 70 °C),[38] and
mesohophilic UPRTs from B. subtillis, BsUPRT (40 % of
retained activity at 52 °C),[38] and Toxoplasma gondii,
TgUPRT(completely inactivated by heating for
5 min at
65 °C).[30]
Figure 4. Temperature and pH dependence on TtUPRT activity and stability.
A) Effect of temperature on TtUPRT activity (●). B) Effect of pH on TtUPRT
activity, (○) sodium acetate 50 mM (pH 4-6), (●) sodium phosphate 50 mM (pH
6-8), (∆) sodium borate 50 mM (pH 8-10). C) Effect of ionic strenght on
TtUPRT activity D) (●). Thermal inactivation of TtUPRT in 20 mM Tris-HCl
buffer, pH 8.0 at different temperatures 50 °C (∆), 60 °C (○), 70 °C (●) and
80 °C (■).
Enzymatic production of natural and non-natural NMPs
In order to assess the potential of TtUPRT as biocatalyst, it was
performed the enzymatic production of different NMP analogues
using different pyrimidine bases as substrates (Table 4).
Interestingly, TtUPRT shows activity towards unusual substrates,
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10.1002/cctc.201701223
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such us thymine, 5-halogenated uracil derivatives, 5-
hidroxymethyluracil and 6-methyluracil (Scheme 1). Among
them, TtUPRT employed these non-natural bases as acceptors
in the following order of preference: Thymine > 5-FUra ≈ 5-ClUra
> 5-BrUra > 5-IUra >> 5-OHMeUra > 6-MetUra. Despite the fact
that several bacterial and protozoan UPRTs, such as uracil
phosphoribosyltransferase from Eschericha coli or Leishmania
donovani, are able to act on different pyrimidine derivatives,
such as 4-thiouracil, 6-azauracil or 5-fluorouracil,[5] none of the
reported UPRTs displayed activity over thymine and the
following non-natural uracil derivatives, 5-chlorouracil, 5-
bromouracil, 5-iodouracil, 5-hydroxymethyluracil and 6-
methyluracil). On the contrary, other uracil derivatives, such as
5-ethyluracil, 6-methyl-2-thiouracil, 6-propyl-2-thiouracil, 2-
methoxy-5-fluorouracil and (E)-5-(2-bromovinyl) uracil, were not
recognized as substrates. Furthermore, neither cytosine
(cytosine, 5-methylcytosine, 5-fluorocytosine and 5-azacytosine)
nor thymine derivatives (trifluorothymine) could be recognized by
TtUPRT.
In 1998, Schumacher et al.[25] reported the structures of UPRT
from T. gondii both in Apo and bonded with uracil and 5-
fluorouracil (PDBs 1bd3, 1bd4 and 1fpU respectively). This work
allowed them to get a better understanding about the substrate
specificity studies previously described by Itzsch and
Tankerslay.[39] The structural alignment of 5-fluorouracil
complexed TgUPRT with uracil complex reveals minor
adjustments in the active-site residues, especially in the base
binding site (Figure 5A). As described for uracil, the molecule
stacks between Met166 and Thr228 and forms 4 hydrogen
bonds with active-site residues, one water mediated. The main
difference between the binding of uracil and 5-fluorouracil is a
small rotation in the latter in order to avoid steric clash with
Ala168 Cβ (the distance between 5-fluoro and Ala168 Cβ is 3.1
Å). Consequently, the failure binding of 5-iodo-, 5-bromo or 5-
chloro is related to the longer carbon-halogen bond and their
larger van der Waals radius which results in steric hindrance
with Ala168 Cβ.
Table 4. Enzymatic production of natural and non-natural NMPs
Pyrimidine basea
Uracil
Conversion (%)
50
18
5-fluorouracil
5-chlorouracil
17
5-bromouracil
12
5-iodouracil
12
5-ethyluracil
n.d.
8
5-hydroxymethyluracil
6-methyluracil
5
Figure 5. A) Superposition of base binding site of Toxoplasma gondii UPRT in
complex with 5-fluorouracil (green) and Thermus thermophilus UPRT (white).
The reduction or absence of enzymatic activity over substituents at position 5
is attributed to steric hindrance of position 5 with Ala168 Cβ in TgUPRT.
However, TtUPRT is able to bind and catalyze these modified uracil
nucleosides. B) Tube mode in B-factor gradient of the TtUPRT. The most
mobile parts of the protein are shown in yellow and red. The turn which lies
residue Tyr192 involved in π-stacking with uracil molecule is the one with the
highest B factors.
6-methyl-2-thiouracil
6-propyl-2-thiouracil
(E)-5-(2-bromovinyl)uracil
Cytosine
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
20
5-methylcytosine
5-fluorocytosine
5-azacytosine
Thymine
However, a different scenario is observed for TtUPRT, which not
only recognizes uracil and 5-fluorouracil but is able to recognize
Trifluorothymine
5-fluoro-2-methoxy-4(1H)pirimidinone
n.d.
n.d.
thymine,
5-chloro-,
5-bromo-,
5-iodouracil
and
5-
hydroxymethyluracil as substrates. Comparison by superposition
with TgUPRT shows an r.m.s.d. of 3.0 and 1.9 Å for tetramer
and monomer, respectively. Comparing base binding site (BBS)
of TtUPRT reveals a conserved site (Figure 5A), although some
differences are observed in the vicinity of BBS residues. These
differences involve a substitution to glycine, which increases the
mobility of these regions (A170G136 and Y227G191,
TgUPRT:TtUPRT). Another substitution observed is F236A200.
Indeed, the region of Y192 is a β-turn and has higher B-factors
indicating an increased flexibility of this region (Figure 5B). As
result of these differences, we could assume an increase of the
active-site size, and therefore TtUPRT, as demonstrated by
enzymatic assays, is able to bind and catalyze 5-uracil
substituents. Nevertheless, substituents larger than hydrogen at
position 6 causes a steric hindrance in the active site, producing
lower or not detected activities of TtUPRT.
aReaction conditions were 3 µg of enzyme in 40 µl at 60 °C for 15-45 min.
Substrate concentrations were 1 mM PRPP, 1 mM modified pyrimidine base,
1.2 mM MgCl2 in 12 mM TrisHCl buffer, pH 8.0.
nd: not detected
These results do not totally agree with previous reports which
state that UPRT displays a strict specificity over uracil and does
27, 38]
not act on cytosine or thymine.[24-25,
Itzsch and
Tankerslay[39] performed a structure-activity study, in which 100
compounds were evaluated as ligands of uracil
phosphoribosyltransferase from Toxoplasma gondii (TgUPRT)
by examining their ability to inhibit UMP synthesis. Their study
reveals that methylene group in position 5 is strongly preferred.
Consequently, large exocyclic substituents at this position
decrease (fluoro) or abolish (bromo) base binding; substituents
larger than hydrogen also abolish binding to UPRT. This
supposed consequence of larger volumes at position 5 causes a
steric hindrance in the active site. Unfortunately, neither of the
aforementioned studies used these substrates in the synthesis
of corresponding NMPs, so we could not know if TgUPRT really
can act on them.
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10.1002/cctc.201701223
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concentrated and stored at 4 °C until its use. Electrophoresis
was carried out on 15 % polyacrylamide slab gel with 25 mM
Tris-HCl buffer, pH 8.6, 0.1 % SDS. Protein concentration was
determined spectrophotometrically by UV absorption at 280 nm
using a ε280= 8940 M-1 cm-1.[40]
ConclusionS
Herein we describe for the first time the full structural and
biochemical characterization of uracil phosphoribosyltransferase
from Thermus thermophilus HB8, TtUPRT, that present
significant catalytic activity in a broad range of pH (from 5.5 to 9)
and temperature interval (from 50 to 80 °C). The thermophilic
origin of TtUPRT shows several associated advantages, such as
an easy purification method and thermostability at high
temperatures (50 to 80 °C). Excitingly, the ability of TtUPRT to
accept position 5-substituted pyrimidines is especially interesting,
since these are positions commonly substituted in nucleotide
analogues used for the treatment of human diseases, which
makes this enzyme attractive for the industrial synthesis of
nucleoside derivatives.
TtUPRT expression and purification for crystallization and
structure determination were carried out according to a different
protocol. For structural determination, TtUPRT was expressed
as a selenomethionine (SeMet)-substituted protein. The plasmid
encoding TtUPRT protein was digested with NdeI and BamHI
and the fragment was inserted into the expression vector pET-
11a linearized with NdeI and BamHI. The recombinant plasmid
was transformed into Escherichia coli B834(DE3)plysS cells and
grown at 37 °C in M9 medium until absorbance of the medium at
600 nm reached 0.4. At this point, 100 mg of L-lysine, 100 mg of
L-phenylalanine, 100 mg of L-threonine, 50 mg of L-isoleucine,
50 mg of L-leucine, and 60 mg of SeMet were added to 1 L of
culture and the cells were grown at 37 °C for a further 1 h,
before inducing the expression with 1 mM IPTG overnight at
25 °C. The cells were harvested by centrifugation. The cell pellet
was suspended in 20 mM Tris-HCl, pH 8.0, containing 0.5 M
Experimental Details
Materials
Cell culture medium reagents were from Difco (St. Louis, MO).
Trimethyl ammonium acetate buffer and 5-phospho-α-D-ribosyl-
1-pyrophosphate were purchased from Sigma-Aldrich (USA). All
other reagents and organic solvents were purchased from
Scharlab (Barcelona, Spain) and Symta (Madrid, Spain). All
natural and non-natural purine bases used were provided by
Carbosynth Ltd. (Compton, United Kingdom).
sodium chloride and
5
mM 2-mercaptoethanol, and
homogenized by ultrasonication. The supernatant of the crude
extract was heated at 70 °C for 12 min and the cell debris and
denatured proteins were removed by centrifugation. The heat-
treated extract was desalted by HiPrep 26/10 desalting column
and applied onto
a Super Q Toyopearl 650M column
equilibrated with 20 mM Tris-HCl, pH 8.0. Proteins were eluted
with a linear gradient of 0−0.3 M sodium chloride. The fractions
containing proteins were then dialyzed against 20 mM Tris-HCl,
pH 8.0, and subjected to Resource Q column (Amersham
Biosciences) equilibrated with 20 mM Tris-HCl, pH 8.0. Proteins
were again eluted with a linear gradient of 0−0.3 M sodium
chloride. The fractions containing proteins were desalted with
HiPrep 26/10 desalting column with 10 mM sodium phosphate,
pH 7.0, and applied onto a Bio-Scale CHT20-I column (Bio-Rad)
equilibrated with 10 mM sodium phosphate, pH 7.0. Proteins
were again eluted with a linear gradient of 10−100 mM sodium
phosphate. The fractions containing proteins were concentrated
by ultrafiltration (Vivaspin) and loaded onto a HiLoad 16/60
Superdex 75 pg column (Amersham Biosciences) equilibrated
with 20 mM Tris-HCl, pH 8.0, containing 0.2 M sodium chloride.
The homogeneity and identity of the purified sample were
ascertained by SDS-PAGE and N-terminal sequence analysis.
Finally, the purified TtUPRT with SeMet substitution was
concentrated to 12.0 mg/mL by ultrafiltration.
Enzyme Cloning, Expression and Purification
Two different protocols were used in this study, one for
biochemical characterization and the other for crystallization and
structure determination.
The encoding ttha1312 gene (uprt gene), which codifies uracil
phosphoribosyltransferase from Thermus thermophilus HB8
(NCBI Reference Sequence: YP_144578.1), was ordered and
purchased from Genscript (Piscataway, USA). The coding
sequence appeared as an NdeI-BamHI fragment subcloned into
the expression vector pET28b (+). The resultant recombinant
vector pET28bTtUPRT provided an N-terminal His6-tagged
fusion with a thrombin cleavage site between the tag and the
enzyme. TtUPRT was expressed in E. coli BL21(DE3) grown in
Luria-Bertani medium at 37 °C with kanamycin 50 μg/mL.
Protein overexpression was induced by adding 0.5 mM
isopropyl-β-D-1-thiogalactopyranoside and the cells further
grown for 10 hours. These were harvested via centrifugation at
3500 x g. The resulting pellet was resuspended in 20 mM Tris-
HCl, pH 8.0, containing 100 mM sodium chloride. Crude extracts
were prepared by ultrasonic cell treatment using a digital sonifier.
The lysate was centrifuged at 17500 x g for 20 min at 4 °C, and
then heated at 70 °C for 20 min. Insoluble material was removed
by centrifugation 17500 x g for 20 min at 4 °C, and the
supernatant was filtered through a 0.22 μm filter (Millipore).
Cleared lysate was loaded onto a 5-mL HisTrap FF column (GE
Healthcare) pre-equilibrated in a binding buffer (20 mM Tris-HCl
buffer, pH 8.0, with 100 mM sodium chloride and 10 mM
imidazole) and the column was washed. Bound proteins were
eluted using a linear gradient of imidazole (from 10 to 500 mM).
Fractions containing TtUPRT were identified by SDS-PAGE,
pooled, concentrated and loaded onto a HiLoad 16/60 Superdex
200 prep grade column (GE Healthcare) pre-equilibrated in 20
mM Tris-HCl, pH 8.0. Fractions with the protein of interest
identified by SDS-PAGE were pooled and the protein was
Analytical ultracentrifugation analysis
Sedimentation velocity experiments for TtUPRT were carried out
in 20 mM Tris-HCl, pH 8.0 at 20 °C and 50,000 × g in an Optima
XL-I analytical ultracentrifuge (Beckman-Coulter Inc.), equipped
with UV-VIS absorbance and Raleigh interference detection
systems, using an An-60Ti rotor and standard (12 mm optical
path) double-sector centre pieces of Epon-charcoal.
Sedimentation profiles were recorded at 292 nm. Sedimentation
coefficient distributions were calculated by least-squares
boundary modelling of sedimentation velocity using the
continuous distribution c(s) Lamm equation model as
implemented by SEDFIT 14.7g.
Baseline offsets were measured afterwards at 200,000 × g. The
apparent sedimentation coefficient of distribution, c(s), and
sedimentation coefficient, s, were calculated from the
sedimentation velocity data using the SEDFIT program.[41] The
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10.1002/cctc.201701223
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FULL PAPER
experimental sedimentation coefficients were corrected to
standard conditions (water, 20 °C, and infinite dilution) using
SEDNTERP software [42] to obtain the corresponding standard s
values (s20,w).
reaction times (15-45 min). Subsequently, the reaction mixture
was processed as described above and the NMP production
was analysed by HPLC.
Kinetic parameters and initial velocity pattern
Crystallization, data collection and structure determination.
The steady-state kinetic parameters, KM kcat and Vmax, were
,
Crystals of TtUPRT were obtained with the oil-microbatch
method using NUNC HLA plates (Nalge Nunc International).
Each crystallization drop was prepared by mixing 1.0 µL of the
precipitant solution (0.2 M ammonium sulfate, 0.1 M sodium
citrate, 0.1 M dioxane, pH 5.5) and 1.0 µL of the protein solution
(SeMet-substituted TtUPRT at 12.0 mg/mL). Then the
crystallization drop was overlaid by 1:1 mixture of silicon/paraffin
oils, allowing a slow evaporation of water in the drop. The
crystals grew to full size in 3−4 days. For the collection of
diffraction data, the crystals were soaked in the precipitant
solution containing 20 % (v/v) glycerol as a cryoprotectant and
flash-frozen with liquid nitrogen. Complete data sets were
collected using a RIGAKU CCD detector at the synchrotron
beam line BL26B1 at SPring-8, Japan. The data were processed
and scaled using the HKL 2000 program suite.[43] Data collection
statistics are given in Table 1.
determined under standard assay conditions at varying
concentrations of one substrate (0.12-17 mM for Uracil, 0.25-32
mM for PRPP), while the concentration of the other substrate
was fixed at constant saturating level (10 mM). Apparent KM, kcat
and kcat/KM values were determined by non-linear regression
assuming Michaelis–Menten kinetics. Calculations were carried
out using the Origin program from Origin Lab Corporation.
Influence of pH, temperature and ionic strength on enzyme
activity
The optimum pH of the His-tag enzyme was initially determined
according to the standard activity assay described above, using
sodium citrate (pH 4-6), sodium phosphate (pH 6-8) and sodium
borate (pH 8-10) as reaction buffers (50 mM). The optimum
temperature was determined using standard assay in 20 - 90 °C
temperature range. A similar approach was used to characterize
the effect of ionic strength on enzyme activity. TtUPRT activity
was measured at different concentrations of sodium chloride,
ranging from 0 to 1 M.
The crystal structure of TtUPRT was determined by the multiple
[44]
anomalous dispersion method
using the anomalous
dispersion effect from selenium atoms in the SeMet-sustituted
protein. The Se-atom coordinates and initial electron density
map were obtained with the SOLVE/RESOLVE program.[45] The
protein model was built and improved manually with the program
QUANTA2000 (Accelrys Inc.) and refined with the CNS
program.[46] The stereo-chemical quality of the model was
checked with the PROCHECK program.[47] The refinement
statistics are summarized in Table 1. The final atomic
coordinates were deposited in the RCSB Protein Data Bank with
the accession code 1v9s. Superposition analysis was performed
using the program LSQKAB[48] in the CCP4 program suite.[49]
Thermal and pH stability of TtUPRT
His-tag TtUPRT was stored at 4 °C in 20 mM Tris-HCl buffer, pH
8.0 for 100 days. Samples were taken periodically, and
enzymatic activity was evaluated. Storage stability was defined
as the relative activity between the first reaction and successive
reactions. Moreover, thermal stability of TtUPRT was performed
by incubating 3 μg of pure His-tag enzyme in 20 mM Tris-HCl
buffer, pH 8.0 at different temperatures (50-90 °C) and different
storage times (15-120 min). After cooling, the samples were
assayed for UPRTase activity under standard conditions.
Enzyme activity assay
The standard activity assay was performed by incubating 10 μL
of free extract or 3 μg of pure His-tag enzyme with 10 mM PRPP,
10 mM uracil (or thymine or cytosine), 12 mM MgCl2 in 12 mM
TrisHCl buffer pH 8.0 in a final volume of 40 μL. The reaction
mixture was incubated at 60 °C for 10 min (300 rpm). Enzyme
was inactivated by adding 40 μL of cold methanol in ice-bath
and heating for 5 min at 100 °C. After centrifugation at 9000 × g
for 5 min, samples were half-diluted with water and the NMP
production was analysed by HPLC to quantitatively measure the
reaction products as described below in the analytical methods.
All determinations were carried out in triplicate and the
maximum error was below 5 %. In such conditions, one
international activity unit (IU) was defined as the amount of
Analytical methods
The production of NMPs was quantitatively measured with an
ACE EXCEL 5 μm CN-ES 250 × 4.6 mm equilibrated with 100 %
trimethyl ammonium acetate at a flow rate of 0.8 mL/min.
Retention times for the reference natural compounds (hereafter
abbreviated according to the recommendations of the IUPAC-
IUB Commission on Biochemical Nomenclature) were as
follows: uracil (Ura), 4.6 min; uridine-5'- monophosphate (UMP),
3.5 min; cytosine (Cyt), 4.1 min; cytidine-5'-monophosphate
(CMP), 3.1 min; thymine (Thy), 7.2 min; thymidine-5'-
monophosphate (TMP), 4.4 min. Retention times for the
reference non-natural bases (hereafter abbreviated according to
the recommendations of the IUPAC-IUB Commission on
Biochemical Nomenclature) were as follows: 5-fluorouracil (5-
FUra), 5.4 min; 5-fluorouridine-5'- monophosphate (5-FUMP),
3.6 min; 5-chlorouracil (5-ClUra), 8.5 min; 5-chlorouridine-5'-
monophosphate (5-ClUMP), 3.9 min; 5-bromouracil (5-BrUra),
10.5 min; 5-bromouridine-5'- monophosphate (5-BrUMP), 4.3
min; 5-iodouracil (5-IUra), 15.0 min; 5-iodouridine-5'-
monophosphate (5-IUMP), 5.3 min; 5-hydroxymethyluracil (5-
OHMeUra), 4.4 min; 5-hydroxymethyluridine-5'-monophosphate
(5-OHMeUMP), 3.6 min; 6-methyluracil (6-MeUra): 6.3 min; 6-
enzyme producing
1 μmol/min of UMP under the assay
conditions.
Enzymatic production of natural and non-natural nucleosides-
5′-monophosphate from pyrimidine bases
Enzymatic production of natural and non-natural NMPs was
carried out by incubating 3 μg of pure His-tag enzyme with 1 mM
PRPP, 1 mM natural or non-natural pyrimidine base and 1.2 mM
MgCl2 in 12 mM Tris-HCl buffer pH 8.0. The reaction mixtures
were incubated at 60 °C and 300 rpm orbital shaking at different
This article is protected by copyright. All rights reserved.

10.1002/cctc.201701223
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FULL PAPER
methyluridine monophosphate (6-MeUMP): 3.7 min; 5-
azacytosine (5-AzaCyt), 3.7 min; 5-fluorocytosine (5-FCyt): 4.0
min; 5-ethyluracil (5-EtUra), 12 min; 6-methyl-2-thiouracil (6-
MetThioUra), 13.5 min; 6-propyl-2-thiouracil (6-PropThioUra),
19.0 min; 5-(2-bromovinyl)uracil, (5-BrVinUra), 26 min; 5-fluoro-
2-methoxy-4(1H) pyrimidinone, (5-FMP), 5 min; 6-azauracil (6-
azaUra), 4.1 min; Trifluorothymine (TFT), 19.0 min. Results were
normalized based on the nucleobase mass balance.
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Keywords: biocatalysis • uracil phosphoribosyltransferase •
thermophiles • nucleoside-5'-monophosphates • crystallization
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10.1002/cctc.201701223
ChemCatChem
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Text for Table of Contents
Authors: Jon del Arco,[a] Javier
Acosta,[a] Humberto M. Pereira,[b]
The enzymatic production of several
Almudena
Perona,[a]
Neratur
K.
O
modified
nucleoside-5'-
Lokanath,[c] Naoki Kunishima, * [d] Jesús
R1
R2
HN
monophosphates has been performed
by uracil phosphoribosyltransferase
from Thermus themophilus,TtUPRT.
In addition, the functional and
structural characterization of TtUPRT
has been reported for first time.
Fernández-Lucas * [a, e]
.
O
NH
P
O
O
Page No. – Page No.
O
P
P
OH
OH
O
Title: Enzymatic production of non-
natural nucleoside-5'-monophosphates
R1
R2
HN
O
O
N
P
O
by
a
novel thermostable uracil
phosphoribosyltransferase
OH OH
This article is protected by copyright. All rights reserved.