Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8

Article abstract of DOI:10.1080/10242422.2017.1313837

Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5′-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg²⁺. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90 °C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis.

Full text of DOI:10.1080/10242422.2017.1313837

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Cloning, expression and biochemical
characterization of xanthine and adenine
phosphoribosyltransferases from thermus
thermophilus HB8
Jon Del Arco, María Martinez, Manuel Donday, Vicente Javier Clemente-
Suarez & Jesús Fernández-Lucas
To cite this article: Jon Del Arco, María Martinez, Manuel Donday, Vicente Javier Clemente-
Suarez & Jesús Fernández-Lucas (2017): Cloning, expression and biochemical characterization
of xanthine and adenine phosphoribosyltransferases from thermus thermophilus HB8, Biocatalysis
and Biotransformation, DOI: 10.1080/10242422.2017.1313837
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Date: 24 April 2017, At: 01:20

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
http://dx.doi.org/10.1080/10242422.2017.1313837
RESEARCH ARTICLE
Cloning, expression and biochemical characterization of xanthine and
adenine phosphoribosyltransferases from thermus thermophilus HB8
a
a
a
a
ꢀ
Jon Del Arco , Marıa Martinez , Manuel Donday , Vicente Javier Clemente-Suarez and
a,b
ꢀ
ꢀ
Jesus Fernandez-Lucas
a
ꢀ
Applied Biotechnology Group, Research and Doctoral School, European University of Madrid, Urbanizacion El Bosque, Madrid, Spain;
b
ꢀ
Grupo de Investigacion en Desarrollo Agroindustrial Sostenible, Ingeniería Agroindustrial, Universidad de la Costa, CUC, Barranquilla,
Colombia
ABSTRACT
ARTICLE HISTORY
Received 24 February 2017
Revised 24 March 2017
Accepted 27 March 2017
Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage
pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in pur-
ine nucleosides-50-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group
from 5-phospho-a-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of
Mg2þ. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis
of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribo-
syltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and
purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2
were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treat-
ment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatog-
raphy. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In
addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a
temperature range from 70 to 90 ꢀC), but they also showed very different behaviour under the
influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high depend-
ence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally,
substrate specificity studies were performed in order to explore their potential as industrial bio-
catalyst for NMPs synthesis.
KEYWORDS
Enzymatic synthesis; purine
nucleosides-50-monophosh-
ate; phosphoribosyltransfer-
ases; thermostable
biocatalysts
Introduction
Serra et al. 2014). These drawbacks increase final pro-
duction costs and lead to a prohibitively expensive
price of the NADs, impeding their biological trials and
studies, as well as limiting their wide therapeutic appli-
cation. Due to the economic and social relevance of
this kind of drugs, the availability of efficient, stereo-
selective and sustainable processes should be very
relevant for the industry.
In this frame, the enzymatic synthesis of nucleo-
sides-50-monophospate (NMPs) is an attractive field
since biotransformations shows many advantages,
such as one-pot reactions under mild conditions, high
stereo- and regioselectivity, and an environmentally
friendly technology. The use of enzymes as industrial
catalysts (white biotechnology) can thus provide novel
and straightforward synthetic schemes and suitable
and efficient methodologies for developing sustainable
industrial processes.
Nucleic acid derivatives, NADs (nucleosides, nucleotides
and nucleobases) are essential metabolites in numerous
biochemical processes. In this way, they have been
widely used for the treatment of several serious human
illnesses, such as cancer or viral diseases among others
(Galmarini et al. 2002; de Clercq 2005; Parker 2009).
Chemical synthesis of NMPs is routinely performed
by 50-phosphorylation of the precursor nucleoside. It
often requires multiple protective group manipulations
to control the regio- and stereochemistry of ribose
phosphorylation and the use of chemical reagents,
such as phosphoryl chloride (POCl3) or phosphorus
pentoxide (P2O5) and organic solvents which are
expensive and environmentally harmful (Asano 2002;
Li et al. 2015). Due to this, chemical synthesis of NMPs
usually provide poor or moderate yields, low product
purity and is also associated with harsh reaction
conditions and waste disposal issues (Asano 2002;
Purine phosphoribosyltransferases belong to the
phosphoribosyltransferases family (PRTs). They catalyse
ꢀ
ꢀ
CONTACT Jesus Fernandez-Lucas
jesus.fernandez2@universidadeuropea.es
Applied Biotechnology Group, European University of Madrid,
ꢀ
ꢀ
Urbanizacion El Bosque, Calle Tajo, s/n, 28670, Villaviciosa de Odon (Madrid), Spain
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
J. D. ARCO ET AL.
Figure 1. Enzymatic synthesis of purine nucleosides-50-monophosphate (NMPs) catalyzed by TtXPRT and TtAPRT2.
the reversible transfer of the 5-phosphoribosyl group
from 5-phospho-a-D-ribosyl-1-pyrophosphate (PRPP) to
N9 in 6-amino or 6-oxopurines, such as adenine (1),
hypoxanthine (2), guanine (3) and xanthine (4), in the
presence of Mg2þ to synthesize adenosine-50-mono-
phosphate, AMP (5), inosine-50-monophosphate, IMP
(6), guanosine-50-monophosphate, GMP (7) or xantho-
sine-50-monophosphate, 50-XMP (8) (el Kouni 2003;
Craig and Eakin 2000) (Figure 1). These kind of
enzymes are essential in the salvage pathways for pur-
ine nucleotide synthesis of all organisms. Due to this,
the use of PRTs as therapeutic targets has been exten-
sively reported (el Kouni 2003). In contrast, only a few
examples about their use as catalysts in NMP synthesis
has been described (Scism et al. 2007, 2010; Valino
et al. 2015; Iglesias et al. 2015; Esipov et al 2016;
Hansen et al 2014).
According to their substrate specificity, purine PRTs
are classified into two general classes: 6-aminopurine
PRTs (APRT), strictly specific for 6-aminopurines, such
as adenine and 6-aminopurine derivatives (Craig and
Eakin 2000; Iglesias et al. 2015; Esipov et al. 2016), and
6-oxopurine PRTs (HPRT, GPRT, XPRT, HGPRT, XGPRT or
HGXPRT) that can recognize different kinds of 6-oxo-
purines, such as hypoxanthine, guanine, xanthine and
other 6-oxopurine analogs (el Kouni 2003; Craig and
Eakin 2000; Scism et al. 2007; Scism & Bachmann 2010;
Iglesias et al. 2015; Valino et al. 2015).
Nowadays, it is very common use of enzymes from
thermophilic organisms in industrial bioprocesses. The
use of thermozymes offers the possibility to use high-
temperature reactions, resulting in a higher solubiliza-
tion of substrates, a diminution of medium viscosity
and an increase in substrate diffusion coefficients,
leading to higher overall reaction rates.
The present work aims to explore the potential of
xanthine phosphoribosyltransferase (XPRT, EC 2.4.2.22)
and adenine phosphoribosyltransferase (APRT, EC
2.4.2.7) from Thermus thermophilus HB8 as biocatalysts
for the synthesis of NMPs. In this sense, we report for
the first time the cloning of the xprt and aprt2 genes
from T. thermophilus (HB8), the expression in
Escherichia coli, purification of the recombinant pro-
teins (TtXPRT and TtAPRT2), as well as their biochem-
ical characterization. In addition, detailed comments
about oligomeric state, substrate specificity and archi-
tecture of active site were included.
Materials and methods
Materials
Cell culture medium reagents were from Difco
(St. Louis, MO). Trimethyl ammonium acetate buffer and
5-phospho-a-D-ribosyl-1-pyrophosphate were purchased
from Sigma-Aldrich (Madrid, Spain). All other reagents
and organic solvents were purchased to Scharlab
(Barcelona, Spain) and Symta (Madrid, Spain). All natural
and non-natural purine bases used in this work were pro-
vided by Carbosynth Ltd. (Compton, United Kingdom).
Enzyme cloning, expression and purification
The encoding ttha0535 (xprt) and ttha1614 (aprt2)
genes, which codifies xanthine phosphoribosyltransfer-
ase, TtXPRT (NCBI Reference Sequence: YP_143801.
1XPRT), and adenine phosphoribosyltransferase,
TtAPRT2 (NCBI Reference Sequence: YP_144880.1) from
T. thermophilus HB8, were ordered and purchased from
Genscript (Piscataway, NJ). The coding sequences
appeared as NdeI-EcoRI fragments subcloned into two
different expression vectors pET28b(þ). The resultant
recombinant
vectors,
pET28bTtXPRT
and
pET28bTtAPRT2, provided two different N-terminal
His6-tagged fusion proteins with a thrombin cleavage
site between the tag and the enzyme. Both enzymes
were expressed in E. coli BL21(DE3) grown in LB
medium at 37 ꢀC supplemented with kanamycin 50 lg/
mL. Protein overexpression was induced by adding
0.5 mM b-D-1-thiogalactopyranoside and the cells

BIOCATALYSIS AND BIOTRANSFORMATION
3
further grown for 4 h. These cells were harvested via
centrifugation at 3500 ꢁ g, and the resulting pellets
were resuspended in 20 mM Tris-HCl buffer, pH 8, con-
taining 100 mM NaCl. Crude extracts were prepared by
ultrasonic cell treatment using a digital sonifier. The
lysates were centrifuged at 17,500ꢁg for 20 min at 4 ꢀC.
After this, lysates were then treated at 70 ꢀC for 20 min.
Insoluble material was removed by centrifugation
17,500ꢁg for 20 min at 4 ꢀC and the supernatant was fil-
tered through a 0.22-lm filter (Millipore). Cleared
lysates were 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 NaCl and 20 mM
imidazole). Bound proteins were eluted using a linear
gradient of imidazole (from 10 to 500 mM). Fractions
containing TtXPRT or TtAPRT2 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 buffer, pH 8.0.
Fractions with the protein of interest identified by SDS-
PAGE were pooled and the protein was concentrated
and stored at 4 ꢀC until its use. Electrophoresis was car-
ried 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 absorp-
tion at 280 nm using e280 ¼ 31,970 Mꢂ1cmꢂ1 for TtXPRT
and e280 ¼ 4470 Mꢂ1cmꢂ1 for TtAPRT2 (Gill and Von
Hippel 1989).
Enzyme activity assay
The standard activity assay was performed by incubat-
ing 10–50 lL of free extracts or 1–5 lg of pure
enzymes with a 40 lL solution containing 10 mM PRPP,
10 mM adenine (TtAPRT) or xanthine (TtXPRT), 12 mM
MgCl2 in 12 mM Tris-HCl buffer pH 8. The reaction mix-
ture was incubated at 60 ꢀC for 10 min (300 rpm).
Enzyme was inactivated by the addition of 40 lL of
cold methanol in icebath and heating for 5 min at
100 ꢀC. After centrifugation at 9000ꢁg for 5 min, sam-
ples were half-diluted with water and the NMP pro-
duction was analysed by HPLC to quantitatively
measure the reaction products as described below in
the analytical methods. All determinations were carried
out by triplicate and the maximum error was below
5%. In such conditions, one international activity unit
(IU) was defined as the amount of enzyme producing
1 lmol/min of the corresponding NMP under the assay
conditions.
Influence of pH and T on enzyme activity
The optimum pH of both enzymes was initially deter-
mined according to the standard activity assay
described earlier, 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 of enzyme activity was determined
using standard assay in a temperature range from 20
to 90 ꢀC.
Analytical ultracentrifugation analysis
Sedimentation velocity for TtUPRT were carried out in
20 mM Tris-HCl buffer, pH 8.0 at 20 ꢀC and 50,000ꢁg
in an Optima XL-I analytical ultracentrifuge (Beckman-
Coulter Inc., Madrid, Spain), equipped with UV-VIS
absorbance and Raleigh interference detection sys-
tems, using an An-60Ti rotor and standard (12-mm
optical path) double-sector centre pieces of Epon-char-
coal. 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 14.7 g.
Baseline offsets were measured afterwards at
200,000ꢁg. The apparent sedimentation coefficient of
distribution, c(s), and sedimentation coefficient s were
calculated from the sedimentation velocity data using
the program SEDFIT (Brown and Schuck 2006). The
experimental sedimentation coefficients were cor-
rected to standard conditions (water, 20 ꢀC and infinite
dilution) using the software SEDNTERP (Holde 1985)
in order to get the corresponding standard s values
(s20,w).
Storage stability
TtXPRT and TtAPRT2 were stored at 4 ꢀC in 20 mM
Tris-HCl buffer, pH 7.0 for 100 days. Periodically, sam-
ples were taken, and enzymatic activity was eval-
uated. Storage stability was defined as the relative
activity between the first and the successive
reactions.
Substrate specificity
Enzymatic synthesis of NMPs using different natural
nucleobases (adenine, hypoxanthine, guanine and xan-
thine) was performed under standard assay conditions.
The reaction mixtures were incubated in different reac-
tion buffers (12 mM sodium citrate pH 6 or 12 mM
sodium borate pH 8.5) at 60 ꢀC and 300 rpm orbital
shaking at different reaction times (5–15 min). After
this, the reaction mixture was processed as previously
described above and the NMP production was ana-
lysed by HPLC.

4
J. D. ARCO ET AL.
Analytical methods
The production of NMPs was quantitatively measured
by HPLC (Agilent 1100 series). Reactions were analysed
with an ACE EXCEL 5 lm CN-ES 250 ꢁ 4.6 mm equili-
brated with 100% trimethyl ammonium acetate at a
flow rate of 0.8 mL/min. Retention times for the refer-
ence natural compounds (hereafter abbreviated
according to the recommendations of the IUPAC-IUB
Commission on Biochemical Nomenclature) were as
follows: adenine (Ade), 10.2 min; adenosine-50-mono-
phosphate (AMP), 5.50 min; guanine (Gua), 4.8 min;
guanosine-50-monophosphate (GMP), 2.8 min; hypoxan-
thine (Hyp), 4.5 min; inosine-50-monophosphate (IMP),
2.5 min; xanthine (Xan), 4.4 min; xanthosine-50-mono-
phosphate (XMP), 2.5 min. In order to confirm the reac-
tion products, commercial AMP, GMP, IMP and XMP
were used as HPLC controls.
Figure 2. Multiple sequence alignment of purine phosphoribo-
syltransferases from Leishmania donovani (LdXPRT), Thermus
thermophilus (TtAPRT1, TtAPRT2 and TtXPRT), Escherichia coli
(EcXGPRT), Mycobacterium tuberculosis (MtUPRT), Escherichia
coli (EcUPRT) and Bacillus subtilis (BsXPRT). Sequences were
aligned by CLUSTALX. Conserved PRPP motif are encircled.
Asterisk ( ), indicates single, fully conserved residues; colon (:),
conservation of strong groups; period (.), conservation of weak
groups.
ꢃ
Results and discussion
genes could code for putative intracellular
phosphoribosyltransferases.
TtXPRT was predicted to contain 154 amino acid
residues and have a relative molecular mass of
Bioinformatic analysis of xanthine and adenine
phosphoribosyltransferases encoding genes of T.
Thermophilus HB8
17.10 kDa
(Protparam,
http://web.expasy.org/prot-
The genomic information of T. thermophilus HB8 has
been analysed and published online (Genbank No.
AP008226). Several genes that potentially encode pur-
ine phosphoribosyltransferases were annotated
throughout the genome. Among these, two ORFs,
ttha0535 (xprt) and ttha1614 (aprt2), which are anno-
tated as putative xanthine phosphoribosyltransferase
(TtXPRT) and a putative adenine phosphoribosyltrans-
ferase (TtAPRT), were subjected to bioinformatic ana-
lysis to identify their physical and chemical properties
and their possible active site architecture. Since other
putative aprt gene (ttha1613) was detected in T. ther-
mophilus HB8 genome, we renamed ttha1613 as aprt1.
BLAST analysis of amino acid sequences (http://
blast.ncbi.nlm.nih.gov/Blast.cgi) reveals that xprt and
aprt2 encodes putative xanthine and adenine phos-
phoribosyltransferases. Multiple sequence alignment of
predicted both enzymes with amino acid sequences of
xanthine and adenine phosphoribosyltransferases from
other thermophilic and mesophilic bacteria and proto-
zoan characterized so far, reveals that both enzymes
display a conserved 13-residue “fingerprint” region,
PRPP binding domain of PRTs type I, typically com-
posed by four hydrophobic residues followed by two
acidic residues (which interact with the vicinal
hydroxyl groups of the ribose phosphate moiety), two
hydrophobic residues, and four small residues
(Figure 2). All these characteristics suggest that these
param/). Moreover, putative TtAPRT2 was predicted to
contain 180 amino acids and a relative molecular mass
of 19.15 kDa.
Production and purification of recombinant
enzymes
The corresponding xprt and aprt2 genes, encoding
TtXPRT and TtAPRT2 were cloned, and overexpressed
in E. coli BL21(DE3) as described in the experimental
section. The recombinant N-terminal His6-tagged pro-
teins were purified by two chromatographic steps.
SDS-PAGE analysis of purified enzymes shows only one
protein band with an apparent molecular mass of
19 kDa for TtXPRT and 21 kDa forTtAPRT2.
These results agree with those from analytical ultra-
centrifugation analysis. Under the conditions assayed,
sedimentation velocity experiments revealed TtXPRT as
a group of four species with experimental sedimenta-
tion coefficients of 3.41 S (s20,w ¼ 3.43 S), 5.84 S
(s20,w ¼ 5.89 S), 13.48 S (s20,w¼13.60 S) and 16.12 S
(s20,w ¼ 16.26) compatible with the theoretical behav-
iour of different oligomeric forms. The major specie
found in solution corresponds to a tetrameric state
(Mw ¼ 76.08 kDa) and is compatible with a single-spe-
cies monomeric model of 19.02 kDa, similar to the
molecular mass calculated from the amino acid
sequence of His6-tagged protein (18.90 kDa). Different

BIOCATALYSIS AND BIOTRANSFORMATION
5
oligomeric states are described for XPRTs. While XPRT
from Streptococcus faecalis and Bacillus subtilis seems
to be a homodimer of 42.00 kDa (Miller et al. 1974;
Arent et al. 2006), XGPRT from E. coli has been
reported as a homotetramer (Vos et al. 1997) after the
determination of its native molecular mass by
Superose-gel filtration chromatography.
On the other hand, experimental data revealed
TtAPRT2 as a single species with an experimental sedi-
mentation coefficient of 3.06 S (s20,w¼3.09) compatible
with a dimer (Mw¼41.66 kDa). This value is compatible
with a single-species monomeric model of 20.83 kDa,
similar to the molecular mass calculated from the
amino acid sequence of His6-tagged protein
(20.95 kDa). This result agrees with other reported bac-
terial, archaea, fungal, protozoan and eukaryotic
APRTs, such as APRT from E. coli (Hochstadt-Ozer and
Stadtman 1971), Sulfolobus solfataricus (Hansen et al.
2014), Saccharomyces cerevisiae (Alfonzo et al. 1997),
Leishmania donovani (Tuttle and Krenitsky 1980) or
human APRT (Holden et al. 1979). In this sense, it
seems that the oligomeric state of APRTS is highly
conserved among different living organisms.
neutral-alkaline conditions, such as XPRTs from
Streptococcus faecalis (Miller et al. 1974) and E. coli
(Vos et al. 1997). Moreover, other non-specific 6-oxo-
purines, such as HGXPRT from Toxoplasma gondii
(Naguib et al. 1995), Bacillus subtilis (Arent et al. 2006)
and Sulfolobus solfataricus (Hansen et al. 2014) display
a similar pattern. In this sense, TtXPRT differs slightly
from other XPRTs.
On the other hand, TtAPRT2 displayed maximum
activity at 80 ꢀC, with more than 80% of retained activ-
ity in a temperature interval from 70 to 90 ꢀC (Figure
3(C)), but contrary to TtXPRT, it could be observed that
TtAPRT2 showed high activity at high pH values (pH
range 8–10) (Figure 3(D)). In this case, TtAPRT2 displays
a similar susceptibility to alkaline conditions than other
mesophilic or thermophilic APRTS, such as APRTS from
Plasmodium falciparum (Queen et al. 1989), Leishmania
donovani (Tuttle and Krenitsky 1980) or Sulfolobus sol-
fataricus (Jensen et al. 2015). These results are very
interesting due to the fact that both proteins, TtXPRT
and TtAPRT2, display optimal activity values at high
temperatures, but TtAPRT2 also displays a non-usual
tolerance in alkaline conditions that is not expected.
Since purine bases usually display poor solubility in
ꢀ
aqueous medium (Fernandez-Lucas et al. 2012),
Biochemical characterization
TtAPRT2 could be an interesting biocatalyst for the
industrial synthesis of purine NMPs using low soluble
bases.
Effect of temperature and pH on the activity of TtXPRT
and TtAPRT2 were studied to select optimal conditions
for their use as biocatalysts in NMPs synthesis.
Experimental results revealed that the enzymatic activ-
ity of TtXPRT and TtAPRT2 presents a strong depend-
ence with temperature, as can be expected for
proteins from hyperthermophilic organisms, but they
also show very different behaviour under the influence
of pH.
The storage of TtXPRT and TtAPRT2 at 4 ꢀC was ana-
lysed to ensure biocatalyst stability. Both enzymes
exhibited more than 85% of retained activity over
more than 70 days (data not shown). Unfortunately,
TtXPRT and TtAPRT2 suffered a high decrease in activ-
ity (20–30%) when they were stored at ꢂ20 ꢀC or
ꢂ80 ꢀC.
On one hand, the temperature profile revealed that
TtXPRT is active (more than 75%) at high temperature
values (more than 75% of retained activity in a tem-
perature range 60–90 ꢀC) showing maximum activity at
80 ꢀC (Figure 3(A)). In addition, recombinant TtXPRT
also displayed high activity (more than 75%) in a nar-
row pH range (5–7), with a maximum activity value at
pH 6. (Figure 3(B)). As it is expected from thermophilic
enzymes, TtXPRT is very active at high temperatures;
unfortunately, there is not any described other strict
xanthine phosphoribosyltransferase from thermophiles
to compare these data. The most similar enzymes are
HGXPRTs from thermoacidophile archaeon Sulfolobus
Substrate specificity
Taking into account all previous results, substrate spe-
cificity studies were carried out under optimal condi-
tions (TtXPRT, pH 6 and 70 ꢀC; TtAPRT2, pH 8.5 and
70 ꢀC). Table 1 summarizes the specific activities of
TtXPRT and TtAPRT2 over adenine, guanine, hypoxan-
thine and xanthine. It can be observed that TtXPRT
recognizes xanthine as an acceptor for the synthesis of
XMP; consequently, TtXPRT has been confirmed as a
xanthine
phosphoribosyltransferase.
Furthermore,
TtXPRT could also slightly recognize hypoxanthine and
guanine as substrates. However, the remaining activity
is negligible; so TtXPRT should not be re-classified as a
HGXPRT. Due to the lack of literature on any specific
thermophilic XPRT so far, TtXPRT is reported as the
first hyperthermophilic XPRT.
solfataricus
and
hyperthermophilic
archaeon
Pyrococcus horikoshii, which are active at high temper-
atures (Hansen et al. 2014; de Souza et al. 2008).
Regarding the effect of pH on enzyme activity, all
described specific XPRTs work reasonably better in

6
J. D. ARCO ET AL.
Figure 3. Temperature and pH dependence on enzymatic activity. (A) Effect of temperature on TtXPRT activity (ꢄ). (B) Effect of pH
on TtXPRT activity, (ꢀ) sodium acetate 50 mM (pH 4–6), (ꢄ) sodium phosphate 50 mM (pH 6–8), (D) sodium borate 50 mM (pH
8–10). (C) Effect of temperature on TtAPRT2 activity (ꢄ). (D) Effect of pH on TtAPRT2 activity, (ꢀ) sodium acetate 50 mM (pH 4–6),
(ꢄ) sodium phosphate 50 mM (pH 6–8), (D) sodium borate 50 mM (pH 8–10).
Table 1. Activity of TtXPRT and TtAPRT2 over different natural
nucleobases.
APRTs are annotated in T. thermophilus HB8 genome
(Genbank No. AP008226), TtAPRT1 (YP_144879.1) and
TtAPRT1 (NCBI Reference Sequence: YP_144880.1).
Recently, Esipov et al. (2016) have described APRT
from T. thermophilus HB27, which is identical to puta-
tive TtAPRT1 from T. thermophilus HB8. Since TtAPRT2
is also encoded in T. thermophilus HB27 genome, it
leads to think that two different APRTS are present in
T. thermophilus, the first one, TtAPRT1, displays an
activity value of 8.8 lM minꢂ1mgꢂ1 (referred to AMP
synthesis) (Esipov et al. 2016), while TtAPRT2 display
an activity of 8000 lMminꢂ1mgꢂ1 (deduced from
Table 1). In this sense, it seems that TtAPRT2 should
play a more important role in purine scavenging in T.
thermophilus HB8 than TtAPRT1. According to multiple
sequence alignment of PRPP sequence motifs of differ-
ent 6-amino and 6-oxopurines PRTs (Figure 2), these
experimental results could be expected considering
Specific activity
Specific activity
Purine base
TtXPRTa (U/mgenz
)
TtAPRT2b (U/mgenz
)
Adenine
Guanine
Hypoxanthine
Xanthine
n.d.
0.10
0.12
1.70
0.32
n.d.
n.d.
n.d.
aReaction conditions: 1.25 lg of enzyme in 40 lL reaction volume.
Substrate concentrations were 10 mM PRPP, 10 mM uracil, 12 mM MgCl2
in 12 mM sodium citrate buffer, pH 6. Reaction time: 10 min, T ¼ 70 ꢀC,
350 r.p.m.
bReaction conditions: 5 lg of enzyme in 40 lL reaction volume. Substrate
concentrations were 10 mM PRPP, 10 mM uracil, 12 mM MgCl2 in 12 mM
sodium borate, pH 8.5. Reaction time: 10 min, T ¼ 70 ꢀC, 350 r.p.m.
n.d.: not detected.
TtAPRT2 could use adenine as acceptor for NMP
synthesis. In contrast, neither guanine nor hypoxan-
thine or xanthine, were recognized as substrates by
TtAPRT2. As mentioned earlier, two different putative

BIOCATALYSIS AND BIOTRANSFORMATION
7
Arent S, Kadziola A, Larsen S, Neuhard J, Jensen KF. 2006.
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distributions by sedimentation velocity analytical ultracen-
trifugation. Biophys J 90:4651–4661.
that TtAPRT1 shows only one of two acidic residues,
which interacts with the vicinal hydroxyl groups of the
ribose phosphate moiety from PRPP. This probably
leads to a less effective binding of PRPP and a subse-
quently decrease in catalytic activity.
Conclusions
In this work, we describe for the first time the cloning,
expression and biochemical characterization of xan-
thine phosphoribosyltransferase, TtXPRT and adenine
phosphoribosyltransferase2, TtAPRT2, from T. thermo-
philus HB8. Biochemical characterization revealed
TtXPRT as a homotetramer with significant activity in a
broad range of temperature pH (from 60 to 90 ꢀC).
Additionally, TtXPRT was also found to exhibit activity
in a pH interval from 5.0 to 7.0. Moreover, experimen-
tal results revealed TtAPRT2 as a homodimer, with a
similar temperature dependence to TtXPRT (optimal
activity in temperature range 70–90 ꢀC), but it displays
a very different behaviour under pH influence (from
7.0 to 9.0). The thermophilic origin of both enzymes
involves several advantages, such as an easy purifica-
tion method and tolerance to high temperatures, what
makes these enzymes attractive biocatalysts for the
industrial synthesis of nucleoside derivatives.
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Acknowledgments
The authors thank European University of Madrid and
Santander Foundation for the financial support of this work.
Moreover, they also thank Dr. Berenguer for his continued
support and enthusiasm for the project.
Disclosure statement
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
This work was supported by grant SAN151610 from the
Santander Foundation and Grant 2014/UEM13 from
European University of Madrid.
Funding
Holden JA, Meredith GS, Kelley WN. 1979. Human adenine
phosphoribosyltransferase. Affinity purification, subunit
structure, amino acid composition, and peptide mapping.
J Biol Chem 254:6951–6955.
This work was supported by grant SAN151610 from the
Santander Foundation and Grant 2014/UEM13 from
European University of Madrid.
Iglesias LE, Lewkowicz ES, Medici R, Bianchi P, Iribarren AM.
2015. Biocatalytic approaches applied to the synthesis of
nucleoside prodrugs. Biotechnol Adv 33:412–434.
Jensen KF, Hansen MR, Jensen KS, Christoffersen S, Poulsen
JCN, Mølgaard A, Kadziola A. 2015. Adenine phosphoribo-
syltransferase from Sulfolobus solfataricus is an enzyme
with unusual kinetic properties and a crystal structure that
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