Recombinant purine nucleoside phosphorylases from thermophiles: Preparation, properties and activity towards purine and pyrimidine nucleosides

Article abstract of DOI:10.1111/febs.12143

Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three 'high molecular mass' purine nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5′-methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate properties were investigated for natural purine nucleosides [adenosine, inosine and their C2′-deoxy counterparts (activity within 50-500 U·mg^-1)], analogues with 2′-amino modified 2′-deoxy-adenosine and -inosine (within 0.1-3 U·mg^-1) as well as 2′-deoxy-2′-fluoroadenosine (9) and its C2′-arabino diastereomer (10, within 0.01-0.03 U·mg ^-1). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2′-deoxyfluoro adenine ribo- and arabino-nucleosides displayed similar substrate properties. Moreover, cytidine and 2′-deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2′-deoxyfluoro adenine nucleosides 9 and 10. C2′-modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2′-deoxyfluoro adenine ribo- and arabino- nucleosides in moderate yield (24%).

Full text of DOI:10.1111/febs.12143

Recombinant purine nucleoside phosphorylases from
thermophiles: preparation, properties and activity
towards purine and pyrimidine nucleosides
1
1
1,2
1,2
3
Xinrui Zhou , Kathleen Szeker , Bernd Janocha , Thomas B o€ hme , Dirk Albrecht ,
4
1
Igor A. Mikhailopulo and Peter Neubauer
1
2
3
4
Laboratory of Bioprocess Engineering, Department of Biotechnology, Technische Universit €a t Berlin, Germany
Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany
Institute of Microbiology, Ernst-Moritz-Arndt-Universit €a t Greifswald, Germany
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus
Keywords
′–deoxyfluoro adenine nucleosides;
Thermostable nucleoside phosphorylases are attractive biocatalysts for the
synthesis of modified nucleosides. Hence we report on the recombinant
expression of three ‘high molecular mass’ purine nucleoside phosphorylases
2
Aeropyrum pernix;
Deinococcus geothermalis;
Geobacillus thermoglucosidasius;
thermostable PNP
(
PNPs) derived from the thermophilic bacteria Deinococcus geothermalis,
Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon
Aeropyrum pernix (5′–methythioadenosine phosphorylase; ApMTAP).
Thermostability studies, kinetic analysis and substrate specificities are
reported. The PNPs were stable at their optimal temperatures (DgPNP,
Correspondence
P. Neubauer, Laboratory of Bioprocess
Engineering, Department of Biotechnology,
Technische Universit €a t Berlin, Ackerstr.
5
erties were investigated for natural purine nucleosides [adenosine, inosine
5 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate prop-
71–76, ACK24, D–13355 Berlin, Germany
and their C2′-deoxy counterparts (activity within 50–500 Uꢀmgꢁ1)], ana-
Fax: +49 3031 427577
logues with 2′-amino modified 2′-deoxy-adenosine and -inosine (within
Tel: +49 3031 472269
ꢁ
1
0
.1–3 Uꢀmg ) as well as 2′-deoxy-2′-fluoroadenosine (9) and its C2′-arabi-
ꢁ1
E-mail: peter.neubauer@tu-berlin.de
Website: http://www.bioprocess.tu-berlin.de
I. A. Mikhailopulo, Institute of Bioorganic
Chemistry, National Academy of Sciences
of Belarus, Acad. Kuprevicha 5/2, 220141
Minsk, Belarus
no diastereomer (10, within 0.01–0.03 Uꢀmg ). Our results reveal that the
structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a
critical role in the phosphorolysis reaction. The implications of this finding
may be helpful for reaction mechanism studies or optimization of reaction
conditions. Unexpectedly, the diastereomeric 2′-deoxyfluoro adenine ribo-
and arabino-nucleosides displayed similar substrate properties. Moreover,
cytidine and 2′-deoxycytidine were found to be moderate substrates of the
prepared PNPs, with substrate activities in a range similar to those deter-
mined for 2′-deoxyfluoro adenine nucleosides 9 and 10. C2′-modified nucle-
osides are accepted as substrates by all recombinant enzymes studied,
making these enzymes promising biocatalysts for the synthesis of modified
nucleosides. Indeed, the prepared PNPs performed well in preliminary
transglycosylation reactions resulting in the synthesis of 2′-deoxyfluoro ade-
nine ribo- and arabino- nucleosides in moderate yield (24%).
Fax: +375 1726 78161
Tel: +375 1726 78148
E-mail: imikhailopulo@gmail.com
(Received 6 December 2012, revised 13
January 2013, accepted 16 January 2013)
doi:10.1111/febs.12143
Abbreviations
Ade, adenine; Ado, adenosine; Ap, Aeropyrum pernix; Cyd, cytidine; dAdo, 2′–deoxyadenosine; dAdo2′F, 2′–deoxy-2′–fluoroadenosine;
2
′F
dAdo , 9–(2-deoxy-2-fluoro- -D-arabinofuranosyl)adenine; dAdo2′NH2, 2′–amino-2′-deoxyadenosine; dCyd, 2′–deoxycytidine;
2
′F
Dg, Deinococcus geothermalis; dIno, 2′–deoxyinosine; dIno2′NH2, 2′–amino-2′–deoxyinoinosine; dUrd , 1–(2-deoxy-2-fluoro- -D-arabinofuranosyl)
uracil; dUrd2′F, 2′–deoxy-2′–fluoroadenosine; Ea, Enterobacter aerogenes; Ec, Escherichia coli; Gs, Geobacillus stearothermophilus;
Gt, Geobacillus thermoglucosidasius; Ino, inosine; IPTG, isopropyl b-D–thiogalactoside; MTAP, 5′–methythioadenosine phosphorylase;
NP, nucleoside phosphorylase; Pf, Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; PyNP, pyrimidine nucleoside phosphorylase;
Ss, Sulfolobus solfataricus; Tt, Thermus thermophilus; Urd, uridine.
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS
1

Purine nucleoside phosphorylases from thermophiles
X. Zhou et al.
Introduction
Base and sugar-modified analogues of natural nucleo-
sides are widely used as pharmaceutical agents for the
treatment of viral infections and cancer [1–3]. Moreover,
the replacement of natural nucleosides by modified
species results in improvement in the biochemical prop-
erties of synthetic oligonucleotides. For example,
C2′–fluorinated nucleosides have been shown to have
favorable properties as components of antisense
oligonucleotides and small interfering RNAs [4–6]. The
chemical synthesis of modified nucleosides is challenged
by stereo- and regioselective requirements, as well as the
need to protect and deprotect sensitive functional
groups. By contrast, enzymes, such as nucleoside phos-
phorylases (NPs) can be used as biocatalysts for the
efficient synthesis of nucleoside analogues under
environmentally friendly conditions [7–11].
However, common disadvantages encountered in bio-
catalysis comprise the instability of many enzymes
under harsh reaction conditions and narrow substrate
spectra limiting the scope of any potential synthetic
reactions. Escherichia coli NP-mediated synthesis of
fluorinated nucleosides represents an example of this
dilemma [12]. The use of enzymes from thermophiles is
a reasonable strategy for overcoming instability prob-
lems [13]. Such thermozymes offer the possibility of per-
forming reactions at high temperature, resulting in
diminution of the viscosity of the medium and increased
substrate diffusion coefficients, and leads to higher over-
all reaction rates. The final yield may be improved
because of the higher solubility of substrates with the
increase in temperature [14]. It is noteworthy that ther-
mozymes are often resistant to pressure and organic sol-
vents [15,16]. They can be expressed in E. coli at high
levels and are easily purified by heat treatment [17,18].
Purine nucleoside phosphorylase (PNP; EC 2.4.2.1)
and 5′–methylthioadenosine phosphorylase (MTAP;
EC 2.4.2.28) catalyze the reversible phosphorolysis of
purine nucleosides, as depicted in Scheme 1. Both
belong to the PNP family [19]. According to Shugar
and co-workers, PNPs can be grouped into two main
classes (for a comprehensive review, see ref. [19]): PNPs
of the first class (‘low molecular mass PNPs’), found
mainly in mammalian tissues and in some bacteria, are
specific for 6–oxo purine nucleosides only; those of the
second class (‘high molecular mass PNPs’) are predomi-
nantly found in bacteria and accept 6–oxo (e.g. inosine
and guanosine) as well as 6–amino (e.g. adenosine) pur-
ine nucleosides as substrates. Because of the broader
substrate specificity, the latter group of enzymes is espe-
cially interesting for synthetic applications. Although
many PNPs have been extensively studied [19],
examples of ‘high molecular mass PNPs’ derived from
thermophiles are limited to those from Geobacillus ste-
arothermophilus (GsPNP, punB gene) [9,20] and Ente-
robacter aerogenes (EaPNP) [21,22]; the MTAP from
Aeropyrum pernix (ApMTAP) [23], Sulfolobus solfatari-
cus (SsMTAP) [24,25] and Pyrococcus furiosus (PfM-
TAP) [26], whereby only GsPNP, EaPNP and
ApMTAP were considered for synthetic applications.
Besides, ApMTAP has been recently applied in synthe-
sis reactions [23], however, the information about the
enzyme’s characteristics is not available.
In this study, we report on: (a) the preparation of
three recombinant ‘high molecular mass’ PNPs derived
from the thermophilic bacteria Deinococcus geother-
malis (DgPNP) and Geobacillus thermoglucosidasius
11955 (GtPNP), as well as from the hyperthermophilic
archaeon A. pernix (ApMTAP); and (b) the investiga-
tion into their physicochemical and substrate specific-
ity. Here, we report on their thermal properties,
kinetic parameters and substrate specificities in regard
to natural purine substrates (inosine, adenosine and
their 2′–deoxy counterparts, 1 and 2, Scheme 1), ana-
logues of natural substrates modified at the C2′ carbon
atom [2′–amino-2′–deoxyadenosine (7), 2′–amino-2′–de-
oxyinosine (8), 2′–deoxy-2′–fluoroadenosine (9) and its
C2′–arabino diastereomer 10 (Scheme 2)].
Our results reveal that 2′–modified nucleosides are
accepted as substrates by all recombinant enzymes stud-
ied, indicating that these enzymes are promising biocat-
alysts for the synthesis of their respective nucleoside
analogues. Indeed, in preliminary experiments for the
synthesis of 2′–deoxyfluoro adenine nucleosides 9 and
10, when the prepared PNPs were applied in combina-
tion with thermostable pyrimidine nucleosides phos-
phorylases (PyNP) [17], using adenine as an acceptor
and the respective 2′–deoxy-2′–fluorouridine (dUrd2′F
)
2′F
and 1-(2-deoxy-2-fluoro-b-D–arabinofuranosyl)uracil (dUrd )
as the pentofuranose residue donors, the formation of
the desired nucleosides was observed with a yield of ~ 24%.
Finally, we tested cytidine (11) and 2′–deoxycytidine
(12) as substrates of the PNPs prepared and found
that these nucleosides showed moderate substrate
activity compared with that of 2′–deoxyfluoro adenine
nucleosides 9 and 10.
Results
Cloning, expression and purification
In order to avoid problems arising from stable second-
ary structures in the 5′–mRNA region [27], the target
2
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

X. Zhou et al.
Purine nucleoside phosphorylases from thermophiles
R
1
N
N
R1
N
O
P
N
N
HO
OH
O
P
O
N
R
2
PNP
O
O
OH
N
H
+
+
N
R2
O
OH
O
O
HO
= OH, R
OH(H)
HO
OH(H)
1
2
3
, R
2'-Deoxy)inosine
, R = NH2, = H
2'-Deoxy)adenosine (dAdo)
, R1 = OH, R2 = NH2
2'-Deoxy)guanosine (dGuo)
1
2
= H
Pi
(Inorganic phosphate)
4, R1 = OH, R2 = H
α
1
-D-(2-Deoxy)ribofuranose
(
(dIno)
(Hypoxanthine, Hyp)
5, R1 = NH2, R2 = H
(Adenine, Ade)
6, R1 = OH, R2 = NH2
(Guanine, Gua)
-phosphate (dRib-1P)
1
R
2
(
(
PNP - purine nucleoside phosphorylase
Scheme 1. Phosphorolysis of purine nucleosides by PNP.
R
NH
2
NH2
N
N
N
N
N
N
HO
N
HO
N
HO
N
O
N
O
O
O
HO
NH2
HO
F
HO
X
7
8
, R = NH2
, R = OH
9, C2'-ribo-F
10, C2'-arabino-F
11, X = OH
12, X = H
Scheme 2. Non-natural substrates of PNPs in this study.
gene sequences were cloned with an N–terminal hexah-
istidine tag.
band related to one of possible oligomerization states
of the ApMTAP monomeric subunits connected by
the disulfide bonds. This assumption was supported by
SDS/PAGE analysis (Fig. 1B, lanes 3 and 6), which
revealed that the oligomeric band was predominant
when the purified ApMTAP was heat-treated in the
absence of dithiothreitol (Fig. 1B, lane 3), whereas the
monomeric band was predominant after dithiothreitol
treatment (Fig. 1B, lane 6). This phenomenon was not
observed for the other proteins. Furthermore, we
found that the higher oligomeric state of ApMTAP
was catalytically active, whereas the monomeric ApM-
TAP was inactive (data not shown).
All three enzymes were functionally expressed in
E. coli BL21. Soluble GtPNP was well expressed under
standard conditions [TB medium, 37 °C, 100 lM
isopropyl b–D–thiogalactoside (IPTG)] as shown in
Fig. 1A (lane 1). For the expression of DgPNP
(
Fig. 1A, lane 3) and ApMTAP (Fig. 1A, lane 5),
ꢀ
EnPresso medium [28] was used at a lower IPTG
concentration (20 lM) to prevent the formation of
insoluble protein aggregates. The final cell densities
varied from A600 = 19 (ApMTAP) to 25 (DgPNP) and
4
2 (GtPNP).
In SDS/PAGE analysis, the purified proteins
Fig. 1A, lanes 2, 4 and 6) showed bands correspond-
(
Thermal characteristics
ing to the theoretically calculated molecular masses of
the monomeric subunits (DgPNP, 30.0 kDa; GtPNP,
The effect of temperature on the activity of the
enzymes was examined between 30 and 99 °C (Fig. 2).
DgPNP showed optimal activity at 55 °C (Fig. 2),
which is slightly higher than the optimal growth tem-
perature (45–50 °C) of D. geothermalis [32]. The stabil-
ity half-life at 60 °C was 1.6 h, whereas 80% of the
activity was retained at 55 °C for 8 h (Fig. 3A).
GtPNP showed an optimal activity at 70 °C (Fig. 2),
which is ~ 15 °C higher than the optimal growth
2
7.6 kDa; and ApMTAP, 27.0 kDa).
However, in the samples of purified ApMTAP, a
second band with a molecular mass of ~ 116 kDa
could be seen. It was confirmed by MS analysis that
this second band also represents ApMTAP (Tables S1
and S2). Because disulfide bond formation is consid-
ered to be a key factor for protein stabilization in ther-
mophilic archaea [29–31], we assumed that the second
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS
3

Purine nucleoside phosphorylases from thermophiles
X. Zhou et al.
A
B
Fig. 1. SDS/PAGE analysis of recombinant
PNPs overexpressed in E. coli BL21 (A)
and the influence of dithiothreitol
treatment on the enzymes (B). (A) Soluble
fraction after incubation of the cell lysis at
65 °C (lane 1), 55 °C (lane 3) and 85 °C
(
lane 5) for 15 min. Purified samples of
GtPNP (lane 2), DgPNP (lane 4) and
ApMTAP (lane 6) after Ni ion-affinity
chromatography and a desalting step. (B)
SDS/PAGE samples of purified enzymes
without dithiothreitol treatment GtPNP
(
(
lane 1), DgPNP (lane 2) and ApMTAP
lane 3); dithiothreitol treatment for
purified GtPNP (lane 4), DgPNP (lane 5)
and ApMTAP (lane 6). M, molecular mass
marker.
temperature (55 °C) of its source microorganism
G. thermoglucosidasius [33] and 10 °C higher than the
temperature optimum of GtPyNP, another NP
enzyme, from the same microorganism [17]. GtPNP
was incubated at 70 °C for 8 h and no significant
activity loss could be observed (Fig. 3B), whereas
GtPyNP at 70 °C had a half-life of only 1.6 h [17].
The activity of ApMTAP increased exponentially at
increased reaction temperatures up to the highest tem-
perature tested (99 °C, Fig. 2), while the source micro-
organism A. pernix shows optimal growth at
temperatures between 90 and 95 °C [34]. ApMTAP is
extremely thermostable with an estimated half-life of
6
9 h at 90 °C (Fig. 3C).
The thermal characteristics of the PNPs studied here
along with other thermostable PNPs characterized
by a broad substrate specificity (recognition of both
6–amino and 6–oxo purine nucleosides) are summa-
rized in Table 1.
Fig. 2. Temperature dependence of the activity of DgPNP, GtPNP
and ApMTAP. The highest reaction rate observed for each enzyme
was set to 100%. Substrate: 1 mM inosine in 50 mM phosphate
buffer (pH 7).
A
B
C
Fig. 3. Thermal stabilities of DgPNP (A), GtPNP (B) and ApMTAP (C). Enzymes were incubated at different temperatures and the residual
activity after defined time intervals was determined under standard assay conditions (substrate: 1 mM inosine in 50 mM phosphate buffer,
pH 7). Enzyme activities before incubation were set to 100%.
4
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

X. Zhou et al.
Purine nucleoside phosphorylases from thermophiles
Table 1. Thermal characteristics of PNPs.
Enzyme
Organism
Thermal stability (t1/2
)
Temp. optimum [°C]
Ref.
EcPNP
Escherichia coli
> 30 min (50 °C); < 30 min (55 °C)
> 8 h (55 °C); 1.7 h (60 °C)
60
55
[35]
DgPNP
EaPNP
Deinococcus geothermalis
Enterobacter aerogenes
Geobacillus thermoglucosidasius
Geobacillus stearothermophilus
Aeropyrum pernix
This study
[21]
a
7 days (60 °C)
60
GtPNP
> 8 h (70 °C); 6.3 h (75 °C)
> 24 h (70 °C)
70
This study
[9,20]
GsPNP
ApMTAP
SsMTAP
PfMTAP
70
69.3 h (90 °C)
> 99
120
125
This study
[25]
Sulfolobus solfataricus
Pyrococcus furiosus
> 2 h (100 °C); 15 min (130 °C)
> 5 h (100 °C); 43 min (130 °C)
[26]
a
Purified enzyme was kept in 30 mM inosine (in 25 mM phosphate buffer) at 60 °C.
Substrate specificity
Moreover, it is noteworthy that ApMTAP is charac-
terized by relatively low specific activities with natu-
ral substrates, but by a relatively broad substrate
The substrate specificities of the enzymes prepared
were studied using a number of natural purine nucleo-
sides [adenosine (Ado), inosine (Ino), 2′–deoxyadeno-
sine (dAdo), 2′–deoxyinosine (dIno)] and 2′-modified
spectrum at the same time, including dAdo
and
′NH2
2
2′F
dIno2′NH2, dAdo2′F and dAdo ; whereas GtPNP is
highly specific for natural substrates but also accepts
modified substrates. DgPNP was the best candidate
for phosphorolysis of the 2′–fluorinated purine nucle-
osides (Fig. 4). Interestingly, GtPNP and ApMTAP
analogues (dAdo
and dIno2′NH2, dAdo2′F and
′NH2
2
2
dAdo ).
′F
Phosphorolysis of purine nucleosides
displayed similar levels of activity with dAdo2′NH2
.
The results show that all three enzymes recognize
both 6–oxopurine (e.g. Ino, dIno, dIno2′NH2) and
However, the replacement of the heterocyclic base
adenine of dAdo2′NH2 with hypoxanthine (resulting
in dIno2′NH2) led to a significant decrease in sub-
strate activity in the case of GtPNP, whereas the
substrate activity towards ApMTAP was slightly
enhanced relative to dAdo2′NH2 (Fig. 4). Notably, a
similar characteristic of the GtPNP activity is
observed in the case of reactions with Ado and Ino,
whereas dAdo and dIno showed reversed dependence
of activities.
6–aminopurine nucleosides (e.g. Ado, dAdo, dAdo2′
NH2) (Fig. 4). Although specific activities for the
same substrate are distinct for each enzyme, it can
be generally stated that natural substrates are signifi-
cantly better accepted than 2′–amino modified sub-
ꢁ1
strates (50–500 versus 0.05–3 Uꢀmg ), whereas the
latter are significantly better accepted than the 2′–flu-
oro substituted compounds (0.01–0.03 Uꢀmgꢁ ).
1
Fig. 4. Activity of the thermostable PNPs with natural and modified substrates. Reaction conditions: 1 mM substrate in 50 mM potassium
phosphate buffer (pH 7) at 55 °C (DgPNP), 70 °C (GtPNP) and 80 °C (ApMTAP). Reactions were stopped by trichloroacetic acid or cold
MeOH (dAdo and dIno) in the initial reaction phase.
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS
5

Purine nucleoside phosphorylases from thermophiles
X. Zhou et al.
Phosphorolysis of pyrimidine nucleosides
shows 86% identity with GsPNP; ApMTAP shows
4
5% identity with SsMTAP.
The amino acid sequence alignment of ‘high molecu-
Araki et al. patented the method for producing
cytidine compounds using recombinant E. coli PNP as
biocatalyst and 2-deoxy-a-D–ribofuranose-1-phosphate
and cytosine as substrates in 100 mM Tris/HCl buffer
lar mass PNPs’ from different thermopiles with E. coli
is shown in Fig. 5. The thermal characteristics and
substrate specificities of these proteins were described
above.
[
36]. Recently, Stepchenko et al. described the phos-
phorolysis of 2′–deoxycytidine (dCyd), 2′–deoxyuridine
dUrd) and 2′–deoxythymidine (dThd) under the con-
The alignment shows that the amino acid residues
known to be involved in the active site of E. coli PNP
and S. solfataricus MTAP [19,24,39] are generally con-
served, with the exception of Met64, Ser90, Cys91,
Ala156, Pro162 and Phe167 in EcPNP which have
variations in the thermostable PNPs (indicated in
boxes in Fig. 5).
(
ditions of recombinant E. coli PNP in 80 mM phos-
phate buffer (pH 7.0) at 15 °C. It was shown that
dCyd and dUrd are good substrates and ~ 50% was
phosphorolyzed after 24 h, whereas substrate activity
of dThd was unexpectedly lower [37]. These data
prompted us to test a number of pyrimidine nucleo-
sides as substrates for the PNPs under investigation.
It was found that substrate efficiency of cytidine (Cyd)
is increasing in the following order: DgPNP (12
The amino acid residues from 62 to 76 of EcPNP
are considered to be highly conserved sequence motifs
[
39] involved in ribose binding [19]. In the thermo-
zymes, especially in ApMTAP (Fig. 5), smaller resi-
dues such as Ile66*, Gly68, Ala71, Ala72, Val73* and
Val74* replace Met64*, Ile66, Cys69, Ser70* and
Ile71* of EcPNP, respectively (residues highlighted in
pink of Fig. 5 are designated by * in the text). More-
over, in or close to the phosphate- and base-binding
sites, similar phenomena can be observed: smaller resi-
dues in ApMTAP (or SsMTAP) Gly25*, Ala44*,
Ala164*, Ala167, Ala172 and Ala173* replace Leu23*,
Val42*, Leu158*, Ser161, Met166 and Phe167* of
EcPNP, respectively. On the one hand, the smaller res-
idues provide more space for the modified substrate
ꢁ
1
ꢁ1
mUꢀmg ) < GtPNP (14 mUꢀmg ) < ApMTAP (36
ꢁ1
mUꢀmg ). Interestingly, in the case of dCyd, the
reverse decrease in substrate activity was observed, viz.
(34 mUꢀmgꢁ ) > GtPNP
1
(17 mUꢀmg
ꢁ1
)
DgPNP
ꢁ
1
> ApMTAP (10 mUꢀmg ). The activities of DgPNP,
GtPNP and ApMTAP with Urd (0.97, 0.83 and
ꢁ
1
4
.21 mUꢀmg , respectively) and thymidine (< 0.05,
ꢁ1
0
be rather low.
.26 and 6.83 mUꢀmg , respectively) were found to
Kinetic parameters
[
38], which may explain the relatively broad substrate
The kinetic parameters of the thermostable PNPs are
summarized in Table 2, along with other literature
data. Km and Vmax were measured in 50 mM potassium
phosphate buffer and typical Michaelis–Menten kinet-
ics were observed. Note that TtPNP, EcPNP, SsM-
TAP and PfMTAP are hexamers, whereas TtPNPII is
a monomer.
spectrum of ApMTAP. On the other hand, the amino
acid composition of thermophilic proteins is thought
to be correlated with their thermostability [13]. Indeed,
compared with EcPNP, ApMTAP contains more
hydrophobic and small residues such as Val (10.4 ver-
sus 8.4%), Leu (12.3 versus 6.3%) and Gly (13.9 ver-
sus 9.6%). Furthermore, it is known that the
substitution of Lys by Arg residues is advantageous at
high temperatures because the Arg d–guanidino moiety
has less reactivity than the Lys amino group due to its
high pKa and resonance stabilization [13]. It can be
observed that the Arg content increases from EcPNP
(5.0%), GtPNP (6.0%), DgPNP (6.9%) to ApMTAP
The results show that the enzymes have a higher
affinity (low Km values) towards adenosine than to
inosine, e.g. 5–8 times higher in the cases of DgPNP
and GtPNP. Similar remarkable differences in the sub-
strate affinity were also described for PNPs from
T. thermophilus [18] and PfMTAP [26]. The kcat values
of the nucleoside phosphorylases assessed for the natu-
ral substrates dIno and dAdo point to a very high cat-
alytic activity of GtPNP (cf. the relevant data in
Fig. 4), provoking reasonable interest to its use in bio-
technological transformations.
(
8.2%), whereas the Lys content decreases from
EcPNP (5.9%), GtPNP (3.4%), ApMTAP (1.6%)
to DgPNP (0.8%).
Among the eight PNPs shown in Fig. 5, only
TtPNPI [18] cannot accept Ado as a substrate, sup-
posedly because of its residues substitutions in the
base-binding sites: Glu156, Phe178 and Asn204
instead of Ala156, Val178 and Asp204 in EcPNP.
Note that these residues are highly conserved in other
PNPs (Fig. 5). The Asp204 (EcPNP) is considered as
Multiple sequence alignment
DgPNP, GtPNP and ApMTAP share a sequence iden-
tity with EcPNP of 54, 55 and 30%, whereas GtPNP
6
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

X. Zhou et al.
Purine nucleoside phosphorylases from thermophiles
Table 2. Kinetic parameters of PNPs on inosine and adenosine. NA, data not available.
ꢁ
1 a
)
(sꢁ1ꢀlMꢁ1
max (Uꢀmgꢁ1
Enzyme
DgPNP
Substrate
K
m
(lM)
k
cat (s
k
cat/K
m
)
V
)
Reaction conditions
Ref.
Inosine
975 ꢂ 65
212 ꢂ 18
1801 ꢂ 288
240 ꢂ 26
137 ꢂ 22
97 ꢂ 14
84 ꢂ 4
43 ꢂ 1
91 ꢂ 2
0.043
0.43
85 ꢂ 2
182 ꢂ 4
540 ꢂ 28
375 ꢂ 14
51 ꢂ 2
44 ꢂ 2
NA
55 °C, 50 mM phosphate, pH 7.0
This study
Adenosine
Inosine
GtPNP
248 ꢂ 13
172 ꢂ 6
23 ꢂ 1
20 ꢂ 1
22 ꢂ 1
7 ꢂ 1
0.14
70 °C, 50 mM phosphate, pH 7.0
80 °C, 50 mM phosphate, pH 7.0
70 °C,100 mM phosphate, pH 7.4
80 °C,100 mM phosphate, pH 7.4
80 °C, 50 mM phosphate, pH 7.0
25 °C, 50 mM phosphate, pH 7.4
This study
This study
[24]
Adenosine
Inosine
0.72
ApMTAP
SsMTAP
PfMTAP
0.17
Adenosine
Inosine
0.21
0.26
Adenosine
Inosine
25 ꢂ 1
0.29
NA
963
1.6
0.0016
0.035
0.014
1.38
NA
[26]
Adenosine
Inosine
109
3.8
NA
TtPNPI
TtPNPII
EcPNP
2080 ꢂ 20
320 ꢂ 20
96 ꢂ 2
29 ꢂ 1
440 ꢂ 3
40 ꢂ 1
33 ꢂ 4
69 ꢂ 1
880 ꢂ 6
[18]
Adenosine
Inosine
b
0.72
10
[38]
b
Adenosine
46 ꢂ 7
0.42
8
a
b
Calculations of kcat are based on the subunit molecular mass of the enzymes. Data not reported but kindly provided by Bennett et al.
[38].
the key base-binding amino acid residue by the pro-
Pyrimidine and purine nucleosides modified at the
C2′ carbon atom of the pentofuranose ring are of
great importance as constituents of artificial oligonu-
cleotides of medicinal potential [41,42]. Among them,
2′-amino-2′-deoxy- and 2′-deoxy-2′-fluoro-b-D–ribo-nu-
cleosides attracted much attention during the last two
decades and a number of oligonucleotides containing
these modified nucleosides at diverse position of the
oligonucleotide chain have been synthesized and their
properties investigated [43–45]. More recently,
2′-deoxy-2′-fluoro-b-D–arabino-nucleosides have been
shown to be of great importance for antisense and low
molecular mass oligonucleotides as potential antican-
cer drugs [6,46,47]. Unfortunately, chemical synthesis
of pyrimidine and especially purine 2′-deoxyfluoro-
ribo- and -arabino-nucleosides, as well as their 2′-am-
inodeoxy counterparts suffers from many drawbacks
despite of enormous progress achieved in this field of
research. The search for enzymatic transformations
that can efficiently replace some of the chemical steps
in the production of these nucleosides using the princi-
ples of ‘green chemistry’ is therefore of great impor-
tance.
tonation of the purine base at N7 accompanied by
hydrogen bonding with the C6 amino group [19].
According to Mikleu ꢀs evi ꢁc et al. [40], mutation of
Arg204 to Asn in EcPNP results in a 1000–fold
decrease in the enzymatic activity, which further pro-
vides evidence for the role of Asp204 in the PNP
specificity of Ado. Asp204 (corresponding to Asp223
in DgPNP, Asp 203 in GtPNP and Asp210 in ApM-
TAP) was conserved in the PNPs studied here and as
a consequence they can well accept Ado as substrate.
However, to explain the difference in the reaction rate
and substrate preference between the studied PNPs
and EcPNP (see Table 2), profound structure studies
of the catalytic site are needed.
Synthesis of fluorinated purine nucleosides
The aforementioned results of this study and data
from previous studies [17] prompted us to investigate
the synthesis of adenine 2′-deoxyfluoro-ribo- and -ara-
2′F
bino-nucleosides, dAdo2′F (17) and dAdo
respectively, using adenine as an acceptor and the
(18),
respective 2′-fluorinated uracil nucleosides, dUrd
(
In our previous work, we described the recombinant
expression and biocatalytic characterization of two
thermostable PyNPs, isolated from G. thermoglucosida-
sius (GtPyNP) and T. thermophilus (TtPyNP) [17]. It
was found that GtPyNP displays very low phosphoro-
2
′F
2
′F
13) and dUrd (14), as donors of the pentofuranose
residues (Scheme 3). Preliminary experiments showed
the formation of: (a) dAdo2′F using TtPyNP and
DgPNP as biocatalysts (55 °C, 24 h) with a yield of
2
′F
2′F
2
3%, and (b) dAdo employing a TtPyNP and ApM-
lytic activity towards dUrd2′F and dUrd , whereas
TAP combination of enzymes (80 °C, 24 h) with a
yield of 24%. These data point to the possibility of
developing a practical synthesis of both 2′-fluorinated
adenine nucleosides, although an optimization of the
reaction conditions is necessary to increase the yield.
TtPyNP shows rather satisfactory activity with both
2′-deoxyfluoro-ribo- and -arabino-nucleosides. This
observation pointed to the possibility of employing
TtPyNP as a biocatalyst in the transglycosylation reac-
tion with adenine as the pentofuranosyl acceptor and
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS
7

Purine nucleoside phosphorylases from thermophiles
X. Zhou et al.
Fig. 5. Multiple sequence alignment of high molecular mass PNP or MTAP from E. coli (EcPNP), Enterobacter aerogenes (EaPNP),
D. geothermalis (DgPNP), G. thermoglucosidasius (GtPNP), G. stearothermophilus (GsPNP), T. thermophilus (TtPNPI), A. pernix (ApMTAP)
and S. solfataricus (SsMTAP), respectively. The scale below is based on the EcPNP sequence reported in the literature [19,24]. The shading
represents the degree of sequence identity. Residues of a representative sequence involved in the active site or in intersubunit contacts
Bzowska et al. [19] and Pugmire et al. [39] are highlighted in pink. The ribose (*), phosphate (+) and base (#) binding sites according to
Bzowska et al. [19] and Cacciapuoti et al. [24] are indicated below the sequences.
2
′F
dUrd2′F or dUrd
ribo- and arabino-pentofuranose.
as donors of the corresponding
arabinofuranosyl)uracil was very low (2%) at 55 °C
[17]. The use of TtPyNP and ApMTAP as a combina-
2
tion of enzymes, and dUrd and adenine as substrates
′F
Here, we studied this reaction testing DgPNP,
GtPNP and ApMTAP as biocatalysts for the coupling
of the intermediates 2′-deoxyfluoro-ribo- and arabino-
1-phosphates with adenine. It was found that DgPNP
catalyzes the formation of the desired dAdo2′F under
2′F
(80 °C, 24 h) resulted in the formation of dAdo with
a yield of 24%; TtPyNP and GtPNP as a combination
of enzymes for the same synthesis resulted in the for-
2′F
mation of dAdo with a yield of 14%. It is obvious
that these results are very promising from the view-
point of their application in the development of practi-
very mild conditions (55 °C, 24 h) with a yield of
4%. It is noteworthy that the undesired chemical
2
transformation of dUrd2′F into O ,2′–anhydro-1-(b-D–
2
2′F
cal syntheses of dAdo2′F and dAdo
.
8
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

X. Zhou et al.
Purine nucleoside phosphorylases from thermophiles
O
NH2
O
P
O
N
NH
N
O
OH
O
P
O
N
O
OH
N
O
NH2
N
H
O
N
H
NH
N
N
N
HO
O
O
OH
O
P
HO
O
O
N
O
OH
O
HO
F
HO
F
HO
F
PyNP
PNP
1
1
3, dUrd2'F (C2'-ribo-F)
4, dUrd2'F (C2'-arabino-F)
15, dRib-1P2F (C2'-ribo-F)
17, dAdo2'F
(C2'-ribo-F)
16, dRib-1P2F (C2'-arabino-F)
8, dAdo2'F (C2'-arabino-F)
1
Scheme 3. Synthesis of fluorinated purine nucleosides by the coupled reactions of PyNP and PNP.
Phosphorolysis of purine nucleosides catalyzed
by GtPyNP and TtPyNP
Table 3. Activity of GtPyNP and TtPyNP with natural and non-
natural nucleosides. Reaction conditions: 1 mM substrate in 50 mM
phosphate buffer at 60 °C (GtPyNP) or 80 °C (TtPyNP).
During the course of the abovementioned studies, we
observed that the thermostable PyNPs, GtPyNP and
TtPyNP, phosphorolytically cleaved not only pyrimi-
dine nucleosides [17], but also various purine nucleo-
sides. To investigate this phenomenon further, we
determined the temperature dependence of PyNP
activity with inosine in order to exclude the possibility
that the PNP activity comes from E. coli PNP. It was
found that temperature dependence of GtPyNP and
TtPyNP activity with inosine (Fig. 6) is similar to that
obtained in experiments with one of the natural sub-
strates, uridine (see Fig. 4A in Ref. [17]). Hence, we
assume that the PNP activity is inherent to the PyNPs
and not the result of contamination with E. coli PNP.
Activity (Uꢀmgꢁ
1
)
Substrate
GtPyNP 60 °C
TtPyNP 80 °C
Urd
51.5
153.7
295.2
0.809
0.900
0.065
0.067
Thd
22.4
Ado
0.458
0.308
0.021
0.012
Ino
dAdo2′NH2
dIno2′NH2
dUrd2′F
a
a
0.002
a,b
ND
0.052
2′F
a
dUrd
0.005
b
Cyd
ND
0.001
a
b
10 mM phosphate buffer. Not observed.
Furthermore, the activities of GtPyNP (at 60 °C) and
TtPyNP (at 80 °C) with the other purine nucleosides
were examined and the results are summarized in
Table 3. It is obvious that both PyNPs display consider-
able activities with Ado and Ino. In fact, TtPyNP
accepts the purine significantly better than 2′–fluorinated
pyrimidine nucleosides. By contrast, the activity with
the natural pyrimidine nucleoside cytidine was extre-
mely low (TtPyNP) or was not detectable (GtPyNP).
Discussion
In this study, we prepared three recombinant PNPs
from thermophiles and studied their properties and
substrate specificity for natural substrates (Ado, dAdo,
Ino, dIno) and three sets of analogues, viz., (a) dAdo
2′
2
′F
and dIno2′NH2, (b) dAdo2′F and dAdo , (c) Cyd
NH2
and dCyd. The reasons for selection of the analogues
are as follows: the first have closest structural relation
of the relevant natural purine nucleosides; the
diastereomeric deoxyfluoro adenine nucleosides also
Fig. 6. Temperature dependence of the activity of GtPyNP and
TtPyNP on inosine. The highest reaction rate observed for each
enzyme was set to 100%. Substrate: 1 mM inosine in 50 mM
potassium phosphate buffer (pH 7).
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS
9

Purine nucleoside phosphorylases from thermophiles
X. Zhou et al.
structurally mimic dAdo. However, it is well known
that the replacement of a hydrogen atom or hydroxyl
group in the sugar moiety of nucleosides gives rise to
derivatives with unexpected physico-chemical and bio-
logical properties. Finally, pyrimidine nucleosides are
not typical substrates of PNP, however, Cyd and dCyd
mimic the pyrimidine fragment of adenine base of
of the phosphate anion and ammonium cation imped-
ing the correct spatial arrangement of the productive
nucleoside/phosphate/enzyme complex, on the other.
The observed dramatic decrease in substrate activity
by going from natural substrates to dAdo2′NH2 (7) and
dIno2′NH2 (8) (2–3 orders of magnitude) prompted us
to: (a) test the substrate activity of both aminodeoxy
nucleosides at different pH values of the reaction mix-
tures (Table S3, Fig. S1), and (b) analyze the
C2′-ammonium cation and C2′-amino forms of the for-
mer by the restricted Hartree–Fock (RHF) ab initio
method using a basis set of STO-3G (HYPER-CHEM, 8.1
release) and the PM3 geometry optimization as start-
ing approximation for the ab initio calculations com-
pared to similar data for Ado (Table S4).
3
N –(b-D–ribofuranosyl)adenine that was shown to be
the substrate of PNP [48–50] and a good substrate
activity of dCyd and 2′-deoxyuridine for E. coli PNP
was also described [36,37].
The role of the vicinal C2′ and C3′ hydroxyl groups
of natural purine nucleosides in the binding site of
PNPs has been discussed in a number of publications
[
38,51,52]. The PNPs studied here and E. coli PNP
contain the same cluster of amino acids (Val–Glu–Met
Glu). In this cluster, the last residue Glu181 of E. coli
Preliminary experiments on the pH dependence on
the substrate properties gave a rather variable picture
of activities (Fig. S1) and pointed to the need to per-
form more detailed studies to obtain meaningful
results. For example, a plot of kcat/Km as a function of
pH is needed to reflect the essential ionizing groups of
the free enzyme that play a role in both substrate
binding and catalytic processing [53].
–
PNP was suggested to play an important role in the
binding of ribose via the vicinal C2′ and C3′ hydroxyl
groups and in facilitating substrate activation by flat-
tering the pentofuranose ring [38]. In a similar way,
one can suggest that the relevant Glu residues of the
enzymes under study [DgPNP (Glu200), GtPNP
(
Glu180) and ApMTAP (Glu187); (Fig. 5)] are
Analysis of the amino and ammonium forms of
dAdo2′NH2 by the ab initio method revealed the essen-
tial differences in the base orientation about the glyco-
syl bond as well as the ribofuranose rings of two
species, on the one hand, and some difference of both
forms from the natural substrate, on the other hand.
Conformational analysis of dAdo2′NH2 (7) in water by
NMR spectroscopy clearly pointed to its high S ↔ N
and anti ↔ syn conformational flexibility [54]. Taking
into account that both functionally competent forms
are in equilibrium under optimal pH values for PNP
functioning, an enzyme can accept the most suitable
conformer for binding. On the whole, these consider-
ations allow us to suggest the involvement of the
C2′-ammonium cation in an interaction with phos-
phate anion as a likely reason for the very low sub-
strate activity of dAdo2′NH2 (7) and dIno2′NH2 (8).
In the case of diastereomeric adenine deoxyfluoro
involved in the ribose binding site. It was previously
shown that E. coli PNP accepts the natural purine
ribo- and 2′-deoxyribo-nucleosides with similar effi-
ciency. The results of this study also showed a similar
trend (Fig. 4), indicating that the C2′ hydroxyl groups
of Ado and Ino are not essential for the enzymatic
reaction. Note that there is a lot of theoretical and
experimental data convincingly demonstrating that
natural ribo- and 2′-deoxyribo-nucleosides have rather
high conformational flexibility, viz., S ↔ N conforma-
tion of the pentofuranose ring and base rotation
around the glycosidic bond. These conformational
peculiarities allow PNPs adopting the required spatial
structure of a natural nucleoside substrate in the cata-
lytic site, which is necessary to form the productive
enzyme-substrate complex.
Compounds of the first and second sets are func-
tionally competent, i.e. contain all the functionality of
inherent natural substrate, purine nucleosides and,
a priori, one can expect their substrate activity for
PNPs to be similar to those of natural substrates.
Indeed, the amino function of dAdo2′NH2 and dIno2′
2
′F
nucleosides dAdo2′F (9) and dAdo
(10), the most
R
R
N
N
N
N
pH > 6.2
HO
N
HO
N
O
N
O
N
is isosteric for the hydroxyl group and can func-
NH2
pH < 6.2
tionally replace the hydroxyl group. The only impor-
tant peculiarity of the amino function vs hydroxyl
group consists in that the former can exist in the
HO
NH3
X
HO
NH2
7
8
, R = NH
, R = OH
2
C2′-ammonium cation
↔
C2′-amino equilibrium
implying the pH dependence of the substrate activity,
on the one hand (Scheme 4), and the ionic interaction
Scheme 4. Equilibrium of C20-ammonium cation and C20-amino at
different pH environment.
1
0
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

X. Zhou et al.
Purine nucleoside phosphorylases from thermophiles
unexpected finding is that both functionally competent
analogues show very similar extremely low (3–4 orders
of magnitude compared with that of Ado) substrate
activity for the all investigated enzymes. The H and F
atoms have similar van der Waals’ radii and thus do not
create any steric hindrances to the substrate binding in
the catalytic center of the investigated enzymes. How-
ever, a fluorine atom, as an isostere for the hydrogen
atom [55], differs dramatically from its isostere owing to
the strongest electronegative character exerting diverse
influences on electronic properties of proximal atoms as
well as on the stereochemistry of the pentofuranose ring.
G. thermoglucosidasius and A. pernix, aiming at the
search for new efficient biocatalysts for the synthesis of
modified nucleosides. The recombinant expression in
E. coli resulted in a high yield of biologically active
enzymes. The PNPs were found to be stable at their
optimal temperatures (DgPNP: optimal activity at 55 °
C retaining thereupon 80% for 8 h; GtPNP: optimal
activity at 70 °C without losing activity for 8 h; ApM-
TAP: activity exponentially rising up to 99 °C). Natural
substrates (Ado, Ino and their 2′-deoxy counterparts)
are effectively phosphorolyzed by all three investigated
ꢁ
1
enzymes (activity level within 50–500Uꢀmg ). The
activity of the enzymes for dAdo and dIno
2
′F
An ab initio analysis of dAdo2′F and dAdo
comparison of the results obtained with those of
and
2′NH2
2′NH2
was found to be approximately two orders of magnitude
lower (within 0.01–3.25 Uꢀmgꢁ ) than their activity for
natural substrates. ApMTAP showed the highest level
of enzymatic activity for dAdo2′NH2 and dIno2′NH2. A
similar further decrease in substrate activity was found
1
X–ray crystallographic data for Ado and dAdo2′F
(
Table S4), as well as the data of the conformational
2
′F
NMR analysis of dAdo2′F [56] and dAdo [57] unex-
pectedly revealed a rather broad stereochemical and
electronic similarity of the 2′-deoxyfluoro-ribo- and
2
′F
ꢁ1
for dAdo2′F and dAdo
DgPNP showed the highest activity for this type of sub-
strate among the enzymes tested here. It should be
(within 0.01–0.03 Uꢀmg ).
-
arabino-nucleosides that is in harmony with the activ-
ity data (Fig. 4). The only reasonable explanation of
the observed very low substrate activity of dAdo
stressed that the substrate activities of dAdo and
2′F
2
′F
2′F
2′F
and dAdo may be connected to the structural pecu-
liarities of the productive complex giving rise to the
glycoside bond cleavage. Bennet et al. suggested flat-
tering of the pentofuranose ring of the substrate (the
C4′–O4′–C1′–C2′ torsion angle strives to 0°) as one of
the most important features of the reaction [38]. As
distinct from the Ado/ara-adenine (Ade) pair, the C2′
fluorine atom of dAdo2′F and dAdo in all likelihood
exerts a much higher energy barrier than that of Ado
and dAdo for flattering of the pentofuranose ring,
which is unexpectedly similar to and independent of
the fluorine ribo- or arabino- configuration.
dAdo are unexpectedly similar and do not depend on
the fluorine atom configuration. Interestingly, cytidine
was phosphorolyzed by the all studied PNPs with an effi-
ciency similar to that of adenine 2′-deoxyfluoro nucleo-
sides. Furthermore, uridine and thymidine were also
phosphorolyzed by the studied PNPs (except for DgPNP,
which cannot accept thymidine as substrate), but the
activity was 6–30 times lower than that for cytidine.
It was found that the previously described thermo-
stable pyrimidine nucleoside phosphorylase TtPyNP
[17] and the present described DgPNP and ApMTAP
can be applied for the transglycosylation reaction of
2′F
2
′F
adenine in the presence of dUrd2′F and dUrd
the formation of the corresponding adenine nucleo-
with
Conclusion
2′F
sides dAdo2′F and dAdo at a yield of ~ 24%.
The investigation of the underlying enzymatic mech-
anisms of the prepared thermostable PNPs and their
Here, we described the preparation of three PNPs from
the thermophilic microorganisms D. geothermalis,
Table 4. Oligonucleotide primers and GenBank number of PNPs in this study.
GenBank
Accession
number
a
Enzyme
DgPNP
Primer sequence (5′–3′)
F:TACTAGGGATCCGTGGCGCGTGTACCGG
R:CAGCATAAGCTTTCACATGCTGTTGGAAGG
F:ACTAGGGATCCTTGAGCATCCATATCGAAG
R:AGCATAAGCTTTTATTCTACGCGAATCGCC
F:ACTAGGGATCCAGGAAGCCGGTTCACCTCG
R:AGCATAAGCTTCTAGACTCCTCCTGTGAGG
ABF45792
AEH47728
NP_147653
GtPNP
ApMTAP
a
Restriction sites for BamHI (GGATCC) and HindIII (AAGCTT) are indicated in bold and the insert-specific sequences are underlined.
F, forward primer; R, reverse primer.
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS
11

Purine nucleoside phosphorylases from thermophiles
X. Zhou et al.
employment for the synthesis of purine nucleosides of
biological interest are in progress.
SDS/PAGE analysis. The molecular mass marker (Sm0431)
was purchased from Fermentas. The purified protein con-
centration was determined by using a NanoDrop1000 Spec-
trophotometer (Thermo Fisher Scientific, Wilmington, ME,
1%
USA) [60] and applying E280 nm
= 6.3 (for DgPNP), or
.8 (for GtPNP and ApMTAP), respectively. The absorp-
Materials and methods
8
tion coefficients were theoretically calculated from the
amino acid sequence (VECTOR NTI software; Invitrogen).
Bacterial strains, cloning, expression and
purification
Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA)
and BL21 (Novagen, Darmstadt, Germany) strains were
used for gene cloning and protein expression, respectively.
The genomic DNA of T. thermophilus HB27 and G. ther-
moglucosidasius 11955 were isolated as previously reported
Enzyme activity assay
All the reactions were performed within 200–1000 lL scale
in Eppendorf tubes, which were incubated in a thermom-
ixer (Biozym, Hessisch Oldendorf, Germany) at 300 rpm.
The standard activity assay (phosphorolysis) was carried
out in potassium phosphate buffer (50 mM; pH 7.0) con-
taining 1 mM purine nucleoside. After 2 min preheating at
[
17]. The genomic DNA of D. geothermalis and A. pernix
were isolated by Marco Casteleijn (Bioprocess Engineering
Laboratory of the University of Oulu, Finland). The target
gene sequences were amplified from genomic DNA by PCR
using Pfu DNA polymerase (Fermentas, Vilnius, Lithua-
nia). The used primer pairs are listed in Table 4.
the corresponding temperature, a certain amount of puri-
ꢁ1
fied enzyme (~ 0.5 lgꢀmL
for natural substrates;
ꢁ1
0
.1 mgꢀmL for modified substrates) was added into the
The PNP encoding genes were cloned via BamHI/Hin-
dIII digestion (FastDigest restriction endonucleases; Fer-
mentas) and subsequent ligation (T4 DNA Ligase; Roche,
Mannheim, Germany) into a modified pCTUT7 vector [17].
In brief, this vector includes an IPTG inducible lac pro-
moter and a hexahistidine coding sequence connected to
the 5′–end of the target gene.
mixture and the reaction was stopped (addition of 1/2 vol
of 10% trichloroacetic acid or methanol) after a defined
time interval so that < 10% of the substrate was converted
to the product. Under these conditions, the reaction rate
was linear as a function of time and enzyme concentration.
After centrifugation (20 000 g, 15 °C, 25 min) the samples
were stored at ꢁ20 °C for following analyses. Negative
controls (without enzyme addition) were performed in par-
allel. The reaction mixtures were analyzed by HPLC as
described in [17]. The substrate conversion was calculated
from the HPLC chromatograms:
DgPNP and ApMTAP were expressed in E. coli BL21 in
ꢀ
EnPresso medium (BioSilta, Oulu, Finland) by the use of
ꢀ
TM
the Enbase technology [58] using Ultra Yield Flasks
TM
and AirOTop seals (both from BioSilta, Oulu, Finland).
The expression of DgPNP and ApMTAP was induced after
overnight cultivation at 37 or 30 °C, respectively, by the
addition of 20 lM IPTG. GtPNP was expressed in E. coli
BL21 cells in Terrific Broth (TB) medium [59] by the induc-
tion of 100 lM IPTG to the culture growing at 37 °C for
½
productꢃ
Conversionð%Þ ¼
ꢄ 100%
½
productꢃ þ ½substrateꢃ
One unit (U) of enzyme activity was defined as the amount
of the enzyme catalyzing the conversion of 1 lmol of
substrate per minute under the respective assay conditions.
2.5 h. Cells were harvested by centrifugation (16 000 g,
5
min, 4 °C) after either 24 h (for EnPressoꢀ medium) or
3.5 h (for TB medium) after induction, respectively.
Cell disruption and protein purification were carried out
Temperature optimum and thermal stability
measurements
as described previously [17]. Briefly, cells were broken by
ultrasonic treatment (UP200S sonicator; Hielscher
Ultrasonics GmbH, Teltow, Germany), then the lysate was
heat-treated at 50 °C (DgPNP), 65 °C (GtPNP) or 85 °C
ApMTAP) for 15 min, and after centrifugation (16 000 g,
0 min, 4 °C) the supernatant containing the target protein
was further purified by metal ion-affinity chromatography
In order to determine temperature optima of the
enzymes, the reaction mixture (1 mM inosine in 50 mM
potassium phosphate buffer, pH 7.0) was preheated for
2 min at different temperatures (from 30 to 99 °C), suit-
ably diluted enzyme solution was added, and the reaction
was stopped by the addition of trichloroacetic acid after
3 min.
(
2
(
Ni-NTA Superflow cartridge; Qiagen, Hilden, Germany)
€
and the HiPrep 26/10 desalting column on an Akta FPLC
system (GE Healthcare, Munich, Germany).
Thermal stability was analyzed by incubating purified
ꢁ
1
The protein purity was checked by SDS/PAGE accord-
ing to a standard protocol [59]. To analyze whether the
PNP aliquots (13–55 lgꢀmL ) in 50 mM phosphate buffer
(pH 7.0) on a PCR machine (Eppendorf, Hamburg, Ger-
many) at the respective temperatures. After defined time
intervals, tubes were withdrawn and cooled on ice. The
residual activity of the incubated enzyme was determined at
disulfide bridge exists in the enzymes, the purified enzymes
1
(
0.5 mgꢀmLꢁ ) were treated with or without the reducing
agent, i.e. 0.1 M dithiothreitol, in the incubation step of
1
2
FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

X. Zhou et al.
Purine nucleoside phosphorylases from thermophiles
5
0 °C for DgPNP, 60 °C for GtPNP and 80 °C for ApM-
Nucleosides and bases
TAP using inosine as a substrate under standard assay con-
ditions. In order to determine the half-life, the residual
activity data over time were fitted to the exponential decay
Ade, Ado and Ino were purchased from Carl Roth (Kar-
lsruhe, Germany); hypoxanthine, dAdo, dIno, Cyt and
dCyt were purchased from Sigma-Aldrich (Steinheim, Ger-
(ꢁk·t)
equation a = a
GmbH, Erkrath, Germany), where k is the first-order deac-
tivation coefficient, a is the residual activity and a is the
original activity before treatment. Finally, the half-life was
0
·e
(SIGMAPLOT 11.0; Systat Software
2
′F
many); dAdo
was purchased from Metkinen Chemistry
(Kuusisto, Finland); dAdo2′NH2, dIno2′NH2 and dAdo2′F
0
were kindly provided by Prof. Alex Azhayev (Metkinen
Chemistry).
ꢁ
1
.
obtained by t1/2 = ln 2·k
Acknowledgements
Kinetic parameters determination
We sincerely thank Dr Marco G. Casteleijn (Biopro-
cess Engineering Laboratory, University of Oulu,
Finland) for providing us with the genomic DNA of
D. geothermalis and A. pernix. We further thank
Prof. Alex Azhayev (Metkinen Chemistry) for the
donation of the substrates of 1-(2-deoxy-2-fluoro-b-D
–arabinofuranosyl)uracil, 2′–amino-2′–deoxyadenosine
and 2′–amino-2′–deoxyinosine. We are thankful to
the Alexander von Humboldt Foundation (Bonn–
Bad–Godesberg, Germany) for computer and pro-
gram facilities used here. We are also indebted to
Prof. Steven E. Ealick (Baker Laboratory, Cornell
University) and Dr William Bill Parker (Southern
Research Institute, Birmingham) for kindly providing
the Vmax data for E. coli PNP. This work is part of
the Cluster of Excellence ‘Unifying Concepts in
Catalysis’ coordinated by the Technische Universit €a t
Berlin. Financial support by the Deutsche Fors-
chungsgemeinschaft (DFG) within the framework of
the German Initiative for Excellence is gratefully
acknowledged (EXC 314).
Reactions were performed in triplicate at least to obtain
the initial velocity at a fixed phosphate concentration
(50 mM, pH 7.0) and the substrate concentration varied
over a 20-fold range (spanning 0.25–5 times the Michaelis–
Menten constant) [61]. Reaction temperatures were: 55 °C
for DgPNP, 70 °C for GtPNP and 80 °C for ApMTAP.
K
m
and Vmax were determined by nonlinear regression
based on the Michaelis–Menten equation (applied in SIGMA-
PLOT 11.0).
Multiple sequence alignment
Protein identities were assessed with the protein basic local
alignment tool (BLAST) of the NCBI web server [62]. The
multiple alignments were constructed using Expresso mode
of T-Coffee server [63], which implemented an automated
identification of suitable structural templates via a BLAST
search against the PDB database [64]. The corresponding
UniProt Protein accession numbers are P0ABP8 (EcPNP),
G0E416 (EaPNP), Q1IY92 (DgPNP), F8CYG4 (GtPNP),
P77835 (GsPNP), Q72IR2 (TtPNPI), Q9YDC0 (ApMTAP)
and P50389 (SsMTAP). The figure of alignment was cre-
ated using Jalview2 [47].
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Supporting information
Additional supporting information may be found in
the online version of this article at the publisher’s web
site:
5
6
Fig. S1. Relative pH-dependent activity of the ther-
mostalbe PNPs with natural and modified substrates.
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FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS