Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: Implications for the clinical efficacy of nucleoside analogues

Article abstract of DOI:10.1042/BJ20112225

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymesmay compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (K_m = 473 μM) and deoxyuridine (K_m = 578 μM), it prefers uridine (K _m =92 μM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NPI class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses. The Authors Journal compilation

Full text of DOI:10.1042/BJ20112225

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Biochem. J. (2012) 445, 113–123 (Printed in Great Britain) doi:10.1042/BJ20112225
113
Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma
hyorhinis: implications for the clinical efficacy of nucleoside analogues
Johan VANDE VOORDE*, Federico GAGO†, Kristof VRANCKEN*, Sandra LIEKENS*1 and Jan BALZARINI*1
*Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, blok x – bus 1030, B-3000 Leuven, Belgium, and †Departamento de Farmacolog´ıa, Universidad de Alcala´,
E-28871 Alcala´ de Henares, Madrid, Spain
In the present paper we demonstrate that the cytostatic and
antiviral activity of pyrimidine nucleoside analogues is markedly
decreased by a Mycoplasma hyorhinis infection and show that
the phosphorolytic activity of the mycoplasmas is responsible
for this. Since mycoplasmas are (i) an important cause of
secondary infections in immunocompromised (e.g. HIV infected)
patients and (ii) known to preferentially colonize tumour tissue in
cancer patients, catabolic mycoplasma enzymes may compromise
efficient chemotherapy of virus infections and cancer. In the
genome of M. hyorhinis, a TP (thymidine phosphorylase)
gene has been annotated. This gene was cloned, expressed
in Escherichia coli and kinetically characterized. Whereas the
mycoplasma TP efficiently catalyses the phosphorolysis of
thymidine (Km = 473 μM) and deoxyuridine (Km = 578 μM), it
prefers uridine (Km = 92 μM) as a substrate. Our kinetic data
and sequence analysis revealed that the annotated M. hyorhinis
TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs
(pyrimidine NPs), and is distinct from the NP-II class TP and NP-
I class UPs (uridine phosphorylases). M. hyorhinis PyNP also
markedly differs from TP and UP in its substrate specificity
towards therapeutic nucleoside analogues and susceptibility
to clinically relevant drugs. Several kinetic properties of
mycoplasma PyNP were explained by in silico analyses.
Key words: antiviral/anticancer activity, Mycoplasma hy-
orhinis, nucleoside analogue, pyrimidine nucleoside phos-
phorylase (PyNP), thymidine phosphorylase (TP), uridine
phosphorylase (UP).
increasing number of patients suffering such immunodeficiencies
[7]. Furthermore, it was shown that some of these prokaryotes, in
particular Mycoplasma hyorhinis, tend to preferentially colonize
tumour tissue in cancer patients [10–17]. Taken together, these
studies indicate that a mycoplasma infection may not only affect
the health of cancer patients or immunocompromised individuals,
but may also compromise the efficacy of chemotherapeutic
treatment.
INTRODUCTION
The treatment of cancer and many viral infections [caused
by e.g. HIV, HSV (herpes simplex virus), Varicella Zoster
virus, cytomegalovirus, hepatitis C virus or hepatitis B virus]
is largely based on the use of nucleoside-derived therapeutics
[1,2]. These molecules mimic the nucleic acid building
blocks and may therefore act as antimetabolites in DNA/RNA
synthesis or as fraudulent substrates for enzymes involved in
nucleoside metabolism. Thus, after enzymatic activation (usually
phosphorylation), they directly or indirectly interfere with the
cellular or viral DNA/RNA synthesis. Owing to the nature of
these drugs they may also be subject to enzymatic inactivation
(e.g. deamination, phosphorolysis or dephosphorylation) by
enzymes involved in nucleo(s)(t)ide catabolism. It has previously
been demonstrated that mycoplasma-derived TP (thymidine
phosphorylase) activity compromises the cytostatic action of
several nucleoside-based chemotherapeutics in cancer cell
cultures [3–6].
Mycoplasmas are the smallest autonomously replicating
organisms and are characterized by the lack of a cell wall and a
strongly reduced genome (600–1200 kb). Many of these bacteria,
belonging to the class of the Mollicutes, have a parasitic lifestyle
and reside in the human body causing asymptomatic infections
[7]. In particular immunocompromised patients (e.g. patients
suffering from AIDS or hypogammaglobulinaemia) are known
to be prone to mycoplasma infections [8,9]. Despite their high
tissue specificity, mycoplasmas are now regularly being isolated
from organs different from their usual habitats owing to the
Previously, we hypothesized that the treatment of patients using
purine and pyrimidine antimetabolite drugs may be optimized by
(i) the elimination of an underlying mycoplasma infection by
antibiotics, (ii) suppression of mycoplasma-encoded enzymes in
human tumour tissue and/or (iii) the development of mycoplasma-
insensitive nucleoside analogue prodrugs [5,6,18]. In the absence
of such approaches, cancer patients may receive suboptimal
chemotherapeutic treatment. In the present paper, we demonstrate
that the M. hyorhinis-derived phosphorolytic activity in cell
cultures is not only responsible for the decreased cytostatic
activity of certain nucleoside analogues, but also for their
decreased antiviral activity towards different human viruses. To
gain further insight into the molecular basis of these observations,
we cloned, expressed and characterized the putative M. hyorhinis-
encoded TP. The kinetic properties of this enzyme shed new
light on pyrimidine nucleoside metabolism in Mollicutes and
reveal a distinct substrate specificity when compared with human
or Escherichia coli TP and UP [Urd (uridine) phosphorylase].
Structural and functional studies revealed two distinct families
of NPs (nucleoside phosphorylases): the NP-I family (containing
purine NP and UP) and the NP-II family [containing TP and
Abbreviations used: ara-T, thymine arabinoside; ara-U, uracil arabinoside; AZT, azidothymidine; BAU, 5-benzylacyclouridine; BVDU, (E)-5-
(2-bromovinyl)-deoxyuridine; BVU, (E)-5-(2-bromovinyl)-uracil; 2-dRib-1-P, 2-deoxyribose-1-phosphate; D4T, stavudine; dThd, thymidine; dUrd, 2ꢀ-
deoxyuridine; 7-DX, 7-deazaxanthine; 5-FU, 5-fluorouracil; GST, glutathione transferase; HSV, herpes simplex virus; NP, nucleoside phosphorylase;
PyNP, pyrimidine nucleoside phosphorylase; Rib-1-P, ribose-1-phosphate; TFT, 5-trifluoromethyl-dUrd; Thy, thymine; TP, thymidine phosphorylase; TPI, TP
inhibitor; UP, uridine phosphorylase; UPP1, human UP1; Ura, uracil; Urd, uridine; VV, vaccinia virus.
1
Correspondence may be addressed to either of these authors (email jan.balzarini@rega.kuleuven.be or sandra.liekens@rega.kuleuven.be).
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J. Vande Voorde and others
PyNPs (pyrimidine NP)] [19]. Our kinetic and computational
sequence analysis data demonstrate that the mycoplasma-encoded
TP activity is due to the presence of an NP-II class PyNP in the cell
cultures that is distinct from the NP-II class TP and NP-I class UP
enzymes. Our findings explain the dramatic effect mycoplasmas
may have on the antiviral and cytostatic efficiency of several
chemotherapeutics in infected human cell cultures.
assembled between the EcoRI and NotI restriction sites of a
pIDTsmart vector (Integrated DNA technologies). The fragment
was subsequently subcloned between the EcoRI and NotI sites
of the pGEX-5X-1 bacterial expression vector (Amersham
Pharmacia) and expressed in E. coli BL21(DE3)pLysS as a
GST (glutathione transferase) fusion protein according to the
procedure described previously [23]. Bacteria were grown for
8 h at 37◦C in LB (Luria–Bertani) medium containing ampicillin
(100 μg/ml) and chloramphenicol (40 μg/ml), diluted 1:30 in
fresh medium and grown overnight under the same conditions.
Next, the culture was diluted 1:10 in fresh medium and incubated
for 2 h at 37◦C. Then cultures were placed at 27◦C for 4 h after
which IPTG (isopropyl-β-D-thiogalactopyranoside) was added
to a final concentration of 0.1 mM to induce the production
of the GST–TP fusion protein. After 15 h of further growth
at 27◦C, cells were pelleted (6000 g for 10 min at 4◦C) and
resuspended in lysis buffer [50 mM Tris/HCl (pH 7.5), 1 mM
dithiothreitol, 5 mM EDTA, 10% glycerol, 1% Triton X-100,
0.1 mM PMSF and 0.15 mg of lysozyme]. Bacterial suspensions
were homogenized and lysed by means of a French Pressure
cell press and ultracentrifuged (20000 rev./min for 15 min at
4◦C using a Beckman Coulter Type 70 T; fixed angle rotor).
GST–TPHyor (henceforth referred to as TPHyor) was purified from
the supernatant using glutathione–Sepharose 4B (Amersham
Pharmacia) following the manufacturer’s instructions. Briefly,
a 50% slurry of glutathione–Sepharose was added to the
bacterial supernatant (2 ml/750 ml of broth), incubated at 4◦C
and then washed three times with 10 bed volumes of lysis
buffer without lysozyme and PMSF. Bound proteins were
eluted in 50 mM Tris/HCl (pH 7.5) containing 0.1% Triton X-
100, 10 mM glutathione and 20% glycerol. SDS/PAGE (10%
gel) revealed a GST-fusion protein of ∼75 kDa (48 kDa for
TPHyor and 25 kDa for GST) (Supplementary Figure S1 at
http://www.BiochemJ.org/bj/445/bj4450113add.htm).
EXPERIMENTAL
Chemicals
Nucleosides, nucleobases, nucleoside analogues and all of the
inorganic compounds were purchased from Sigma–Aldrich unless
stated otherwise. TPI {a potent TP inhibitor/5-chloro-6-(1-
[2-iminopyrrolidinyl]methyl)Ura hydrochloride, where Ura is
uracil} [20] was kindly provided by Professor Vern Schramm (Al-
bert Einstein College of Medicine, New York, NY, U.S.A.). BAU
(5-benzylacyclouridine) was purchased from RNDCHEM. 7-DX
(7-deazaxanthine) was synthesized as described previously [21].
Cell cultures
MCF-7 human breast carcinoma cells were kindly provided by
Professor Godefridus Peters (VU University Medical Center,
Amsterdam, The Netherlands). MCF-7 cells were infected with
M. hyorhinis (strain number A.T.C.C. 17981) resulting in a
chronically infected cell line henceforth referred to as MCF-
7.Hyor. All of the cells were maintained in DMEM (Dulbecco’s
modified Eagle’s medium; Invitrogen) with 10% foetal bovine
serum (Biochrom), 10 mM Hepes and 1 mM sodium pyruvate
(Invitrogen). Cells were grown at 37◦C in a humidified incubator
with a gas phase of 5% CO2.
Biological assays
The antiviral assays were based on inhibition of virus-induced
cytopathicity in MCF-7 and MCF-7.Hyor cell cultures. Cells were
seeded in 96-well plates (Thermo Fisher Scientific) at 20000
cells/well and were allowed to proliferate for 24 h at 37◦C.
The cells were then exposed to fresh medium containing 100
CCID50 (50% cell culture infectious dose) of virus [VV (vaccinia
virus), HSV-1 (strain KOS), or HSV-2 (strain G)] and different
concentrations of the test compound in the presence or absence
of TPI (10 μM). The cells were incubated at 37◦C and viral cyto-
pathicity was recorded as soon as it reached completion in the
control virus-infected cell cultures that were not treated with
the test compounds. In addition, the antiviral activity of 5-iodo-
dUrd (where dUrd is 2ꢀ-deoxyuridine) against VV was compared
in MCF-7 and MCF-7.Hyor cells that were pretreated for 4 days
with 1μg/ml tetracycline, an antibiotic targeting mycoplasmas.
To compare the cytostatic activity of nucleoside analogues in
mycoplasma-infected and control cancer cell lines, MCF-7 and
MCF-7.Hyor cells were seeded in 48-well plates (Thermo Fisher
Scientific) at 10000 cells/well. After 24 h, an equal volume of
fresh medium containing the test compounds was added. On day
5, cells were trypsinized and counted in a Coulter counter. The
IC50 value was defined as the compound concentration required
to reduce cell proliferation by 50%.
Enzyme assays
Determination of the pH and temperature optima
The TPHyor-mediated phosphorolysis of dThd (thymidine) was
assayed under different pH and temperature conditions. dThd
(100 μM) was incubated in the presence of the enzyme (9 nM)
at 37◦C with varying pH buffer conditions (pH = 5.5–8.5).
Reactions were carried out in a total volume of 500 μl of
phosphorolysis buffer (10 mM Tris/HCl, 1 mM EDTA, 150 mM
NaCl and 200 mM potassium phosphate). At different time
points (0, 20, 40 and 60 min), 100 μl fractions were withdrawn,
transferred to an Eppendorf tube and heated at 95◦C for 3 min to
inactivate the enzyme. Next, the samples were rapidly cooled on
ice for 15 min and cleared by centrifugation at 16000 g for 15 min.
Thy (thymine) was separated from dThd on a reverse phase RP-
8 column (Merck) and quantified by HPLC analysis (Alliance
2690, Waters). The separation was performed by a gradient
from 100% buffer B [50 mM NaH2PO4 (Acros Organics) and
5 mM heptane sulfonic acid (pH 3.2)] to 75% buffer B and 25%
acetonitrile (BioSolve) (10 min linear gradient of 100% buffer B
to 98% buffer B and 2% acetonitrile; 10 min linear gradient to
90% buffer B and 10% acetonitrile; 5 min linear gradient to
75% buffer B and 25% acetonitrile; and 5 min linear gradient
to 100% buffer B followed by equilibration at 100% buffer B for
10 min). UV-based detection of dThd was performed at 266 nm.
The TPHyor-mediated phosphorolysis of dThd was also compared
after incubation at 20◦C and at 37◦C in a similar assay carried
out in TP buffer [10 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl
and 200 mM potassium phosphate (pH 7.6)].
Purification of TPHyor (M. hyorhinis TP)
In the recently published M. hyorhinis HUB-1 genome [22],
a TP gene, but no UP gene, was found. Therefore a codon-
optimized DNA sequence encoding the TPHyor was synthetically
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Characterization of M. hyorhinis pyrimidine nucleoside phosphorylase
115
formation of dThd and (d)Urd was quantified by HPLC analysis
as described above.
Determination of TPHyor substrate specificity
To study the phosphorolysis of different nucleosides and
nucleoside analogues by TPHyor, different potential substrates
(100 μM) were exposed to the enzyme (45 nM) and incubated
at 37◦C in TP buffer in a total volume of 300 μl. At different time
points (0, 10, 30 and 60 min), 65 μl fractions were withdrawn,
transferred and processed as described above. Nucleobases and
nucleosides were separated by HPLC analysis as described above
and for each product UV-based detection was performed at the
specific wavelength of optimal absorption. Separation of BVDU
[(E)-5-(2-bromovinyl)-dUrd] from its respective base BVU [(E)-
5-(2-bromovinyl)-Ura] was performed by a linear gradient from
98% buffer C [1 mM potassium phosphate buffer, (pH 5.5)]
and 2% buffer D [1 mM potassium phosphate buffer (pH 5.5) and
80% methanol] to 20% buffer C and 80% buffer D. After
injection of the samples, 98% buffer C and 2% buffer D was run
for 10 min before the start of the gradient (10 min linear gradient
from 2% to 80% buffer D). After 5 min running at 80% buffer
D, a 5 min linear gradient to 98% buffer C and 2% buffer D was
performed followed by equilibration at 98% buffer C for 5 min.
Inhibition assays
In the assays where the inhibitory effect of the TP inhibitors 7-
DX and TPI and the UP inhibitor BAU was evaluated, a variety
of inhibitor concentrations, including 500 μM, 250 μM, 100 μM,
25 μM and 0 μM (control), were added to a reaction mixture that
contained 100 μM substrate (dThd or Urd) in TP buffer containing
2 mM Pi. Next, the reaction mixture was exposed to different
phosphorolytic enzymes [TPHyor, human TP, E. coli TP or UPP1
(human UP1; derived from tumour tissue)] and, after a 20 min
incubation at 37◦C, the substrate degradation was determined by
HPLC as described above.
Bioinformatics and computational in silico analysis
Protein alignments and pairwise alignment scores were calculated
using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
The one-to-one threading method implemented in the Phyre2
server [24] provided different TPHyor models using as templates
the three-dimensional structures of several TPs and PyNPs
that have been solved by X–ray crystallography and are
deposited in the PDB. The computer graphics program PyMOL
(http://www.pymol.org/) was used for molecular visualization and
superimposition.
Kinetic assays
The enzymatic activity of TPHyor towards different substrates
(dThd, dUrd, Urd, 5-fluoro-dUrd, 5-iodo-dUrd, 5-fluoro-Urd
and 5-iodo-Urd) was evaluated. The nucleoside-to-nucleobase
conversion at varying concentrations of substrate was studied
in a reaction containing 9 nM enzyme incubated in TP buffer
at 37◦C for 20 min. For each substrate, at least 10 different
concentrations in the following range were assayed: dThd
and dUrd, 100–6000 μM; Urd, 25–2000 μM; 5-fluoro-dUrd,
25–1000 μM; and 5-iodo-dUrd, 5-fluoro-Urd and 5-iodo-
Urd, 25–500 μM. In the kinetic assays where the enzymatic
activity of TPHyor was evaluated at varying concentrations of Pi
(10 different concentrations in the range of 0.1–50 mM), the
nucleoside substrate was kept fixed at a concentration of 10× Km.
After incubation, samples were processed and analysed by HPLC
as described above. The Michaelis constant (Km) and turnover
number (kcat) were determined by means of non-linear regression
analysis (using GraphPad Prism5).
RESULTS
M. hyorhinis infection compromises the biological activity of
therapeutic nucleoside analogues
Human breast carcinoma MCF-7 cells were infected with M.
hyorhinis (designated MCF-7.Hyor) and used to study the effect
of the mycoplasma infection on the antiviral/antiproliferative
activity of various nucleoside analogues.
The antiviral activity of 5-halogenated dThd analogues,
including the clinically approved antiherpetic agents 5-iodo-dUrd
(idoxuridine) and BVDU (Brivudin) against different viruses was
determined in MCF-7 and MCF-7.Hyor cell cultures. As shown in
Table 1, the inhibitory activity of the drugs against VV, HSV-1 and
HSV-2 infection was decreased by 6–55 fold in MCF-7.Hyor cell
cultures when compared with mycoplasma-free control MCF-7
cells. The antiviral activity of the compounds could be rescued
by pretreating the cells for 4 days with tetracycline (1μg/ml),
an antibiotic targeting mycoplasmas (results not shown). The
antiviral activity could also be fully restored upon administration
of 10 μM TPI, a powerful TP inhibitor (Table 1). Since MCF-7
cells show a very low level of endogenous TP expression
(undetectable by Western blot analysis) [25], the data indicate
that mycoplasma-encoded TP, expressed in the MCF-7.Hyor cell
cultures, is responsible for the decreased antiviral activity of the
drugs. In contrast, the activity of BVDU, an antiherpetic agent
that is used to treat Varicella Zoster virus infections and known to
be an excellent substrate for both human and E. coli-derived TP
[26–28], was not compromised in the mycoplasma-infected cell
cultures. Instead, BVDU became 5- and 3-fold more inhibitory
against VV and HSV-2 respectively in mycoplasma-infected cell
cultures, and this increased activity was lost upon addition of TPI.
We also investigated the cytostatic activity of fluoropyrimidines
in mycoplasma-infected and -free cell cultures. We previously
demonstrated that the decreased cytostatic activity of halogenated
dThd analogues could be rescued by elimination of the
mycoplasmas using an antibiotic or by the addition of TPI to
Competition experiments
To determine the substrate preference of TPHyor, dThd and Urd
(each at 100 μM) were exposed in one reaction to 9 nM TPHyor
.
After incubation at 37◦C in TP buffer, fractions were collected at
0, 20, 40 and 60 min and processed as described above.
To study whether both substrates are mutually exclusive,
TPHyor-mediated phosphorolysis of a fixed concentration of dThd
(100 μM) was studied in the presence of different concentrations
of Urd (1 mM, 0.5 mM, 0.25 mM, 0.1 mM and 0 mM) and vice
versa. After a 30 min incubation of the substrates with the enzyme
(9 nM) at 37◦C in TP buffer, samples were processed as described
above.
Anabolic activity of TPHyor
The M. hyorhinis TP-mediated coupling of 2-dRib-1-P (2-
deoxyribose-1-phosphate) to Thy and Ura and the coupling
of Rib-1-P (ribose-1-phosphate) to Ura was studied. Sugars
(1 mM) and nucleobases (100 μM) were incubated at 37◦C
for 20 min in the presence of 9 nM TPHyor in TP buffer with
varying concentrations of inorganic phosphate (Pi = 50 mM;
5 mM; 0.5 mM and 0 mM) in a total volume of 200 μl. The
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J. Vande Voorde and others
Table 1 Inhibitory activity of dThd analogues against viral infection in
MCF-7 and MCF-7.Hyor cells
+
Results are the means S.D. of two independent experiments.
−
(a) VV infection
EC50 (μM)
MCF-7
Alone
MCF-7.Hyor
Alone
Compound
+ TPI
+ TPI
+
+
+
+
5-Chloro-dUrd
5-Bromo-dUrd
5-Iodo-dUrd
BVDU
2.8 1.1
1.6 0.3
2.2 0.7
1.6 0.2
47.6 4.0
48.5 3.3
1.6 0.7
1.9 0.1
−
−
−
−
+
+
+
+
−
−
−
−
+
+
+
1.8 0.6
2.9 1.1
ꢀ100
2.7 1.2
−
−
−
+
+
47.4 3.7
ꢀ44.7
10.2 1.4
ꢀ100
−
−
(b) HSV-1 (KOS) infection
EC50 (μM)
MCF-7
Alone
MCF-7.Hyor
Alone
Compound
+ TPI
+ TPI
+
+
+
+
5-Chloro-dUrd
5-Bromo-dUrd
5-Iodo-dUrd
BVDU
7.7 3.2
1.5 0.6
8.2 1.7
1.5 0.6
44.7
44.7
0
0
3.5 1.1
1.1 0.4
−
−
−
+
−
+
−
+
+
+
−
−
−
+
+
+
1.8 0.5
1.9 0.1
47.6 4.0
1.3 0.2
−
−
−
−
+
+
+
+
0.04 0.02
0.02 0.006
0.04 0.03
0.02 0.01
−
−
−
−
(c) HSV-2 (G) infection
EC50 (μM)
MCF-7
Alone
MCF-7.Hyor
Alone
Compound
+ TPI
+ TPI
+
+
+
+
5-Chloro-dUrd
5-Bromo-dUrd
5-Iodo-dUrd
BVDU
8.7 1.4
8.5 1.9
1.9 0.1
51.0 5.7
46.6 3.3
1.6 0.7
1.9 0.1
−
+
−
−
−
+
+
+
2.0
0
−
−
−
−
+
+
+
+
2.1 0.2
2.0
0
63.1 31.9
2.0 0.3
−
−
+
−
−
+
Figure 1 pH and temperature dependence of TPHyor-mediated dThd
phosphorolysis
+
+
1.5 0.6
5.0 1.7
0.5 0.3
4.0
0
−
−
−
−
(A) pH-dependent dThd (100 μM) degradation after a 60 min incubation in the presence of
TPHyor. (B) Temperature-dependent dThd (100 μM) degradation after a 60 min incubation in the
the infected cell cultures [3,6]. The antiproliferative activity
of 5-fluorouridine, being a poor substrate for human- or E.
coli-derived TP and human UPP1 (results not shown), was
decreased by 20-fold in mycoplasma-infected MCF-7.Hyor cells
+
presence of TPHyor. Results are the means S.D. of two independent experiments.
−
Table 2 Substrate specificity of TPHyor for pyrimidine nucleosides and
nucleoside analogues
+
(IC50 = 0.226 0.126 μM) when compared with control
−
+
cells (IC50 = 0.011 0.003 μM). However, the cytostatic
−
Substrate for TPHyor
No substrate for TPHyor
activity of this compound could also be fully restored upon
administration of TPI (10 μM), suggesting that mycoplasma-
encoded TP has unique characteristics, distinct from its
mammalian and E. coli counterparts, and would deserve further
investigation and kinetic characterization.
Natural pyrimidine nucleosides
dThd
dUrd
Cytidine
Deoxycytidine
Urd
Pyrimidine nucleoside analogues
5-Trifluoromethyl-dUrd
5-Fluoro-dUrd
5-Chloro-dUrd
5-Bromo-dUrd
5-Iodo-dUrd
2ꢀ,2ꢀ-Difluoro-2ꢀ-deoxycytidine
2ꢀ,2ꢀ-Difluoro-2ꢀ-deoxyuridine
Determination of substrate specificity of TPHyor
Ara-U
Ara-T
BVDU*
AZT
D4T
Substrate specificity of TPHyor for natural pyrimidine nucleosides
The M. hyorhinis gene responsible for the TP activity was
cloned and the enzyme was expressed and purified as described
(see the Experimental section). The phosphorolysis of dThd
catalysed by the purified TPHyor was found to be optimal at
pH 7.5 (Figure 1A) and at a temperature of 37◦C (Figure 1B).
To determine the substrate specificity of TPHyor towards natural
pyrimidine nucleosides, different nucleosides were exposed to
the enzyme and phosphorolysis was monitored. TPHyor cata-
lyses the conversion of dThd and dUrd, but also, surprisingly, of
Urd into their respective bases (Thy and Ura) and phosphorylated
sugars (2-dRib-1-P and Rib-1-P). Neither cytidine nor 2ꢀ-
deoxycytidine were substrates for the enzyme (Table 2). The
Michaelis constant (Km) and the turnover number (kcat) were
determined for different substrates using non-linear regression
5-Fluoro-Urd
5-Iodo-Urd
6-Azauridine
*Very poor phosphorolysis of BVDU was observed at the highest enzyme concentration.
analysis and are displayed in Table 3. The specificity constant
(kcat/Km) was calculated as an estimate for the catalytic efficiency
of the enzyme. Remarkably, the TPHyor-mediated phosphoro-
lysis of Urd (Km = 92 μM, kcat/Km = 0.092) was found to
be almost twice as efficient when compared with dThd
(Km = 473 μM, kcat/Km = 0.046) and dUrd (Km = 578 μM,
kcat/Km = 0.043).
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Table 3 Kinetic parameters of TPHyor
+
The Km and kcat values for the natural substrates ( S.E.M.) were computationally determined
−
using non-linear regression analysis (using GraphPad Prism 5) from data obtained in four
(dThd and dUrd) or three (Urd) independent experiments. The kinetic parameters for the other
compounds were derived from data obtained in two independent experiments.
Substrate
Km (μM)
kcat (s− 1
)
kcat/Km [(s · μM)− 1
]
Natural substrates
dThd
dUrd
+
+
473 25
578 29
21.6 0.5
24.6 0.5
0.046
0.043
0.092
−
−
+
+
−
+
−
+
Urd
92
8
8.5 0.2
−
−
Nucleoside analogues
5-Fluoro-dUrd
5-Iodo-dUrd
5-Fluoro-Urd
5-Iodo-Urd
Pi
+
−
+
+
169
9
11.9 0.2
0.070
0.066
0.130
0.093
−
+
144 25
9.5 0.6
−
−
+
+
47
69
4
7
6.1 0.1
−
+
−
−
+
6.4 0.2
−
+
+
Co-substrate dThd
Co-substrate Urd
797 107
17.5 0.6
0.022
0.022
−
−
+
+
388 43
8.6 0.2
−
−
When dThd or Urd were added at fixed saturating substrate
concentrations and the inorganic phosphate concentration was
varied in the reaction mixture, an identical phosphorolytic efficacy
(kcat/Km = 0.022) was found for inorganic phosphate in the
presence of either nucleoside substrate (Table 3).
Substrate specificity of TPHyor for nucleoside analogues
Next, the substrate specificity and kinetic parameters for a
variety of antiviral and antitumour nucleoside analogues were
determined (Tables 2 and 3). TFT (5-trifluoromethyl-dUrd) and
the 5-halogenated dThd and Urd analogues were found to
be efficient substrates for TPHyor-mediated phosphorolysis. In
contrast, BVDU, an excellent substrate for human and E. coli
TP, was only poorly recognized by the mycoplasma-derived
enzyme (<5% BVU formation from BVDU compared with
∼66% Thy formation from dThd after a 10 min incubation
under identical reaction conditions). Also ara-T (Thy arabinoside)
and ara-U (Ura arabinoside), and the anti-HIV agents AZT
(azidothymidine/zidovudine) and D4T (stavudine) which are not
a substrate for human and E. coli TP, were not converted by
TPHyor. Whereas the glycosidic bond of the riboside analogue
5-fluoro-Urd was efficiently cleaved by this enzyme, the
cytostatic antimetabolite drug 6-azauridine was not susceptible to
phosphorolysis by TPHyor. As expected, the difluorinated cytidine
analogue gemcitabine (2ꢀ,2ꢀ-difluoro-2ꢀdeoxycytidine) as well as
its deaminated metabolite 2ꢀ,2ꢀ-difluoro-2ꢀ-deoxyuridine were not
cleaved either (Table 2).
Overall, the 5-halogenated thymidine analogues 5-fluoro-dUrd
(floxuridine) and 5-iodo-dUrd (idoxuridine), which represent
clinically approved drugs for the treatment of cancer and
herpesvirus infections respectively, were found to be better
substrates for TPHyor than dThd. Likewise, TPHyor-catalysed
phosphorolysis of the Urd analogues 5-fluoro-Urd and 5-iodo-
Urd was found to proceed more efficiently when compared
with Urd. In general, Urd and its analogues were found to be
preferred substrates over dThd and its analogues (Table 3).
Figure 2 Competition for TPHyor-mediated phosphorolysis
(A) TPHyor-mediated metabolite formation from an equimolar mixture of Urd and dThd. (B)
TPHyor-mediated phosphorolysis of a fixed dThd (100 μM) concentration in the presence of
varying Urd concentrations after a 30 min incubation. (C) TPHyor-mediated phosphorolysis of a
fixed Urd (100 μM) concentration in the presence of varying dThd concentrations after a 30 min
+
incubation. Results are the means S.D. of two independent experiments.
−
mutually exclusive or can be concomitantly used as a substrate
by the enzyme. In a first set of experiments, dThd and Urd were
mixed at equimolar concentrations (100 μM) and simultaneously
incubated with the enzyme. Metabolite formation was then
monitored as a function of time. As shown in Figure 2(A),
both dThd and Urd were time-dependently converted into their
respective base with Urd being more efficiently cleaved than
dThd. After a 60 min concomitant incubation of both substrates
with the enzyme, ∼90% of the supplied Urd and only ∼50%
of the supplied dThd were converted. In addition, Ura and Thy
were found to be coupled again with the (deoxy)ribose moieties
(derived from Urd and dThd) in an anabolic reaction mediated by
TPHyor, resulting in the formation of 2ꢀ-deoxyuridine (2-dRib-1-P
and Ura) and thymine riboside (Rib-1-P and Thy).
In a second set of experiments, a fixed concentration of dThd
(100 μM) was incubated for 30 min with the enzyme in the
presence of different Urd concentrations. In analogy, 100 μM Urd
was incubated in the presence of different dThd concentrations
for 30 min. Urd dose-dependently decreased the phosphorolysis
of dThd (Figure 2B). This inhibition occurred more efficiently
dThd and Urd compete for phosphorolysis by TPHyor
Since the enzymatic parameters demonstrated a more efficient
TPHyor-mediated phosphorolysis of Urd when compared with
dThd, we next investigated whether both natural substrates are
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Table 4 Inhibition of different phosphorolytic enzymes by inhibitors of
human TP and UP
+
Results are the means S.D. of two independent experiments.
−
IC50 (μM)
Substrate = dThd
Inhibitor TPHyor Human TP
Substrate = Urd
TPHyor
E. coli TP
UPP1
+
+
−
+
−
+
TPI
7-DX
BAU
0.003 0.001 0.007 0.001 0.007 0.001 0.005 0.0007 ꢀ500
−
+
−
−
−
−
+
−
Figure 3 Anabolic activity of TPHyor
+
+
+
60
5
151 33
108 0.7
30
3
301 71
−
+
>500
>500
>500
>500
0.89 0.02
−
TPHyor-mediated Urd formation from uracil (100 μM) and Rib-1-P (1mM); dUrd formation from
uracil (100 μM) and 2-dRib-1-P (1 mM); and dThd formation from thymine (100 μM) and
2-dRib-1-P (1 mM) in the presence of varying Pi concentrations after a 20 min incubation at
◦
+
37 C. Results are the means S.D. of two independent experiments.
−
respiratory tract [22] and strain MCLD derived from a primary
human melanoma cell culture [29]). The protein sequence of
TP was found to be highly conserved in both M. hyorhinis
strains (identical sequence, with the exception of Ser240 in HUB-1
compared with Phe240 in MCLD). In contrast, no putative UP gene
was annotated in these mycoplasma strains.
than the decreased Urd phosphorolysis in the presence of dThd
(Figure 2C). Taken together, these results strongly indicate that
dThd and Urd compete for the same substrate-binding site.
A multiple sequence alignment of TPHyor (Figure 4A) with
similar enzymes whose three-dimensional structures have been
solved and are deposited in the PDB revealed that TPHyor
has sequence identities of 35% and 40% respectively, with
E. coli (over 440 amino acids) and human TP (over 273 amino
acids) and of 46% and 42% respectively, with the PyNP of
Geobacillus stearothermophilus (over 433 amino acids) and
Thermus thermophilus (over 423 amino acids). In all of these
enzymes the residues making up the phosphate-binding site and
the thymidine-binding site are highly conserved. For this reason
our homology-built models of TPHyor (Supplementary Figure S2A
at http://www.BiochemJ.org/bj/445/bj4450113add.htm), which
basically differ in the ligand-dependent degree of closure of the
active site, are considered to be very reliable (the Ramachandran
Z-score of − 1.87 obtained from WhatCheck [30] means that phi
and psi angles for all residues are within the expected ranges
for well refined structures). In contrast, a multiple sequence
alignment of TPHyor with human UPP1 and E. coli UP (Figure 4B)
showed much lower sequence similarity between TPHyor and
uridine phosphorylases (pairwise alignment score of 6% and 3%
respectively), whereas human UPP1 and E. coli UP sequences
show a similarity score of 21%.
Urd formation is superior to dThd formation in the anabolic
direction of the TPHyor reaction
The TPHyor-mediated coupling of Ura to Rib-1-P and Ura and
Thy to 2-dRib-1-P was studied. Urd was more efficiently formed
than dUrd and dThd (Figure 3). These findings are in line with the
enzymatic parameters determined for the individual substrates and
the results obtained in the competition experiments (see above).
Nucleoside formation was also found to be highly dependent
on the concentration of Pi present in the reaction mixture, i.e.
increasing concentrations of Pi dose-dependently inhibit the TP-
catalysed nucleoside formation (Figure 3).
Differential inhibition of TPHyor by known specific TP and UP
inhibitors
The human TP inhibitors TPI and 7-DX were examined for their
capacity to inhibit the phosphorolysis of dThd (catalysed by
TPHyor, human TP or E. coli TP) and Urd (catalysed by TPHyor
or UPP1). As shown in Table 4, TPI inhibits the phosphorolysis
of dThd catalysed by TP of either mycoplasma, human or E. coli
origin in the lower nanomolar range (IC50 = 3 nM, 7 nM and 7 nM
respectively). TPI also efficiently inhibits the phosphorolysis of
Urd catalysed by TPHyor (IC50 = 5 nM), but does not inhibit the
phosphorolysis of Urd by UPP1 (IC50ꢀ500 μM) (Table 4). The
The almost identical amino acid composition and putative
architecture of the active site of TPHyor with respect to human TP
(as shown in Supplementary Figure S3 at http://www.BiochemJ.
org/bj/445/bj4450113add.htm) can account for the comparable
inhibition of these two enzymes by TPI. In fact, the Arg202, Ser217
and Lys221 side chains that hydrogen bond to the uracil base of TPI
in its crystallographic complex with human TP [31], as well as the
hydrophobic side chains of Leu148 and Ile218 that stack against
purine-derived TP inhibitor 7-DX was found to inhibit TPHyor
-
mediated dThd and Urd phosphorolysis in the micromolar range
(IC50 = 60 μM and 30 μM respectively). A similar inhibitory
activity of 7-DX was found against human- and E. coli-derived
TP (IC50 = 151 μM and 108 μM respectively, but a 10-fold
decreased activity was observed towards UPP1-mediated Urd
the nucleobase ring, are also present in TPHyor (Arg168, Ser183
,
Lys187, Leu114 and Ile184 respectively) (Supplementary Figure
S2B). The same can be said about the catalytic His116 in human
TP and His82 in TPHyor, which are positionally and functionally
equivalent to His85 in E. coli TP [32]. These marked similarities,
however, cannot explain why TPHyor recognizes Urd as a better
substrate than dThd, given that Urd is a very poor substrate, if at
all, for human TP.
phosphorolysis (IC50 = 300 μM) when compared with TPHyor
.
The well-known UP inhibitor BAU was found to inhibit only
the phosphorolysis of Urd catalysed by UPP1 (IC50 = 0.89 μM),
but affected neither the efficiency of mycoplasma-, human- or
E. coli-catalysed dThd phosphorolysis (IC50>500 μM) nor the
phosphorolysis of Urd by TPHyor. These results indicate that even
though TPHyor preferably shows Urd phosphorylase activity, the
enzyme behaves catalytically more similar to the TPs.
Interestingly, Lys108 in the phosphate-binding pocket of
TPHyor (Figure 5) is positionally equivalent to Lys108
in G. stearothermophilus PyNP (PDB code 1BRW), Lys108 in
Staphylococcus aureus PyNP (PDB code 3H5Q) and Lys107
in T. thermophilus (PDB code 2DSJ), but is replaced by Met142 and
Met111 respectively, in human (PDB codes 1UOU and 2WK6)
and E. coli (PDB codes 2TPT and 1AZY) TP. Likewise, the
Computer-assisted molecular modelling
The complete M. hyorhinis genome was recently published
for two different strains (strain HUB-1 isolated from swine
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Characterization of M. hyorhinis pyrimidine nucleoside phosphorylase
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Figure 4 Multiple sequence alignment of TPHyor with PDB entries
(A) TP from E. coli (PDB code 2TPT) and a human source (PDB code 1UOU) and PyNP from T. thermophilus (PDB code 2DSJ), S. aureus (PDB code 3H5Q) and G. stearothermophilus (PDB
code 1BRW). Important catalytic residues are formatted in grey and the most significant differences in the substrate-binding site are boxed. (B) UP from E. coli (PDB code 1TGY) and UPP1 (PDB
code 3EUE). Black boxes indicate conserved amino acid residues compared with TPHyor. The symbols underneath each block stand for identical amino acids (*), conserved substitutions (:) and
semi-conserved substitutions (.).
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J. Vande Voorde and others
also possess TP activity display a much lower (∼25–160-fold)
associated TP than UP activity [33].
However, early studies on pyrimidine nucleoside phos-
phorylases isolated from e.g. G. stearothermophilus and
Haemophilus influenzae reported comparable TP and UP
activities [34,35], as now also shown for TPHyor. Such prokaryotic
enzymes indeed do not discriminate at the 2ꢀ-position of the
ribose, and are considered as a separate and well-defined
class of pyrimidine nucleoside cleaving enzymes, referred to
as PyNP (EC. 2.4.2.2) and ranked within the NP-II family of
phosphorolytic enzymes. A similar observation was made for the
UP/TP activity of the parasite Giardia lamblia [36]. PyNP and TP
are known to display significant sequence similarity and similar
physical properties [19]. X-ray crystallography revealed indeed
that the three-dimensional structures of G. stearothermophilus
PyNP and E. coli TP are very similar [37,38].
From our model of TPHyor it is apparent that this enzyme
is structurally related to the PyNP subfamily (NP-II) rather
than to the UP subfamily (NP-I) of nucleoside phosphorylases
(to which human and E. coli UP belong) [19] (Figure 4). We
therefore believe that TPHyor, due to both its structural nature and
catalytic properties, should be annotated as a NP-II class PyNP to
distinguish it from the NP-II class TP and NP-I class UP enzymes.
The data of the present study indicate that one and the same
active site in TPHyor/PyNP is responsible for the phosphorolysis
of both dThd and Urd, which is in agreement with the PyNP
properties of the enzyme: (i) increasing dThd concentrations
decrease Urd phosphorolytic activity and vice versa; (ii) an
equimolar mixture of dThd and Urd exposed to TPHyor/PyNP
results in the concomitant formation of dUrd (formed from
Urd-derived Ura and dThd-derived 2-dRib-1-P) and the thymine
riboside (formed from Urd-derived Rib-1-P and dThd-derived
thymine); and (iii) the specific TP inhibitor TPI, being inactive
against UPP1, is equally effective in inhibiting TPHyor/PyNP-
catalysed phosphorolysis of dThd and Urd.
Inhibitor studies also reveal that, despite its efficient
multifunctional activity on dThd/Urd substrates, the nature of
TPHyor/PyNP is much more similar to other TP and PyNP than to
UP. TPI is the most potent and selective transition state inhibitor
of human TP reported so far. It is currently the subject of clinical
trials in combination with TFT to prevent premature breakdown
of this anticancer drug to its inactive base [39–41]. We found that
TPI is an equally highly potent inhibitor of E. coli TP, human TP
and TPHyor/PyNP that does not inhibit human UPP1. However, by
blocking the TPHyor/PyNP enzyme activity, TPI also annihilates
the concomitant UP activity of TPHyor/PyNP. Conversely, BAU, a
well-known UP inhibitor that selectively inhibits UPP1 and E. coli
UP without affecting TP activity [42,43], did not abrogate the UP
(and TP) activity of TPHyor/PyNP and thus discriminates between
the mycoplasma UP activity of TPHyor/PyNP and the human (and
E. coli) UP activity.
Although TPHyor/PyNP was expressed as a recombinant GST-
fusion protein, we felt that tagging the nucleoside phosphorylase
does not compromise its phosphorolytic activity. Indeed, the
specificity constants (kcat/Km) of dThd phosphorolysis by both
human TP and human TP-GST were determined using linear
regression analysis and were found to be very similar (human TP,
kcat/Km = 0.036; human TP–GST, kcat/Km = 0.030). Thus the pre-
sence of GST in human TP did not substantially affect the
kinetic properties of the enzyme. The catalytic efficiency of GST-
tagged TPHyor/PyNP was found to be in the same range when
compared with previously characterized orthologue NPs. The
turnover number (kcat) of TPHyor/PyNP was determined as 21.6,
24.6 and 8.5 s− 1 for dThd, dUrd and Urd respectively. This
compares reasonably well with the turnover numbers of human TP
Figure 5 Model of the active site of TPHyor
Detail of the active site of the homology-built model of TPHyor showing the putative positions for
the nucleoside substrate (dThd) and the attacking phosphate.
position of the neighbouring Thr92 in TPHyor is occupied in the
latter two enzymes by a serine (Ser126 in human TP and Ser95
in E. coli TP). It is therefore likely that the nature of the amino
acid at these two positions has an influence on transition state
formation and/or stabilization of the enzyme–substrate complex
during catalysis.
For BAU, there are two X-ray crystal structures available for
its complex with E. coli UP (PDB code 1U1C) and UPP1 (PDB
code 3EUF). The active sites of both enzymes are at the interface
between two monomers that, in turn, are part of the ‘trimer of
dimers’ that is seen in the crystal structure of the bacterial enzyme.
However, many of the residues of E. coli UP and UPP1 that
interact with BAU are different in TPHyor, and explain why BAU
selectively inhibits the E. coli UP and UPP1, but not the UP
activity of TPHyor. The fact that these two UPs are completely
different enzymes from a structural point of view, containing
different active site topologies to the TP enzymes, is in agreement
with our kinetic observations.
DISCUSSION
To the best of our knowledge the gene annotated as TPHyor is the
first mycoplasma-encoded phosphorylase to be cloned, expressed
and characterized. We provide evidence that the presence of TPHyor
in cell cultures due to a mycoplasma infection negatively affects
the efficiency of nucleoside analogues used in the treatment
of virus infections and cancer. The enzyme shows an unusual
substrate selectivity since it catalyses not only the phosphorolysis
of dThd and dUrd, but also Urd, which surprisingly turned out
to be the preferred substrate. Thus, although the amino acid
alignment of TPHyor with human TP, E. coli TP, UPP1 and E.
coli UP revealed a markedly higher similarity of TPHyor with
TP than with UP, the enzyme shows a superior (∼2-fold) UP
catalytic activity. Extensively studied TP enzymes that display,
besides TP activity, also UP activity (i.e. TP from human liver,
human placenta and mouse liver) have a much lower (∼90–200-
fold) UP than TP activity, and vice versa, those UP enzymes that
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Characterization of M. hyorhinis pyrimidine nucleoside phosphorylase
121
for dThd (kcat = 9.4 s− 1) and for H. influenzae PyNP (kcat = 11.1,
6.4 and 52.9 s− 1 for dThd, dUrd and Urd respectively) [35,44].
The kinetic characterization of TPHyor/PyNP sheds new light on
nucleotide metabolism in mycoplasmas. A potent phosphorolysis
of Urd in extracts of mycoplasmas has been described previously
[45,46]. Originally this activity was attributed to the assumed
presence of mycoplasma-encoded UP. This UP activity
(measured, for example, by the increased formation of [14C]Ura
from [14C]Urd or the decreased incorporation of [3H]Urd in the
RNA of infected cell cultures) was even proposed as a tool for
the identification of mycoplasma infections in cells [47,48]. In
contrast with the annotation of a TP and a purine nucleoside
phosphorylase gene in the genome of different mycoplasma
strains [22,49], no UP gene has been annotated in mycoplasmas
to the best of our knowledge. This was also pointed out by Bizarro
and Schuck [50] who, in the framework of an in silico study on
the purine and pyrimidine nucleotide metabolism in Mollicutes,
suggested a TP to be responsible for UP activity. Indeed, the
results obtained in the present study indicate that not a UP, but
instead a PyNP is responsible for the release of uracil from Urd
in mycoplasma cultures. These findings are also in agreement
with our data on the cytostatic activity of 5-fluoro-Urd that is
markedly decreased in mycoplasma-infected cell cultures, but
efficiently restored in the presence of TPI. The phosphorolytic
activity of TPHyor/PyNP against 5-fluoro-Urd could be efficiently
blocked by TPI in a cell-free assay (results not shown) pointing
again to the UP activity of TPHyor/PyNP being responsible for
5-fluoro-Urd degradation. The occurrence of a multisubstrate
enzyme in mycoplasmas fits the minimal cell and genome
concept of mycoplasmas, being the smallest autonomously
replicating bacteria, rarely exceeding a genome of 1 Mb [51–53].
Surprisingly, the antiherpetic drug BVDU, a well-known and
excellent substrate for human and E. coli TP [26], was hardly
acted upon by TPHyor/PyNP. This finding is in agreement with
our observation that mycoplasma infection does not compromise
the biological (antiviral) activity of BVDU in vitro. Instead, the
antiviral activity of BVDU against VV and HSV-2 was found to
be slightly (3–5-fold) enhanced in the presence of TPHyor/PyNP.
This effect, which could be reversed by administration of
the TP inhibitor TPI, may be explained by the TPHyor/PyNP-
mediated depletion of intracellular dThd pools, which most likely
gives BVDU a competitive advantage for phosphorylation to its
biologically active 5ꢀ-monophosphate metabolite in mycoplasma-
infected cells. Our modelling studies with TPHyor/PyNP provide
a partial explanation for the unexpectedly different behaviour
of BVDU as a potential substrate for TPHyor/PyNP, human TP
and E. coli TP in so far as the 2-bromovinyl group at the 5ꢀ-
position of the uracil ring might be too bulky to fit into the
active site of TPHyor/PyNP due to the presence of the phenyl
ring of Phe207, which is positionally equivalent to the smaller
Val241 in the human TP enzyme. However, the corresponding
Phe210 in the complex of E. coli TP with TPI has been shown
to change its rotameric state relative to that found in other PyNPs
[38]. On the other hand, TPI inhibits TPHyor/PyNP as efficiently
as it inhibits human TP despite the fact that it contains a 5-
Cl substituent on the uracil base that is bioisosteric with the
methyl group of thymine in dThd. The observation that BVDU
is not a good substrate for TPHyor/PyNP is important in view
of the fact that BVU, the free base of this antiviral agent, is a
potent inhibitor of DHP (dihydropyrimidine dehydrogenase) [54–
56]. Inhibition of the latter enzyme prevents further catabolism
of Ura and Thy and, more importantly, of the anticancer agent
5-FU (5-fluorouracil), leading to the accumulation of 5-FU and
its concomitant life-threatening toxicity [57–59]. Accordingly,
a mycoplasma infection will not affect DHP activity in cancer
patients treated with BVDU and 5-FU, and thus will not be
expected to increase 5-FU toxicity.
BVDU became somewhat more active against VV and HSV-2,
but not HSV-1, infection in the presence of mycoplasmas. This
can be explained by the markedly different potencies of antiviral
activity of BVDU against the particular viruses and/or different
targets of inhibition by BVDU. Indeed, BVDU is far more
inhibitory to HSV-1 (EC50 = 0.04 μM) than to HSV-2 and VV
(EC50 = 1.5 and 47 μM respectively). The molecular mechanisms
of action of BVDU against HSV-1 are inhibition of HSV-1 DNA
polymerase and incorporation into viral DNA after metabolic
conversion into its 5ꢀ-mono- and 5ꢀ-di-phosphates by HSV-1
thymidine kinase, and to its 5ꢀ-triphosphate derivative by cellular
enzymes. Instead both VV and HSV-2 encode a thymidine kinase
that can only convert BVDU into its 5ꢀ-monophosphate derivative,
which is known to inhibit thymidylate synthase [60,61]. Therefore
it is well possible that thymidylate synthase is the molecular target
of BVDU for HSV-2 and VV inhibition. In this case, the dTMP
(and dTDP and dTTP) pools might be further decreased, resulting
in a more favourable inhibition of the virus infection by the lack
of sufficient dTTP for DNA synthesis in the presence of PyNP-
expressing mycoplasmas.
The present study may have important implications for the
treatment of cancer patients or patients suffering from viral
infections with nucleoside analogues. Mycoplasmas are known
to reside in the human body, causing asymptomatic infections.
Also, it is widely accepted that particularly immunocompromised
patients, such as those suffering from HIV infections, are
prone to secondary infections by mycoplasmas, which are then
also found in organs different from their usual habitat [7].
Additionally, an increasing number of studies report on the high
preferential colonization of human tumours by mycoplasmas [10–
17]. Taking these facts into consideration, our findings indicate
that the treatment of patients with nucleoside analogues may be
compromised by the expression of catabolic enzymes with broad
substrate specificity, such as those encoded by mycoplasmas, and
could be optimized by the co-administration of a mycoplasma-
targeting antibiotic or specific enzyme inhibitors. The unique
substrate specificity of TPHyor/PyNP towards natural nucleosides
and nucleoside analogues indicates that the design of such a
specific mycoplasma PyNP inhibitor could be a feasible goal. We
would suggest extending the structure of TPI to allow interaction
with the phosphate-binding site in TPHyor/PyNP where Lys108 and
Thr92 are found instead of Met142 and Ser126 in human TP. These
conserved amino acid differences may underlie the substrate
preferences reported above and could be exploited for specific
inhibitor design.
AUTHOR CONTRIBUTION
Johan Vande Voorde participated in the design of the study, carried out the cell culture
experiments, sequence alignments and enzyme experiments and participated in the writing
of the paper; Federico Gago performed sequence alignments and modelling; Kristof
Vrancken contributed to the cloning of the TPHyor/PyNP gene and to the purification of
the enzyme; Sandra Liekens and Jan Balzarini designed and supervised the study and
participated in the writing of the paper. All of the authors read and approved the final paper
prior to submission.
ACKNOWLEDGEMENTS
We thank Christiane Callebaut, Eef Meyen, Kristien Minner, Leentje Persoons, Ria Van
Berwaer and Peter Vervaeke for their excellent technical assistance, Dr Tarmo Roosild
(Nevada Cancer Institute, Las Vegas, NV, U.S.A.) for generously providing human uridine
phosphorylase and Professor Vern Schramm (Albert Einstein College of Medicine, New
York, NY, U.S.A.) for kindly providing TPI.
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23 Liekens, S., Herna´ndez, A. I., Ribatti, D., De Clercq, E., Camarasa, M. J., Pe´rez-Pe´rez,
M. J. and Balzarini, J. (2004) The nucleoside derivative 5ꢀ-O-trityl-inosine (KIN59)
suppresses thymidine phosphorylase-triggered angiogenesis via a noncompetitive
mechanism of action. J. Biol. Chem. 279, 29598–29605
24 Kelley, L. A. and Sternberg, M. J. (2009) Protein structure prediction on the web: a case
study using the Phyre server. Nat. Protoc. 4, 363–371
FUNDING
This work was supported by the Institute for the Promotion of Innovation through Science
andTechnologyinFlanders(IWT-Vlaanderen)(toJ.V.V.), the‘FondsvoorWetenschappelijk
Onderzoek (FWO) Vlaanderen’ [grant number G.0486.08] and the K.U. Leuven [grant
number PF 10/18].
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Received 23 December 2011/30 March 2012; accepted 4 April 2012
Published as BJ Immediate Publication 4 April 2012, doi:10.1042/BJ20112225
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Biochem. J. (2012) 445, 113–123 (Printed in Great Britain) doi:10.1042/BJ20112225
SUPPLEMENTARY ONLINE DATA
Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma
hyorhinis: implications for the clinical efficacy of nucleoside analogues
Johan VANDE VOORDE*, Federico GAGO†, Kristof VRANCKEN*, Sandra LIEKENS*1 and Jan BALZARINI*1
*Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, blok x – bus 1030, B-3000 Leuven, Belgium, and †Departamento de Farmacolog´ıa, Universidad de Alcala´,
E-28871 Alcala´ de Henares, Madrid, Spain
Figure S1 Purity evaluation of the M. hyorhinis TP–GST fusion protein
Three different volumes of the purified enzyme preparation (10 μl, 3 μl and 1 μl; 0.17 μg/μl)
were analysed using SDS/PAGE to evaluate the purity of the sample. Proteins were stained using
Bio-SafeTM Coomassie G-250 Stain (Bio-Rad Laboratories). Molecular mass is given on the
left-hand side in kDa.
1
Correspondence may be addressed to either of these authors (email jan.balzarini@rega.kuleuven.be or sandra.liekens@rega.kuleuven.be).
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J. Vande Voorde and others
Figure S2 Homology model of TPHyor in complex with TPI
(A) Stereoview of a cartoon representation of TPHyor (C atoms in dark green) in complex with TPI (C atoms in blue). (B) Detailed view of (A). The uracil base of TPI (C atoms, cyan; N atoms, blue;
and O atoms, red) is sandwiched between the hydrophobic side chains of Leu114 (above) and Ile184 (below), and held in place by hydrogen bonds (yellow broken lines) to the side chains of Arg168
,
Ser183 and Lys187 (stabilized by the carboxylate of Asp161), whereas the amino group on the pyrrolidine ring forms a strong hydrogen bond with the backbone carbonyl of Ser83. Phe207 appears in the
foreground, just in front of the 5-chlorine substituent on the uracil that is isosteric with the methyl group of thymine in the dThd substrate (not shown).
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Characterization of M. hyorhinis pyrimidine nucleoside phosphorylase
Figure S3 Sequence alignment and secondary structure prediction for TPHyor using human TP as the template
The predicted (Phyre2 server) secondary structure (green α-helices and blue β-sheets) of TPHyor closely resembles the known secondary structure of human TP.
Received 23 December 2011/30 March 2012; accepted 4 April 2012
Published as BJ Immediate Publication 4 April 2012, doi:10.1042/BJ20112225
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