Nucleoside phosphorylases from clostridium perfringens in the synthesis of 2',3'-dideoxyinosine

Article abstract of DOI:10.1080/15257771003741422

Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an antiviral drug 2',3'-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases, 2',3'-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%. Copyright

Full text of DOI:10.1080/15257771003741422

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Nucleoside Phosphorylases from
Clostridium Perfringens in the Synthesis
of 2′,3′-Dideoxyinosine
S. Christoffersen a , I. Serra b , M. Terreni b & J. Piškur a
a Department of Cell and Organism Biology , Lund University , Lund,
Sweden
b Dipartimento di Chimica Farmaceutica and Italian Biocatalysis
Center , University of Pavia , Pavia, Italy
Published online: 11 Jun 2010.
To cite this article: S. Christoffersen , I. Serra , M. Terreni & J. Piškur (2010) Nucleoside
Phosphorylases from Clostridium Perfringens in the Synthesis of 2′,3′-Dideoxyinosine, Nucleosides,
Nucleotides and Nucleic Acids, 29:4-6, 445-448, DOI: 10.1080/15257771003741422
To link to this article: http://dx.doi.org/10.1080/15257771003741422
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Nucleosides, Nucleotides and Nucleic Acids, 29:445–448, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257771003741422
NUCLEOSIDE PHOSPHORYLASES FROM CLOSTRIDIUM
PERFRINGENS IN THE SYNTHESIS OF 2ꢀ,3ꢀ-DIDEOXYINOSINE
S. Christoffersen,1 I. Serra,2 M. Terreni,2 and J. Pisˇkur1
1Department of Cell and Organism Biology, Lund University, Lund, Sweden
2Dipartimento di Chimica Farmaceutica and Italian Biocatalysis Center,
University of Pavia, Pavia, Italy
Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for
2
their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an
antiviral drug 2ꢁ,3ꢁ-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases,
2ꢁ,3ꢁ-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%.
Keywords
Nucleoside phosphorylase; Clostridium perfringens; biocatalysis; 2ꢁ,3ꢁ-
dideoxyinosine; anti-viral drugs
INTRODUCTION
Nucleoside phosphorylases (NPs) catalyze the cleavage of (deoxy)
ribonucleosides to give the free base and ribose 1-phosphate or deoxyri-
bose 1-phosphate, respectively.[1] Apart from its biomedical application,[2]
this class of enzymes is of interest because they can be used as biocatalysts for
the enzymatic synthesis of nucleoside analogues. In fact, NPs catalyze both
the phosphorolytic and synthetic reactions, and in the presence of a sec-
ond nucleobase (sugar acceptor) catalyze the synthesis of a new nucleoside.
Such transglycosylation reaction has been used for the one-pot synthesis of
several nucleosides starting from cheap, commercially available nucleosides
(“sugar donors”) and modified bases (“acceptor bases”).[3,4] In this work, we
explored the Clostridium perfringens NCTC8449 genome, identified five pu-
tative genes and subsequently subcloned and overexpressed four NPs. The
recombinant enzymes were subsequently used to synthesize the nucleoside
analogue 2ꢁ,3ꢁ-dideoxyinosine (ddI), a reverse transcriptase inhibitor which
Address correspondence to J. Piˇskur, Department of Cell and Organism Biology, Lund University,
So¨lvegatan 35, 22362 Lund, Sweden. E-mail: Jure.Piskur@cob.lu.se
445

446
S. Christoffersen et al.
is used in combination with other drugs in the highly active antiretroviral
therapy (HAART).
MATERIALS AND METHODS
Cloning and Purification
Three putative NPs genes encoding DeoD(1) (Gene ID 989708),
DeoD(2) (Gene ID 988651), and PunA (Gene ID 990873) were subcloned
from genomic DNA (kindly provided by A.R. Clausen) into TOPO 151 vec-
tor (Invitrogen Paisley, UK) giving the following plasmids, P967, P969, and
P968, respectively. The plasmids were overexpressed as fusion proteins with
a N-terminal His6 tag, and purified as reported by Knecht et al.[5] The to-
tal protein content of the soluble fraction was measured by the Bradford
method.
Purine NP Activity Assays
Xanthine oxidase coupled assay was used to test NPs towards inosine
and 2ꢁ-deoxyinosine. The assay was optimized from a previously reported
assay.[6] The activity was calculated from the increase in absorbance at 293
nm using 11.5 as millimolar extinction coefficient. The activity on guanine
and adenine nucleosides was assayed following the increase in absorbance at
260 nm. The amount of free base formed was deduced by using a millimolar
extinction coefficient of 1.316 and 5.4 for adenine and guanine, respectively.
HPLC-Based Assays
The reaction took place in 50 mM potassium buffer pH 7.5 (10 ml)
containing 5 mM of nucleoside substrate, and was started by the addition of
the enzyme, kept under mechanical stirring and stopped by ultra filtration
of the enzymes with the 10 kDa MWCO filter devices. The reaction was
monitored by HPLC. The column was a RP18 Lichrocart 250 × 4.6, 5 µm
(Merck, Darmstadt, Germany); eluent, 0.01 M potassium phosphate buffer
pH 4.6 and methanol 90% (97:3); flow, 1 ml/min; T = 35◦C; λ = 260 nm.
One-Pot Synthesis of ddI
A solution of 10 mM potassium buffer pH 7.5 (1.5 ml) containing 5 mM
of 2ꢁ,3ꢁ-dideoxyuridine (ddU) and 5 mM of hypoxanthine was prepared.
Reaction was started by the addition of the enzymes (2.4 U of UP and 40
U of either DeoD (1) or PunA). The reaction was kept under mechanical
stirring and stopped by ultra filtration of the enzymes with 10 kDa MWCO
filter devices. The reaction was monitored by HPLC as described above.

Clostridium perfringens in Nucleoside Phosphorylases
447
TABLE 1 Kinetic parameters of Clostridium Deod (1) and PunA
Enzyme
Substrate
Vmax (U/mg)
kcat (s−1
)
Km (µM)
kcat/Km (M−1s−1
)
DeoD (1)
Inosine
16
29.5
8
6.6
12
4.8
4.3
41.5
31.1
n.d.
7.9
14.5
3.9
3.2
6.1
2.7
2.4
23.1
17.4
n.d.
900
1300
400
400
200
279
400
249
174
n.d.
8777
10769
9750
8000
3050
9677
5988
92771
100000
n.d.
2ꢁ-deoxyinosine
Guanosine∗
2ꢁ-deoxyguanosine∗
Adenosine∗
PunA
Inosine
2ꢁ-deoxyinosine
Guanosine∗
2ꢁ-deoxyguanosine∗
Adenosine∗
U: µmol min−1
.
∗Double reciprocal plot calculation.
n.d. = not detected.
RESULTS AND DISCUSSION
The C. perfringens JCTC8449 genome codes for five putative NPs. We
cloned, overexpressed and characterized four of them with the exception
of pyrimidine nucleoside phosphorylase (DeoA). DeoD (1) and PunA be-
long to purine specific NPases (Table 1). One NP, DeoD (2), showed very
poor activity towards all the substrates tested and was not subjected to fur-
ther investigations. Clostridium uridine phosphorylase (UP, Gene ID 988640,
overexpressed from plasmid P966) is described elsewhere (in preparation).
For DeoD(1) Km values were 400 µM for guanosine and 2ꢁ-deoxyguanosine
and 200 µM for adenosine, while for inosine and 2ꢁ-deoxyinosine Km values
were higher: 900 and 1300 µM, respectively (Table 1). However, the latter
compounds had higher kcat values. Therefore, all substrates exhibited sim-
ilar catalytic efficiency (kcat/Km). PunA was specific for nucleosides based
on guanine or hypoxanthine as base, the Km values for all these compounds
were in the range of 174 to 400 µM, but phosphorolysis of hypoxanthine
nucleosides occurred to almost one tenth of guanine nucleosides (Table 1).
The enzymes were tested for their activity also towards ddI and showed a
specific activity of 0.09 and 0.02 U/mg for DeoD(1) and PunA, respectively
(Table 2). One pot bi-enzymatic synthesis of 2ꢁ,3ꢁ-dideoxyinosine was carried
TABLE 2 Specific activities of Clostridium NPases on 2ꢁ,3ꢁ-dideoxyriboside substrates (as determined by
the HPLC assay)
Enzyme
ddU (U/mg)
ddI (U/mg)
UP
DeoD(1)
PunA
1.9
—
—
—
0.09
0.02
U: µmol min−1
.

448
S. Christoffersen et al.
out with these two NPs and with UP that accepts 2ꢁ,3ꢁ-dideoxyuridine. Com-
bining Clostridium UP with either PunA or DeoD(1), we achieved after 24
hours about 30% yield of ddI.
In conclusion, we successfully cloned and overexpressed four C. per-
fringens NPs, and showed that two of them accept different native purine
substrates, including ddI (Tables 1 and 2). We could run a one-pot synthesis
approach [3,4] with two pairs of enzymes, PunA and UP, and DeoD(1) and UP,
using ddU and hypoxanthine as substrates and converting them into ddI. In
future, the yields could easily be optimized, for example in terms of reagents
concentrations, pH, temperature and by using immobilized biocatalysts.[6]
REFERENCES
1. Pugmire, M.J.; Ealick, S.E. Structural analyses reveal two distinct families of nucleoside phosphorylases.
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2005, 5, 1259–1274.
3. Prasad, A.K.; Trikha, S.; Parmar, V.S. Nucleoside synthesis mediated by glycosyl transferring enzymes.
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acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines.
EMBO J. 2002, 21, 1873–1880.
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