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Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [α-32P]d/rNTP

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Abstract

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d2CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k m and k cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α-32P]rNTP and [α-32P]dNTP.
ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 6, pp. 734–738. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © A.Yu. Skoblov, G.V. Mikoulinskaia, S.A. Taran, A.I. Miroshnikov, S.A. Feofanov, Yu.S. Skoblov, 2009, published in Bioorganicheskaya Khimiya, 2009,
Vol. 35, No. 6, pp. 816–821.
734
INTRODUCTION
The phosphorylation of 5' nucleotides to the corre-
sponding nucleoside 5' diphosphates plays a key role in
the process of the intracellular metabolism of precursors
of nucleic acid biosynthesis. This process is catalyzed by
specific enzymes called nucleoside monophosphate
kinases (ATP: nucleoside 5'-monophosphate transferase
(NMP kinases).
2
Over 50 years of intense study, much
information on the properties of these enzymes, their
mechanism of action, and their potential use in various
areas has been accumulated. This information was sys-
tematized in several reviews [1, 2].
As a rule, bacterial and eukaryotic NMP kinases are
highly specific to the heterocyclic base of the substrate,
whereas the specificity to sugar is expressed to a much
lesser degree.
In
E. coli
cells, five nucleoside monophosphate
kinases were found: adenylate kinase (EC 2.7.4.11),
thymidylate kinase (EC 2.7.4.9), guanylate kinase
(EC 2.7.4.8), cytidylate kinase, and uridylate kinase
(EC 2.7.4.14) [3, 4]. Human tissues contain thymidy-
late kinase, cytidylate–uridylate kinase, five isoforms
of adenylate kinase, and several guanylate kinases [5].
Enzymes encoded by bacteriophage genomes
occupy a special place among NMP kinases. For the
provision of the need for nucleotides, which increase in
the presence of bacteriophages, first of all, in thymidyl-
based nucleotides, many bacteriophages generate the
synthesis of their own monophosphate kinases. Due to
a small genome size, phages cannot “afford” the encod-
ing of the four enzymes. Therefore, phage monophos-
phate kinases often manifest a rather wide substrate
specificity, which, as a rule, correlates with the nucle-
otide composition of phage DNA. In particular, bacte-
riophages T2, T4, and T6, whose DNA contains hydro-
hymethylated cytidine, produce dNMP kinases
(EC 2.7.4.12) that can phosphorylate hydrohymethy-
lated (but not common) dCMP along with dGMP and
dTMP [6, 7].
T5 bacteriophage can also induce the synthesis of
dNMP kinase (EC 2.7.4.13). This enzyme is active
towards all four canonical substrates dAMP, dCMP,
dGMP, and dTMP. Its wide substrate specificity can be
explained by an increased need for dNMP induced by
infection. This specificity is especially low for T5
phage because it does not use a pool of cell nucleotides
for the synthesis of its own DNA [8].
Bessman’s group was the first to isolate and charac-
terize dNMP kinase from
E. coli
infected with T5 [9].
Unfortunately, as the enzyme content was very small,
Substrate Specificity of T5 Bacteriophage Deoxyribonucleoside
Monophosphate Kinase and Its Application for the Synthesis
of [
α
-
32
P
]d/rNTP
A. Yu. Skoblov
a
, G. V. Mikoulinskaia
b
, S. A. Taran
b
, A. I. Miroshnikov
a
,
S. A. Feofanov
b
, and Yu. S. Skoblov
a
,1
a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10,
Moscow, 117997 Russia
b
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Division, Russian Academy of Sciences, pr. Nauki 6,
Pushchino, Moscow oblast, 142290 Russia
Received April 21, 2009; in final form, May 5, 2009
Abstract
—Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown
to catalyze the phosphorylation of both d
2
CMP and ribonucleotides AMP, GMP, and CMP, but does not phos-
phorylate UMP. For natural acceptors of the phosphoryl group,
k
m
and
k
cat
were found. The applicability of T5
dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the syn-
thesis of [
α
-
32
P]rNTP and [
α
-
32
P]dNTP.
Key words: T5 bacteriophage deoxyribonucleoside monophosphate kinase, [
α
-
32
P]rNTP and [
α
-
32
P]dNTP,
synthesis
DOI:
10.1134/S1068162009060090
1
Corresponding author; phone: +7 (495) 336-2641; e-mail:
sur@ibch.ru.
2
Abbreviations: d
2
CMP, 2',3'-dideoxycytidine 5'-phosphate; PEI,
polyethyleneimine; PEP, phosphoenolpyruvate.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 35
No. 6
2009
SUBSTRATE SPECIFICITY OF T5 BACTERIOPHAGE DEOXYRIBONUCLEOSIDE 735
the authors failed to purify the enzyme to a homoge-
nous state. However, they determined the main param-
eters: optimum pH, potential substrates and the affinity
to them, and the dependence of activity versus the ions
of various bivalent metals.
The development of the gene engineering technique
supported the identification and cloning of the
dnk
gene
encoding T5 dNMP kinase, implemented its expression
in
E. coli
, and obtained considerable amounts of the
highly purified enzyme [10]. In this work, we report the
results of studies on the substrate specificity of T5
dNMP kinase and the use of this enzyme for the synthe-
sis of [
α
-
32
P]dNTP and [
α
-
32
P]rNTP.
RESULTS AND DISCUSSION
The unusually wide substrate specificity of T5
dNMP kinase towards phosphoryl group acceptors
makes this enzyme very attractive for application in the
enzymatic syntheses of nucleoside 5'-triphosphates.
Evidently, all natural dNMP can be phosphorylated
with T5 dNMP kinase. This potential was realized in
early studies [11]. However, the phosphorylation of
ribonucleotides with this enzyme was not studied in
detail.
We studied the kinetic properties of T5 dNMP
kinase for the phosphorylation of various substrates by
the spectrophotometrical determination of Michaelis
constants (
k
m
) and catalytic constants (
k
cat
) [12]. As is
seen in the table, the enzyme as a phosphoryl group
acceptor demonstrated the maximal affinity to dCMP
(
k
m
0.037). It is noteworthy that the
k
m
and
k
cat
values
for the process of phosphorylation of all natural dNMP
were similar and only varied by a few times. At the
same time, the kinetic parameters of ribonucleotide
phosphorylation dramatically differed:
k
m
of AMP,
GMP, and CMP reactions differed by three times,
whereas
k
cat
differed by 100 times. It is noteworthy that
the pH optima for dAMP–AMP and dCMP–CMP pairs
were the same and were close to a value of 7.0 (data not
shown).
The data on UMP phosphorylation are not given in
the table, since the enzyme activity with UMP as an
acceptor of the phosphoryl group could not be mea-
sured by optical methods. For the determination of the
applicability of T5 dNMP kinase to be used for UMP
phosphoryration, the substrates labeled with phospho-
rus-32 with a high specific activity were used (see the
Experimental section). The use of substrates labeled
with phosphorus-32 essentially increased the sensitiv-
ity of the method both for the case of [[
γ
-
32
P]ATP as a
phosphoryl donor and for the case of [5'-
32
P]UMP as its
acceptor. As UMP phosphorylation turned out to be
very ineffective, the testing of [[
γ
-
32
P]ATP as a donor
yielded ambiguous results. Therefore, the activity of T5
dNMP kinase was tested using [5'-
32
P]UMP with a spe-
cific activity of 3000 Ci/mmol (Fig. 1). In this case, the
rate of UMP phosphorylation was 1 pmol/min. The
reaction mixture contained 0.4 U T5 dNMP kinase (see
the Experimental section). When considering that a
0.4-U enzyme catalyzes the conversion of 0.4
μ
mol
dCMP per minute, one can conclude that the efficacy of
UMP phosphorylation is extremely low. A direct com-
parison of UMP and dCMP phosphorylation rates is
inconsistent, because the concentrations of substrate–
acceptor phosphoryl groups are incomparable (4
μ
mol
and 1 mM, respectively, see the Experimental section).
However, one must accept that the 10
4
- to 10
5
-fold dif-
ference in the reaction rates is very significant.
As a whole, the phenomenon of T5 dNMP kinase
specificity towards natural dNMP is unique: the kinetic
parameters of the phosphorylation of dCMP–CMP or
dAMP–AMP pairs are close; whereas
k
cat
for the
Kinetic characteristics of T5 bacteriophage dNMP kinase in
the reactions with different phosphoryl acceptors and ATP as
a phosphoryl donor determined by the spectrophotometrical
method
Substrate
K
m
, mM
k
cat
, c
–1
dAMP 0.275 58
rAMP 0.367 31
dGMP 0.267 43.2
rGMP 0.81 0.29
dCMP 0.037 24.7
rCMP 0.39 5.6
dTMP 0.190 38.7
dUMP 4.2 10
rATP (c dAMP) 0.042 58
123
[5'-
32
P]UMP
[
α
-
32
P]UDP
Fig. 1.
An autoradiograph of a chromatogram on PEI cellu-
lose of [
α
-
32
P]UMP phosphorylation catalyzed by T5
dNMP kinase after incubation for 0 (
1
, control), 15 (
2
), and
60 min (
3
). The portion of [
α
-
32
P]UDP in aliquot
3
deter-
mined using a phosphorimager was 10% of the total activity
of the reaction mixture.
736
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 35
No. 6
2009
SKOBLOV et al.
dGMP–GMP pair differs by 200 times, and for the
dUMP–UMP pair, by more than 10
4
times.
It is difficult enough to understand the reason for
such dramatic differences in the efficacies of dUMP
and UMP phosphorylation in view of the phosphoryla-
tion potential towards other natural nucleotides. One
can assume that these crucial differences result from
the fact that UMP phosphorylation may be involved in
the regulation of the synthesis of the ribonucleoside
triphosphate pool in a phage-infected cell. However,
the molecular mechanism of such a specificity cannot
be comprehended without deep insight into the struc-
ture of the active site of the enzyme and its interaction
with the substrates. Similar specificity was found for
neither of the known NMP kinases isolated from bacte-
rial, plant, or animal cells because the specificity of
these enzymes towards phosphoryl group acceptors is
determined by the nucleotide heterocyclic base and, to
a lesser degree, the carbohydrate moiety of the mole-
cule [1].
Unfortunately, our attempts to phosphorylate 5-sub-
stituted derivatives of cytidyl 5'-nucleotides failed.
Even with the great excess of the enzyme phosphoryla-
tion of 5-substituted (biotinylated and fluorescent)
dCMP derivatives (see the structures) with the use of
[[
γ
-
32
P]ATP as a phosphoryl donor, the phosphoryl was
not detected. At the same time, unlike
E. coli
CMP
kinase, T5 dNMP kinase effectively phosphorylated
2',3'-dideoxycytidine 5'-phosphate (Fig. 2). This prop-
erty of T5 dNMP kinase is especially interesting in
view of the potential technological use of the enzyme
for ddNTP syntheses. We plan to study these opportu-
nities in the future.
HO3S
NN
SO3H
ONH
N
N
NH2
OO
O
OH
PO
O
O
ONH
N
N
NH2
OO
O
OH
PO
O
O
H
N
S
O
NH
HN
O
()(b)
+
Structures of 5-substituted 2'-deoxycytidine 5-phosphate derivatives: (a) a fluorescent derivative
containing a CY-5 dye and (b) biotinyl derivative.
[
β
-
32
P]d
2
CDP
12345
[
γ
-
32
P]ATP
[
β
-
32
P]dCDP
Fig. 2.
A TLC autoradiograph on PEI cellulose of aliquots of d
2
CMP and dCMP phosphorylation catalyzed by T5 dNMP kinase (
2
and
3
, respectively) and
E. coli
CMP kinase (
4
and
5
, respectively);
1,
[
γ
-
32
P]ATP (control).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 35
No. 6
2009
SUBSTRATE SPECIFICITY OF T5 BACTERIOPHAGE DEOXYRIBONUCLEOSIDE 737
Taking into consideration the capacity of T5 dNMP
kinase to phosphorylate both dNMP and rNMP, we used
the enzyme for the preparative syntheses of [5'-32P]dATP
and [5'-32P]rATP. The synthetic conditions for [5'-32P]dATP
and [5'-32P]rATP from the corresponding [5'-32P]mono-
phosphates are described in the Experimental section.
The two-step phosphorylation, namely, T5 dNMP
kinase-catalyzed phosphorylation followed by a reac-
tion with piruvate kinase and PEP, was performed in
situ without the isolation of intermediate products. We
synthesized [5'-32P]dATP, [5'-32P]dGTP, [5'-32P]dCTP,
and [5'-32P]TTP, as well as [5'-32P]ATP, [5'-32P]GTP
and [5'-32P]CTP, in 80–90% yields and specific activi-
ties of 3–4 kCi/mmol. The attempt to use T5 dNMP kinase
for the preparative phosphorylation of [5'-32P]UMP failed.
A reasonable product yield was not achieved even in
the presence of considerable excesses of the enzyme
after several hours of incubation.
It is noteworthy that the synthesis catalyzed by T5
dNMP kinase and piruvate kinase was performed in the
presence of a small excess of ATP and a significant
excess of the enzymes and PEP. Such a ratio of the com-
ponents in the reaction mixture is characteristic of the
enzymatic synthesis of phosphorus-labeled com-
pounds. It enables successive NMP phosphorylation by
the common mixing of all reagents in a reaction con-
tainer, a reduction of the reaction time, and the simpli-
fication of the chromatographic purification of the
product. The target product was isolated from the reac-
tion mixture by reverse-phase HPLC in ion-pair mode.
The use of [5'-32P]ATP as a donor of the phosphoryl
group in the phosphorylation of dATP allowed us to
maintain the molar activity of [5'-32P]ATP.
Thus, by using T5 dNMP kinase for the synthesis of
nucleoside 5'-triphosphates labeled with phosphorus-32 at
the α position, we simplified the synthesis with the
retention of high product yields. Apparently, the
obtained results can be used for the synthesis of similar
compounds labeled with phosphorus-33. In general, the
technological approach of the replacement of low-spe-
cific E. coli NMP kinases by a “universal” enzyme capa-
ble of effectively phosphorylating nucleoside 5'-mono-
phosphates is promising and, when properly developed,
can expand into other technological processes where
d/rNMP need phosphorylation.
EXPERIMENTAL
Materials. Tris, phosphoenol piruvate (PEP), NADH
(reduced), piruvate kinase, and lactate dehydrogenase con-
taining piruvate kinase were from Sigma; plates with PEI
cellulose were from Merck; [γ-32P]ATP (radioactive con-
centration of 10 mCi/ml, molar activity of 4000 Ci/mmol)
was purchased from TsKP Phosphor, Russian Academy of
Sciences. d2CMP was synthesized from 2',3'-dideoxycyti-
dine and phosphorus oxychloride as described in [13]. All
[5'-32P]rNMP and [5'-32P]dNMP were prepared as
described in [14] with a molar activity of 3000 Ci/mmol.
E. coli CMP kinase was isolated according to [4] with a
specific activity of 4 U/mg. T5 dNTP kinase was obtained
as described in [10].
Determination of the Activities of Nucleoside
Monophosphate Kinases
The enzymatic activity of T5 dNMP kinase was
determined using two methods. 1) Spectrophotometri-
cally by the oxidation of NADH. Constants Km and kcat
were determined as described in [9]. The final reaction
mixture 1 ml in volume contained 50 mM Tris-HCl
(pH 7.5), 80 mM KC1, 8 mM MgCl2, 2 mM EDTA,
0.8 mM PEP, 0.2 mM Äíê, 0.1 mM NADH, 2 mM
NMP or dNMP, 20 U lactate dehydrogenase containing
piruvate kinase, and the tested enzyme (0.02–0.2 U).
The reaction was performed in an acryl cuvette at 25°C
for 3–5 min. The rate of NADH oxidation was mea-
sured spectrophotometrically by decreasing the optical
absorption at 340 nm. 1 U was taken as the enzyme
amount capable of catalyzing the conversion of 1 μmol
of dCMP per minute at 25°C. 2) By the phosphoryla-
tion of nucleotides with [γ-32P]ATP as a donor of a
phosphoryl group. The reaction mixture 25 μl in vol-
ume contained a 50 mM Tris–HCl buffer (pH 7.6),
5 mM MgCl2, 1 mM nucleoside 5'-monophosphate,
0.1 M KCl, 0.1 mM ATP, 1 μCi [γ-32P]ATP, and 0.05 U
of the tested enzyme. The mixture was incubated at
37°C. Aliquots (0.5 μl) were taken out in varied time
intervals and loaded on PEI cellulose plates. TLC was
performed in 0.5 M KH2PO4. After the chromatogra-
phy, the plate was dried and the products on PEI cellu-
lose plates were visualized using a Packard Cyclone
Storage Phosphor System.
The activity of T5 dNMP kinase in the reaction with
UMP. The reaction mixture 25 μl in volume contained
a 50-mM Tris–HCl buffer (pH 7.6), 5 mM MgCl2,
0.1 M KCl, 0.1 mM ATP, 300 μCi [5'-32P]ATP, and
0.4 U T5 dNMP kinase. The mixture was incubated at
37°C. Aliquots (0.5 μl) were taken out at 20 and 60 min
and loaded on PEI cellulose plates. TLC was performed
in 0.5 M KCl. The products on PEI cellulose plates
were visualized using a Packard Cyclone Storage Phos-
phor System.
Synthesis of [
α
-32P]dCTP. A mixture of [5'-32P]dCTP
(10 mCi, 2.5–3 nmol), 5 U T5 dNMP kinase, and 5 U
piruvate kinase was added to the reaction mixture
(100 μl) containing a 50-mM Tris–HCl buffer (pH 8.0),
5 mM MgCl2, 0.2 M KCl, 0.05 mM ATP, 5 mM dithio-
threitol, and 5 mM PEP. The mixture was incubated at
37°C for 30 min. For the determination of the reaction
yield, an aliquot (0.2–0.3 μl) was analyzed by TLC on
a PEI cellulose plate in a 0.5-M potassium phosphate
buffer (pH 4.0). The plate was dried and visualized
using a phosphorimager or autoradiography. The prod-
uct yield was 90% relative to radioactivity. The target
product was isolated by reverse-phase HPLC in ion-
pair mode on a C-18 column in a gradient of ethanol in
50 mM triethylammonium bicarbonate. The final yield
738
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009
SKOBLOV et al.
of [α-32P]dCTP was 7.5 mCi (75% per the starting
compound).
Synthesis of [
α
-32P]ATP. A mixture of [5'-32P]AMP
(5 mCi, 1.5–2 nmol), 5 U T5 dNMP kinase, and 5 U
piruvate kinase was added to the reaction mixture
(100 μl) containing a 50-mM Tris–HCl buffer (pH 8.0),
5 mM MgCl2, 0.2 M KCl, 5 mM dithiothreitol, 0.1 mM
dATP, and 5 mM PEP. The mixture was incubated at
37°C for 30 min. For determination of the reaction
yield, an aliquot (0.2–0.3 μl) was analyzed by TLC on
a PEI cellulose plate in a 0.5-M potassium phosphate
buffer (pH 4.0). The plate was dried and visualized
using a phosphorimager or autoradiography. The prod-
uct yield was 90% by radioactivity. The target product
was isolated by reverse-phase HPLC in ion-pair mode
on a C-18 column in a gradient of ethanol in 50 mM tri-
ethylammonium bicarbonate. The final yield of
[α-32P]ATP was 4.5 mCi (90% per the starting com-
pound).
CONCLUSIONS
The enzyme can effectively phosphorylate AMP,
dAMP, GMP, dGMP, CMP, dCMP, d2CMP, TMP, and
dUMP, but not UMP, and can be used for the prepara-
tive synthesis of the corresponding nucleoside 5'-triph-
osphates labelled with radioactive isotopes.
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research, project no. 08-04-00653-a.
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Crude 3'-azido-3'-deoxythymidine-5'-phosphate (AZT-P), obtained from direct phosphorylation of 3'-azido-3'-deoxythymidine (azidothymidine, AZT), was separated and purified by isocratic preparative high-performance liquid chromatography. The components in a 2.5-g load of crude AZT-P, obtained from work-up of the phosphorylation reaction, were separated in 50 min to give 1.8 g of 99.5% pure AZT-P. AZT-P was analyzed by high-performance liquid chromatography and by high-resolution nuclear magnetic resonance (1H, 13C, 31P) spectroscopy. The practical and rapid preparative chromatographic method is being applied to the purification of AZT-P and other antiretroviral dideoxynucleotides, used as intermediates in the synthesis of target-directed experimental drugs for the treatment of AIDS.
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A technique is proposed for isolation of nucleosidemonophosphate kinases--AMP-kinase (EC 2.7.4.11), GMP-kinase (EC 2.7.4.8), CMP-kinase (EC 2.7.4.14), UMP-kinase (EC 2.7.4.14) and TMP-kinase (EC 2.7.4.9)--from E. coli MRE-600. It involves cell destroying, precipitation of nucleic acids with polyethyleneimine, fractionation with ammonium sulphate followed by chromatography on different carriers (DEAE-Toyopearl-650 M, Matrex gel Blue A, Matrex gel Red A). The technique enables all the five enzymes to be obtained separately and without contaminations with nucleotide dephosphorylating enzymes. For all the enzymes the pH optimum was found to range from 6.5 to 8.0, and Mg2+ ions were found to be the best activator for all the enzymes studied. The substrate specificity was investigated with respect to acceptors and donors of the phosphate groups. The enzymes showed strict specificity to the heterocyclic base of the acceptor phosphate group. AMP-, GMP- and CMP-kinases phosphorylated the corresponding deoxynucleoside monophosphates less effectively than ribonucleoside monophosphates. ATP was found to be the most effective phosphate donor for all the enzymes under study.