From the Department of Biological Chemistry, University of Copenhagen, Sølvgade 83, DK 1307 Copenhagen K, Denmark
Received for publication, August 29, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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The overlapping yaaG and
yaaF genes from Bacillus subtilis were cloned
and overexpressed in Escherichia coli. Purification of the
gene products showed that yaaG encoded a homodimeric
deoxyguanosine kinase (dGK) and that yaaF encoded a
homodimeric deoxynucleoside kinase capable of phosphorylating both
deoxyadenosine and deoxycytidine. The latter was identical to a
previously characterized dAdo/dCyd kinase (Møllgaard, H. (1980)
J. Biol. Chem. 255, 8216-8220). The purified
recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the
preferred phosphate donor with a Km value of 6 µM compared with 36 µM for ATP. In
addition, the Km for dGuo was 0.6 µM
with UTP but 6.5 µM with ATP as phosphate donor. The
combination of these two effects makes UTP over 50 times more efficient
than ATP. Initial velocity and product inhibition studies indicated
that the reaction with dGuo and UTP as substrates followed an Ordered
Bi Bi reaction mechanism with UTP as the leading substrate and
UDP the last product to leave. dGTP was a potent competitive inhibitor
with respect to UTP. Above 30 µM of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.
Biosynthesis of 2'-deoxyribosyl groups occurs solely through
reduction of the 2'-hydroxyl group of ribonucleoside di- or
triphosphates, catalyzed by ribonucleotide reductases (1, 2). In
addition, a number of organisms possess deoxynucleoside kinases that
provide a salvage pathway for the utilization of preformed
deoxynucleosides as precursors of DNA. Because the cytotoxicity of a
large variety of deoxynucleoside analogs depends on the conversion of
these compounds to the corresponding deoxynucleotide analogs,
characterization of the substrate specificity and regulation of
deoxynucleoside kinases from various sources have received considerable
attention (3).
Thymidine kinase (dTK),1
which can generally use both Thd and dUrd as substrates, is widely
distributed in both prokaryotes and eukaryotes. In contrast, only
relatively few genera have been shown to express deoxyguanosine kinase
(dGK), deoxycytidine kinase (dCK), and deoxyadenosine kinase (dAK)
activities. From mammalian tissues four deoxyribonucleoside kinases,
TK1, TK2, dCK, and dGK, with overlapping substrate specificities, have
been characterized (for review see Ref. 3). TK1 and TK2 are
pyrimidine-specific, phosphorylating Thd and dUrd. TK2 can in addition
use dCyd as a substrate (4-6). dCK phosphorylates dCyd, dAdo,
and dGuo (7, 8), and the mitochondrial dGK is specific for dGuo and
dAdo (9, 10). A deoxynucleoside kinase with a very different substrate specificity was recently characterized from Drosophila
melanogaster. In this organism a single homodimeric enzyme
(Dm-dNK) is capable of phosphorylating all four
deoxyribonucleosides, although with widely different efficiencies
(11-13).
Among eubacteria only two genera, Lactobacilli (14) and
Bacilli (15), have been shown to phosphorylate all four
deoxynucleosides, whereas it has been established that a number of
bacteria, including Escherichia coli and Salmonella
enterica serovar Typhi, are lacking dGK, dAK, and dCK activities
(16). In Lactobacillus acidophilus strain R26, a strain that
appears to lack a functional ribonucleotide reductase (14), the
deoxynucleoside kinase activities are organized as three enzymes. In
addition to a separate dTK, the remaining three activities are located
on two heterodimeric proteins, dGK/dAK and dCK/dAK, with each subunit
being highly specific for the individual substrates, and specifically
feedback inhibited by its respective dNTP end-product (17). The three
subunits of the two heterodimeric enzymes are encoded by two tandem
genes, dak and dgk, where dak encodes
the dAK subunit of both enzymes, and dgk encodes both dGK
and dCK. The only difference between the amino acid sequences of the
two latter subunits is that dCK lacks the N-terminal amino acid
residues 2 and 3. The mechanism responsible for the co- or post-translational deletion event has not yet been identified (18). In
contrast, the organization of the activities in Bacillus subtilis is quite different. dTK and dGK are genetically distinct and different from the enzyme that phosphorylates dAdo and dCyd (15).
The present report concerns the molecular cloning of two overlapping
B. subtilis genes encoding two homodimeric enzymes, dGK and
dAK/dCK, and describes the purification and characterization of the
recombinant dGK overexpressed in E. coli. The dAK/dCK enzyme appeared to be identical to the deoxyadenosine/deoxycytidine kinase previously purified and characterized from B. subtilis
(15).
Materials
Tris (TRIZMA base), BSA, nucleotides, and nucleosides were
obtained from Sigma-Aldrich, Denmark, and 2',3'-dideoxynucleosides were
gifts from Dr. H. G. Ihlenfeldt, Roche Molecular Biochemicals. Phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase were
from of Roche Molecular Biochemicals. Restriction endonucleases and T4
DNA ligase were purchased from New England BioLabs, and [8-3H]-2'-deoxyguanosine was from Moravek Biochemicals.
Dyematrex Blue A and Red A were from Amicon, and Sephacryl S-300 was
from Amersham Pharmacia Biotech. DE-81 ion exchange paper was from Whatman Ltd., United Kingdom. SDS-PAGE low range molecular weight standards were purchased from Bio-Rad Laboratories.
Methods
Bacterial Strains and Growth Conditions--
E. coli
SØ5110 (MC1061 cdd::Tn10 (19)) was
used as host strain and pUC18 as cloning vector throughout. Cells were
grown at 37 °C in LB medium (20). When required, ampicillin was
added to the medium at 100 µg/ml.
DNA Techniques--
The methods used for preparing chromosomal
DNA and for transforming E. coli have been described
previously by Sambrook et al. (21). Plasmids were isolated
from E. coli by the alkaline/SDS lysis procedure (22).
Fragments from digested plasmid DNA were isolated and purified from
agarose gels by the QIAquick gel extraction kit (Qiagen, Germany).
Endonuclease digestion and ligation of DNA was done according to the
recommendations of the suppliers. DNA was sequenced by the chain
termination method (23) on polymerase chain reaction (PCR) products,
using the BigDye Terminator cycle sequencing kit (PE Applied
Biosystems, Warrington, United Kingdom) and an ABI Prism 310 genetic
analyzer (PE Applied Biosystems).
Plasmid Constructions--
The overlapping yaaG and
yaaF genes were amplified from genomic B. subtilis DNA by PCR using Vent polymerase (New England BioLabs Inc.). The 5'-primer, BsdNT4
(5'-GAAGGTCGGATCCTGACGCACG-3'), was designed to contain a
BamHI site (underlined) 35 bp upstream of the
translational start codon of yaaG. The 3'-primer, BsdNT5 (5'-GTTTTGAAGCTTGCATTCGGTGCGGCG-3'), was designed to
contain a HinDIII (underlined) 40 bp downstream
of the yaaF stop codon. The resulting amplicon was digested
with BamHI and HindIII and ligated into similarly
digested pUC18, yielding pRA1. Because the reading frames of
yaaG and the vector-borne lacZ' happened to be
the same on pRA1, the BamHI site of the plasmid (located between the lac promoter and the cloned fragment) was
opened, filled in with the Klenow fragment of E. coli DNA
polymerase, and religated. The lacZ' reading frame of the
resulting plasmid, pRA8, was changed, and the BamHI site was
replaced by a unique ClaI site (see Fig. 1). Plasmid pRA5
was obtained by subcloning the 825-bp
PstI-HindIII fragment of pRA8 into pUC18. Plasmid
pRA9 was obtained by cloning the 713-bp EcoRI fragment into
the EcoRI site of pUC18. Plasmids with the correct
orientation relative to the lac promoter were identified by
PstI digestion (see Fig. 1).
Separation of dGK and dAK/dCK--
A 1-liter culture of E. coli SØ5110 containing plasmid pRA8 was grown overnight at
37 °C in LB medium containing ampicillin, harvested by
centrifugation, and subjected to sonication in 15 ml of 50 mM Tris-HCl, pH 7.8 (buffer A). Following centrifugation at
7000 × g for 10 min at 4 °C, the supernatant was
centrifuged at 100,000 × g for 1 h in a Beckman
L8-80 ultracentrifuge. The supernatant was subjected to ammonium
sulfate fractionation, and the precipitate formed between 45 and 70%
saturation was redissolved and dialyzed overnight against buffer A. The
dialyzed fraction was applied to a Dyematrex RedA column (1.5 × 16 cm), washed with 10 volumes of buffer A, and the column was eluted
with 5 volumes of a linear KCl gradient (0-2 M) in buffer
A. Fractions with dNK activity were pooled, concentrated by ammonium
sulfate precipitation, redissolved, dialyzed against buffer A, and
applied to a Dyematrex Blue A column (1.5 × 16 cm). The column
was washed with 10 volumes of buffer A and eluted as above. Fractions
34-37, containing the highest dGK activity, and fractions 46-51,
containing the highest dAK/dCK activity, were pooled separately,
concentrated by ammonium sulfate precipitation, redissolved, and
dialyzed against buffer A.
Purification of dGK--
One liter of an early stationary phase
culture of E. coli SØ5110/pRA9 in LB medium containing
ampicillin was harvested by centrifugation and washed once with 0.9%
NaCl, and the resultant cell pellet was suspended in 15 ml of buffer A
containing 5 mM dithiothreitol and 1 mM EDTA.
The suspension was subjected to sonication followed by centrifugation
to remove cell debris. The supernatant was further centrifuged for
1 h at 100,000 × g in an ultracentrifuge, and the
100,000 × g supernatant was applied to a Dyematrex
Blue A column (1.5 × 16 cm). The column was washed with 5 volumes
of buffer A followed by 30 ml of a linear gradient of KCl (0-0.2
M) in buffer A. The activity was recovered by subsequent isocratic elution with 0.2 M KCl in buffer A. Fractions
containing dGK activity were pooled and applied to a phenyl-Sepharose
Hi Performance column (1.5 × 15 cm). The column was washed with
50 ml of 25 mM Tris-HCl, pH 7.8, followed by 50 ml of 5 mM Tris-HCl, pH 7.8, 20% 2-propanol, 5% ethanediol, and
subsequently eluted with 1% Triton X-100. Fractions with the highest
specific activity were pooled, dialyzed against buffer A, and
concentrated by ultrafiltration using a Centriprep centrifugal
concentrator (Amicon, Inc.). The concentrated enzyme was stored in 50%
(v/v) glycerol at Enzyme Assays--
Deoxyguanosine kinase activity was measured
with one of two procedures. Assay 1 measured the conversion
of [3H]dGuo to [3H]dGMP. The reaction
mixture contained 50 mM Tris-HCl, pH 8.5, variable
concentrations of [3H]dGuo and nucleoside triphosphate, 5 mM MgCl2, and 1 mg/ml BSA in a final volume of
50 µl. The reaction was started by addition of enzyme and performed
at 37 °C. Four 10-µl aliquots of the reaction mixture were taken
at different times after addition of enzyme. These were mixed with 5 µl of 1.5 M formic acid to stop the reaction, and the
entire mixture was pipetted onto DE-81 filter discs and dried. The
discs were washed in deionized water for 30 min and dried, and the
bound radioactivity was eluted from the filters with 1 ml of 0.1 M HCl, 0.2 M KCl and counted in a Tri-Carb
liquid scintillation analyzer (Packard Instruments, Inc., Downers
Grove, IL) using 5 ml of Ecoscint A scintillation fluid (National
Diagnostics). The amount of enzyme used in the assays was such that
less than 10% of the substrate was converted to product during the
reaction. All samples were taken within the linear part of the
reaction, and initial velocities were determined from the slope of the
lines. Assay 2 measured spectrophotometrically the oxidation
of NADH coupled enzymatically to the phosphorylation of nucleoside to nucleotide. The reaction mixture (500 µl) contained 50 mM
Tris-HCl, pH 7.8, 100 mM KCl, 5 mM
MgCl2, 1.5 mM NTP, 0.1-0.4 mM
nucleoside, 0.5 mM phosphoenolpyruvate, 0.18 mM
NADH, 2 units of pyruvate kinase (10 units when UTP or CTP was used as
phosphate donor), 2.5 units of lactate dehydrogenase, and enzyme. The
reaction was started by addition of nucleoside, and the decrease in
absorbance at 340 nm was followed at 37 °C. One enzyme unit is
defined as the amount of enzyme that catalyzes the formation of 1 µmol of dGMP per min. Protein was determined by the method of Lowry
et al. (24) using BSA as a standard.
Data Treatment--
Km and
Vmax values were obtained by fitting
steady-state kinetic data to the rate equation for a sequential Bi Bi mechanism with the Biosoft program UltraFit 3.0 for Macintosh. Ki values were calculated from secondary plots of
inhibitor concentration versus intercept or slope of the
double-reciprocal plots using linear least-squares regression analysis
to determine the best-fit line describing the data.
Mass Spectrometry--
The mass spectrometry was performed
by Dr. Jette Wagtberg Sen at the Statens Seruminstitut, Copenhagen,
using a Mariner electrospray time-of-flight spectrometer (PerSeptive Biosystems).
Denaturing Polyacrylamide Gel Electrophoresis--
Protein
samples were incubated for 5 min at 100 °C in 50 mM
Tris-HCl, pH 8.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue and applied on a 15% polyacrylamide-SDS gel (25). Gels were run at 40 mA
for 70 min and silver-stained (26).
Cloning of the B. subtilis yaaG and yaaF Genes--
The amino acid
sequences of dAK and dGK from L. acidophilus, deduced from
the nucleotide sequence of the dak/dgk tandem genes (27),
were used to search the B. subtilis genome sequence (28) for
open reading frames encoding putative deoxynucleoside kinases. Two
genes, yaaG and yaaF, were identified at
coordinates 22497-23767 bp, which encoded putative polypeptides with
25-32% amino acid sequence identity to the L. acidophilus
deoxynucleoside kinases. The nucleotide sequence indicated that the TGA
stop codon of yaaG overlapped 2 bp with the ATG start codon
of yaaF, and that both open reading frames were preceded by
typical B. subtilis Shine-Dalgarno sequences (29). The
deduced amino acid sequences of the two gene products were 30% identical.
The putative yaaG/yaaF operon was amplified from B. subtilis genomic DNA by PCR and cloned into pUC18, resulting in
pRA8 (Fig. 1). Subsequently,
yaaG and yaaF were subcloned separately in pUC18 using the internal PstI and EcoRI sites of pRA8,
yielding plasmids pRA9 and pRA5, respectively (Fig. 1). In all
constructs the cloned genes were transcribed from the lac
promoter (lacP) of the vector and translated from the native
ribosomal binding regions. DNA sequencing of the cloned fragments of
pRA9 and pRA5 confirmed the published genomic sequence and showed that
they encoded potential polypeptides of 207 and 217 amino acid residues,
respectively. SDS-PAGE of total proteins from cultures of E. coli harboring pRA9 or pRA5 revealed a major band with a mobility
corresponding to a 24-kDa polypeptide, in accordance with the molecular
masses of the yaaG and yaaF gene products as
deduced from the nucleotide sequence (data not shown).
Identification of the yaaG and yaaF Gene Products--
Crude
cellular extracts of E. coli SØ5110 harboring pRA8, pRA9,
and pRA5 were assayed for deoxynucleoside kinase activity with dGuo,
dAdo, and dCyd as substrates (Table I).
The results suggested that yaaG encoded a kinase specific
for deoxyguanosine, whereas the yaaF gene product was
capable of phosphorylating both deoxyadenosine and deoxycytidine.
To establish whether all three kinase activities were part of a single
heterooligomeric enzyme in conditions where both yaaG and
yaaF were present in the cells, crude extracts of E. coli SØ5110/pRA8 were subjected to both ammonium sulfate
fractionation and dye-affinity chromatography. Table
II shows that the kinase activities for
dGuo, dAdo, and dCyd copurified upon ammonium sulfate precipitation and
on RedA dye chromatography. However, on a BlueA dye column
deoxyguanosine kinase activity eluted clearly before the deoxyadenosine
and deoxycytidine kinase activities. Together with the results
presented in Table I, this indicated that the gene products of
yaaG and yaaF formed two physically distinct enzymes, dGK and dAK/dCK. Preliminary characterization of the recombinant dAK/dCK from the BlueA dye column indicated that it was
identical to the deoxyadenosine/deoxycytidine kinase previously purified and characterized from B. subtilis (15).
Purification of Recombinant dGK--
Having established that
deoxyguanosine kinase was encoded by the yaaG gene, the
recombinant enzyme was purified from cells of E. coli
SØ5110 harboring pRA9. Because preliminary experiments showed that
ammonium sulfate precipitation resulted in heavy losses, this step was
avoided. The final procedure adopted involved fractionation by BlueA
dye affinity chromatography and separation according to hydrophobicity
on a phenyl-Sepharose column producing an enzyme that was >99% pure
as judged by SDS-PAGE, with an overall yield of 25% (Table
III).
Molecular Weight--
The molecular mass of the subunit was
determined to be 24,147 Da by electrospray mass spectrometry (data not
shown), in accordance with the molecular mass of the yaaG
gene product deduced from the DNA sequence (24,145 Da). The molecular
mass of purified recombinant dGK was about 49 kDa as estimated by gel
filtration on a Sephacryl S-300 column equilibrated with 50 mM Tris-HCl, pH 7.8, suggesting that the enzyme was a
homodimer under the conditions employed.
Stability--
In the presence of 50% v/v glycerol, concentrated
solutions of the enzyme (>1 mg/ml) could be stored for at least 1 year
at pH Optimum--
With saturating concentrations of dGuo and UTP,
dGK showed a broad pH optimum centered around pH 9. At pH 7.5 and 11.5 more than 80% of maximal activity was still observed. At pH 6.0, 60% activity remained, whereas the enzyme was completely inactive below pH
5.6 (data not shown).
Metal Ion Requirement--
As observed for most kinases, dGK
required Mg2+ for activity, suggesting that the true
phosphate donor is MgNTP2 Phosphate Acceptor Specificity--
The specificity of the enzyme
toward the nucleoside substrate was determined using the coupled assay
and GTP as the phosphate donor. Naturally occurring ribo- and
deoxyribonucleosides as well as certain nucleoside analogs were tested.
As shown in Table IV, the enzyme is
highly specific toward 6-oxopurine 2'-deoxynucleosides. Only marginal
activity was observed with guanosine, and neither 2',3'-dideoxynucleosides nor hypoxanthine arabinoside served as substrates.
Phosphate Donor Specificity--
Previous studies with dAK/dCK
from B. subtilis indicated that GTP and dGTP were the
preferred phosphate donors with both dAdo and dCyd as substrates; ATP
and UTP were only half as effective (15). Preliminary experiments with
dGK indicated that all naturally occurring ribo- and
deoxyribonucleoside triphosphates, with the exception of dGTP, were
active as phosphate donors. Under the conditions used (20 µM dGuo and 25 µM NTP) UTP was slightly
more efficient than the other NTPs. With dGTP no measurable activity was observed (data not shown).
Steady-state Kinetics--
Initial velocity experiments were
carried out at varying concentrations of the four individual
ribonucleoside triphosphates and fixed concentrations of dGuo. For each
of the NTPs, double reciprocal plots of the initial rate as a function
of the NTP concentration for various fixed dGuo concentrations yielded
a series of intersecting lines, diagnostic of a sequential reaction mechanism (data not shown). The data obtained were fitted to the rate
equation for a sequential Bi Bi mechanism. The kinetic constants obtained (Table V) showed that the true
Vmax values for the various NTPs were quite
similar, whereas large variations in the true Km
values for the four NTPs were observed. The
kcat/Km measure of substrate
efficiency indicated a preference for UTP as phosphate donor by a
factor of 4-6. Furthermore, the data indicated that the
Km for dGuo was highly dependent on the nature of
the phosphate donor (Table V). In the presence of UTP the true
Km for dGuo was 0.6 µM compared with
6.5 with ATP. This synergism between the high affinity for UTP and the
low Km for dGuo with UTP as phosphate donor makes
UTP 50-60 times more efficient than ATP as a phosphate donor. In all
further kinetic studies UTP was employed as phosphate donor.
Product inhibition studies with dGuo and UTP as substrates
were performed to determine the binding
order of substrates and products (Fig.
2, A-D; Table
VI). The secondary plots of the slopes or
intercepts versus the concentration of inhibitor were in all cases linear (see insets in Fig. 2, A-D). With
UTP as the variable substrate, inhibition by UDP was competitive (Fig.
2A), whereas inhibition by dGMP was noncompetitive with
Kis being of the same order of magnitude as
Km for UTP, and Kii about 25-fold
higher (Fig. 2B). With dGuo as the variable substrate, both
products produced noncompetitive inhibition (Fig. 2, C and D). This inhibition pattern suggested that dGK followed a
sequential ordered reaction mechanism in which UTP had to bind before
dGuo, and dGMP was leaving the enzyme complex before UDP. Such an
ordered reaction mechanism predicted uncompetitive
inhibition by dGMP with UTP as variable substrate, under conditions of
full saturation with respect to the second substrate, dGuo. Because of
substrate inhibition exerted by dGuo (see below) this prediction could
not be tested. Whether the reaction with other NTPs as phosphate donors followed the same ordered reaction mechanism was not investigated.
Substrate inhibition was observed by dGuo at concentrations above 30 µM. As shown in Fig. 3, the
inhibition was exclusively observed with UTP as phosphate donor, and it
was found to be linear (Fig. 3, inset). This indicated that
the inhibition resulted from the formation of a dead-end complex of
dGuo with a form of the enzyme that it is not supposed to react with
(30). The inability of high UTP concentrations to reverse the
inhibition (data not shown) suggested that dGuo, the second substrate
in the ordered reaction, combined with the E·UDP complex to
form the dead-end complex.
Inhibition of dGK by dGTP--
As mentioned above, dGTP was the
only naturally occurring NTP that did not function as phosphate donor
for dGK. In contrast, dGTP was a potent competitive inhibitor of the
enzyme with respect to UTP, with a Ki value of 0.4 µM (data not shown). The pattern of dGTP inhibition with
dGuo being the variable substrate was noncompetitive at lower dGuo
concentrations with Kii and Kis
values of 1.6 and 0.7 µM, respectively.
The genetic and enzymatic basis for the ability of B. subtilis to phosphorylate dCyd, dAdo, and dGuo was established by
cloning and overexpressing the overlapping yaaG and
yaaF genes in E. coli, an organism unable to
phosphorylate deoxyribonucleosides other than dThd (16). Simultaneous
expression of both genes yielded two separable enzymes, one specific
for dGuo and the other capable of phosphorylating both dCyd and dAdo.
Expression of each gene separately showed that yaaG encoded
dGK and yaaF encoded dCK/dAK, and that both enzymes were
homodimeric proteins. Preliminary studies indicated that dCK/dAK was
identical to the previously characterized deoxyadenosine/deoxycytidine
kinase of B. subtilis (15). Based on these findings we
propose that the yaaG and yaaF genes be renamed as dgk and dak, respectively.
The recombinant Bs-dGK was purified to homogeneity and
characterized. Compared with other enzymes with deoxyguanosine
kinase activity the selectivity of Bs-dGK for the nucleoside
substrate is unique. Only 6-oxopurine 2'-deoxyribonucleosides (dGuo and dIno) are phosphorylated by the enzyme at significant rates, with a
Km for dGuo of 0.6 µM and a
kcat of 1.4 s In contrast to its strict phosphate acceptor requirement,
Bs-dGK can use most NTPs as phosphate donors. However, the
results presented in Table V indicate that UTP is the preferred
phosphate donor. Compared with the other NTPs the efficiency of UTP
results from a 6-fold lower Km for UTP than for the
other NTPs and a much lower Km for dGuo when UTP is
employed as donor. The steady-state kinetic analysis of the reaction
with dGuo and UTP indicated that the reaction follows an Ordered Bi Bi
mechanism with UTP as the leading substrate and release of UDP last
(Scheme 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of specific plasmids. All
plasmids are derivatives of pUC18. The thin line represents
pUC18 DNA. The boxes pertain to the amplified B. subtilis DNA present in the individual plasmids. The position of
relevant restriction sites are indicated by vertical lines
and designated as follows: E, EcoRI;
C, ClaI; P, PstI;
H, HindIII. lacP indicates the
vector-borne lac promoter. The nucleotide sequence at the
junction between yaaF and yaaG is shown below
pRA8 with the start codon of yaaF underlined and
the stop codon of yaaG marked by a line
above.
Deoxynucleoside kinase activities in crude extracts of E. coli
harboring various plasmids
Separation of recombinant dAK/dCK and dGK from E. coli harboring
pRA8
Purification of B. subtilis recombinant dGK
20 °C without loss of activity. Upon dilution for assays the enzyme was highly unstable at 0 °C unless BSA (1 mg/ml) was added to
the dilution buffer. In the presence of BSA the diluted enzyme (>1
µg/ml) was stable for months at 0 °C.
. Excess Mg2+ did
not inhibit the enzyme. Thus, all assays were performed with Mg2+ at 5 mM to ensure that all NTP present in
the reaction was complexed with magnesium.
Nucleoside specificity of dGK
Kinetic constants for Bs-dGK with different NTPs as phosphate donors
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Fig. 2.
Product inhibition studies.
Double-reciprocal plots of product inhibition experiments.
Insets are replots of slopes and intercepts against
inhibitor concentrations. A, inhibition by UDP at various
UTP concentrations; B, inhibition by dGMP at various UTP
concentrations; C, inhibition by UDP at various dGuo
concentrations; D, inhibition by dGMP at various dGuo
concentrations. Inhibitor concentrations in micromolar are shown at the
end of each line. 1/v is
(units/mg) 1.
Product inhibition of dGK
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Fig. 3.
Substrate inhibition by dGuo with various
NTPs as phosphate donors. The inset shows a replot of
the data from the UTP inhibition curve as 1/v
versus [dGuo]. The concentration of the NTPs was 100 µM.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. The homodimeric
mammalian mitochondrial dGK, which in addition to dGuo and dIno can use
dAdo as a substrate, shows Km and
kcat values of 7.6 µM and 0.002 s
1, respectively, with dGuo as a substrate (9), and the
homodimeric human dCK, which uses dCyd, dAdo, and dGuo, phosphorylates
dGuo with a Km of 150-430 µM and a
kcat of 0.4-6.0 s
1 (7, 31).
Recently, a monomeric multisubstrate dNK from Drosophila melanogaster (Dm-dNK) was characterized (12). Its
Km for dGuo was found to be very high (654 µM), but at the same time the enzyme displayed the
highest kcat reported for any deoxynucleoside kinase (18 s
1). The specificity constant
(kcat/Km) for dGuo
phosphorylation by Bs-dGK is thus 102 to
104 higher than the corresponding values of the various
eukaryotic deoxyguanosine kinases. Expression in E. coli of
the L. acidophilus dgk gene, encoding the dGK subunit of the
heterodimeric dAK/dGK, results in the production of a loosely
associated homodimeric dGK, with significant similarity to
Bs-dGK regarding its specificity toward the nucleoside
substrate. However, the specificity constant of this "unnatural"
dGK is only 1/20th of that of Bs-dGK (18).
It was previously observed that UTP, CTP, and dTTP were more efficient phosphate donors than ATP for the bovine liver mitochondrial dGK at physiological pH (33), and that UTP was the preferred phosphate donor for human dCK with dCyd and dAdo as acceptors (34, 35). More recently, a detailed kinetic analysis of human dCK with dCyd and UTP was reported (36). It showed that the preference for UTP as phosphate donor was primarily due to large differences in the true Km values for the phosphate donor with values of 1.2 and 54 µM for UTP and ATP, respectively. Only a 2-fold difference in the true Km values for dCyd was observed (0.5 and 1.0 µM with UTP and ATP, respectively). The kinetic mechanism of dCyd phosphorylation also appeared to depend on the phosphate donor showing a random Bi Bi reaction sequence with ATP but an ordered addition-random release sequence with UTP (31, 36). Unfortunately, the kinetic properties of the enzyme with dAdo or dGuo as substrates were not investigated.
Most deoxynucleoside kinases are end-product-inhibited by the corresponding dNTP, and it has been shown in a number of cases that the dNTP inhibitor behaves like a bisubstrate analog (17, 37, 38). The kinetics of dGTP inhibition and the observation that all NTPs and dNTPs except dGTP function as phosphate donors, suggest that dGTP inhibition of Bs-dGK is due to binding of the inhibitor to both the deoxynucleoside acceptor site and the triphosphate binding site of UTP. The intracellular amounts of nucleoside triphosphates in B. subtilis have been measured previously (39). For UTP and dGTP they were estimated to be 0.15 and 1.6 nmol/mg of dry weight, which corresponds to 188 µM and 2 mM respectively.2 Insertion of these values, the Ki value for dGTP of 0.4 µM, and the Km for UTP of 6 µM, into the rate equation for competitive inhibition shows that the enzyme would be inhibited by ~60% under these conditions, indicating that this feedback control may be of physiological importance.
Recently, the nucleotide sequence of the entire Deinococcus
radiodurans genome was determined (40). It revealed that this Gram-positive bacterium contains two overlapping genes encoding putative polypeptides with 32 and 35% identity to the yaaG
and yaaF genes of B. subtilis, respectively.
Thus, D. radiodurans may represent a third genus of
eubacteria possessing dGuo, dAdo, and dCyd kinase activities. It is
noteworthy that homologous genes have so far not been identified in
Gram-negative bacteria.
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ACKNOWLEDGEMENTS |
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We thank L. Stauning for expert technical assistance and Dr. M. Willemoës for numerous enlightening discussions.
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FOOTNOTES |
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* This work was supported by the Danish Natural Science Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.: 45-35-32-20-02;
Fax: 45-35-32-20-40; E-mail: neuhard@mermaid.molbio.ku.dk.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M007918200
2 The calculations are based on the assumption that 1 mg of bacterial dry weight corresponds to 2 × 108 cells and that the volume of one cell is 4 µm3.
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ABBREVIATIONS |
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The abbreviations used are: dTK, thymidine kinase; BSA, bovine serum albumin; Bs-dGK, Bacillus subtilis deoxyguanosine kinase; dAK, deoxyadenosine kinase; dCK, deoxycytidine kinase; dGK, deoxyguanosine kinase; Ds-dNK, Drosophila melanogaster deoxynucleoside kinase; PAGE, polyacrylamide gel-electrophoresis; PCR, polymerase chain reaction; bp, base pair(s).
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