Plant Physiol. (1998) 117: 245-254
Cloning, Expression in Escherichia coli, and
Characterization of Arabidopsis thaliana UMP/CMP
Kinase1
Lan Zhou,
François Lacroute, and
Robert Thornburg*
Department of Biochemistry and Biophysics, Iowa State University,
Ames, Iowa 50011 (L.Z., R.T.); and Centre de Génétique
Moléculaire, Centre Nationale de La Recherche Scientifique, 22 Avenue de la Terrace, 91198 Gif sur Yvette, cedex France (F.L.,
R.T.)
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ABSTRACT |
A cDNA encoding the
Arabidopsis thaliana uridine 5
-monophosphate
(UMP)/cytidine 5-monophosphate (CMP) kinase was isolated by
complementation of a Saccharomyces cerevisiae ura6
mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA
was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli.
Following proteolytic digestion, the plant UMP/CMP kinase was purified
and analyzed for its structural and kinetic properties. The mass,
N-terminal sequence, and total amino acid composition agreed with the
sequence and composition predicted from the cDNA sequence. Kinetic
analysis revealed that the UMP/CMP kinase preferentially uses ATP
(Michaelis constant [Km] = 29 µm when UMP is the other substrate and
Km = 292 µm when CMP is the
other substrate) as a phosphate donor. However, both UMP
(Km = 153 µm) and CMP
(Km = 266 µm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is
6.5. P1, P5-di(adenosine-5) pentaphosphate was
found to be a competitive inhibitor of both ATP and UMP.
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INTRODUCTION |
Pyrimidines are intimately involved in the physiology of cells.
They participate at multiple levels in intermediary and secondary metabolism from nucleotide and macromolecule biosynthesis to the biosynthesis of complex carbohydrates and the metabolic regulation of
intermediary metabolism. All pyrimidines within the cell are derived
from UMP, which arises either from the de novo pyrimidine biosynthetic
pathway or from salvage of preformed pyrimidines. UMP/CMP kinase
converts uridine and cytidine monophosphates into the corresponding
uridine and cytidine diphosphates. Because all pyrimidines are derived
from UMP, UMP kinase is the first committed step and one of the central
enzymes in the further anabolism of pyrimidine nucleotides.
The importance of pyrimidine monophosphokinases to cell physiology has
been firmly established. In both bacteria and yeast, the roles of
pyrimidine monophosphokinases with respect to cell proliferation and
physiology have been widely studied. In Escherichia coli,
the product of the UMP kinase gene (pyrH/smbA) has been shown to influence cell proliferation. Yamanaka et al. (1992)
, working
with the mukB gene, isolated a suppressor of mukB
that they termed smbA. The smbA phenotype is
pleiotropic. First, the smbA mutant ceased macromolecular
synthesis, was hypersensitive to SDS, and showed a novel morphological
phenotype under nonpermissive conditions. Later, it was found that the
wild-type smbA gene is identical to the pyrH gene
and that the smbA2 mutant protein encodes an unstable UMP
kinase with impaired catalytic and regulatory functions.
In yeast, mutations in the UMP kinase gene have been shown to cause a
conditional lethal phenotype (Liljelund and Lacroute, 1986). When grown
at the nonpermissive temperature, the UTP and CTP pools decline to 10%
of their wild-type levels, which affects both RNA and protein synthesis
and ultimately results in cell death. Complementation of this mutant
permitted the first isolation of a eukaryotic UMP kinase gene
(Liljelund and Lacroute, 1986). This is the same mutant that we have
complemented in this study to isolate the Arabidopsis
thaliana UMP/CMP kinase cDNA.
Other pyrimidine monophosphokinases are also important in normal
cellular physiology. The cdc8 mutant of Saccharomyces
cerevisiae was isolated as a cell-cycle-deficient mutant that was
defective in nuclear division (Newlon and Fangman, 1975). When
incubated at the restrictive temperature, the cdc8 mutant
cells arrest in S phase with a typical dumbbell morphology. This
mutation also produces other pleiotropic effects, including inhibition
of normal cellular DNA replication (Birkenmeyer et al., 1984),
involvement in error-prone repair (Prakash et al., 1979; Baranowska and
Zuk, 1991), and involvement in repair of single-stranded breaks
(Baranowska et al., 1990). The CDC8 gene was isolated
(Birkenmeyer et al., 1984; Kuo and Campbell, 1983) and found to encode
dTMP kinase (Jong et al., 1984). Known suppressors of the
CDC8 gene have also been isolated and characterized. One of
these, SOC8, encodes the UMP kinase gene ura6
(Jong et al., 1993). Thus, nucleotide monophosphokinases can have
profound effects on cellular morphology and physiology.
Substrate utilization by eukaryotic UMP kinases has also received much
study (Weismüller et al., 1990; Jong et al., 1993; Müller-Dieckmann and Schultz, 1994). Whereas all eukaryotic
enzymes have specificity for both UMP and CMP, the yeast enzyme also
has specificity for AMP. Indeed, the yeast UMP kinase has such a high affinity for AMP that the ura6 gene has been isolated as a
multicopy suppressor of yeasts deficient in adenylate kinase (Schricker et al., 1992). The yeast UMP kinase gene also complements the E. coli adenylate kinase enzyme. In contrast, the cellular slime mold
(Dictyostelium discoideum) enzyme does not complement either the yeast or the E. coli adenylate kinase mutants. UMP
kinase is also required for the metabolic activation of several
important anti-tumor drugs, including 5-fluorouracil and Ara-C
(Seagrave and Reyes, 1987). Consequently, UMP kinase plays a central
and very important role in pyrimidine anabolism.
In addition to studies of the eukaryotic enzyme, the prokaryotic enzyme
has received much attention (Valentin-Hansen, 1978; Yamanaka et al.,
1992; Serina et al., 1995, 1996). Because of this, significant
differences between the enzymatic activities of the prokaryotic and
eukaryotic enzymes have been identified. The prokaryotic enzyme is
allosterically regulated by both GTP and UTP (Serina et al., 1995). GTP
functions to stimulate the enzyme when there is an overabundance of
purine triphosphates, and UTP down-regulates the enzyme when pyrimidine
triphosphates have accumulated to a high level. In addition, UMP kinase
is further regulated by divalent metal ions in a novel mechanism
related to metal-free-UTP binding (Serina et al., 1996). The binding of metal-free UTP causes a gel-sol transition that affects the state of
UMP kinase aggregation and, subsequently, the enzyme activity.
In humans, UMP kinase is associated with an autoimmune deficiency that
results in susceptibility to respiratory infections such as invasive
Hemophilus influenzae type B disease in Alaskan Eskimos
(Petersen et al., 1985) and South American Indians (Gallango and
Suinaga, 1978; Gallango et al., 1978). This underexpression of UMP
kinase results in a syndrome similar to the immune defect resulting
from adenosine deaminase deficiency (Giblett et al., 1974), which is
thought to be due to the toxic build-up of substrates.
In plants, UMP kinase has received only marginal study. UMP kinase is
elevated during seedling development (Deng and Ives, 1972; Mazus and
Buchowicz, 1972) and fruit ontogeny (Rudd and Fites, 1972; Deng and
Ives, 1975). Because UMP kinase is likely to be as important in plants
as it is in microorganisms, we have isolated the cDNA for the A. thaliana UMP/CMP kinase, expressed the coding region in E. coli, and characterized the resulting plant enzyme.
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MATERIALS AND METHODS |
The pGEX-4T-3 and glutathione-Sepharose 4B were purchased from
Pharmacia. The vector pT7-Blue was from Novagen (Madison, WI). Enterokinase was from Biozyme Laboratories (San Diego, CA). Restriction enzymes, T4 ligase, and Taq polymerase were from Promega.
All other enzymes and reagents were obtained from Sigma unless
otherwise noted. The Arabidopsis thaliana cDNA library in a
yeast transformation vector was previously described (Minet et al.,
1992). Oligonucleotides were synthesized at the Iowa State University
(Ames) Nucleic Acid Facility.
Strains
Saccharomyces cerevisiae FL100a was the wild-type
strain used for all yeast-related manipulations. The S. cerevisiae ura6 strain was derived from FL100a and displayed a
conditional thermosensitive and 5-fluorouracil-resistant phenotype
(Liljelund and Lacroute, 1986). The strain used in these studies was
also auxotrophic for His and Trp. Genotypes of the yeast strains were
determined by plating at either the permissive or restrictive
temperature on media lacking various ingredients. The Escherichia
coli strain XL1-Blue was used for all bacterial manipulations.
Yeast Methods
Yeast transformation was conducted as described previously (Gietz
et al., 1992). S. cerevisiae auxotrophic mutants were
complemented by A. thaliana cDNAs as previously described
(Minet et al., 1992). To rescue the plasmids from yeast, a loopful of
yeast cells was suspended in 0.4 mL of 10 mm Tris, pH 8.0, 1 mm EDTA, and 0.4 m NaCl and vortexed (2 min)
with 200 µL of glass beads. Following vortexing, an equal volume of
phenol/chloroform (1:1) was added and the vortexing was repeated. After
the sample was centrifuged, 250 µL of supernatant was removed and DNA
was precipitated by the addition of 500 µL of ethanol. The sample was
washed with 70% ethanol, and the DNA was resuspended in water and used
for electroporation of E. coli (Ausubel et al., 1993).
Recombinant DNA Methods
DNA-sequencing reactions were performed using the Prism Dye-Deoxy
cycle sequencing kit (Applied Biosystems). The reactions were run on a
DNA sequencer (Prism 377, Perkin-Elmer). Sequencing was initiated from
known vector sequences. On the basis of these runs, oligonucleotide
primers specific to the A. thaliana UMP/CMP kinase gene
sequence were constructed. Both strands were completely sequenced.
The 606-bp UMP kinase-coding region was PCR amplified from the pAt-URA6
clone using a pair of oligonucleotides, LZ2080
(5-GCGGATCCGATGACGATGACAAGATGGGATCTGTTGATGCTGCT-3) and LZ2081
(5-CGGCTCGAGCTACTAGGCTTCAACCTTCTCAGC-3). The PCR product was
cloned into the pT7-Blue(R) T-vector to form the vector pRT379. These
oligonucleotide primers introduced into the PCR produced unique
BamHI and XhoI sites at the ends of the PCR
fragment, an enterokinase site for cleavage of the GST fusion protein,
and an additional stop codon. Following cloning of the PCR product in
pRT379, the sequence of the coding region was confirmed by completely
sequencing the insert. The expression vector pRT380 was subsequently
prepared by inserting the 637-bp BamHI/XhoI
fragment from pRT379 into the BamHI/XhoI sites of
pGEX-4T-3. Again, the sequence of the coding region was confirmed by
completely sequencing the insert.
Expression in E. coli
The vector pRT380 was transformed into E. coli
XL1-Blue for the production of the fusion protein. To induce the fusion protein, 5 mL of overnight culture was diluted into 500 mL of 2YT
medium (1.6% bactotryptone, 1.0% yeast extract, and 0.5% NaCl, pH
6.0) and grown at 37°C until mid-log phase. IPTG was then added to a
final concentration of 1 mm. Growth continued for 3 h
at 37°C. Cells were harvested by centrifugation at 4000g for 10 min. The cell pellet was stable when stored at 
20°C.
Purification of UMP/CMP Kinase
Frozen cells were thawed in 10 mL of PBS containing 25 mg of
lysozyme. After 30 min at room temperature, the cells were sonicated on
ice. DNase I (2 mg) in 0.8 m MgCl2
was added and the cell sonicate was incubated at room temperature for
10 min. Cellular debris were removed by centrifugation at 12,000g for
40 min, and the supernatant was used to resuspend 0.3 mL of packed
PBS-washed glutathione-Sepharose beads. The beads and supernatant were
incubated overnight at room temperature on a rotating wheel. The beads
were removed by centrifugation and washed with PBS until the
supernatant was clear (usually five times). The fusion protein could be
eluted by the addition of 10 mm glutathione. However, for
most studies, the UMP/CMP kinase domain was removed from the bound
fusion protein by resuspending the beads in 0.35 mL of enterokinase
buffer (25 mm Tris, pH 7.5, and 10 mm
CaCl2) and digesting with 1000 units of
enterokinase until completion at room temperature on a rotating wheel.
After digestion, the supernatant was recovered by centrifugation and
applied to a reverse-phase C18 HPLC column
(250 × 10 mm, i.d.; Vydac, Hesperia, CA). The column was eluted
at a rate of 4 mL min
1 with a programmed
elution profile. Solvent A was 0.1% trifluoroacetic acid in water and
solvent B was 0.08% trifluoroacetic acid in acetonitrile. The enzyme
was eluted by a series of linear gradients: 0 to 5 min, gradient from
20 to 40% solvent B; 5 to 23 min, gradient from 40 to 43% solvent B;
the column was cleaned and equilibrated for further separations by two
gradients: 23 to 26 min, gradient from 43 to 100% solvent B; and 26 to
28 min, gradient back to 20% solvent B. The UMP/CMP kinase was eluted
at 41% solvent B. Following elution, the enzyme fraction was dialyzed
against 20 mm Mes, pH 6.5, at 4°C for 24 h and then
concentrated to about 0.5 mL using a centrifugal concentrator
(Centricon-10, Amicon, Beverly, MA).
Protein Methods
Protein concentration was determined by the method of Bradford
(1976) with BSA as a standard. SDS-PAGE was performed according to the
method of Laemmli (1970). N-terminal amino acid analysis was performed
in the Iowa State University Protein Facility (Ames) by sequential
Edman degradation on a protein sequencer (model 477A, Applied
Biosystems) and a protein analyzer (model 120A, Applied Biosystems).
Amino acid composition was performed with phenyl
isothiocyanate-derivatized amino acids following hydrolysis of purified
UMP/CMP kinase in 6 n HCl.
Matrix-assisted laser-desorption ionization MS was used for determining
molecular mass. Protein samples of 0.5 to 1.0 µL containing about 0.5 to 1 µg of protein were loaded with 0.5 µL of freshly prepared
3,5-dimethoxy-4-hydroxy cinnamic acid matrix onto a time-of-flight mass
analyzer (Lasermat 2000 MALDI, Finnigan, Madison, WI). The collected
data were analyzed using data processing software (Lasermat 2000, Finnigan). Lysozyme was used as an internal calibration standard.
Enzymatic activity was determined spectrophotometrically by measuring
the formation of ADP and UDP at 23°C with a coupled-enzyme assay
(Agarwal et al., 1978). Quantitation was performed by following the
decrease of NADH A340. The initial-rate
data were analyzed for kinetic mechanisms (Fromm, 1975) by using a
computer program written in the MINITAB language with an 
-value of
2.0 (Siano et al., 1975).
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RESULTS |
Cloning by Complementation
Yeast genetic crosses were made between
ura2 and
ura6-15(Ts) to provide progeny blocked
in de novo pyrimidine biosynthesis as well as in the conversion of UMP
into UDP. The inclusion of the ura2
mutation provided a better screen for
ura6-15(Ts). These progeny were
unable to make pyrimidines via the de novo pyrimidine biosynthetic
pathway, but could be rescued at the permissive temperature by the
addition of uracil through the salvage pathway. Strain
ura6-15A, which has an FL100a,
ura6-15Ts,ura2,trp,his
genotype, was used for all transformation experiments.
The yeast strain was transformed with an Arabidopsis cDNA library in
the vector pFL61 (Minet et al., 1992). Approximately 54,000 individual
transformants were screened for colonies at the restrictive
temperature. Two colonies were found from the 54,000 transformants. DNA
was isolated from each of these yeast strains and E. coli
was transformed by electroporation. Both colonies yielded the identical
cDNA clone when analyzed by DNA sequence analysis. After growth in
E. coli, both clones were reisolated and used to transform
the original ura6-15A strain to verify that the clones
produced the URA+ phenotype. The plasmid
containing this clone was termed pAt-ura6.
Analysis of the UMP/CMP Kinase mRNA
The DNA sequence of the pAt-ura6 insert is presented in Figure
1. This presumptive Arabidopsis UMP/CMP
kinase cDNA sequence has been deposited in GenBank as accession no.
AF000147 and is 895 nucleotides long. It shows a 60-nucleotide
5-untranslated region, a coding region of 606 nucleotides, and a
175-nucleotide 3-untranslated region extending to the
poly(A+) site at position 874. This cDNA did not
show a typical polyadenylation signal. The closest to the consensus is
AATTTT, which is duplicated at positions 836 to 841 and 853 to 858. DNA
matrix analysis revealed no significant regions of internal duplication
or inverted repeats within the cDNA sequence.
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| Figure 1.
cDNA sequence of the A. thaliana
UMP/CMP kinase. The nucleotide number is presented at the end of each
row. The deduced amino acid sequence is presented below the DNA
sequence and the amino acids are numbered above the DNA sequence.
Asterisks indicate the amber (UAG) codon.
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The translation start codon correlates well with the rules of Kozak
(1986). The sequence at the translation start differs from the
eukaryotic consensus at only a single nucleotide (ACAATGG). Consequently, this cDNA is expected to be translated very efficiently.
Analysis of the UMP/CMP Kinase Protein
The deduced amino acid sequence of the Arabidopsis UMP/CMP kinase
is also presented in Figure 1. The protein is 202 amino acids long.
There is a pair of conserved Cys residues that are shared among all of
the eukaryotic UMP kinases. However, it has been proposed that the
conserved Cys residue found at position 31 in the Arabidopsis protein
has a free SH group (Weismüller et al., 1990).
The Arabidopsis UMP/CMP kinase amino acid sequence is very similar to
UMP kinases from other eukaryotic sources (Fig.
2). UMP kinases have been isolated from
yeast (S. cerevisiae), a cellular slime mold (D. discoideum), and a mammal (Sus scrofa). Each of these
enzymes shows 47 to 53% amino acid identity with each other. It was
interesting that 32.2% of the amino acid residues are identical among
all of the four proteins. If conservative substitutions are permitted,
then the homology among the four proteins increases to 47.5%.
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| Figure 2.
Comparison of pyrimidine monophosphokinases. A,
Comparison of the amino acid identity between the various pyrimidine
monophosphate kinases was performed using the PileUp program from the
Genetics Computer Group (Madison, WI). For this analysis, both UMP
kinases and TMP kinases were included. UMP kinases were included from archebacterial, eubacterial, and eukaryotic (plant, mammal, yeast, and
mold) sources. The GenBank accession numbers for the sequences used in
this study are as follows: Saccharomyces pombe TMP
kinase, L04126; S. cerevisiae TMP kinase, K02116;
Homo sapiens TMP kinase, L16991; Thermus
aquaticus UMP kinase, X83598; E. coli UMP
kinase, X78809; S. cerevisiae UMP kinase, M69295;
S. scrofa UMP kinase, D29655; D. discoideum UMP kinase, M34568; and A. thaliana
UMP/CMP kinase, AF000147. B, Alignment of amino acid sequences of
eukaryotic UMP kinases from a plant (A. thaliana), a
mammal (S. scrofa), a yeast (S. cerevisiae), and a cellular slime mold (D. discoideum). Identical residues are marked by dots ( ) and
conservative substitutions are indicated with plus signs (+).
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There is a conserved region near the N terminus that is maintained in
nucleotide-binding proteins (Möller and Amons, 1985). This
sequence, GGPGS/AGK, is
also preserved in the eukaryotic nucleotide monophosphokinases. In
crystallographic studies of adenylate kinase, Pai et al. (1977) found
that this loop anchors the 
-phosphate moiety of ATP.
Expression in E. coli
After characterization of the Arabidopsis UMP/CMP kinase cDNA, we
constructed a vector to express the Arabidopsis protein in E. coli. The structure of the GST fusion protein used in this work is
shown in Figure 3A. The nucleotide
sequence of the GST-kinase and the kinase-vector junction in pRT380 are
presented in Figure 3B. Because the plant UMP/CMP kinase does not
contain an N-terminal signal sequence, the vector was designed so that
enterokinase would cleave at the exact N terminus of the enzyme to
yield the full-length UMP/CMP kinase protein.
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| Figure 3.
Sequence of portions of the construct pRT380. A,
Structural model of the GST-kinase fusion protein showing the amino
acid sequence at the site of fusion. B, Nucleotide and translated amino acid sequences of the pRT380 construct at the site of GST-UMP/CMP kinase fusion and the 3 end of the fusion protein. The sequences up
through the BamHI site are from the pGEX-4T-3 vector and
encode the C terminus of the GST. The aspartate-rich sequence extending from the BamHI site to the Met is the site of
enterokinase recognition. Cleavage occurs after the Lys and is
indicated by the arrow. The sequence Met-Gly-Ser-Val-Asp represents the
first five amino acids of the Arabidopsis UMP/CMP kinase. The sequence
of the remainder of the protein is identical to that presented in
Figure 1. After the stop codon, we introduced a second TAG stop codon
and an XhoI site. Downstream of the XhoI
site is the pGEX-4T-3 vector.
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After transfer of the vector to XL1-Blue cells, the induction of these
cells by IPTG resulted in the accumulation of a 51-kD protein (Fig.
4, lane 2) that was not present in host
cells without plasmid (data not shown) or uninduced cells containing
pRT380 (Fig. 4, lane 1). After sonication of the IPTG-induced cells, enzymatic assays showed the presence of a new UMP kinase activity that
was not present in control cells (data not shown). This new UMP kinase
activity arises from the GST-kinase fusion protein.
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| Figure 4.
SDS-PAGE of UMP/CMP kinase. All samples were
analyzed on a 12% polyacrylamide gel and stained with Coomassie
brilliant blue R-250. Lane 1, Cell sonicates of E. coli
XL1-Blue cells containing the plasmid pRT380 with no added IPTG; lane
2, cell sonicates of E. coli XL1-Blue cells containing
the plasmid pRT380 after the addition of IPTG; lane 3, GST-UMP/CMP
kinase fusion protein eluted from glutathione-Sepharose beads; lane 4, GST-UMP/CMP kinase fusion protein eluted from glutathione-Sepharose
beads and then digested with enterokinase; lane 5, digestion of
GST-UMP/CMP kinase fusion protein with enterokinase while fusion
protein was still attached to the beads; lane 6, HPLC purified UMP/CMP
kinase. Molecular mass markers used were: phosphorylase
b, 97.4 kD; BSA, 66 kD; ovalbumin, 45 kD; carbonic
anhydrase, 31 kD; trypsin inhibitor, 21.5 kD; and lysozyme, 14.4 kD.
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Purification of UMP/CMP Kinase
The various steps in the purification of the enzyme were monitored
by SDS-PAGE, as shown in Figure 4. A 51-kD protein was observed upon
induction of the cells containing pRT380 with IPTG (Fig. 4, compare
lane 2 [induced] with lane 1 [uninduced]). This fusion protein
could be selectively bound and eluted from the glutathione-Sepharose
beads (Fig. 4, lane 3). Enterokinase digestion of the fusion protein
resulted in the conversion of the 51-kD protein into two subunits (Fig.
4, lane 4). For most studies, however, the GST-kinase fusion protein
was proteolyzed while still bound to glutathione-Sepharose beads, which
releases the 22.5-kD protein while the 29-kD protein remains bound to
the glutathione-Sepharose (Fig. 4, lane 5). After enzymatic cleavage of
the fusion protein the UMP/CMP kinase was purified by HPLC. A typical
HPLC elution profile is shown in Figure
5. The eluted UMP/CMP kinase protein was
collected, dialyzed, and concentrated. An aliquot of the
purified UMP/CMP kinase after HPLC elution is shown in Figure 4, lane
6.
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| Figure 5.
HPLC purification of UMP/CMP kinase. The
GST-UMP/CMP kinase fusion protein was digested with enterokinase while
still attached to the glutathione-Sepharose beads. The eluate was
applied to a 250- × 10-mm, i.d., reverse-phase C18 column.
A214 was monitored. The eluted protein peak
containing UMP/CMP kinase activity was collected (B = 41%). The
hatched area under the peak illustrates the collected pool of UMP/CMP
kinase.
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About 0.5 to 1 µg of the HPLC-purified UMP/CMP kinase was subjected
to MS (Fig. 6). The subunit molecular
mass of the purified UMP/CMP kinase [MH]+ peak
was 22,405 ± 185 D (n = 4). Secondary peaks
[MH2]2+ and
[2MH]+ were identified at 11,286 ± 94 and
44,894 ± 333 D. Therefore, the subunit molecular mass found from
the average of these masses, 22, 448 ± 44 D, is in good agreement
with the molecular mass of UMP/CMP kinase predicted from the cDNA
(22,482 D).
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| Figure 6.
Typical mass spectrum of A. thaliana UMP/CMP kinase. The [MH2]2+
peak is at 11,286, the [MH]+ peak is at 22,405, and the
[2MH]+ peak is at 44,984.
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Characterization of the Purified UMP/CMP Kinase Protein
To confirm that the expressed protein was indeed the UMP/CMP
kinase, the N terminus of the purified protein was sequenced. The
N-terminal sequence MGSVDAANGSGKKPT was found to be identical to the
amino acid sequence deduced from the cDNA. This sequence also indicates
that the enterokinase cuts exactly at the predicted site, leaving no
extra amino acids at the N terminus.
The amino acid composition of the expressed protein was also
determined. About 1 µg of HPLC-purified UMP/CMP kinase was hydrolyzed in 6 n HCl for 65 min at 150°C (PICO-TAG Workstation,
Waters). The free amino acids were derivatized under basic conditions
with phenyl isothiocyanate in a derivatizer (model 420A, Applied
Biosystems) and separated on a narrow-bore C18
column. The phenylthiocarbamyl chromophore was detected at
A254. Quantitation was performed from the
A254 and comparison with a norleucine
internal standard. The total amino acid composition was determined
independently two times (data not shown). Gln and Asn could not be
detected because both amino acids were converted to the corresponding
carboxylic acids. Therefore, the resulting Asp and Glu were the sum of
Asp and Asn (Asx) and of Glu and Gln (Glx), respectively. The number of
Trp and Cys residues could not be determined by this method because
they were destroyed during acid hydrolysis. There was good agreement
between the independent analyses. A 
2 analysis
indicated with greater than 99% confidence that the composition found
in the purified protein was the same as that of the predicted protein,
confirming that the purified protein was the Arabidopsis UMP/CMP
kinase.
Initial Rate Studies
Once we were convinced that the purified protein was indeed the
Arabidopsis UMP/CMP kinase, the kinetic parameters of this purified
protein were determined (Table I). The
enzyme can utilize CMP as a phosphate donor almost as well as it does
UMP. The Km for UMP was 5- to 6-fold higher
than that for ATP. Therefore, the enzyme binds ATP much more tightly
than UMP. However, the Km for CMP was about
equal to the Km for ATP, indicating that the enzyme binds CMP as tightly as ATP. The
kcat obtained for each substrate was
relatively the same.
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Table I.
Kinetic parameters of A. thaliana UMP/CMP kinase
The standard enzyme reaction contained 50 mm Mes, pH 6.5, 50 mm KCl, 2 mm MgCl2, 1 mm PEP, 0.2 mm NADH, 3.5 units of pyruvate kinase, and 5 units of lactate dehydrogenase in a final volume of 1 mL.
When ATP was used as the variable substrate, UMP and CMP were fixed at
400 and 450 µm, respectively. When UMP or CMP were used
as the variable substrate, ATP-Mg2+ was fixed at 300 µm for UMP and at 800 µm for CMP. The
reaction was started by the addition of UMP/CMP kinase. The change in
A340 was recorded. One unit of UMP kinase is
defined as the amount of enzyme that catalyzed the formation of 1 µmol of UDP or CDP per min.
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pH Dependence of the Enzyme
The values of kcat were determined at
a series of different pH values from 5.5 to 8.0. All pH buffers
contained both 50 mm Mes and 50 mm Hepes. When
ATP was the variable substrate, the concentration of UMP was fixed at
400 µm. When UMP was the variable substrate, the
concentration of ATP was fixed at 300 µm. The values of
kcat were obtained at pH 5.5, 6.5, 7.0, 7.5, and 8.0 when ATP or UMP was the variable substrate (data not
shown). These analyses showed that optimal activity was achieved at pH
6.5 for both ATP and UMP.
Thermal Stability
The enzyme was heated for 10 min at various temperatures between
30 and 100°C. Residual activity was determined with 300 µm ATP and 400 µm UMP (data not shown).
One-half of the enzymatic activity was maintained when the enzyme was
heated at 58°C. Although still high, this is about 10°C lower than
that of the E. coli enzyme.
Kinetic Mechanism
To better understand the kinetic mechanism, enzyme assays were
conducted in which one substrate was varied at different fixed concentrations of other substrates. Double-reciprocal plots of these
data showed that, when the ATP concentration was varied at different
fixed levels of UMP, a family of lines intersecting in the second
quadrant was obtained (Fig. 7A). A
similar family of lines was obtained for various 1/UMP concentrations
at different fixed levels of ATP (Fig. 7B). These families of lines are
theoretical, obtained from Equation 1 when n = 1. The
data obtained from the experimental evaluation matches the theoretical
family of lines for both ATP and UMP. Based on the closeness of this
fit, we conclude that the enzyme fits a random Bi-Bi mechanism. The
data fit the random Bi-Bi mechanism shown in Equation 1 (Fromm, 1975):
![<FR><NU>1</NU><DE>V</DE></FR> = <FR><NU>1</NU><DE>V<SUB>m</SUB></DE></FR>[1 + <FR><NU>K<SUB>a</SUB></NU><DE>A<SUP>n</SUP></DE></FR> + <FR><NU>K<SUB>b</SUB></NU><DE>B</DE></FR> + <FR><NU>K<SUB>ia</SUB>K<SUB>b</SUB></NU><DE>A<SUP>n</SUP>B</DE></FR>]](/content/vol117/issue1/fulltext/245/img001.gif)
|
(1)
|
where V, Vm, A,
B, Ka, Kb,
and Kia represent the initial velocity,
maximum velocity, concentration of free ATP, concentration of free UMP,
Michaelis constant for ATP, Michaelis constant for UMP, and
dissociation constant for ATP, respectively. The Hill coefficient for
ATP is represented by n. Kib is the
dissociation constant for UMP and substitutes into Equation 1 in place
of Kia. Kia and
Kib were determined experimentally.
Kia (for ATP) = 14.9 ± 1.3 µm and Kib (for UMP) = 110.2 ± 8.7 µm.
View larger version (19K):
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| Figure 7.
Analysis of kinetic mechanism. A,
Double-reciprocal plot of initial velocity versus ATP concentrations.
For these experiments the concentrations of UMP were 40 ( ), 60 (+),
90 ( ), and 120 µm ( ). The lines are theoretical
based on Equation 1 where n = 1. The points were
experimentally determined. B, Double-reciprocal plot of initial
velocity versus UMP concentrations. For these experiments, the
concentrations of ATP were 15 (), 25 (+), 40 (), and 65 µm (). The lines are theoretical based on Equation 1
where n = 1. The points were experimentally
determined.
|
|
Alternative Substrates
To examine the specificity of the UMP/CMP kinase in more detail,
various phosphate donors and acceptors were evaluated for their ability
to function in this enzyme assay. To evaluate the phosphate acceptors,
300 µm ATP was used as the donor with different monophosphate acceptors at 400 µm. Only UMP and CMP are
effective phosphate acceptors. Neither orotidine monophosphate nor TMP
were effective as a phosphate acceptor. The presence of the 2 hydroxyl on the Rib moiety is also important because dUMP and dCMP are 30-fold
less active than their ribosyl analogs (data not shown). None of the
purine monophosphates tested (AMP, GMP, inosine monophosphate, and
xanthine monophosphate) functioned effectively as a phosphate acceptor.
Similarly, other nucleotide triphosphates were examined for their
ability to function as phosphate donors to UMP. For evaluation, 300 µm phosphate donors were used with UMP at 400 µm. In this analysis, only ATP and dATP were effective
phosphate donors, with dATP only half as effective as ATP. Other purine triphosphates (GTP, dGTP, inosine triphosphate, and xanthine
triphosphate) were 20- to 30-fold less active than ATP. Pyrimidine
triphosphates (UTP, CTP, dCTP, and deoxyribothymine triphosphate) were
70-fold less active than ATP.
It is known that the E. coli UMP kinase is allosterically
regulated by both GTP and UTP (Serina et al., 1995). With the E. coli enzyme, 100 µm GTP activates the enzyme 5-fold
and 100 µm UTP down-regulates the enzyme 5-fold. We
therefore explored whether these nucleotides affected the activity of
the Arabidopsis enzyme. Neither of these nucleotides significantly
affected the activity of the Arabidopsis UMP/CMP kinase, even at
extremely high levels. At 3 mm GTP enzyme activity
increased by 25%, but this is much lower than the 5-fold activation of
the E. coli enzyme that occurs with lower levels of GTP
(Serina et al., 1995). Conversely, UTP also affects the UMP/CMP kinase
activity but, again, only by 25% at UTP concentrations as high as 3 mm. Therefore, whereas GTP and UTP both affect the plant
enzyme, the effect of each of these nucleotides even at very high
levels is significantly less than the effect on the prokaryotic enzyme.
Kinetics of Ap5A Inhibition
We also examined the inhibition of the Arabidopsis UMP/CMP kinase
with the bifunctional inhibitor Ap5A (Fig.
8). The inhibitor concentration required
for 50% inhibition was determined by fixing ATP at 300 µm and UMP at 400 µm and varying the
Ap5A concentration between 0 and 100 µm. The
inhibitor concentration for 50% displacement of Ap5A on
the Arabidopsis enzyme was 14 µm, which is lower than that found on the enzyme from either D. discoideum
(Weismüller et al., 1990) or E. coli (Serina et al.,
1995). The inhibition mechanism of Ap5A was determined by
fixing one substrate at a saturating concentration and varying the
concentration of the other substrate at different fixed concentrations
of Ap5A. The families of lines shown in Figure 8 are
theoretically fit to Equation 2 (Fromm, 1975) and the points were
experimentally derived.
![<FR><NU>1</NU><DE>V</DE></FR> = <FR><NU>1</NU><DE>V<SUB>m</SUB></DE></FR>[1 + <FR><NU>K<SUB>a</SUB></NU><DE>A</DE></FR><FENCE>1 + <FR><NU>I</NU><DE>K<SUB>i</SUB></DE></FR></FENCE>]](/content/vol117/issue1/fulltext/245/img002.gif)
|
(2)
|
where I, Ka, and
Ki represent the concentration of free
Ap5A, the Michaelis constant for ATP (or UMP), and the
inhibition constant for Ap5A, respectively. The inhibition
by Ap5A when ATP was the variable substrate is shown in
Figure 8A, and the inhibition of Ap5A when UMP was the
variable substrateis shown in Figure 8B. These data clearly demonstrate
that Ap5A is a competitive inhibitor of both ATP and UMP.
The Ki values for ATP and UMP were 1.20 ± 0.02 and 6.53 ± 0.03 µm, respectively.
View larger version (17K):
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| Figure 8.
Kinetics of Ap5A
inhibition. A, Double-reciprocal plot of initial velocity
versus ATP concentrations in the presence of Ap5A
inhibitor. UMP concentrations were fixed at 400 µm. The concentrations of Ap5A were 0 (), 2 (+), 4 (), and 6 µm (). The lines are theoretical based on Equation 2.
The points were experimentally determined. B, Plot of reciprocal of
initial velocity of UMP/CMP kinase versus reciprocal of UMP
concentrations in the presence of Ap5A inhibitor. ATP
concentrations were fixed at 300 µm. The concentrations
of Ap5A were 0 (), 8 (+), 16 (), and 24 µm (). The lines are theoretical based on Equation 2.
The points were experimentally determined.
|
|
| |
DISCUSSION |
We have isolated the cDNA encoding the A. thaliana
UMP/CMP kinase by complementation of an S. cerevisiae UMP
kinase mutant. Complementation of known yeast mutants is a very
effective way to obtain eukaryotic genes. Following isolation, the
plant cDNA was characterized by sequencing. The full-length cDNA
encodes a 202-amino acid protein that is closely related to UMP kinases from other eukaryotic sources. The plant enzyme, however, did not share
high identity with the bacterial or archebacterial UMP kinases. In
previous studies, the E. coli UMP kinase was found to belong
to the aspartokinase family (Serina et al., 1995).
When the intracellular location of the Arabidopsis UMP kinase was
examined using the online analysis tool PSORT (Nakai and Kanehisa,
1992), a cytosolic localization for the enzyme was predicted. A
cytosolic localization has also been found for the rice adenylate kinase enzyme (Kawai and Uchimiya, 1995). The cytosolic localization is
also consistent with the finding that uridine nucleotides are predominantly located in the cytosol (Dancer et al., 1990).
After characterization of the cDNA, the coding region was expressed by
fusing it to GST. Following expression, the fusion protein was cleaved
and the Arabidopsis UMP/CMP kinase was purified by HPLC. The molecular
mass, amino acid composition, and N-terminal sequence of the expressed
protein all demonstrated that it was indeed UMP/CMP kinase. When
examined for enzyme activity, the purified enzyme was found to have
both UMP kinase and CMP kinase activity. Kinetic parameters were
determined for the plant enzyme and several differences from the
E. coli enzyme were noted.
First, the E. coli enzyme shows remarkable thermal stability
in the absence of protective agents, being stable up to 65°C. Although relatively thermostable, the Arabidopsis enzyme did not show
the same degree of stability. Second, the plant enzyme is not
allosterically regulated in the same way that the prokaryotic enzyme is
regulated. At concentrations that dramatically affect the enzyme
activity of the E. coli UMP kinase, neither GTP nor UTP have
a significant effect on the plant enzyme. Indeed, at 30 times the level
that affects the bacterial enzyme, the plant enzyme is affected by only
about 25%. Bourne et al. (1991) have identified a pair of sequences,
Asp-77-His-Met-Gly-80 and Thr-165-Lys-Val-Asp-168, that are conserved
in GTP-binding proteins. Both of these sequences are conserved in the
E. coli UMP kinase (Serina et al., 1995). However, neither
of them is present in the Arabidopsis UMP/CMP kinase. Thus, by both
sequence identity and experimental observation, allosteric regulatory
sites are not present in the plant enzyme, and the Arabidopsis enzyme
seems to be both functionally and structurally different from the
prokaryotic enzyme.
The Km values for UMP and CMP indicate that
the Arabidopsis enzyme can utilize both pyrimidine monophosphates
equally well as phosphate acceptors. This is similar to the enzyme from
rat Novikoff ascites tumors (Orengo and Maness, 1978), but the mouse enzyme utilizes UMP nearly twice as effectively as CMP as a phosphate acceptor (Andersen, 1978a). The deoxy forms are almost totally ineffective as phosphate acceptors in the Arabidopsis enzyme. Two
important conclusions can be drawn from this finding. First, a
different enzyme must be responsible for the conversion of dCMP into
dCDP. This is different from the mouse and the Tetrahymena pyriformis enzyme, in which dCMP can also act as a phosphate
acceptor (Andersen, 1978b). Second, because the nucleotide
monophosphate-binding pocket does not discriminate between UMP and CMP,
the exclusion of deoxynucleotides results not only in the exclusion of
dCMP but also in the exclusion of dUMP. Thus, the conversion of dUMP into dUDP does not occur, thereby forcing the conversion of dUMP into
TMP by thymidylate synthase. Furthermore, TMP is ineffective as a
phosphate acceptor, indicating that, as in yeast (Jong et al., 1984), a
separate TMP kinase must also exist.
Structural studies of adenylate kinase from both E. coli
(Müller and Schulz, 1992) and beef heart (Diederichs and Schulz, 1991) revealed that the 2 hydroxyl of the phosphate acceptor forms a
strong hydrogen bond with an -chain carboxyl. It is likely that
similar interactions are required in the plant UMP/CMP kinase, thereby
biasing the specificity against the deoxynucleotides.
Eukaryotic UMP kinases in general have a higher specificity for ATP as
the phosphate donor, with dATP effective at about 10% the level of ATP
(Orengo and Maness, 1978). This was also found for the plant enzyme.
Other nucleotide triphosphates were essentially ineffective as
phosphate donors.
The plant UMP kinase has a high degree of identity with other
eukaryotic UMP kinases and are expected to share significant structural
identity. The UMP kinase enzyme from yeast has been purified and
characterized (Ma et al., 1990). The crystal structure of this enzyme
has been solved with substrates in place (Müller-Dieckmann and
Schulz, 1994, 1995). The substrates are held in position by numerous
favorable contacts with the protein. Most of these contacting residues
are conserved between the yeast enzyme and the plant enzyme.
Structural studies have also demonstrated that the UMP-binding
pocket of the yeast enzyme is of sufficient size to accommodate an AMP
moiety (Müller-Dieckmann and Schulz, 1994, 1995). This explains
the high activity of the yeast UMP kinase for AMP. The finding that
Ap5A is a competitive inhibitor of UMP with a micromolar Ki indicates that the Arabidopsis enzyme
has a UMP-binding pocket that is also sufficiently large to accommodate
an AMP moiety. However, the Arabidopsis enzyme has less than 0.5%
activity with AMP. Therefore, the structure of the UMP-binding pocket
of the Arabidopsis enzyme will be particularly interesting to
understand. The radiography-crystallographic studies of the yeast UMP
kinase have failed to explain the specificity of this enzyme for UMP. Those residues that have been shown to line the uracil-binding pocket
of the yeast UMP kinase (Ala-47, Leu-51, Ile-75, Val-76, Thr-81,
Phe-105, Arg-107, and Gln-111) are, with only one exception (Asn
substitutes for Gln), completely conserved in the Arabidopsis enzyme.
Note that this same substitution (Asn for Gln) is found in the D. discoideum enzyme, which also shows a high degree of substrate
discrimination for UMP over AMP (Weismüller et al., 1990).
The enzymatic mechanism is also relatively well understood for the
yeast enzyme. The transition state of phosphoryl transfer is maintained
by a scaffold of interactions, including the
C
-backbones of the CORE domains and a series
of six positively charged residues (Lys-29, Arg-52, Arg-107, Arg-142,
Arg-148, and Arg-159) positioned to coordinate the phosphates. All of
these residues are conserved in the Arabidopsis enzyme. The interaction of the substrate-fixed phosphates with the yeast UMP kinase is virtually identical to E. coli adenylate kinase
(Müller-Dieckmann and Schultz, 1994), indicating the widespread
and general conservation of this enzymatic mechanism. Because of the
high degree of conservation in these important contacting residues, it
is probable that the Arabidopsis enzyme also shares this enzymatic
mechanism.
| |
FOOTNOTES |
1
This research was supported by grant no.
91-37301-6208 from the U.S. Department of Agriculture. This is paper
no. J-17416 from the Iowa Agriculture and Home Economics Experiment
Station.
*
Corresponding author; e-mail thorn{at}iastate.edu; fax
1-515-294-0453.
Received September 8, 1997;
accepted February 10, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Ap5A, P1,
P5-di(adenosine-5) pentaphosphate.
GST, glutathione
S-transferase.
IPTG, isopropyl-
-d-thiogalactoside.
| |
ACKNOWLEDGMENTS |
We thank Dr. Michelle Minet for her help with yeast molecular
biology techniques and Dr. Herbert Fromm for his help with analysis of
the kinetics of UMP/CMP kinase. We would also like to thank Dr. Roger
S. Goody (MPI fuer Molekulare Physiologie, Reinlanddamm) for kindly
supplying the Ap5A.
| |
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Abstract
Introduction