(Received for publication, October 10, 1995; and in revised form, November 21, 1995)
From the
CMP kinase from Escherichia coli is a monomeric protein
of 225 amino acid residues. The protein exhibits little overall
sequence similarities with other known NMP kinases. However, residues
involved in binding of substrates and/or in catalysis were found
conserved, and sequence comparison suggested conservation of the global
fold found in adenylate kinases or in several CMP/UMP kinases. The
enzyme was purified to homogeneity, crystallized, and analyzed for its
structural and catalytic properties. The crystals belong to the
hexagonal space group P6, have unit cell parameters a = b = 82.3 Å and c =
60.7 Å, and diffract x-rays to a 1.9 Å resolution. The
bacterial enzyme exhibits a fluorescence emission spectrum with maximum
at 328 nm upon excitation at 295 nm, which suggests that the single
tryptophan residue (Trp
) is located in a hydrophobic
environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with
CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast
with CMP/UMP kinase from Dictyostelium discoideum, an enzyme
active on CMP or UMP but much less active on the corresponding
deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this
family of proteins that might explain inhibition of enzyme activity by
excess of nucleoside monophosphate.
Nucleoside monophosphate (NMP) ()kinases represent an
ubiquitous family of catalysts playing a key role in the cell
metabolism including synthesis of RNA and DNA molecules (Anderson,
1973; Neuhard and Nygaard, 1987). They catalyze the reversible transfer
of phosphoryl group from a NTP (in general ATP) to a NMP. Adenylate
kinase represents the best known member of NMP kinase family (Noda,
1973). The adk gene from a great number of living cells was
cloned and sequenced, the corresponding protein was purified, and
numerous variants obtained by site-directed mutagenesis were
characterized for catalytic or structural properties (Tsai and Yan,
1991; Bârzu and Gilles, 1993). Much less is known
on the other members of NMP kinase family. Apparently they belong to
the adenylate kinase paradigm (Liljelund et al., 1989;
Wiesmüller et al., 1990; Konrad, 1992;
Müller-Dieckmann and Schulz, 1994) exhibiting
sequence similarities and related three-dimensional structure. However,
UMP kinase from Escherichia coli and probably from other
enteric bacteria deviate from this paradigm. The protein encoded by the pyrH gene (Smallshaw and Kelln, 1992) does not display any
sequence similarity to known NMP kinase but belongs to the
aspartokinase family (Serina et al., 1995). Moreover, it has
an oligomeric structure and is subject to complex regulatory control by
GTP and UTP (Serina et al., 1995). Because UMP kinase from E. coli has an absolute specificity for UMP as substrate, the
existence in this bacterium of at least one other enzyme acting
specifically on CMP was postulated. Fricke et al.(1995) showed
that the mssA gene from E. coli, whose function was
identified as suppressing the conditional lethal phenotype of certain smbA mutants (Yamanaka et al., 1992, 1994), is
identical to the cmk gene. The smbA gene itself,
shown to be essential for cell proliferation, is identical to the pyrH gene (Smallshaw and Kelln, 1992; Serina et al.,
1995). These surprising observations prompted us to undertake a
detailed biochemical analysis of CMP kinase from E. coli,
purified after overexpression of the corresponding gene.
Figure 1:
Alignment of amino acid sequences in 11
forms of NMP kinases. Identical and similar residues expressed in
one-letter codes are indicated in black and gray
boxes, whereas strictly conserved residues shown to play a role in
catalysis are marked by asterisks. The black points identify the Cys and Trp
residues, whose
role is mentioned under ``Results.'' The preferential trypsin
cleavage site is marked by an arrow, and the secondary
structure elements are mentioned under the alignment. The proteins are
(from top to bottom): E. coli CMP kinase, B. subtilis CMP kinase, M. leprae CMP kinase, D. discoideum CMP/UMP kinase, yeast UMP kinase, pig muscle adenylate kinase 1,
bovine mitochondrial adenylate kinase 3, yeast adenylate kinase 2, E. coli adenylate kinase, Bordetella pertussis adenylate kinase, and B. subtilis adenylate
kinase.
Figure 2: SDS-PAGE (12.5%) of fractions obtained during the purification of CMP kinase from E. coli. Lane 1, bacterial extract (25 µg of protein); lane 2, blue Sepharose chromatography (16 µg of protein); lane 3, pure enzyme after Ultrogel AcA54 chromatography (25 µg of protein). The arrows indicate the standard proteins: a, phosphorylase a (94,000); b, bovine serum albumin (68,000); c, ovalbumin (43,000); d, carbonic anhydrase (30,000); e, soybean trypsin inhibitor (20,100); and f, lysozyme (14,400).
Figure 3: Electrospray ionization mass spectrum of CMP kinase from E. coli. The molecular mass was calculated from the multiply charged molecular ion envelope (24617.0 ± 2.3 daltons). The minor series correspond to a CMP kinase dimer (molecular mass, 49234.8 ± 5.4 daltons).
CMP kinase from E.
coli has a single Cys residue (Cys) that is conserved
in adenylate kinase 1 and UMP kinase from D. discoideum but
not in CMP kinase from B. subtilis and M. leprae.
This residue reacted with DTNB under native conditions at low rates
(0.2 SH/mol enzyme at pH 7.4 and 10 min of incubation at room
temperature). In the presence of SDS, the protein reacted rapidly with
DTNB, the thionitrobenzoate/protein ratio being
0.9. It seems
therefore that the single thiol of CMP kinase is less exposed to the
solvent. The same is true for the single Trp residue (Trp
,
according to the numbering in protein sequence). Upon excitation at 295
nm, E. coli CMP kinase exhibits a fluorescence emission
spectrum with maximum at 328 nm. Guanidinium hydrochloride at
concentrations higher than 0.6 M shifted the fluorescence
maximum to higher wavelengths with no decrease of the maximum
amplitude. The midpoint transition for CMP kinase from E. coli is at 0.9 M of guanidinium hydrochloride (Fig. 4).
Figure 4:
Fluorescence analysis of CMP kinase
denaturation by guanidinium hydrochloride. CMP kinase (25 µg/ml,
1 µM) in 50 mM Tris-HCl (pH 7.4) was
incubated for 12 h in various concentrations of guanidinium
hydrochloride (GdmCl), and then the fluorescence spectrum of
the protein was recorded as indicated under ``Experimental
Procedures.''
Thermal denaturation experiments indicated that CMP kinase was
half-inactivated at 52 °C (not shown). The first order rate
constant (3.1 10
s
)
of inactivation of CMP kinase by TPCK-trypsin was in good agreement
with the decrease in absorption of the enzyme band scanned after
SDS-PAGE and Coomassie Blue staining. ATP (as well as ADP, CDP, CTP,
and to a lesser extent CMP) exerted significant protection against
proteolysis (Fig. 5). The N-terminal sequencing of various
peptides after electroblot transfer onto nitrocellulose membrane filter
suggested that TPCK-trypsin cleaves CMP kinase from its C-terminal end,
rich in lysine and arginine residues. The fragment marked with an asterisk in Fig. 5that is resistant to further
proteolytic cleavage corresponds most probably to a C-terminal
truncated form of CMP kinase ending with amino acids
AHRR
.
Figure 5: Proteolysis of E. coli CMP kinase by TPCK-trypsin and protection by ATP. CMP kinase at 1 mg/ml in 50 mM Tris-HCl (pH 7.4) was incubated at 4 °C with TPCK-trypsin (2 µg/ml) in the absence (lanes 1-4) or the presence of 1 mM ATP (lanes 5-8). At different time intervals, 5 s (lanes 1 and 5), 2.5 min (lanes 2 and 6), 5 min (lanes 3 and 7), and 10 min (lanes 4 and 8), 10-µl aliquots were withdrawn, boiled with electrophoresis buffer, and analyzed by SDS-PAGE (12.5%) and Coomassie Blue staining. The molecular weight standards are the same as those described in the legend to Fig. 2.
Figure 6: Photomicrograph of a CMP kinase crystal. The maximum dimension is 0.7 mm.
Figure 7: Screened zero level precession photograph obtained from a CMP kinase crystal. Reflections along 00l (i.e. the vertical axis) appear only when l = 2n. The circumference of the diffraction pattern corresponds to approximately 3 Å resolution.
Like
other NMP kinases, CMP kinase from E. coli was inhibited by
high concentrations of nucleotides. The reaction rate in these cases
was fitted with the equation: v = V
S/(K
+ S + S
/K
), which
allowed calculation of the V
, K
, and K
with different pairs
of nucleoside mono- and triphosphates. The apparent K
for ATP with different NMPs varied within a factor of 2 (between
0.038 and 0.08 mM). The apparent K
for
various NMPs was not very much dependent on the chemical nature of the
phosphate donor. The kinetic parameters in the reverse reaction were
the following: K
= 0.025
mM; K
= 0.052
mM, and V
= 410 units/mg protein.
Both ADP and CDP exerted a slight inhibitory effect over 0.3 mM (calculated K
values,
3 mM).
Figure 8:
Binding of Ant-dATP to CMP kinase from E. coli (left) and displacement of the analog by ATP (right) as determined from fluorescence experiments. A 2
µM solution of Ant-dATP in 50 mM Tris-HCl (pH
7.4), 100 mM NaCl, and 2 mM MgCl was
supplemented with 2-20 µM CMP kinase. Then, the CMP
kinase-Ant-dATP complex was titrated with increasing concentrations of
ATP or ADP. One data point corresponds to fluorescence intensities
integrated over a total time of 8 s. Prot.,
protein.
De novo synthesis and recycling of nucleotides in bacteria and eukaryotes are quite well understood processes. It is generally assumed that phosphorylation to nucleoside diphosphates is a specific reaction and that NMP kinases represent a homogeneous family of catalysts sharing similar primary and three-dimensional structure with adenylate kinases. It was therefore a surprise to discover that UMP kinase in E. coli is an enzyme of completely different descent being related to aspartokinases rather than to adenylate kinases (Serina et al., 1995). In addition to the highly specific UMP kinase, enteric bacteria contain two other pyrimidine NMP kinases: a TMP kinase and a CMP kinase. Mutants defective in the two enzyme activities were isolated and characterized either in E. coli or in S. typhimurium (Beck et al., 1974; Blinkley and Kuempel, 1986). However, only recently Fricke et al.(1995), corroborating previous works on an E. coli gene specifying a 25-kDa polypeptide (Pedersen et al., 1984) and recent works on a mssA gene (Yamanaka et al., 1994) (from multicopy suppressor of smbA), demonstrated that cmk and mssA are identical genes. The known E. coli cmk gene is not essential (contrary to the adk or pyrH genes), but this may be due to the presence of a second cmk gene, as found in Haemophilus influenzae, a close relative of E. coli (Fleischmann et al., 1995). Cytidine nucleotides have a special situation in the nucleotide metabolism because CTP results in the de novo pathway from UTP and not from the corresponding monophosphate and diphosphate precursors. This is particularly important because deoxyribonucleotides result by reduction of the corresponding ribonucleoside diphosphates. Scavenging of CMP and production of CDP are therefore steps of major importance for DNA synthesis. This accounts for the observation that the DNA replication rate is reduced in cmk mutants where CMP and dCMP accumulate at high levels. The fact that in high gene copy number the cmk/mssA gene can suppress defects in the smbA/pyrH gene (Yamanaka et al., 1994) implies that E. coli CMP kinase is endowed with a residual UMP kinase activity, which indeed was the case as shown in this paper.
Although CMP is not produced in the de novo pathway, it might accumulate either from CTP during the synthesis of phospholipids or from the hydrolytic cleavage of mRNA. Therefore, the physiological role of CMP kinase is to recycle CMP to CDP, which is either rapidly phosphorylated by the unspecific nucleoside-diphosphate kinase to CTP or reduced to dCDP. In bacteria CDP (as well as ADP, UDP, or GDP) can also result from phosphorolytic cleavage of mRNA by polynucleotide phosphorylase (EC 2.7.7.8) (Carpousis et al., 1994; Py et al., 1994). It therefore seems worth looking for a possible link between these two CDP-producing enzymes, i.e. CMP kinase and polynucleotide phosphorylase. The cmk gene is located in the E. coli chromosome as the first gene of an operon comprising the rpsA gene (Fricke et al., 1995). In this organism, the rpsA gene product (protein S1) promotes translation initiation by binding to the 5` end of the mRNA molecule and enhancing the recognition of the ribosome binding site upstream of the start codon. Consulting of the data base present at the Institute for Genomic Research site (http://www.tigr.org), we found that the same organization holds for one of the cmk genes present in H. influenzae (Fleischmann et al., 1995). Surprisingly, the same is also true for B. subtilis, a Gram-positive organism, described as not possessing a ribosomal S1 protein. Comparison of S1 sequence with data libraries revealed that it possessed an internal repetition motif of 69 residues that is also present in polynucleotide phosphorylase (Regnier et al., 1987). In addition, further analysis demonstrates that this motif is also present in a RNA helicase molecule. This permits us to propose that the primary function of S1 is to present RNA molecules to polynucleotide phosphorylase, so that they can be degraded efficiently from their 3` end. This is consistent with the newly discovered complex of RNA degradation, comprising polynucleotide phosphorylase (Carpousis et al., 1994). In E. coli, this function has evolved, as a side effect, to that of presenting the mRNA to the ribosome, under a conformation adapted to translation initiation. The selection pressure linked to this function has associated S1 to the cmk gene product, because this ends in the same general function, generation of CDP (Company et al., 1991).
Sequence comparison of E. coli CMP kinase
with other members of the NMP kinase family showed few overall sequence
similarities. However, the protein seems to conserve the same global
fold as found in the NMP kinases whose three-dimensional structure was
already solved (Müller-Dieckmann and Schulz, 1995;
Vonrhein et al., 1995). Molecular modelling of E. coli CMP kinase showed that Cys and Trp
are
buried in the protein core at the interface of the helix
1 and the
-strand, in agreement with experiments of intrinsic fluorescence
of the protein or reactivity toward DTNB. These two amino acid residues
are conserved in UMP/CMP kinase from D. discoideum as
Cys
and Trp
. Because the latter enzyme has a
second Cys residue at the position 119 and was readily inactivated by
DTNB (Wiesmüller et al., 1990), we might
deduce that the DTNB-sensitive thiol group in the D. discoideum enzyme corresponds to Cys
. In the same way we can
deduce that Arg
in E. coli CMP kinase that is
exposed to the solvent in nucleotide-free form of protein is stacked
against the substrate base rings in the nucleotide-complexed CMP
kinase, explaining the protection of enzyme against trypsin digestion
by ATP or ADP.
Another residue found conserved as threonine in
adenylate kinases (Thr in pig muscle adenylate kinase 1
and Thr
in E. coli enzyme) and as alanine in
yeast (Ala
), D. discoideum (Ala
), or
porcine brain (Ala
) UMP/CMP kinase (Okajima et
al., 1995) deserves some comments. These residues were suggested
by several authors (Müller-Dieckmann and Schulz,
1995; Okajima et al., 1993) to play a role in recognition of
the heterocycle and therefore to contribute to the substrate
specificity of NMP kinases. Contrary to expectations, in the E.
coli, B. subtilis, and M. leprae CMP kinase the
same position is occupied by Ser/Thr residues, which are characteristic
to the adenylate kinase family. In fact, site-directed mutagenesis of
Ala
to Thr in D. discoideum UMP/CMP kinase (
)did not change the substrate specificity of the slime mold
enzyme. The determination of the three-dimensional structure of the CMP
kinase from E. coli is expected to answer more precisely all
these questions and also to explain differences in substrate
specificity as compared with the enzymes from yeast or from D.
discoideum (Wiesmüller et al., 1995).