Publications

• The Rv1712 Locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially phosphorylates dCMP.

  Thum C., Schneider C.Z., Palma M.S., Santos D.S., Basso L.A.

  The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A ... >> More

  The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed. << Less

  J. Bacteriol. 191:2884-2887(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

  Bucurenci N., Sakamoto H., Briozzo P., Palibroda N., Serina L., Sarfati R.S., Labesse G., Briand G., Danchin A., Barzu O., Gilles A.M.

  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 suggeste ... >> More

  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(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A 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 (Trp30) 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. << Less

  J Biol Chem 271:2856-2862(1996) [PubMed] [EuropePMC]

• The human adenylate kinase 9 is a nucleoside mono- and diphosphate kinase.

  Amiri M., Conserva F., Panayiotou C., Karlsson A., Solaroli N.

  Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate k ... >> More

  Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate kinase family of enzymes and accordingly named the protein adenylate kinase 9 (AK9). Initially a full-length cDNA of a hypothetical protein containing a predicted adenylate kinase domain was identified and subsequently cloned and expressed in Escherichia coli. The substrate specificity of the recombinant protein showed that the enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP as phosphate donor, while only AMP and CMP were phosphorylated when GTP was the phosphate donor. The kinetic parameters of AK9 were determined for AMP, dAMP and CMP with ATP as phosphate donor. Interestingly, in addition to the diphosphate products, a nucleoside diphosphate kinase (NDPK) activity was also present with subsequent triphosphates formed. With ATP or GTP as phosphate donor it was possible to detect the production of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and TTP as enzymatic products from the corresponding diphosphate substrates. A number of previously characterized adenylate kinases were also tested and found to possess a broad phosphotransferase activity similar to AK9. These enzymes are accordingly suggested to be regarded as nucleoside mono- and diphosphate kinases with catalytic activities possibly determined by local substrate concentrations. << Less

  Int. J. Biochem. Cell Biol. 45:925-931(2013) [PubMed] [EuropePMC]

  This publication is cited by 23 other entries.

• The cmk gene encoding cytidine monophosphate kinase is located in the rpsA operon and is required for normal replication rate in Escherichia coli.

  Fricke J., Neuhard J., Kelln R.A., Pedersen S.

  A gene encoding a polypeptide of 25 kDa is located immediately upstream of the gene for ribosomal protein S1, rpsA. In high gene copy number, this gene, mssA, was previously found to suppress defects in smbA, which is now known to be identical to pyrH, encoding UMP kinase. We show here that the 25 ... >> More

  A gene encoding a polypeptide of 25 kDa is located immediately upstream of the gene for ribosomal protein S1, rpsA. In high gene copy number, this gene, mssA, was previously found to suppress defects in smbA, which is now known to be identical to pyrH, encoding UMP kinase. We show here that the 25-kDa polypeptide comprises CMP kinase and propose that the gene be designated cmk. In a strain deleted for cmk, the pools of CMP and dCMP were elevated approximately 30-fold. We constructed a plasmid from which synthesis of CMP kinase was regulated by the lac promoter-operator and measured the synthesis rates for RNA and DNA after induction in the delta cmk/lacPO-cmk+ strain. A specific increase in the rate of DNA synthesis was observed. Further analyses showed that the replication elongation rate was halved in the delta cmk strain, most likely caused by the reductions of the dCTP and dTTP pools to 30 and 70%, respectively, of the levels in the parental strain, but that this was compensated for by a doubling in the frequency of initiation. The delta cmk strain is viable at 37 degrees C but cold sensitive. The cold sensitivity may be related to defects in the synthesis of phospholipids or lipopolysaccharides. In addition to the physiological studies, the region upstream of cmk was sequenced, and 120 codons with strong homology to an uncharacterized protein of the speB operon were identified. << Less

  J. Bacteriol. 177:517-523(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Sugar specificity of bacterial CMP kinases as revealed by crystal structures and mutagenesis of Escherichia coli enzyme.

  Bertrand T., Briozzo P., Assairi L., Ofiteru A., Bucurenci N., Munier-Lehmann H., Golinelli-Pimpaneau B., Barzu O., Gilles A.M.

  Bacterial cytidine monophosphate (CMP) kinases are characterised by an insert enlarging their CMP binding domain, and by their particular substrate specificity. Thus, both CMP and 2'-deoxy-CMP (dCMP) are good phosphate acceptors for the CMP kinase from Escherichia coli (E. coli CMPK), whereas euka ... >> More

  Bacterial cytidine monophosphate (CMP) kinases are characterised by an insert enlarging their CMP binding domain, and by their particular substrate specificity. Thus, both CMP and 2'-deoxy-CMP (dCMP) are good phosphate acceptors for the CMP kinase from Escherichia coli (E. coli CMPK), whereas eukaryotic UMP/CMP kinases phosphorylate the deoxynucleotides with very low efficiency. Four crystal structures of E. coli CMPK complexed with nucleoside monophosphates differing in their sugar moiety were solved. Both structures with CMP or dCMP show interactions with the pentose that were not described so far. These interactions are lost with the poorer substrates AraCMP and 2',3'-dideoxy-CMP. Comparison of all four structures shows that the pentose hydroxyls are involved in ligand-induced movements of enzyme domains. It also gives a structural basis of the mechanism by which either ribose or deoxyribose can be accommodated. In parallel, for the four nucleotides the kinetic results of the wild-type enzyme and of three structure-based variants are presented. The phosphorylation rate is significantly decreased when either of the two pentose interacting residues is mutated. One of these is an arginine that is highly conserved in all known nucleoside monophosphate kinases. In contrast, the other residue, Asp185, is typical of bacterial CMP kinases. It interacts with Ser101, the only residue conserved in all CMP binding domain inserts. Mutating Ser101 reduces CMP phosphorylation only moderately, but dramatically reduces dCMP phosphorylation. This is the first experimental evidence of a catalytic role involving the characteristic insert of bacterial CMP kinases. Furthermore, this role concerns only dCMP phosphorylation, a feature of this family of enzymes. << Less

  J Mol Biol 315:1099-1110(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates.

  Liou J.-Y., Dutschman G.E., Lam W., Jiang Z., Cheng Y.-C.

  Pyrimidine nucleoside monophosphate kinase [UMP/CMP kinase (UMP/CMPK);EC 2.7.4.14] plays a crucial role in the formation of UDP, CDP, and dCDP, which are required for cellular nucleic acid synthesis. Several cytidine and deoxycytidine analogues are important anticancer and antiviral drugs. These d ... >> More

  Pyrimidine nucleoside monophosphate kinase [UMP/CMP kinase (UMP/CMPK);EC 2.7.4.14] plays a crucial role in the formation of UDP, CDP, and dCDP, which are required for cellular nucleic acid synthesis. Several cytidine and deoxycytidine analogues are important anticancer and antiviral drugs. These drugs require stepwise phosphorylation to their triphosphate forms to exert their therapeutic effects. The role of UMP/CMPK for the phosphorylation of nucleoside analogues has been indicated. Thus, we cloned the human UMP/CMPK gene, expressed it in Escherichia coli, and purified it to homogeneity. Its kinetic properties were determined. UMP and CMP proved to be far better substrates than dCMP. UMP/CMPK used all of the nucleoside triphosphates as phosphate donors, with ATP and dATP being the best donors and CTP being the poorest. Furthermore, UMP/CMPK was able to phosphorylate all of the deoxycytidine analogue monophosphates that we tested. The relative efficiency was as follows: arabinofuranosyl-CMP > dCMP > beta-L-2',3'-dideoxy-3'-thia-CMP > Gemcitabine monophosphate > beta-D-2',3'-dideoxy-CMP; beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluoro-CMP; beta-L-2',3'-dideoxy-5-fluoro-3'-thia-CMP > beta-L-2',3'-dideoxy-CMP > beta-L-dioxolane-CMP. By comparing the relative V(max)/K(m) values of D- and L-form dideoxy-CMP, we showed that this kinase lacked stereoselectivity. Reducing agents, such as DTT, 2-mercaptoethanol, and thioredoxin, were able to activate this enzyme, suggesting that its activity may be regulated by redox potential in vivo. UMP/CMPK localized predominantly to the cytoplasm. In addition, 196-amino acid UMP/CMPK was the actual form of UMP/CMPK, rather than the 228-amino acid form as suggested before. << Less

  Cancer Res. 62:1624-1631(2002) [PubMed] [EuropePMC]

• Structural and catalytic properties of CMP kinase from Bacillus subtilis: a comparative analysis with the homologous enzyme from Escherichia coli.

  Schultz C.P., Ylisastigui-Pons L., Serina L., Sakamoto H., Mantsch H.H., Neuhard J., Barzu O., Gilles A.M.

  CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in ... >> More

  CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7-to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect. << Less

  Arch. Biochem. Biophys. 340:144-153(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.