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Yungchi Cheng - One of the best experts on this subject based on the ideXlab platform.

  • modulation of human UMP cmp kinase affects activation and cellular sensitivity of deoxycytidine analogs
    Biochemical Pharmacology, 2010
    Co-Authors: Jiehyuan Liou, Chihhung Hsu, Hui-ru Lai, Wei-ling Chang, Mei-ju Hsieh, Yu-chun Huang, Yungchi Cheng
    Abstract:

    Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

  • Modulation of human UMP/CMP kinase affects activation and cellular sensitivity of deoxycytidine analogs
    Biochemical pharmacology, 2009
    Co-Authors: Jiehyuan Liou, Chihhung Hsu, Hui-ru Lai, Wei-ling Chang, Mei-ju Hsieh, Yu-chun Huang, Yungchi Cheng
    Abstract:

    Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

  • phosphorylation of cytidine deoxycytidine and their analog monophosphates by human UMP cmp kinase is differentially regulated by atp and magnesium
    Molecular Pharmacology, 2005
    Co-Authors: Chihhung Hsu, Jiehyuan Liou, Ginger E Dutschman, Yungchi Cheng
    Abstract:

    Human UMP/CMP kinase (cytidylate kinase; EC 2.7.4.14) is responsible for phosphorylation of CMP, UMP, and deoxycytidine monophosphate (dCMP) and also plays an important role in the activation of pyrimidine analogs, some of which are clinically useful anticancer or antiviral drugs. Previous kinetic data using recombinant or highly purified human UMP/CMP kinase showed that dCMP, as well as pyrimidine analog monophosphates, were much poorer substrates than CMP or UMP for this enzyme. This implies that other unidentified mechanisms must be involved to make phosphorylation of dCMP or pyrimidine analog monophosphates inside cells by this enzyme possible. Here, we reevaluated the optimal reaction conditions for human recombinant human UMP/CMP kinase to phosphorylate dCMP and CMP (referred as dCMPK and CMPK activities). We found that ATP and magnesium were important regulators of the kinase activities of this enzyme. Free magnesium enhanced dCMPK activity but inhibited CMPK activity. Free ATP or excess ATP/magnesium, on the other hand, inhibited dCMPK but not CMPK reactions. The differential regulation of dCMPK versus CMPK activities by ATP or magnesium was also seen in other 2′-deoxypyrimidine analog monophosphates (deoxyuridine monophosphate, 5-fluorodeoxyuridine monophosphate, 1-β-d-arabinofuranosylcytosine monophosphate, and gemcitabine monophosphate) versus their ribose-counterparts (UMP and 5-fluorouridine monophosphate), in a similar manner. The data suggest that the active sites of human UMP/CMP kinase for dCMP and for CMP cannot be identical. Furthermore, enzyme inhibition studies demonstrated that CMP could inhibit dCMP phosphorylation in a noncompetitive manner, with Ki values much higher than its own Km values. We thus propose novel models for the phosphorylation action of human UMP/CMP kinase.

  • atp and magnesium differentially regulate ribose and 2 deoxyribose cytidine and its analogue monophosphate phosphorylation by human UMP cmp kinase
    Cancer Research, 2004
    Co-Authors: Chihhung Hsu, Jiehyuan Liou, Ginger E Dutschman, Yungchi Cheng
    Abstract:

    Proc Amer Assoc Cancer Res, Volume 45, 2004 2964 UMP/CMP kinase (EC 2.7.4.14), which is responsible for phosphorylation of CMP, UMP, and dCMP, is crucial for nucleic acid synthesis and DNA repair. It also plays an important role in the activation of several cytidine or deoxycytidine analogues, some of which are clinically useful anticancer or antiviral drugs. Previous kinetic data using recombinant or highly purified human enzyme by us or others showed that dCMP, as well as other deoxycytidine analogue monophosphates, are relatively poor substrates for human UMP/CMP kinase. Under identical ATP and magnesium (Mg2+) condition (ATP/Mg2+= 8/8 mM), dCMP could only be phosphorylated with a > 200-fold less efficiency than CMP (referring to dCMPK activity Vs. CMPK activity). This raises a question how dCMP and these deoxycytidine analogue- monophosphates can be phosphorylated by this enzyme in vivo , giving higher intracellular concentrations of CMP and UMP. In the current study, we re-evaluate the optimal reaction conditions for recombinant human UMP/CMP kinase in phosphorylating dCMP and CMP. The data showed that ATP and Mg2+ affected dCMPK and CMPK activities very differently. While Mg2+ was crucial for kinase activity, free Mg2+ enhanced the dCMPK activity but inhibited the CMPK activity. Although ATP served as a good phosphate donor for this enzyme reaction, free ATP or excess ATP-Mg2+ showed inhibition of dCMPK activity, but no inhibition of CMPK activity. The differential regulation of dCMPK versus CMPK activities by ATP or Mg2+ was also seen in other 2’-deoxyribose-cytidine or uridine analogue monophosphates (dUMP, 5FdUMP, and ara-CMP) versus their ribose-counterparts (UMP, 5FUMP), in a similar manner. Further, under the optimal reaction condition for dCMP (when ATP/Mg2+= 0.1/2 mM), the relative efficiency for this enzyme to phosphorylate dCMP can be improved for 493± 25%, compared to when ATP/Mg2+= 8/8 mM. Our data suggest that the active sites of human UMP/CMP kinase for dCMP and for CMP cannot be identical. We hypothesize that this enzyme might be able to phosphorylate dCMP and deoxycytidine analogue monophosphates, such as gemcitabine, ara-C, or lamivudine monophosphates, in vivo through compartmentation of substrates or conformational change of enzyme either by post-translational modification, multimeric formation, or by interaction with other cellular factors. (This work was supported by NIH grants R01CA63477 & R01AI38204.)

  • characterization of human UMP cmp kinase and its phosphorylation of d and l form deoxycytidine analogue monophosphates
    Cancer Research, 2002
    Co-Authors: Jiehyuan Liou, Ginger E Dutschman, Wing Lam, Zaoli Jiang, Yungchi Cheng
    Abstract:

    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 > β-l-2′,3′-dideoxy-3′-thia-CMP > Gemcitabine monophosphate > β-d-2′,3′-dideoxy-CMP; β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluoro-CMP; β-l-2′,3′-dideoxy-5-fluoro-3′-thia-CMP > β-l-2′,3′-dideoxy-CMP > β-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.

Alan G Marshall - One of the best experts on this subject based on the ideXlab platform.

Jiehyuan Liou - One of the best experts on this subject based on the ideXlab platform.

  • modulation of human UMP cmp kinase affects activation and cellular sensitivity of deoxycytidine analogs
    Biochemical Pharmacology, 2010
    Co-Authors: Jiehyuan Liou, Chihhung Hsu, Hui-ru Lai, Wei-ling Chang, Mei-ju Hsieh, Yu-chun Huang, Yungchi Cheng
    Abstract:

    Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

  • Modulation of human UMP/CMP kinase affects activation and cellular sensitivity of deoxycytidine analogs
    Biochemical pharmacology, 2009
    Co-Authors: Jiehyuan Liou, Chihhung Hsu, Hui-ru Lai, Wei-ling Chang, Mei-ju Hsieh, Yu-chun Huang, Yungchi Cheng
    Abstract:

    Deoxycytidine analogs are an important class of clinically active antiviral and anticancer agents. The stepwise phosphorylation of these analogs to triphosphate metabolites is crucial for biological action. Human UMP/CMP kinase (UMP/CMPK; cytidylate kinase; EC 2.7.4.14) is thought to be responsible for phosphorylation of UMP, CMP, and dCMP and may also play an important role in the activation of pyrimidine analogs. However, no evidence has verified this notion in intact cells. In this study we explored the functional roles of UMP/CMPK in natural pyrimidine synthesis and metabolism of deoxycytidine analogs, as well as 5-FU in HeLa S3 and HCT8 cells. The amounts of UMP/CMPK protein in different cell lines correlated with UMP, CMP, and dCMP kinase activities and amounts of UMP/CMPK RNA. Modulation of UMP/CMPK by overexpression or down-regulation had no impact on natural pyrimidine nucleotides and cell growth. However, down-regulating UMP/CMPK expression by siRNA led to a decrease in the formation of the triphosphate metabolites, resulting in cellular resistance to these analogs. More diphosphate and triphosphate metabolites of deoxycytidine analogs were detected and cellular sensitivity to these agents was increased in the UMP/CMPK-overexpressing cells. This study indicates that the second step enzyme (UMP/CMPK) is responsible for the phosphorylation of pyrimidine analogs and also has an impact on cellular sensitivity to these analogs in those cell lines.

  • phosphorylation of cytidine deoxycytidine and their analog monophosphates by human UMP cmp kinase is differentially regulated by atp and magnesium
    Molecular Pharmacology, 2005
    Co-Authors: Chihhung Hsu, Jiehyuan Liou, Ginger E Dutschman, Yungchi Cheng
    Abstract:

    Human UMP/CMP kinase (cytidylate kinase; EC 2.7.4.14) is responsible for phosphorylation of CMP, UMP, and deoxycytidine monophosphate (dCMP) and also plays an important role in the activation of pyrimidine analogs, some of which are clinically useful anticancer or antiviral drugs. Previous kinetic data using recombinant or highly purified human UMP/CMP kinase showed that dCMP, as well as pyrimidine analog monophosphates, were much poorer substrates than CMP or UMP for this enzyme. This implies that other unidentified mechanisms must be involved to make phosphorylation of dCMP or pyrimidine analog monophosphates inside cells by this enzyme possible. Here, we reevaluated the optimal reaction conditions for human recombinant human UMP/CMP kinase to phosphorylate dCMP and CMP (referred as dCMPK and CMPK activities). We found that ATP and magnesium were important regulators of the kinase activities of this enzyme. Free magnesium enhanced dCMPK activity but inhibited CMPK activity. Free ATP or excess ATP/magnesium, on the other hand, inhibited dCMPK but not CMPK reactions. The differential regulation of dCMPK versus CMPK activities by ATP or magnesium was also seen in other 2′-deoxypyrimidine analog monophosphates (deoxyuridine monophosphate, 5-fluorodeoxyuridine monophosphate, 1-β-d-arabinofuranosylcytosine monophosphate, and gemcitabine monophosphate) versus their ribose-counterparts (UMP and 5-fluorouridine monophosphate), in a similar manner. The data suggest that the active sites of human UMP/CMP kinase for dCMP and for CMP cannot be identical. Furthermore, enzyme inhibition studies demonstrated that CMP could inhibit dCMP phosphorylation in a noncompetitive manner, with Ki values much higher than its own Km values. We thus propose novel models for the phosphorylation action of human UMP/CMP kinase.

  • atp and magnesium differentially regulate ribose and 2 deoxyribose cytidine and its analogue monophosphate phosphorylation by human UMP cmp kinase
    Cancer Research, 2004
    Co-Authors: Chihhung Hsu, Jiehyuan Liou, Ginger E Dutschman, Yungchi Cheng
    Abstract:

    Proc Amer Assoc Cancer Res, Volume 45, 2004 2964 UMP/CMP kinase (EC 2.7.4.14), which is responsible for phosphorylation of CMP, UMP, and dCMP, is crucial for nucleic acid synthesis and DNA repair. It also plays an important role in the activation of several cytidine or deoxycytidine analogues, some of which are clinically useful anticancer or antiviral drugs. Previous kinetic data using recombinant or highly purified human enzyme by us or others showed that dCMP, as well as other deoxycytidine analogue monophosphates, are relatively poor substrates for human UMP/CMP kinase. Under identical ATP and magnesium (Mg2+) condition (ATP/Mg2+= 8/8 mM), dCMP could only be phosphorylated with a > 200-fold less efficiency than CMP (referring to dCMPK activity Vs. CMPK activity). This raises a question how dCMP and these deoxycytidine analogue- monophosphates can be phosphorylated by this enzyme in vivo , giving higher intracellular concentrations of CMP and UMP. In the current study, we re-evaluate the optimal reaction conditions for recombinant human UMP/CMP kinase in phosphorylating dCMP and CMP. The data showed that ATP and Mg2+ affected dCMPK and CMPK activities very differently. While Mg2+ was crucial for kinase activity, free Mg2+ enhanced the dCMPK activity but inhibited the CMPK activity. Although ATP served as a good phosphate donor for this enzyme reaction, free ATP or excess ATP-Mg2+ showed inhibition of dCMPK activity, but no inhibition of CMPK activity. The differential regulation of dCMPK versus CMPK activities by ATP or Mg2+ was also seen in other 2’-deoxyribose-cytidine or uridine analogue monophosphates (dUMP, 5FdUMP, and ara-CMP) versus their ribose-counterparts (UMP, 5FUMP), in a similar manner. Further, under the optimal reaction condition for dCMP (when ATP/Mg2+= 0.1/2 mM), the relative efficiency for this enzyme to phosphorylate dCMP can be improved for 493± 25%, compared to when ATP/Mg2+= 8/8 mM. Our data suggest that the active sites of human UMP/CMP kinase for dCMP and for CMP cannot be identical. We hypothesize that this enzyme might be able to phosphorylate dCMP and deoxycytidine analogue monophosphates, such as gemcitabine, ara-C, or lamivudine monophosphates, in vivo through compartmentation of substrates or conformational change of enzyme either by post-translational modification, multimeric formation, or by interaction with other cellular factors. (This work was supported by NIH grants R01CA63477 & R01AI38204.)

  • characterization of human UMP cmp kinase and its phosphorylation of d and l form deoxycytidine analogue monophosphates
    Cancer Research, 2002
    Co-Authors: Jiehyuan Liou, Ginger E Dutschman, Wing Lam, Zaoli Jiang, Yungchi Cheng
    Abstract:

    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 > β-l-2′,3′-dideoxy-3′-thia-CMP > Gemcitabine monophosphate > β-d-2′,3′-dideoxy-CMP; β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluoro-CMP; β-l-2′,3′-dideoxy-5-fluoro-3′-thia-CMP > β-l-2′,3′-dideoxy-CMP > β-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.

Dominique Deville-bonne - One of the best experts on this subject based on the ideXlab platform.

  • 5',6'-Nucleoside Phosphonate Analogues Architecture: Synthesis and Comparative Evaluation towards Metabolic Enzymes
    ChemMedChem, 2011
    Co-Authors: Franck Gallier, Dominique Deville-bonne, Julie A.c. Aleandre, Chahrazade El Amri, Suzanne Peyrottes, Christian Périgaud
    Abstract:

    Nucleoside phosphonates have been designed as stable 5‚ -mononucleotide mimics and are nowadays considered a potent class of antiviral agents. Within cells, they must be metabolised to the corresponding diphosphate to exert their biological activity. In this process, the first phosphorylation step, catalysed by nucleoside monophosphate kinases (NMP kinases), has been proposed as a bottleneck. Herein, we report the synthesis of a series of ribonucleoside phosphonate derivatives isosteric to 5‚ -mononucleotides, with different degrees of flexibility within the 5‚ ,6 ‚ -CC bond, as well as different polarities, through the introduction of hydroxy groups. The influence of these modifications on the capacity of the compounds to act as substrates for appropriate human NMP kinases, involved in nucleic acids metabolism, has been investigated. Low flexibility, as well as an absence of hydroxy groups within the riboseˆphosphorus architecture, is critical for efficient phosphotransfer. Among the series of pyrimidine analogues, one derivative was shown to be phosphorylated by human UMP-CMP kinase, with rates similar to those of dUMP and even better than dCMP.

  • Enantioselectivity of human AMP, dTMP and UMP-CMP kinases.
    Nucleic Acids Research, 2007
    Co-Authors: Julie A C Alexandre, Béatrice Roy, Dimitri Topalis, Sylvie Pochet, Christian Périgaud, Dominique Deville-bonne
    Abstract:

    l-Nucleoside analogues such as lamivudine are active for treating viral infections. Like d-nucleosides, the biological activity of the l-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases. We have therefore investigated the capacity of l-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only d-(d)AMP. l-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than d-dTMP. Both l-dUMP and l-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2'-azido derivatives of dUMP and dUMP while, unexpectedly, the 2'-azido-d-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of d-(d)NMP to their respective kinases could account for the differences in interactions of the l-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for l-dT.

  • Reaction of human UMP‐CMP kinase with natural and analog substrates
    European journal of biochemistry, 2003
    Co-Authors: Claudia Pasti, Michel Veron, Anne Marie Gilles, Sarah Gallois-montbrun, Hélène Munier-lehmann, Dominique Deville-bonne
    Abstract:

    UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.

Anna Karlsson - One of the best experts on this subject based on the ideXlab platform.

  • human UMP cmp kinase 2 a novel nucleoside monophosphate kinase localized in mitochondria
    Journal of Biological Chemistry, 2008
    Co-Authors: Yunjian Xu, Magnus Johansson, Anna Karlsson
    Abstract:

    Abstract Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis.

  • the drosophila melanogaster UMP cmp kinase cdna encodes an n terminal mitochondrial import signal
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Sophie Curbo, Magnus Johansson, Marjan Amiri, Fariba Foroogh, Anna Karlsson
    Abstract:

    Drosophila melanogaster cells express a multi-substrate deoxyribonucleoside kinase that phosphorylates both purine and pyrimidine deoxyribonucleosides. The subsequent phosphorylation step is catalyzed by nucleoside monophosphate kinases. We have cloned and characterized the D. melanogaster UMP-CMP kinase (Dm.UMP-CMP kinase) to further study the nucleotide metabolizing enzymes in these cells. The kinase, encoded by the dak1 gene, was approximately 60% similar to the human UMP-CMP kinase and predominantly phosphorylated CMP, dCMP, and UMP. Expression analysis showed that the Dm.UMP-CMP kinase mRNA was constitutively expressed throughout the Drosophila development. The open-reading frame of the Dm.UMP-CMP kinase cDNA was extended in the 5'-region compared to UMP-CMP kinases of other species. The extended region encoded an N-terminal sequence with properties characteristic of a mitochondrial import signal. Expression of the enzyme in fusion with the green fluorescent protein verified that the N-terminal signal targeted the enzyme to mitochondria. This is the first time a mitochondrial pyrimidine nucleoside monophosphate kinase has been cloned from any organism and we discuss the implication of this finding for deoxyribonucleoside salvage in both Drosophila and other organisms.

  • phosphorylation of deoxycytidine analog monophosphates by UMP cmp kinase molecular characterization of the human enzyme
    Molecular Pharmacology, 1999
    Co-Authors: R Van Rompay, Magnus Johansson, Anna Karlsson
    Abstract:

    Phosphorylation of deoxycytidine analogs by cellular enzymes is a prerequisite for the activity of these compounds. We have investigated the kinetic parameters for the phosphorylation of 1-beta-D-arabinofuranosylcytosine (araC) and 2', 2'-difluorodeoxycytidine (dFdC) to their diphosphate forms catalyzed by human UMP-CMP kinase. We cloned the cDNA of this enzyme to enable characterization of the recombinant protein, determine its expression in different tissues, and determine the chromosome location of the gene. We showed that the recombinant UMP-CMP kinase phosphorylated CMP, dCMP, and UMP with highest efficiency and dUMP, AMP, and dAMP with lower efficiency. The monophosphates of araC and dFdC were shown to be phosphorylated with similar efficiency as dCMP and CMP. We further showed, in a combined enzymatic assay, that human deoxycytidine kinase and UMP-CMP kinase together phosphorylated araC, dFdC, and 2',3'-dideoxycytidine to their diphosphate forms. Northern blot analysis showed that the UMP-CMP kinase mRNA was ubiquitously present in human tissues as a 3.9-kb transcript with highest levels in pancreas, skeletal muscle, and liver. The human UMP-CMP kinase gene was localized to chromosome 1p34.1-1p33 by radiation hybrid analysis. We further expressed the UMP-CMP kinase as a fusion protein to the green fluorescent protein in Chinese hamster ovary cells, and showed that the fusion protein was located in the cytosol and nucleus.