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Jure Piskur - One of the best experts on this subject based on the ideXlab platform.
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two thymidine kinases and one multisubstrate Deoxyribonucleoside kinase salvage dna precursors in arabidopsis thaliana
FEBS Journal, 2012Co-Authors: Anders R Clausen, Wolfgang Knecht, Michael P B Sandrini, Birgitte Munchpetersen, Lenart Girandon, Ashfaq Ali, Elzbieta Rozpedowska, Erik Andreasson, Jure PiskurAbstract:Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (dNKs) salvage Deoxyribonucleosides by transfer of a phosphate group to the 5' of a Deoxyribonucleoside. This salvage pathway is well characterized in mammals but in contrast little is known about how plants salvage Deoxyribonucleosides. We show that during salvage, Deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding mono-phosphate compounds with a surprising preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome we identified two types of genes that could encode enzymes which are involved in the salvage of Deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1-like genes (AtTK1a and AtTK1b) and deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, but AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. Our results point out a crucial role for salvage of thymidine during early plant development. © 2012 The Authors Journal compilation © 2012 FEBS. (Less)
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Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Anders R Clausen, Michael P B Sandrini, Oonagh Shannon, Lars Bjorck, Jure PiskurAbstract:Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial Deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising alternative for combating pathogenic bacteria.
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Deoxyribonucleoside kinases two enzyme families catalyze the same reaction
Trends in Biochemical Sciences, 2005Co-Authors: Michael P B Sandrini, Jure PiskurAbstract:Mammals have four Deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native Deoxyribonucleoside substrates, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions.
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animal Deoxyribonucleoside kinases forward and retrograde evolution of their substrate specificity
FEBS Letters, 2004Co-Authors: Jure Piskur, Wolfgang Knecht, Michael P B Sandrini, Birgitte MunchpetersenAbstract:Deoxyribonucleoside kinases, which catalyse the phosphorylation of Deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.7.1.145), which subsequently, through remodelling of a limited number of amino acid residues, gained a broad substrate specificity.
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structural basis for feedback inhibition of the Deoxyribonucleoside salvage pathway studies of the drosophila Deoxyribonucleoside kinase
Biochemistry, 2003Co-Authors: Nils Egil Mikkelsen, Kenth Johansson, Wolfgang Knecht, Jure Piskur, Birgitte Munchpetersen, Andreas Karlsson, Gorm Andersen, Hans EklundAbstract:Deoxyribonucleoside kinases are feedback inhibited by the final products of the salvage pathway, the Deoxyribonucleoside triphosphates. In the present study, the mechanism of feedback inhibition is presented based on the crystal structure of a complex between the fruit fly Deoxyribonucleoside kinase and its feedback inhibitor deoxythymidine triphosphate. The inhibitor was found to be bound as a bisubstrate inhibitor with its nucleoside part in the nucleoside binding site and with its phosphate groups partially occupying the phosphate donor site. The overall structure of the enzyme--inhibitor complex is very similar to the enzyme--substrate complexes with deoxythymidine and deoxycytidine, except for a conformational change within a region otherwise directly involved in catalysis. This conformational change involves a magnesium ion, which is coordinated in the inhibitor complex to the phosphates and to the primary base, Glu52, that normally is positioned close to the 5'-OH of the substrate deoxyribose.
Birgitte Munchpetersen - One of the best experts on this subject based on the ideXlab platform.
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isolation of a novel protein p12 from adult drosophila melanogaster that inhibits Deoxyribonucleoside and protein kinase activities and activates 3 5 exonuclease activity
Nucleosides Nucleotides & Nucleic Acids, 2016Co-Authors: Louise Slot Christiansen, Leif Sondergaard, Gabriella Christina Van Zanten, Dvora Berenstein, Marianne Lauridsen, Soren Kjaerulff, Birgitte MunchpetersenAbstract:ABSTRACTWe have previously found that Drosophila melanogaster only has one Deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural Deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10–13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. Howeve...
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non viral Deoxyribonucleoside kinases diversity and practical use
Journal of Genetics and Genomics, 2015Co-Authors: Louise Slot Christiansen, Birgitte Munchpetersen, Wolfgang KnechtAbstract:Deoxyribonucleoside kinases (dNKs) phosphorylate Deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate Deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of great medical interest. However, during the last 20 years, research on dNKs has gone into non-mammalian organisms. In this review, we focus on non-viral dNKs, in particular their diversity and their practical applications. The diversity of this enzyme family in different organisms has proven to be valuable in studying the evolution of enzymes. Some of these newly discovered enzymes have been useful in numerous practical applications in medicine and biotechnology, and have contributed to our understanding of the structural basis of nucleoside and nucleoside analogue activation.
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two thymidine kinases and one multisubstrate Deoxyribonucleoside kinase salvage dna precursors in arabidopsis thaliana
FEBS Journal, 2012Co-Authors: Anders R Clausen, Wolfgang Knecht, Michael P B Sandrini, Birgitte Munchpetersen, Lenart Girandon, Ashfaq Ali, Elzbieta Rozpedowska, Erik Andreasson, Jure PiskurAbstract:Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (dNKs) salvage Deoxyribonucleosides by transfer of a phosphate group to the 5' of a Deoxyribonucleoside. This salvage pathway is well characterized in mammals but in contrast little is known about how plants salvage Deoxyribonucleosides. We show that during salvage, Deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding mono-phosphate compounds with a surprising preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome we identified two types of genes that could encode enzymes which are involved in the salvage of Deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1-like genes (AtTK1a and AtTK1b) and deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, but AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. Our results point out a crucial role for salvage of thymidine during early plant development. © 2012 The Authors Journal compilation © 2012 FEBS. (Less)
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animal Deoxyribonucleoside kinases forward and retrograde evolution of their substrate specificity
FEBS Letters, 2004Co-Authors: Jure Piskur, Wolfgang Knecht, Michael P B Sandrini, Birgitte MunchpetersenAbstract:Deoxyribonucleoside kinases, which catalyse the phosphorylation of Deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.7.1.145), which subsequently, through remodelling of a limited number of amino acid residues, gained a broad substrate specificity.
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structural basis for feedback inhibition of the Deoxyribonucleoside salvage pathway studies of the drosophila Deoxyribonucleoside kinase
Biochemistry, 2003Co-Authors: Nils Egil Mikkelsen, Kenth Johansson, Wolfgang Knecht, Jure Piskur, Birgitte Munchpetersen, Andreas Karlsson, Gorm Andersen, Hans EklundAbstract:Deoxyribonucleoside kinases are feedback inhibited by the final products of the salvage pathway, the Deoxyribonucleoside triphosphates. In the present study, the mechanism of feedback inhibition is presented based on the crystal structure of a complex between the fruit fly Deoxyribonucleoside kinase and its feedback inhibitor deoxythymidine triphosphate. The inhibitor was found to be bound as a bisubstrate inhibitor with its nucleoside part in the nucleoside binding site and with its phosphate groups partially occupying the phosphate donor site. The overall structure of the enzyme--inhibitor complex is very similar to the enzyme--substrate complexes with deoxythymidine and deoxycytidine, except for a conformational change within a region otherwise directly involved in catalysis. This conformational change involves a magnesium ion, which is coordinated in the inhibitor complex to the phosphates and to the primary base, Glu52, that normally is positioned close to the 5'-OH of the substrate deoxyribose.
Wolfgang Knecht - One of the best experts on this subject based on the ideXlab platform.
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non viral Deoxyribonucleoside kinases diversity and practical use
Journal of Genetics and Genomics, 2015Co-Authors: Louise Slot Christiansen, Birgitte Munchpetersen, Wolfgang KnechtAbstract:Deoxyribonucleoside kinases (dNKs) phosphorylate Deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate Deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of great medical interest. However, during the last 20 years, research on dNKs has gone into non-mammalian organisms. In this review, we focus on non-viral dNKs, in particular their diversity and their practical applications. The diversity of this enzyme family in different organisms has proven to be valuable in studying the evolution of enzymes. Some of these newly discovered enzymes have been useful in numerous practical applications in medicine and biotechnology, and have contributed to our understanding of the structural basis of nucleoside and nucleoside analogue activation.
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two thymidine kinases and one multisubstrate Deoxyribonucleoside kinase salvage dna precursors in arabidopsis thaliana
FEBS Journal, 2012Co-Authors: Anders R Clausen, Wolfgang Knecht, Michael P B Sandrini, Birgitte Munchpetersen, Lenart Girandon, Ashfaq Ali, Elzbieta Rozpedowska, Erik Andreasson, Jure PiskurAbstract:Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (dNKs) salvage Deoxyribonucleosides by transfer of a phosphate group to the 5' of a Deoxyribonucleoside. This salvage pathway is well characterized in mammals but in contrast little is known about how plants salvage Deoxyribonucleosides. We show that during salvage, Deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding mono-phosphate compounds with a surprising preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome we identified two types of genes that could encode enzymes which are involved in the salvage of Deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1-like genes (AtTK1a and AtTK1b) and deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, but AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. Our results point out a crucial role for salvage of thymidine during early plant development. © 2012 The Authors Journal compilation © 2012 FEBS. (Less)
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animal Deoxyribonucleoside kinases forward and retrograde evolution of their substrate specificity
FEBS Letters, 2004Co-Authors: Jure Piskur, Wolfgang Knecht, Michael P B Sandrini, Birgitte MunchpetersenAbstract:Deoxyribonucleoside kinases, which catalyse the phosphorylation of Deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.7.1.145), which subsequently, through remodelling of a limited number of amino acid residues, gained a broad substrate specificity.
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structural basis for feedback inhibition of the Deoxyribonucleoside salvage pathway studies of the drosophila Deoxyribonucleoside kinase
Biochemistry, 2003Co-Authors: Nils Egil Mikkelsen, Kenth Johansson, Wolfgang Knecht, Jure Piskur, Birgitte Munchpetersen, Andreas Karlsson, Gorm Andersen, Hans EklundAbstract:Deoxyribonucleoside kinases are feedback inhibited by the final products of the salvage pathway, the Deoxyribonucleoside triphosphates. In the present study, the mechanism of feedback inhibition is presented based on the crystal structure of a complex between the fruit fly Deoxyribonucleoside kinase and its feedback inhibitor deoxythymidine triphosphate. The inhibitor was found to be bound as a bisubstrate inhibitor with its nucleoside part in the nucleoside binding site and with its phosphate groups partially occupying the phosphate donor site. The overall structure of the enzyme--inhibitor complex is very similar to the enzyme--substrate complexes with deoxythymidine and deoxycytidine, except for a conformational change within a region otherwise directly involved in catalysis. This conformational change involves a magnesium ion, which is coordinated in the inhibitor complex to the phosphates and to the primary base, Glu52, that normally is positioned close to the 5'-OH of the substrate deoxyribose.
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mosquito has a single multisubstrate Deoxyribonucleoside kinase characterized by unique substrate specificity
Nucleic Acids Research, 2003Co-Authors: Wolfgang Knecht, Gitte Petersen, Michael P B Sandrini, Leif Sondergaard, Birgitte Munchpetersen, Jure PiskurAbstract:In mammals four Deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural Deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for Deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural Deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine Deoxyribonucleosides than the fruit fly multisubstrate Deoxyribonucleoside kinase (EC 2.7.1.145). In addition, Ag-dNK could also phosphorylate some medically interesting nucleoside analogs, like stavudine (D4T), 2-chloro-deoxyadenosine (CdA) and 5-bromo-vinyl-deoxyuridine (BVDU). Although the biological significance of multisubstrate Deoxyribonucleoside kinases and their diversity among insects remains unclear, the observed variation provides a whole range of applications, as species specific and highly selective targets for insecticides, they have a potential to be used in the enzymatic production of various (di-)(deoxy-)ribonucleoside monophosphates, and as suicide genes in gene therapy.
Michael P B Sandrini - One of the best experts on this subject based on the ideXlab platform.
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two thymidine kinases and one multisubstrate Deoxyribonucleoside kinase salvage dna precursors in arabidopsis thaliana
FEBS Journal, 2012Co-Authors: Anders R Clausen, Wolfgang Knecht, Michael P B Sandrini, Birgitte Munchpetersen, Lenart Girandon, Ashfaq Ali, Elzbieta Rozpedowska, Erik Andreasson, Jure PiskurAbstract:Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (dNKs) salvage Deoxyribonucleosides by transfer of a phosphate group to the 5' of a Deoxyribonucleoside. This salvage pathway is well characterized in mammals but in contrast little is known about how plants salvage Deoxyribonucleosides. We show that during salvage, Deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding mono-phosphate compounds with a surprising preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome we identified two types of genes that could encode enzymes which are involved in the salvage of Deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1-like genes (AtTK1a and AtTK1b) and deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, but AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. Our results point out a crucial role for salvage of thymidine during early plant development. © 2012 The Authors Journal compilation © 2012 FEBS. (Less)
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Deoxyribonucleoside kinases activate nucleoside antibiotics in severely pathogenic bacteria
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Anders R Clausen, Michael P B Sandrini, Oonagh Shannon, Lars Bjorck, Jure PiskurAbstract:Common bacterial pathogens are becoming progressively more resistant to traditional antibiotics, representing a major public-health crisis. Therefore, there is a need for a variety of antibiotics with alternative modes of action. In our study, several nucleoside analogs were tested against pathogenic staphylococci and streptococci. We show that pyrimidine-based nucleoside analogs, like 3'-azido-3'-deoxythymidine (AZT) and 2',2'-difluoro-2'deoxycytidine (gemcitabine), are specifically activated by the endogenous bacterial Deoxyribonucleoside kinases, leading to cell death. Deoxyribonucleoside kinase-deficient Escherichia coli strains become highly susceptible to nucleoside analogs when they express recombinant kinases from Staphylococcus aureus or Streptococcus pyogenes. We further demonstrate that recombinant S. aureus deoxyadenosine kinase efficiently phosphorylates the anticancer drug gemcitabine in vitro and is therefore the key enzyme in the activation pathway. When adult mice were infected intraperitoneally with a fatal dose of S. pyogenes strain AP1 and afterwards received gemcitabine, they failed to develop a systemic infection. Nucleoside analogs may therefore represent a promising alternative for combating pathogenic bacteria.
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Deoxyribonucleoside kinases two enzyme families catalyze the same reaction
Trends in Biochemical Sciences, 2005Co-Authors: Michael P B Sandrini, Jure PiskurAbstract:Mammals have four Deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native Deoxyribonucleoside substrates, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions.
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animal Deoxyribonucleoside kinases forward and retrograde evolution of their substrate specificity
FEBS Letters, 2004Co-Authors: Jure Piskur, Wolfgang Knecht, Michael P B Sandrini, Birgitte MunchpetersenAbstract:Deoxyribonucleoside kinases, which catalyse the phosphorylation of Deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.7.1.145), which subsequently, through remodelling of a limited number of amino acid residues, gained a broad substrate specificity.
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mosquito has a single multisubstrate Deoxyribonucleoside kinase characterized by unique substrate specificity
Nucleic Acids Research, 2003Co-Authors: Wolfgang Knecht, Gitte Petersen, Michael P B Sandrini, Leif Sondergaard, Birgitte Munchpetersen, Jure PiskurAbstract:In mammals four Deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural Deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for Deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural Deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine Deoxyribonucleosides than the fruit fly multisubstrate Deoxyribonucleoside kinase (EC 2.7.1.145). In addition, Ag-dNK could also phosphorylate some medically interesting nucleoside analogs, like stavudine (D4T), 2-chloro-deoxyadenosine (CdA) and 5-bromo-vinyl-deoxyuridine (BVDU). Although the biological significance of multisubstrate Deoxyribonucleoside kinases and their diversity among insects remains unclear, the observed variation provides a whole range of applications, as species specific and highly selective targets for insecticides, they have a potential to be used in the enzymatic production of various (di-)(deoxy-)ribonucleoside monophosphates, and as suicide genes in gene therapy.
Anna Karlsson - One of the best experts on this subject based on the ideXlab platform.
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progressive loss of mitochondrial dna in thymidine kinase 2 deficient mice
Human Molecular Genetics, 2008Co-Authors: Xiaoshan Zhou, Magnus Johansson, Nicola Solaroli, Mia Bjerke, James B Stewart, Bjorn Rozell, Anna KarlssonAbstract:Deficient enzymatic activity of the mitochondrial Deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.
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the drosophila melanogaster ump cmp kinase cdna encodes an n terminal mitochondrial import signal
Biochemical and Biophysical Research Communications, 2003Co-Authors: Sophie Curbo, Magnus Johansson, Marjan Amiri, Fariba Foroogh, Anna KarlssonAbstract: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.
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substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human Deoxyribonucleoside kinases and ribonucleoside kinases
Pharmacology & Therapeutics, 2003Co-Authors: An R. Van Rompay, Magnus Johansson, Anna KarlssonAbstract:Structural analogues of nucleosides, nucleoside analogues (NA), are used in the treatment of cancer and viral infections. Antiviral NAs inhibit replication of the viral genome, whereas anticancer NAs inhibit cellular DNA replication and repair. NAs are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. The Deoxyribonucleoside kinases (dNK) and ribonucleoside kinases (rNK) catalyze the first phosphorylation step, converting Deoxyribonucleosides and ribonucleosides to their corresponding monophosphate form. The dNKs have been studied intensively, whereas the rNKs have not been as thoroughly investigated. This overview is focused on the substrate specificity, tissue distribution, and subcellular location of the mammalian dNKs and rNKs and their role in the activation of NAs.
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cloning and characterization of the multisubstrate Deoxyribonucleoside kinase of drosophila melanogaster
Journal of Biological Chemistry, 1999Co-Authors: Magnus Johansson, An R Van Rompay, B Degreve, Jan Balzarini, Anna KarlssonAbstract:A Drosophila melanogaster Deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural Deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926–3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus Deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.