The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells

Birte Plitzko, Gudrun Ott, Debora Reichmann, Colin J Henderson, C Roland Wolf, Ralf Mendel, Florian Bittner, Bernd Clement, Antje Havemeyer

    Research output: Contribution to journalArticle

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    Abstract

    The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a stand-alone protein but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5) it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime-prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knockout in mice. Using heme-free apo CYB5 the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.
    Original languageEnglish
    Pages (from-to)20228-20237
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume288
    Issue number28
    DOIs
    Publication statusPublished - 2013

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    Cytochromes b5
    Metabolism
    Cells
    Heme
    Protein Isoforms
    Enzymes
    Genes
    Cytochrome-B(5) Reductase
    Hemeproteins
    Proteins
    Mammals
    Molybdenum
    HEK293 Cells
    Prodrugs
    Electron Transport
    RNA Interference
    Polymorphism
    Catalysis
    Knockout Mice
    Small Interfering RNA

    Cite this

    Plitzko, Birte ; Ott, Gudrun ; Reichmann, Debora ; Henderson, Colin J ; Wolf, C Roland ; Mendel, Ralf ; Bittner, Florian ; Clement, Bernd ; Havemeyer, Antje. / The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 28. pp. 20228-20237.
    @article{bc59e8e79f5743c4bdf5f5d683794d90,
    title = "The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells",
    abstract = "The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a stand-alone protein but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5) it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime-prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knockout in mice. Using heme-free apo CYB5 the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.",
    author = "Birte Plitzko and Gudrun Ott and Debora Reichmann and Henderson, {Colin J} and Wolf, {C Roland} and Ralf Mendel and Florian Bittner and Bernd Clement and Antje Havemeyer",
    year = "2013",
    doi = "10.1074/jbc.M113.474916",
    language = "English",
    volume = "288",
    pages = "20228--20237",
    journal = "Journal of Biological Chemistry",
    issn = "0021-9258",
    publisher = "American Society for Biochemistry and Molecular Biology",
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    The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells. / Plitzko, Birte; Ott, Gudrun; Reichmann, Debora; Henderson, Colin J; Wolf, C Roland; Mendel, Ralf; Bittner, Florian; Clement, Bernd; Havemeyer, Antje.

    In: Journal of Biological Chemistry, Vol. 288, No. 28, 2013, p. 20228-20237.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells

    AU - Plitzko, Birte

    AU - Ott, Gudrun

    AU - Reichmann, Debora

    AU - Henderson, Colin J

    AU - Wolf, C Roland

    AU - Mendel, Ralf

    AU - Bittner, Florian

    AU - Clement, Bernd

    AU - Havemeyer, Antje

    PY - 2013

    Y1 - 2013

    N2 - The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a stand-alone protein but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5) it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime-prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knockout in mice. Using heme-free apo CYB5 the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.

    AB - The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a stand-alone protein but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5) it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime-prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knockout in mice. Using heme-free apo CYB5 the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.

    U2 - 10.1074/jbc.M113.474916

    DO - 10.1074/jbc.M113.474916

    M3 - Article

    C2 - 23703616

    VL - 288

    SP - 20228

    EP - 20237

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 28

    ER -