Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

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Abstract

Aldehyde oxidoreductase from Desulifovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH2 ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.
Original languageUnknown
Pages (from-to)7990-7998
JournalJournal of the American Chemical Society
Volume131
Issue number23
Publication statusPublished - 1 Jan 2009

Keywords

    Cite this

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    title = "Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System",
    abstract = "Aldehyde oxidoreductase from Desulifovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH2 ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.",
    keywords = "linear, direct, enzymes, crystal-structure, reductase, xanthine-oxidase, 4-hydroxybenzoyl-coa, plot, iron-sulfur, protein, arsenite, maximum-likelihood, inhibited, family, mechanism, forms, mononuclear, molybdenum",
    author = "Moura, {Jos{\'e} Jo{\~a}o Galhardas de} and Gonzalez, {Pablo Javier} and Moura, {Isabel Maria Andrade Martins Galhardas de} and Santos-silva, {Teresa Sacadura} and Rom{\~a}o, {Maria Jo{\~a}o}",
    year = "2009",
    month = "1",
    day = "1",
    language = "Unknown",
    volume = "131",
    pages = "7990--7998",
    journal = "Journal of the American Chemical Society",
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    TY - JOUR

    T1 - Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

    AU - Moura, José João Galhardas de

    AU - Gonzalez, Pablo Javier

    AU - Moura, Isabel Maria Andrade Martins Galhardas de

    AU - Santos-silva, Teresa Sacadura

    AU - Romão, Maria João

    PY - 2009/1/1

    Y1 - 2009/1/1

    N2 - Aldehyde oxidoreductase from Desulifovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH2 ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

    AB - Aldehyde oxidoreductase from Desulifovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH2 ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

    KW - linear

    KW - direct

    KW - enzymes

    KW - crystal-structure

    KW - reductase

    KW - xanthine-oxidase

    KW - 4-hydroxybenzoyl-coa

    KW - plot

    KW - iron-sulfur

    KW - protein

    KW - arsenite

    KW - maximum-likelihood

    KW - inhibited

    KW - family

    KW - mechanism

    KW - forms

    KW - mononuclear

    KW - molybdenum

    M3 - Article

    VL - 131

    SP - 7990

    EP - 7998

    JO - Journal of the American Chemical Society

    JF - Journal of the American Chemical Society

    SN - 0002-7863

    IS - 23

    ER -