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Research ArticleArticle

Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver to Escherichia coli Heterologous Protein Expression

Martin Mahro, Catarina Coelho, José Trincão, David Rodrigues, Mineko Terao, Enrico Garattini, Miguel Saggu, Friedhelm Lendzian, Peter Hildebrandt, Maria João Romão and Silke Leimkühler
Drug Metabolism and Disposition October 2011, 39 (10) 1939-1945; DOI: https://doi.org/10.1124/dmd.111.040873
Martin Mahro
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Catarina Coelho
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José Trincão
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David Rodrigues
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Mineko Terao
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Enrico Garattini
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Miguel Saggu
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Friedhelm Lendzian
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Peter Hildebrandt
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Maria João Romão
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Silke Leimkühler
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Abstract

Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 Å. The crystals belong to space group P1, and two dimers are present in the unit cell.

Footnotes

  • This work was supported by the Cluster of Excellence “Unifying Concepts in Catalysis” coordinated by the Technische Universität Berlin; and Fundação para a Ciência e Tecnologia, Portugal [Grant SFRH/BD/37948/2007] (to C.C.) and Project [PTDC/QUI/64733/2006]. The exchange of researchers among laboratories involved in the work was funded by the Deutscher Akademischer Austauschdienst Programm-GRICES program.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.111.040873.

  • ABBREVIATIONS:

    AOX
    aldehyde oxidase
    Moco
    molybdenum cofactor
    XOR
    xanthine oxidoreductase
    XO
    xanthine oxidase
    XDH
    xanthine dehydrogenase
    MCSF
    Moco sulfurase
    mAOX
    mouse aldehyde oxidase
    mMCSF
    mouse Moco sulfurase
    Ni-NTA
    nickel-nitrilotriacetic acid
    PAGE
    polyacrylamide gel electrophoresis
    DCPIP
    2,6-dichlorophenolindophenol
    ICP-MS
    inductively coupled plasma mass spectrometry
    CD
    circular dichroism
    ESRF
    European Synchrotron Radiation Facility.

  • Received May 25, 2011.
  • Accepted June 24, 2011.
  • Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 39 (10)
Drug Metabolism and Disposition
Vol. 39, Issue 10
1 Oct 2011
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Research ArticleArticle

CHARACTERIZATION OF MOUSE ALDEHYDE OXIDASE 3

Martin Mahro, Catarina Coelho, José Trincão, David Rodrigues, Mineko Terao, Enrico Garattini, Miguel Saggu, Friedhelm Lendzian, Peter Hildebrandt, Maria João Romão and Silke Leimkühler
Drug Metabolism and Disposition October 1, 2011, 39 (10) 1939-1945; DOI: https://doi.org/10.1124/dmd.111.040873

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Research ArticleArticle

CHARACTERIZATION OF MOUSE ALDEHYDE OXIDASE 3

Martin Mahro, Catarina Coelho, José Trincão, David Rodrigues, Mineko Terao, Enrico Garattini, Miguel Saggu, Friedhelm Lendzian, Peter Hildebrandt, Maria João Romão and Silke Leimkühler
Drug Metabolism and Disposition October 1, 2011, 39 (10) 1939-1945; DOI: https://doi.org/10.1124/dmd.111.040873
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