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Research ArticleArticle

Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli

Joshua F. Alfaro, Carolyn A. Joswig-Jones, Wenyun Ouyang, Joseph Nichols, Gregory J. Crouch and Jeffrey P. Jones
Drug Metabolism and Disposition December 2009, 37 (12) 2393-2398; DOI: https://doi.org/10.1124/dmd.109.029520
Joshua F. Alfaro
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Carolyn A. Joswig-Jones
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Wenyun Ouyang
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Joseph Nichols
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Gregory J. Crouch
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Jeffrey P. Jones
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Abstract

Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.

Footnotes

  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM84546].

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

    doi:10.1124/dmd.109.029520

  • XO
    xanthine oxidase
    AO
    aldehyde oxidase
    AOX1
    aldehyde oxidase 1
    PCR
    polymerase chain reaction
    PAGE
    polyacrylamide gel electrophoresis
    MS/MS
    mass spectrometry
    KIE
    kinetic isotope effect.

    • Received July 15, 2009.
    • Accepted September 2, 2009.
  • Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 37 (12)
Drug Metabolism and Disposition
Vol. 37, Issue 12
1 Dec 2009
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Research ArticleArticle

Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli

Joshua F. Alfaro, Carolyn A. Joswig-Jones, Wenyun Ouyang, Joseph Nichols, Gregory J. Crouch and Jeffrey P. Jones
Drug Metabolism and Disposition December 1, 2009, 37 (12) 2393-2398; DOI: https://doi.org/10.1124/dmd.109.029520

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Research ArticleArticle

Purification and Mechanism of Human Aldehyde Oxidase Expressed in Escherichia coli

Joshua F. Alfaro, Carolyn A. Joswig-Jones, Wenyun Ouyang, Joseph Nichols, Gregory J. Crouch and Jeffrey P. Jones
Drug Metabolism and Disposition December 1, 2009, 37 (12) 2393-2398; DOI: https://doi.org/10.1124/dmd.109.029520
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