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Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

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Title: Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.
Authors: Silvia Schumann, Mineko Terao, Enrico Garattini, Miguel Saggu, Friedhelm Lendzian, Peter Hildebrandt, Silke Leimkühler
Source: PLoS ONE, Vol 4, Iss 4, p e5348 (2009)
Publisher Information: Public Library of Science (PLoS), 2009.
Publication Year: 2009
Collection: LCC:Medicine
LCC:Science
Subject Terms: Medicine, Science
More Details: Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.
Document Type: article
File Description: electronic resource
Language: English
ISSN: 1932-6203
Relation: http://europepmc.org/articles/PMC2671166?pdf=render; https://doaj.org/toc/1932-6203
DOI: 10.1371/journal.pone.0005348
Access URL: https://doaj.org/article/1dd54ff12e3c40da9b0ab9d7846881d2
Accession Number: edsdoj.1dd54ff12e3c40da9b0ab9d7846881d2
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  Data: Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.
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  Data: PLoS ONE, Vol 4, Iss 4, p e5348 (2009)
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  Data: Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.
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