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URN: urn:nbn:de:kobv:517-opus-45030
URL: http://opus.kobv.de/ubp/volltexte/2010/4503/


Schumann, Silvia ; Terao, Mineko ; Garattini, Enrico ; Saggu, Miguel ; Lendzian, Friedhelm ; Hildebrandt, Peter ; Leimkühler, Silke

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

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Kurzfassung in Englisch

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 Eschericia 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.

Institut: Institut für Biochemie und Biologie
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: c Postprint
Schriftenreihe: Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, ISSN 1866-8372
Bandnummer: paper 134
Quelle: PLoS one 4 (2009), 4, Art. e5348, DOI: 10.1371/journal.pone.0005348
Sprache: Englisch
Erstellungsjahr: 2009
Publikationsdatum: 19.07.2010
Bemerkung:
The article was originally published by PUBLIC LIBRARY SCIENCE:
PLoS one. - 4 (2009), 4, Art. e5348 (9 S.)
ISSN 1932-6203
DOI 10.1371/journal.pone.0005348
Lizenz: Dieses Werk ist unter einer Creative Commons-Lizenz lizenziert.
Lizenz-Logo  Creative Commons - Attribution 3.0 unported


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