Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.
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.
Schumann
Silvia
S
Universität Potsdam, Institut für Biochemie and Biologie, Potsdam, Germany.
Terao
Mineko
M
Garattini
Enrico
E
Saggu
Miguel
M
Lendzian
Friedhelm
F
Hildebrandt
Peter
P
Leimkühler
Silke
S
D042931:Aldehyde Oxidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics; D013045:Species Specificity; D013379:Substrate Specificity; D014968:Xanthine Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D018698:Glutamic Acid; D014968:Xanthine Dehydrogenase; D042931:Aldehyde Oxidase