RESUMEN
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.
Asunto(s)
Aldehído Oxidasa/química , Aldehído Oxidasa/genética , Aldehído Oxidasa/metabolismo , Sustitución de Aminoácidos , Animales , Dominio Catalítico/genética , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Ácido Glutámico/química , Técnicas In Vitro , Cinética , Ratones , Modelos Químicos , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodobacter capsulatus/enzimología , Rhodobacter capsulatus/genética , Especificidad de la Especie , Especificidad por Sustrato , Xantina Deshidrogenasa/química , Xantina Deshidrogenasa/genética , Xantina Deshidrogenasa/metabolismoRESUMEN
Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alphabeta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (alphabeta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alphabeta) subunits has to precede Moco insertion. In an (alphabeta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alphabeta) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (alphabeta)(2) heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (alphabeta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.