Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

Abstract

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.

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

“…Thus, hAOX1 was purified in a form in which 30% was active. This is consistent with other reports of the purification of AOX1 and AOX3 from mouse (Schumann et al, 2009;Mahro et al, 2011) or of XDH purified from a baculovirus insect cell system (Nishino et al, 2002). Thus, apparently when heterologous expression systems are used, the terminal sulfido ligand seems to be the limiting step to obtain a fully active mammalian enzyme.…”

“…The line widths and positions of the FeS signals at lower temperatures were affected by magnetic interactions. The g tensors obtained and line widths for FeSI and FeSII of hAOX1 are almost identical to the values published previously for mAOX1 (Schumann et al, 2009) or mAOX3, indicating high structural similarity between the proteins as expected from amino acid sequence identities of 83% and 61%, respectively.…”

“…The three bands were analyzed by matrix-assisted laser desorption ionization peptide mapping and identified to be the degradation products of hAOX1. Similar bands also were observed in purified mAOX3 (Mahro et al, 2011) and AOX1 protein preparations of mouse and rat origin (Kundu et al, 2007;Schumann et al, 2009). These products were never detectable upon native PAGE (Kundu et al, 2007;Mahro et al, 2011) and fast protein liquid chromatography.…”

“…A prerequisite for the functional characterization of the identified SNPs was the optimization of the system used for the purification of a catalytically active hAOX1 protein (Alfaro et al, 2009). For this purpose, recombinant hAOX1 was expressed as an N-terminal His 6 tag fusion protein in E. coli TP1000 using the protocol described for mAOX1 (Schumann et al, 2009) with slight modifications. The protein was purified using nickel-nitrilotriacetic acid, anion exchange, and size-exclusion chromatography.…”

A Single Amino Acid Substitution Confers High Cinchonidine Oxidation Activity Comparable with That of Rabbit to Monkey Aldehyde Oxidase 1

“…However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.Aldehyde oxidase (AO, EC 1.2.3.1) is a major member of the xanthine oxidase family belonging to the molybdo-flavoenzymes (MFEs) together with xanthine oxidoreductase (XOR; xanthine dehydrogenase form, EC 1.1.1.204; xanthine oxidase form, EC 1.1.3.22) (Beedham, 1985(Beedham, , 1987(Beedham, , 1998(Beedham, , 2002Kitamura et al, 2006;Garattini et al, 2008;Schumann et al, 2009). Aldehyde oxidase 1 (AOX1) consists of a homodimer with a subunit molecular mass of approximately 150 kDa.…”