Functional expression of two Arabidopsis aldehyde oxidases in the yeast Pichia pastoris

To investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO1 and AAO2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO1 and AAO2 proteins gave activity bands with the same mobilities on native gel electrophoresis and exhibited the same substrate preferences on zymograms with 8 aldehydes as those of AO alpha and AO gamma in Arabidopsis seedlings, respectively. Furthermore, anti-AAO1 and anti-AAO2 antibodies, which specifically recognize the seedling AO alpha and AO gamma, respectively, reacted with the AAO1 and AAO2 proteins produced in P. pastoris, respectively. These results indicate that these AO proteins are accurately produced in the yeast system, as in Arabidopsis seedlings. Using AO preparations from P. pastoris, the enzymatic properties of Arabidopsis AO alpha and AO gamma were investigated. AOa showed a relatively wide substrate specificity for 7 aldehydes tested, with high affinity to benzaldehyde and indole-3-aldehyde, while AO gamma could most efficiently oxidize naphthaldehyde. AOa was strongly inhibited by iodoacetate and KCN, while AO gamma was inhibited not only by iodoacetate and KCN but also by 2-mercaptethanol, dithiothreitol, menadion, and estradiol. AO alpha and AO gamma showed the highest activity at around 65 and 50 degrees C, respectively, and exhibited pH dependence around pH 8.0. These results indicate that the two AO isoforms in Arabidopsis seedlings have different enzymatic properties and may have different physiological roles in vivo.