Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver to Escherichia coli Heterologous Protein Expression

Abstract

Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N1-meth-ylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the me-tabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is be-lieved that they recognize distinct substrates and carry out differ-ent physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofac-tor, two distinct [2Fe-2S] clusters, and one FAD. We purified th