PT - JOURNAL ARTICLE AU - Martin Mahro AU - Catarina Coelho AU - José Trincão AU - David Rodrigues AU - Mineko Terao AU - Enrico Garattini AU - Miguel Saggu AU - Friedhelm Lendzian AU - Peter Hildebrandt AU - Maria João Romão AU - Silke Leimkühler TI - Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver to <em>Escherichia coli</em> Heterologous Protein Expression AID - 10.1124/dmd.111.040873 DP - 2011 Oct 01 TA - Drug Metabolism and Disposition PG - 1939--1945 VI - 39 IP - 10 4099 - http://dmd.aspetjournals.org/content/39/10/1939.short 4100 - http://dmd.aspetjournals.org/content/39/10/1939.full SO - Drug Metab Dispos2011 Oct 01; 39 AB - Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N1-methylnicotinamide 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 metabolism 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 believed that they recognize distinct substrates and carry out different 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 cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 Å. The crystals belong to space group P1, and two dimers are present in the unit cell.