PT - JOURNAL ARTICLE AU - Martin Mahro AU - Catarina Coelho AU - Jose Trincao AU - David Rodrigues AU - Mineko Terao AU - Enrico Garattini AU - Saggu Miguel AU - Friedhelm Lendzian AU - Peter Hildebrandt AU - Maria Joao Romao AU - Silke Leimkuehler TI - Characterization and crystallization of mouse Aldehyde Oxidase 3 (mAOX3): from mouse liver to E. coli heterologous protein expression AID - 10.1124/dmd.111.040873 DP - 2011 Jun 24 TA - Drug Metabolism and Disposition PG - dmd.111.040873 4099 - http://dmd.aspetjournals.org/content/early/2011/06/24/dmd.111.040873.short 4100 - http://dmd.aspetjournals.org/content/early/2011/06/24/dmd.111.040873.full AB - Aldehyde oxidase (AOX) is characterized by a broad substrate specificity oxidizing aromatic aza-heterocycles, e.g. N1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal and vanillin. In the past decade, AO has been increasingly recognized 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 homologues 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 of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor (Moco), two distinct [2Fe-2S] clusters and one FAD. We purified the AOX homologue (mAOX3) from mouse liver 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, while the native protein is 100% active. Spectroscopic characterizations showed that the FeSII is not completely assembled in mAOX3. Additionally, both proteins were crystallized. The best crystals were from the native mAOX3 and diffracted beyond 2.9Å;. The crystals belong to space group P1 and two dimers are present in the unit cell.