title: 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-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.
authors.affiliation: Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Mahro
authors.forename: Catarina
authors.initials: C
authors.lastname: Coelho
authors.forename: José
authors.initials: J
authors.lastname: Trincão
authors.forename: David
authors.initials: D
authors.lastname: Rodrigues
authors.forename: Mineko
authors.initials: M
authors.lastname: Terao
authors.forename: Enrico
authors.initials: E
authors.lastname: Garattini
authors.forename: Miguel
authors.initials: M
authors.lastname: Saggu
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Lendzian
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Romão
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism / Q000379:methods; D003460:Crystallization / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; C443116:Aox3 protein, mouse