title: Purification and mechanism of human aldehyde oxidase expressed in Escherichia coli.
abstract: Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.
authors.affiliation: Department of Chemistry, Washington State University, Pullman, WA 99163, USA.
authors.forename: Joshua F
authors.initials: JF
authors.lastname: Alfaro
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Joswig-Jones
authors.forename: Wenyun
authors.initials: W
authors.lastname: Ouyang
authors.forename: Joseph
authors.initials: J
authors.lastname: Nichols
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Crouch
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Jones
mesh_terms: D042931:Aldehyde Oxidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D014969:Xanthine Oxidase; C526436:AOX1 protein, human; D042931:Aldehyde Oxidase