title: Expression and characterization of functional dog flavin-containing monooxygenase 1.
abstract: A full-length dog (beagle) flavin-containing monooxygenase 1 (FMO1) cDNA (dFMO1) was obtained from liver by reverse transcription-polymerase chain reaction. The amino acid sequence of dFMO1 was 89% homologous to human FMO1. Using a baculovirus expression system in Sf-9 insect cells, dFMO1 was expressed to protein levels of 0.4 nmol/mg, as determined by immunoquantitation. The flavin content of the expressed enzyme was consistent with immunodetectable dFMO1 protein levels. Expressed dFMO1 catalyzed NADPH-dependent methyl p-tolyl sulfide oxidation, with K(m) and V(max) values of 98.6 microM and 63.8 nmol of S-oxide formed/min/mg of protein, respectively. By comparison, human FMO1 showed similar values of 87.1 microM (K(m)) and 51.0 nmol/min/mg (V(max)). Activity for dFMO1 showed characteristic pH dependence, with a 4.5-fold increase in S-oxidase activity as the incubation pH increased from 7.6 to 9.0. Human FMO1 also showed an increase in reaction rate with pH but a somewhat lower optimum of 8.0 to 8.4. dFMO1 also catalyzed imipramine N-oxidation, with a K(m) of 4.7 microM and a V(max) of 82.1 nmol/min/mg of protein. This enzyme displayed other characteristics of FMO enzymes, with rapid depletion of enzyme activity upon heating in the absence of NADPH. Protein levels of 74 pmol of dFMO1/mg of microsomal protein were determined for a pooled liver microsome sample, suggesting that this enzyme is a major canine hepatic monooxygenase. In conclusion, the expression and characterization of catalytically active dFMO1 will allow the role of this enzyme in the metabolism of xenobiotics to be determined.
authors.affiliation: Global Drug Metabolism, Pharmacia Corporation, Kalamazoo, Michigan 49007, USA. jeffrey.c.stevens@pharmacia.com
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Stevens
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Melton
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Zaya
authors.forename: Leslie C
authors.initials: LC
authors.lastname: Engel
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D015870:Gene Expression; D015151:Immunoblotting; D007313:Insecta / Q000166:cytology; D008297:Male; D010105:Oxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)