Vol. 63, Issue 2, 271-275, February 2003

ACCELERATED COMMUNICATION
Expression and Characterization of Functional Dog Flavin-Containing Monooxygenase 1

Jeffrey C. Stevens, Roger J. Melton, Matthew J. Zaya, and Leslie C. Engel

Global Drug Metabolism, Pharmacia Corporation, Kalamazoo, Michigan (J.C.S., M.J.Z.) and Creve Coeur, Missouri (R.J.M., L.C.E.)

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 µM and 63.8 nmol of S-oxide formed/min/mg of protein, respectively. By comparison, human FMO1 showed similar values of 87.1 µM (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 µM 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.