RT Journal Article
SR Electronic
T1 Expression and Characterization of Functional Dog Flavin-Containing Monooxygenase 3
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1987
OP 1990
DO 10.1124/dmd.109.027714
VO 37
IS 10
A1 Andrew J. Lickteig
A1 Rochelle Riley
A1 Roger J. Melton
A1 Beverly A. Reitz
A1 H. David Fischer
A1 Jeffrey C. Stevens
YR 2009
UL http://dmd.aspetjournals.org/content/37/10/1987.abstract
AB Mammalian flavin-containing monooxygenase (FMO) enzymes catalyze oxidation at nucleophilic, heteroatom centers and are important for drug, xenobiotic, and endogenous substrate metabolism. In human liver, human FMO3 (hFMO3) is the most abundant FMO isoform and is known to contribute to the hepatic clearance of a variety of clinical drugs. The purpose of the current study was to express and compare the dog (beagle) FMO3 (dFMO3) to hFMO3. A full-length dFMO3 cDNA was obtained from liver by reverse transcription-polymerase chain reaction. Using a baculovirus expression system in Spodoptera frugiperda insect cells, dFMO3 was expressed to protein levels of 0.50 nmol/mg, as determined by liquid chromatography-fluorescence detection. Expressed dFMO3 displayed Michaelis-Menten kinetics, catalyzing NADPH-dependent N-oxidation of benzydamine, with Km and Vmax values of 18.6 μM and 0.63 nmol N-oxide formed/min/nmol of enzyme, respectively. Benzydamine N-oxidation catalyzed by hFMO3 showed values of 42.6 μM (Km) and 3.56 nmol/min/nmol of enzyme (Vmax). Human FMO3 was observed to catalyze the S-oxidation of sulindac sulfide, with respective Km and Vmax values of 69.3 μM and 35.4 nmol/min/nmol of enzyme. dFMO3 also catalyzed sulindac sulfide S-oxidation with 6.8 nmol/min/nmol of enzyme being the highest velocity observed. Finally, Western blot analysis indicated protein expression levels of dFMO3 in pooled dog liver and lung microsomes to be 27 and 9 pmol/mg, respectively. In summary, dFMO3 appears to be a functional enzyme expressed at appreciable levels in liver, but one with some kinetic properties that are substantially different from its human homolog hFMO3. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics