Expression and characterization of functional dog flavin-containing monooxygenase 3.
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 K(m) and V(max) values of 18.6 microM and 0.63 nmol N-oxide formed/min/nmol of enzyme, respectively. Benzydamine N-oxidation catalyzed by hFMO3 showed values of 42.6 microM (K(m)) and 3.56 nmol/min/nmol of enzyme (V(max)). Human FMO3 was observed to catalyze the S-oxidation of sulindac sulfide, with respective K(m) and V(max) values of 69.3 microM 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.
Lickteig
Andrew J
AJ
Pharmacokinetics, Dynamics and Metabolism, Chesterfield, MO 63017, USA. andrew.lickteig@pfizer.com
Riley
Rochelle
R
Melton
Roger J
RJ
Reitz
Beverly A
BA
Fischer
H David
HD
Stevens
Jeffrey C
JC
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D005260:Female; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007313:Insecta; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010641:Phenotype
D018076:DNA, Complementary; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)