Expression and characterization of functional dog flavin-containing monooxygenase 1.
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.
Stevens
Jeffrey C
JC
Global Drug Metabolism, Pharmacia Corporation, Kalamazoo, Michigan 49007, USA. jeffrey.c.stevens@pharmacia.com
Melton
Roger J
RJ
Zaya
Matthew J
MJ
Engel
Leslie C
LC
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
D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)