Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3.
In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.
Lang
D H
DH
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
Yeung
C K
CK
Peter
R M
RM
Ibarra
C
C
Gasser
R
R
Itagaki
K
K
Philpot
R M
RM
Rettie
A E
AE
D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007422:Intestines / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000196:embryology / Q000201:enzymology; D008744:Methylamines / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D016877:Oxidants / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*
D008744:Methylamines; D016877:Oxidants; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); C005855:trimethyloxamine