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 monooxygena se 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expr ession vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane p rotein. These four isoforms, as well as purified rabbit FMO2, and elev en heterologously expressed human P450 isoforms were examined for thei r capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), u sing a new, specific HPLC method with radiochemical detection. Human F MO3 was by far the most active isoform, exhibiting a turnover number o f 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the oth er monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/mi n under these conditions. Human fetal liver, adult liver, kidney and i ntestine microsomes were screened for TMA oxidation, and only human ad ult liver microsomes provided substantial TMAO formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formati on by recombinant human FMO3, employing three different analytical met hods, resulted in a K-m of 28 +/- 1 mu M and a V-max of 36.3 +/- 5.7 n mol TMAO/nmol FMO3/min. The K-m determined in human liver microsomes r anged from 13.0 to 54.8 mu M. Therefore, at physiological pH, human FM O3 is a very specific and efficient TMA N-oxygenase, and is likely res ponsible for the metabolic clearance of TMA in vivo in humans. In addi tion, this specificity provides a good in vitro probe for the determin ation of FMO3-mediated activity in human tissues, by analyzing TMAO fo rmation at pH 7.4 with TMA concentrations not higher than 0.5 mM. (C) 1998 Elsevier Science Inc.