Mammalian microsomal flavin-containing monooxygenases (FMOs) catalyze the oxygenation of a diverse range of nucleophilic drugs and dietary constituents. This enzyme system is a multigene family, consisting of a minimum of four members in humans: FMO1, FMO3, FMO4 and FMO5. The hog liver (FMO1) form has been studied intensively over the last twenty years, however, it is clear that FMO1 is not expressed significantly in adult human liver. A dominant form of FMO in adult human liver is FMO3, which the investigators have found to be expressed in microsomes at levels comparable to those of the major human liver P450, CYP3A4. In light of these observations, it would appear that the vast body of information available on the substrate specificity, toxicological and potential physiological significance of FMO1 is not particularly relevant to human xenobiotic metabolism. Consequently, it becomes important to understand the substrate specificity, tissue distribution and regulation of expression of FMO3 and the other more recently identified adult human liver forms such as FMO4 and FMO5. The goals of this research proposal are to express human FMO isoforms with the baculovirus-insect cell system, use the purified proteins to study tissue-specific expression, define the role that these hepatic FMO isoforms play in the metabolism of endogenous and exogenous compounds in human, and identify the structural basis for their substrate selectivities.
The Specific Aims of the research are as follows: 1. Purify and characterize recombinant forms of human FMO from a baculovirus expression system. 2. Quantitate the levels of expression of each protein in human liver and intestinal microsomes. 3. Define structural features of substrates which promote efficient oxygenation by the individual forms. 4. Identify active site residues of the FMOs responsible for conferring their individual substrate specificities. These studies will permit them to test their central hypotheses - I) that human liver FMOs preferentially metabolize small, non-aromatic compounds of dietary origin, and II) that the substrate specificity of the major human liver forms of the enzyme are controlled by their C-terminal domains. In addition, the data obtained will establish structure-activity relationships for the human FMOs which may be used to predict the FMO-mediated metabolism of new chemical entities in humans. Finally, these studies will provide a rational basis for the future development of in vivo metabolic probes for human FMOs which are invaluable to the long-term goal of understanding the physiological function and regulation of this enzyme system.
| Yeung, Catherine K; Adman, Elinor T; Rettie, Allan E (2007) Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria. Arch Biochem Biophys 464:251-9 |
