(Verbatim from the application): The long term objective of this research is to understand the structure-function relationships of the human flavin-containing monooxygenase (FMOs). This enzyme family is comprised of at least five isoforms which catalyze the oxidative metabolism of a diverse array of nucleophilic drugs and dietary constituents. This is usually a benign process whereby polar metabolic products are readily excreted in the urine. However, genetic defects in the major human liver isoform, FMO3, have serious consequences for affected individuals who suffer from an inability to metabolize the dietary constituent, trimethylamine. This causes the metabolic disorder known as """"""""fish-odor syndrome"""""""" which results often in societal calumny. The physiological function of the other five isoforms is not known, nor is it understood why small, relatively polar substrates like trimethylamine are selectively """"""""gated"""""""" to FMO3. Benefits to public health can be expected to arise if sufficient structural information were available for the human FMO enzymes to enable prediction of substrates and pathways of metabolism for drugs and other xenobiotics, thereby guiding new drug design and development. In the present proposal we intend to develop structural models for human FMO3. Two approaches will be taken: (1) to develop homology models based on existing three-dimensional structures of potentially related flavoproteins such as glutathione reductase, (2) to evaluate bacterial cyclohexanone monooxygenases (CMOs) as an alternative structural template for the mammalian enzymes and to subsequently crystallize this enzyme. The detailed structural information that will ensue will permit construction of second-generation homology models for the human FMO isoforms.
The Specific Aims of this proposal are: (A) Express, purify and characterize histidine-tagged human FMO3 and CMO from an E. coli expression system. (B) Assess functional parallels between FMO and CMO by determining the scope of FMO3-catalyzed Baeyer-Villiger reactions and CMO-catalyzed N-oxygenation reactions. (C) Challenge the developing homology model for FMO3 by identifying common amino acid residues in human FMO3 and CMO that are critical to FAD-binding and NADPH specificity. (D) Determine the mechanism underlying loss of function in FMO3 variants associated with trimethylaminuria as a further test of the model. (E) Prepare diffraction quality crystals of CMO.
| 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 |
