Abstract

To characterize the contribution of amino acid 360 to the functional activity of the human flavin-containing monooxygenase form 3 (FMO3) and form 1 (FMO1) in the oxygenation of drugs and chemicals, we expressed four FMO3 variants (i.e., Ala³⁶⁰-FMO3, His³⁶⁰-FMO3, Gln³⁶⁰-FMO3, and Pro³⁶⁰-FMO3) and one FMO1 variant (i.e., Pro³⁶⁰-FMO1) and compared them to wild-type enzymes (Leu³⁶⁰-FMO3 and His³⁶⁰-FMO1, respectively). The amino acid substitutions were introduced into wild-type FMO3 or FMO1 cDNA by site-directed mutagenesis. The thermal stability of variants of Leu³⁶⁰ FMO3 was also studied, and the thermal stability was significantly different from that of wild-type FMO3. The influence of different substrates to modulate the catalytic activity of FMO3 variants was also examined. Selective functional substrate activity was determined with mercaptoimidazole, chlorpromazine, and 10-[(N,N-dimethylaminopentyl)-2-(trifluoromethyl)]phenothiazine. Compared with wild-type FMO3, the Ala³⁶⁰-FMO3 and His³⁶⁰-FMO3 variants were less catalytically efficient for mercaptoimidazole S-oxygenation. N-Oxygenation of chlorpromazine was significantly less catalytically efficient for His³⁶⁰-FMO3 compared with wild-type FMO3. Human Pro³⁶⁰-FMO1 was significantly more catalytically efficient at S-oxygenating mercaptoimidazole and chlorpromazine compared with wild-type FMO1. The data support the mechanism that the Pro³⁶⁰ loci affect thermal stability of FMO3. Because different amino acids at position 360 affect substrate oxygenation in a unique fashion compared with that of FMO3 stimulation, we conclude that the mechanism of stimulation of FMO3 is distinct from that of enzyme catalysis. A molecular model of human FMO3 was also constructed to help explain the results. The increase in catalytic efficiency observed for Pro³⁶⁰ in human FMO3 was also observed when the His of FMO1 was replaced by Pro at loci 360.