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This Article Full Text Full Text (PDF) All Versions of this Article: dmd.106.010827v1 dmd.106.010827v2 34/12/1995    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Borbás, T. Articles by Cashman, J. R. Search for Related Content PubMed PubMed Citation Articles by Borbás, T. Articles by Cashman, J. R.

Investigation of Structure and Function of a Catalytically Efficient Variant of the Human Flavin-Containing Monooxygenase Form 3

Human BioMolecular Research Institute, San Diego, California (J.Z., M.A.C., J.R.C.); and Division of Pharmacology and Drug Safety, Gedeon Richter Ltd., Budapest, Hungary (T.B., I.L.)

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.