Expression in Escherichia coli of the flavin-containing monooxygenase D (form II) from adult human liver: determination of a distinct tertiary amine substrate specificity.
The cDNA for a major component of the family of flavin-containing monooxygenases (FMOs) present in adult human liver (i.e., HLFMO-D) has been cloned and expressed in a prokaryotic system. Escherichia coli strain NM522 was transformed with pTrcHLFMO-D, and the HLFMO-D cDNA was expressed under the control of the Trc promoter. A variety of tertiary amine substrates [i.e., chlorpromazine and 10-[(N,N-dimethylamino)alkyl]- 2-(trifluoromethyl)phenothiazines] were efficiently oxygenated by HLFMO-D cDNA expressed in E. coli or by adult human liver microsomes. Approximate dimensions of the substrate binding channel for both adult human liver microsomal FMO and cDNA-expressed HLFMO-D were apparent from an examination of the N-oxygenation of a series of 10-[(N,N-dimethylamino)alkyl]-2-(trifluoromethyl)phenothiazines. The substrate regioselectivity studies suggest that adult human liver FMO form D possesses a distinct substrate specificity compared with form A FMO from animal hepatic sources. It is likely that the substrate specificity observed for cDNA-expressed adult human liver FMO-D may have consequences for the metabolism and distribution of tertiary amines and phosphorus- and sulfur-containing drugs in humans and may provide insight into the physiologic substrate(s) for adult human liver FMO.
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
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Lomri
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Yang
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JR
Cashman
D000818:Animals; D001483:Base Sequence; D002746:Chlorpromazine / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D010640:Phenothiazines / Q000378:metabolism; D011387:Prokaryotic Cells / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D014170:Transformation, Genetic
D007527:Isoenzymes; D010640:Phenothiazines; D004247:DNA; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D002746:Chlorpromazine