Expression in Escherichia coli of the flavin-containing monooxygenase D (form II) from adult human liver: determination of a distinct tertiary amine substrate specificity

Chem Res Toxicol. Jul-Aug 1993;6(4):425-9. doi: 10.1021/tx00034a006.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Chlorpromazine / metabolism
  • Cloning, Molecular
  • DNA / biosynthesis
  • Escherichia coli / enzymology*
  • Humans
  • Isoenzymes / biosynthesis
  • Isoenzymes / genetics
  • Kinetics
  • Liver / enzymology*
  • Microsomes, Liver / enzymology
  • Molecular Sequence Data
  • Oxygenases / biosynthesis*
  • Oxygenases / genetics
  • Phenothiazines / metabolism
  • Prokaryotic Cells / metabolism
  • Substrate Specificity
  • Swine
  • Transformation, Genetic

Substances

  • Isoenzymes
  • Phenothiazines
  • DNA
  • Oxygenases
  • dimethylaniline monooxygenase (N-oxide forming)
  • Chlorpromazine