Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1).

The flavin-containing monooxygenases (FMOs) are a family of flavoenzymes and contain one molecule of FAD per monomer. In order to demonstrate where FMO interacts with FAD, four mutants for the rat liver FMO1 protein were expressed in yeast and characterized. All four mutants were immunochemically similar to the unmodified form, although the contents of FAD in all four mutants were much lower than that in the unmodified form. Interestingly, the mutant generated by changing the first glycine of the proposed FAD-binding domain (GxGxxG) to alanine revealed catalytic activities, but was lower than those seen with the unmodified form. The conversion of the first glycine to alanine markedly increased and decreased the Km and Vmax values for imipramine N-oxidation, respectively. The other three mutants (RFMOm2, RFMOm3, and RFMOm4) were catalytically inactive. Our results suggest that three glycines, especially the second and third glycines, in the proposed FAD-binding domain were necessary for FMO to show catalytic activities. Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity of FMO1 were investigated. The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamine. The Km and Vmax values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respectively.

[1]  K. Itagaki,et al.  Expression and Characterization of a Modified Flavin-containing Monooxygenase 4 from Humans* , 1996, The Journal of Biological Chemistry.

[2]  I. Phillips,et al.  Differential developmental and tissue-specific regulation of expression of the genes encoding three members of the flavin-containing monooxygenase family of man, FMO1, FMO3 and FM04. , 1996, European journal of biochemistry.

[3]  R. Philpot,et al.  Cloning and sequencing of flavin-containing monooxygenases FMO3 and FMO4 from rabbit and characterization of FMO3. , 1994, The Journal of biological chemistry.

[4]  J. Cashman,et al.  A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities. , 1994, Archives of biochemistry and biophysics.

[5]  T. Kamataki,et al.  Rat liver flavin-containing monooxygenase (FMO): cDNA cloning and expression in yeast. , 1993, Biochimica et biophysica acta.

[6]  R. Philpot,et al.  Cloning, sequencing, distribution, and expression in Escherichia coli of flavin-containing monooxygenase 1C1. Evidence for a third gene subfamily in rabbits. , 1993, The Journal of biological chemistry.

[7]  R. Philpot,et al.  Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains. , 1993, The Journal of biological chemistry.

[8]  R. Philpot,et al.  Molecular genetics of the flavin-dependent monooxygenases. , 1993, Pharmacogenetics.

[9]  D. Ziegler.  Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. , 1993, Annual review of pharmacology and toxicology.

[10]  R. Philpot,et al.  Guinea pig or rabbit lung flavin-containing monooxygenases with distinct mobilities in SDS-PAGE are allelic variants that differ at only two positions. , 1992, Pharmacogenetics.

[11]  S. Povey,et al.  Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family. , 1992, The Biochemical journal.

[12]  T. Kronbach,et al.  Properties of expressed and native flavin-containing monooxygenases: evidence of multiple forms in rabbit liver and lung. , 1991, Molecular pharmacology.

[13]  J. Ozols.  Multiple forms of liver microsomal flavin-containing monooxygenases: complete covalent structure of form 2. , 1991, Archives of biochemistry and biophysics.

[14]  S. Povey,et al.  Cloning, primary sequence, and chromosomal mapping of a human flavin-containing monooxygenase (FMO1). , 1991, The Journal of biological chemistry.

[15]  K. Douglas,et al.  Evidence for gene duplication forming similar binding folds for NAD(P)H and FAD in pyridine nucleotide‐dependent flavoenzymes , 1991, FEBS letters.

[16]  H. Yamada,et al.  Multiplicity of liver microsomal flavin-containing monooxygenase in the guinea pig: its purification and characterization. , 1990, Archives of biochemistry and biophysics.

[17]  J. Ozols.  Covalent structure of liver microsomal flavin-containing monooxygenase form 1. , 1990, The Journal of biological chemistry.

[18]  E. Hodgson,et al.  The flavin-containing monooxygenase enzymes expressed in rabbit liver and lung are products of related but distinctly different genes. , 1990, The Journal of biological chemistry.

[19]  D. Ziegler,et al.  The flavin-containing monooxygenase expressed in pig liver: primary sequence, distribution, and evidence for a single gene. , 1990, Biochemistry.

[20]  P. Backlund,et al.  Expression of rat liver S-adenosylhomocysteinase cDNA in Escherichia coli and mutagenesis at the putative NAD binding site. , 1989, The Journal of biological chemistry.

[21]  J. Ozols.  Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens. , 1989, Biochemical and biophysical research communications.

[22]  D. Ziegler,et al.  Flavin-containing monooxygenases: catalytic mechanism and substrate specificities. , 1988, Drug metabolism reviews.

[23]  D. Williams,et al.  Rabbit lung flavin-containing monooxygenase. Purification, characterization, and induction during pregnancy. , 1985, Molecular pharmacology.

[24]  E. Hodgson,et al.  Identification of distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the mouse and rabbit. , 1985, Biochemical and biophysical research communications.

[25]  R. Wierenga,et al.  INTERACTION OF PYROPHOSPHATE MOIETIES WITH ALPHA-HELIXES IN DINUCLEOTIDE BINDING-PROTEINS , 1985 .

[26]  D. Williams,et al.  Rabbit lung flavin-containing monooxygenase is immunochemically and catalytically distinct from the liver enzyme. , 1984, Biochemical and biophysical research communications.

[27]  E. Hodgson,et al.  Purification of the flavin-containing monooxygenase from mouse and pig liver microsomes. , 1984, The International journal of biochemistry.

[28]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[29]  Wim G. J. Hol,et al.  Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant , 1983, Nature.

[30]  T. Kimura,et al.  Purification of mixed-function amine oxidase from rat liver microsomes. , 1983, Biochemical and biophysical research communications.

[31]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .

[32]  F. Guengerich,et al.  Immunochemical comparison and quantitation of microsomal flavin-containing monooxygenases in various hog, mouse, rat, rabbit, dog, and human tissues. , 1982, Molecular pharmacology.

[33]  R. Schirmer,et al.  Glutathione reductase from human erythrocytes: amino-acid sequence of the structurally known FAD-binding domain. , 1981, European journal of biochemistry.

[34]  J. Cashman,et al.  Microsomal oxidation of thiobenzamide. A photometric assay for the flavin-containing monooxygenase. , 1981, Biochemical and biophysical research communications.

[35]  L. Poulsen,et al.  The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies. , 1979, The Journal of biological chemistry.

[36]  W. Hol,et al.  Crystal structure of p-hydroxybenzoate hydroxylase. , 1979, Journal of molecular biology.

[37]  G. Schulz,et al.  The structure of the flavoenzyme glutathione reductase , 1978, Nature.

[38]  E. Faeder,et al.  A rapid micromethod for determination of FMN and FAD in mixtures. , 1973, Analytical biochemistry.

[39]  Y. Oshima,et al.  Isolation and Characterization of Acid Phosphatase Mutants in Saccharomyces cerevisiae , 1973, Journal of bacteriology.

[40]  D. Ziegler,et al.  Microsomal oxidase. IV. Properties of a mixed-function amine oxidase isolated from pig liver microsomes. , 1972, Archives of biochemistry and biophysics.

[41]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.