title: Human flavin-containing monooxygenase form 3: cDNA expression of the enzymes containing amino acid substitutions observed in individuals with trimethylaminuria.
abstract: Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individuals diagnosed with trimethylaminuria excrete relatively large amounts of trimethylamine in their urine, sweat, and breath, and this results in a fishy odor characteristic of trimethylamine. Activity of the human flavin-containing monooxygenase (FMO) has been proposed to be deficient in trimethylaminuria patients causing a decrease in the metabolism of trimethylamine that results in a fishy body odor. Cohorts of Australian, American, and British individuals suffering from trimethylaminuria have been identified. The human FMO3 cDNA was amplified from lymphocytes of affected patients. We report preliminary evidence of substitutions detected by screening of the cDNA and genomic DNA. The variant human FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-directed mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia coli and compared with wild type human FMO3 maltose-binding proteins (FMO3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine, tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated the three amine substrates. Human Lys158 Ile66 FMO3-MBP, Glu158 Ile66 FMO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all constructed as mutants identified as possible FMO3 variants responsible for trimethylaminuria and were found to be inactive as N-oxygenases. The results suggest that mutations at codons 66 and 153 of FMO3 can cause trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal allele frequencies in a number of control Australian and North American samples studied. The Lys158 to Glu158 human FMO3 polymorphism does not decrease trimethylamine N-oxygenation for the cDNA-expressed enzyme and thus does not appear to be causative of trimethyaminuria. The data show that the functional activity of human FMO3 can be significantly altered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria.
authors.affiliation: Seattle Biomedical Research Institute, Washington 98109, USA. jcashman@sbri.org
authors.forename: J R
authors.initials: JR
authors.lastname: Cashman
authors.forename: Y A
authors.initials: YA
authors.lastname: Bi
authors.forename: J
authors.initials: J
authors.lastname: Lin
authors.forename: R
authors.initials: R
authors.lastname: Youil
authors.forename: M
authors.initials: M
authors.lastname: Knight
authors.forename: S
authors.initials: S
authors.lastname: Forrest
authors.forename: E
authors.initials: E
authors.lastname: Treacy
mesh_terms: D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008661:Metabolism, Inborn Errors / Q000652:urine*; D008744:Methylamines / Q000652:urine*; D009154:Mutation; D009812:Odorants; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D018076:DNA, Complementary; D008744:Methylamines; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); C023336:trimethylamine