HUMAN FLAVIN-CONTAINING MONOOXYGENASE FORM-3 - CDNA EXPRESSION OF THEENZYMES CONTAINING AMINO-ACID SUBSTITUTIONS OBSERVED IN INDIVIDUALS WITH TRIMETHYLAMINURIA

Trimethylaminuria is an autosomal recessive human disorder affecting a small part of the population as an inherited polymorphism. Individual s 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 deficien t in trimethylaminuria patients causing a decrease in the metabolism o f trimethylamine that results in a fishy body odor. Cohorts of Austral ian, American, and British individuals suffering from trimethylaminuri a have been identified. The human FMO3 cDNA was amplified from lymphoc ytes of affected patients. We report preliminary evidence of substitut ions detected by screening of the cDNA and genomic DNA. The variant hu man FMO3 cDNA was constructed from wild type human FMO3 cDNA by site-d irected mutagenesis as maltose-binding protein fusions. Five distinct human FMO3 mutants were expressed as fusion proteins in Escherichia co li and compared with wild type human FMO3 maltose-binding proteins (FM O3-MBP) for the N-oxygenation of 10-[(N,N-dimethylamino)pentyl]-2-(tri fluoromethyl) tyramine, and trimethylamine. Human Lys158 FMO3-MBP and, to a greater extent, human Glu158 FMO3-MBP efficiently N-oxygenated t he three amine substrates. Human Lys158 Ile66 FMOS-MBP, Glu158 Ile66 F MO3-MBP, Lys158 Leu153 FMO3-MBP, and Glu158 Leu153 FMO3-MBP were all c onstructed 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 ca use trimethylaminuria in humans. We observed a common polymorphism of Lys to Glu at codon 158 of FMO3 that segregated with almost equal alle le frequencies in a number of control Australian and North American sa mples studied. The Lys158 to Glu158 human FMO3 polymorphism does not d ecrease trimethylamine N-oxygenation for the cDNA expressed enzyme and thus does not appear to be causative of trimethyaminuria. The data sh ow that the functional activity of human FMO3 can be significantly alt ered by amino acid changes that have been observed in individuals with clinically diagnosed trimethylaminuria.