Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria.
Impaired conversion of trimethylamine to trimethylamine N-oxide by human flavin containing monooxygenase 3 (FMO3) is strongly associated with primary trimethylaminuria, also known as 'fish-odor' syndrome. Numerous non-synonymous mutations in FMO3 have been identified in patients suffering from this metabolic disorder (e.g., N61S, M66I, P153L, and R492W), but the molecular mechanism(s) underlying the functional deficit attributed to these alleles has not been elucidated. The purpose of the present study was to determine the impact of these disease-associated genetic variants on FMO3 holoenzyme formation and on steady-state kinetic parameters for metabolism of several substrates, including trimethylamine. For comparative purposes, several common allelic variants not associated with primary trimethylaminuria (i.e., E158K, V257M, E308G, and the E158K/E308G haplotype) were also analyzed. When recombinantly expressed in insect cells, only the M66I and R492W mutants failed to incorporate/retain the FAD cofactor. Of the remaining mutant proteins P153L and N61S displayed substantially reduced (<10%) catalytic efficiencies for trimethylamine N-oxygenation relative to the wild-type enzyme. For N61S, reduced catalytic efficiency was solely a consequence of an increased K(m), whereas for P153L, both K(m) and k(cat) were altered. Similar results were obtained when benzydamine N-oxygenation was monitored. A homology model for FMO3 was constructed based on the crystal structure for yeast FMO which places the N61 residue alone, of the mutants analyzed here, in close proximity to the FAD catalytic center. These data demonstrate that primary trimethylaminuria is multifactorial in origin in that enzyme dysfunction can result from kinetic incompetencies as well as impaired assembly of holoprotein.
Yeung
Catherine K
CK
University of Washington School of Pharmacy, Department of Medicinal Chemistry, Box 357610, Seattle, WA 98195, USA.
Adman
Elinor T
ET
Rettie
Allan E
AE
D000595:Amino Acid Sequence; D020022:Genetic Predisposition to Disease / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D008661:Metabolism, Inborn Errors / Q000235:genetics* / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D008744:Methylamines; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)