Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria

Abstract

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.

Figure 1

Reaction schematic for FMO3 probe substrates. (A) Methyl p-tolyl sulfide, (R)- and (S)- enantiomers of methyl p-tolyl sulfoxide are indicated. (B) Benzydamine. (C) Trimethylamine.

Figure 2

(A) Coomassie-stained SDS-PAGE gel (B) Western Blot of wild type and mutant FMO3-expressing insect cell membranes. 10μg of insect cell membranes were loaded in each lane of a 9% acrylamide resolving gel. Lane 1, molecular weight markers in kDa; lanes 2 and 12, purified FMO3 standard; lane 3, uninfected insect cell membranes; lane 4, wild type FMO3; lane 5, N61S; lane 6, M66I; lane 7, P153L; lane 8, E158K; lane 9, E308G; lane 10, 158K/308G; lane 11, R492W.

Figure 3

Eadie-Hofstee plots for wild-type FMO3 ( ), P153L (▲) and N61S (●) catalyzed TMA N-oxygenation.

Figure 4

Sequence alignment used to build FMO3 homology model prepared using ESPript 2.2 from Expasy Web server [26]. FMO3 sequence is repeated top and bottom for ease of comparison with structural template (24% homology with 2GV8, at top) and with yeast, drosophila, and other human FMOs(26%, 27%, 55%, 57% and 57% homology) (at bottom). Conserved residues are both in black, and starred underneath. Boxed residues are semi-conserved. Motifs are indicated, as are mutated residues characterized in this study.

Figure 5

FMO3 structural model based on yeast FMO. Model is shown with FAD cofactor and O₂ from yeast FMO structure, and benzydamine (green) superposed on substrate (methimazole) found in yeast FMO structure. Sites of mutation are designated by colored spheres at alpha carbons, pink for those not significantly affecting reactivity, yellow for those which cannot bind FAD, and magenta for those which show significant change in reactivity. White ribbon depicts regions most similar to yeast FMO. Blue, insert at residues 217-295. Red, additional residues at C-terminus. Other motifs depicted in color (cyan, FATGY; yellow, nucleotide binding motifs; purple, FxGxxxHxGxxI/F)