A Mutation in the Flavin-containing Monooxygenase 3 Gene and its Effects on Catalytic Activity for N-oxidation of Trimethylamine In Vitro

Abstract

To clarify the mutation of the flavin-containing monooxygenase (FMO) 3 gene causing fish-odor syndrome, we analyzed the FMO3 gene of a Thai subject who possibly suffered from fish-odor syndrome. A novel mutation, a single-base substitution from G to A at the position of 265 (G265A), was identified in exon 3. The mutation caused an amino acid substitution from valine to isoleucine at residue 58 (V58I). The mutated FMO3 protein with V58I exhibited the reduced trimethylamine N-oxidase activity when it was expressed in E. coli. The V(max)/K(m) value for the activity of the mutant-type FMO3 was about 5 times lower than that for the wild-type FMO3.

Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population

“…The study participants collected their urine samples as described previously [22]. Urinary TMA and TMAO concentrations were determined by gas chromatography using a flame ionization detector as described previously [23]. Urinary concentrations of free TMA or total TMA (µmol/mL of urine) were corrected for creatinine excretion (mmol/mL) [22].…”

“…The sequence of the complete human FMO3 gene described in GenBank (Accession Number AL021026) was used as a reference. Polymerase chain reaction (PCR) for the all exons and exon-intron junctions of the human FMO3 gene was conducted in a 25 µL reaction mixture containing 50 ng of genomic DNA, 1.0 U LA-Taq DNA polymerase (Takarabio, Shiga, Japan), LA-PCR buffer, 2.0 mM MgCl 2 , 0.2 mM dNTPs, 5.0 pmol of each sense and antisense primer reported previously [23]. The PCR conditions consisted of an initial denaturation at 94°C for 1 min, following by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s. The PCR products were directly sequenced on both strands using an ABI bigdye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) with the sequencing primers [20].…”

“…Genotyping analysis for the novel g.30398 C>T mutation (Arg500Stop FMO3) in exon 9 was also carried out by a PCR-RFLP method with amplified DNA with the primers FMO3-9S and FMO3-9AS [23] and digestion by BssSI at 37 °C for 2 h.…”

“…The FMO3 cDNA used was previously modified by a PCR procedure [23] using a 5'-primer (5'-AAAAAGCTTACCATGGGGAAGAAAG-3', that introduced an Nco I site prior to the start codon) and a 3'-primer (5'-CTAGAGAAGCTTATGATTAGGTCAACAC-3', that introduced a Hind III site downstream of the stop codon). The full-length DNA sequence was confirmed again using DNA re-sequencing of both strands.…”

“…The full-length DNA sequence was confirmed again using DNA re-sequencing of both strands. To produce Arg205Cys FMO3, site directed mutagenesis was performed by the primer-directed enzymatic amplification method [23]. Briefly, G-base at position 706 bp in the FMO3 cDNA was substituted by a T-base using the primers, 5'-AGAACTCAGCtGCACAGCAGA-3' and 5'-TCTGCTGTGCaGCTGAGTTCT-3', to introduce the single nucleotide substitution, that coded for Arg205Cys in exon 5.…”