A family study of compound variants of flavin-containing monooxygenase 3 (FMO3) in Japanese subjects found by urinary phenotyping for trimethylaminuria

Variants of Flavin-Containing Monooxygenase 3 Found in Subjects in an Updated Database of Genome Resources

“…Variant FMO3 cDNAs were prepared as previously described (Shimizu et al, 2019b;Shimizu et al, 2021a;Shimizu et al, 2023) using a QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with the designated 43 sets of primers (Table 2) and the FMO3 wild-type cDNA. Recombinant FMO3 proteins were expressed in Escherichia coli strain JM109 using the pTrc99A vector system (Pharmacia Biotechnology, Milwaukee, WI, USA) (Shimizu et al, 2019b;Shimizu et al, 2021a;Shimizu et al, 2023).…”

“…Variant FMO3 cDNAs were prepared as previously described (Shimizu et al, 2019b;Shimizu et al, 2021a;Shimizu et al, 2023) using a QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with the designated 43 sets of primers (Table 2) and the FMO3 wild-type cDNA. Recombinant FMO3 proteins were expressed in Escherichia coli strain JM109 using the pTrc99A vector system (Pharmacia Biotechnology, Milwaukee, WI, USA) (Shimizu et al, 2019b;Shimizu et al, 2021a;Shimizu et al, 2023). The amounts of variant FMO3 proteins (0.1-200 pmol/mg protein) in the bacterial membrane fractions were determined immunochemically using an anti-FMO3 antibody (ab126790, Abcam, Cambridge, UK) and a human FMO3 standard (Corning, Woburn, MA, USA) (Fig.…”

“…The amounts of variant FMO3 proteins (0.1-200 pmol/mg protein) in the bacterial membrane fractions were determined immunochemically using an anti-FMO3 antibody (ab126790, Abcam, Cambridge, UK) and a human FMO3 standard (Corning, Woburn, MA, USA) (Fig. 1) as described previously (Shimizu et al, 2019b;Shimizu et al, 2021a;Shimizu et al, 2023).…”

“…Benzydamine and trimethylamine N-oxygenation rates by recombinant FMO3 proteins in the bacterial membranes (1.0 pmol equivalent FMO3) fortified with an NADPH-generating system and 1000 µM benzydamine (Sigma-Aldrich, St Louis, MO) and 25-1000 µM trimethylamine (Fujifilm Wako Pure Chemical, Osaka, Japan) in a final volume of 0.10 mL of 50 mM potassium phosphate buffer (pH 8.4) (Nagashima et al, 2009) at 37°C for 10 min in triplicate were evaluated as described previously (Shimizu et al, 2015;Shimizu et al, 2019b;Shimizu et al, 2021a;Shimizu et al, 2023). Michaelis-Menten kinetic parameters for the N-oxygenation of trimethylamine by recombinant human FMO3 variant proteins (nmol product formation/min/nmol FMO3, i.e., min -1 ) were calculated by nonlinear regression (mean ± standard error, n = 6 substrate concentrations, in triplicate determinations) using Prism 9 software (GraphPad Software, San Diego, CA, USA).…”

“…These were already tested variants FMO3 p.Phe43Ile (Shimizu et al, 2015), p.Pro153GlnfsTer14 (Shimizu et al, 2019b), and p.Pro282Leu (Shimizu et al, 2019a) in addition. Among the novel 19 amino-acid-substituted FMO3 variants, FMO3 p.Met260Lys was recently reported in a compound variant found during phenotype-genotype analysis (Shimizu et al, 2023).…”