1. Oxidative metabolism of seleno-L-methionine to L-methionine selenoxide by flavin-containing monooxygenases
Renee J Krause, Steven C Glocke, Anna Rita Sicuri, Sharon L Ripp, Adnan A Elfarra Chem Res Toxicol. 2006 Dec;19(12):1643-9. doi: 10.1021/tx0601915.
The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-l-methionine (SeMet) to l-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. The formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean +/- S.D.; n = 4) of 0.91 +/- 0.29 mM and a Vmax value of 44 +/- 8.0 nmol MetSeO/mg protein/min. The inclusion of 1-benzylimidazole, superoxide dismutase, or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, the formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest that FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions.
2. Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: immunochemical and kinetic evidence for FMO3 being the major catalyst
R J Krause, S L Ripp, P J Sausen, L H Overby, R M Philpot, A A Elfarra Arch Biochem Biophys. 1996 Sep 1;333(1):109-16. doi: 10.1006/abbi.1996.0370.
Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mM methionine, the d-isomer of methionine sulfoxide was preferentially detected over the l-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mM methionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. The Vmax/K(m) ratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine and S-benzyl-L-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinct S-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings that the methionine S-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBC S-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionine K(m) determined with the purified methionine S-oxidase was 3.4 mM, whereas the apparent methionine K(m) determined with the purified SBC S-oxidase was 48 mM. The methionine sulfoxide d:l diastereomeric ratio obtained with methionine S-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBC S-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.
3. Flavin-containing monooxygenase (FMO)-dependent metabolism of methionine and evidence for FMO3 being the major FMO involved in methionine sulfoxidation in rabbit liver and kidney microsomes
R J Duescher, M P Lawton, R M Philpot, A A Elfarra J Biol Chem. 1994 Jul 1;269(26):17525-30.
Methionine was a substrate for cDNA-expressed rabbit flavin-containing monooxygenase (FMO) 1, FMO2, and FMO3, while incubations with membrane fractions containing cDNA-expressed FMO5 did not lead to the detection of methionine sulfoxide; Km values with FMO1, FMO2, and FMO3 were about 48.0, 30.0, and 6.5 mM, respectively. With FMO3 methionine d-sulfoxide was formed in nearly 8-fold higher concentrations than the l-diastereomer, whereas with FMO1 and FMO2, the d:l diastereomeric ratios were approximately 1.5:1 and 0.7:1, respectively. These results provide evidence for methionine being the first identified endogenous compound metabolized to diastereomeric sulfoxides by flavin-containing monooxygenases. The Km values for methionine sulfoxidation in rabbit liver and kidney microsomes (3.7 and 6.0 mM, respectively) were more comparable to the Km value obtained with FMO3 than FMO1 or FMO2. This result provides evidence that FMO3 is the major FMO isoform involved in methionine sulfoxidation in rabbit liver and kidney microsomes. Further evidence for this hypothesis is provided by the finding that methionine d-sulfoxide was also the preferred product in rabbit liver and kidney microsomes by nearly 8:1 and 6:1 over the l-diastereomer, respectively.
