Characterization of the MethionineS-Oxidase Activity of Rat Liver and Kidney Microsomes: Immunochemical and Kinetic Evidence for FMO3 Being the Major Catalyst

doi:10.1006/abbi.1996.0370

Archives of Biochemistry and Biophysics

Volume 333, Issue 1, 1 September 1996, Pages 109-116

https://doi.org/10.1006/abbi.1996.0370 Get rights and content

Abstract

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 mmmethionine, thed-isomer of methionine sulfoxide was preferentially detected over thel-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mmmethionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. TheV_max/K_mratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine andS-benzyl-l-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinctS-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 methionineS-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBCS-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionineK_mdetermined with the purified methionineS-oxidase was 3.4 mm, whereas the apparent methionineK_mdetermined with the purified SBCS-oxidase was 48 mm. The methionine sulfoxided:ldiastereomeric ratio obtained with methionineS-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBCS-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.

References (0)

Cited by (35)

Regulated methionine oxidation by monooxygenases
2017, Free Radical Biology and Medicine
Citation Excerpt :
However, most of the monooxygenases can produce H2O2 under “uncoupling” conditions, i.e. the nonproductive decay of the hydroperoxide intermediate [59,65–68] (Fig. 2, step e). While mammalian FMOs have been extensively characterized as detoxifying enzymes, it has been shown that FMO1, FMO2, and FMO3 possess catalytic activity toward methionine, generating MetO [56,69–72]. Interestingly, the MetO formed in liver, kidney, and lung microsomal fraction shows enrichment for the R isomer, pointing to a catalytic mechanism rather than a direct reaction with H2O2.
Protein function can be regulated via post-translational modifications by numerous enzymatic and non-enzymatic mechanisms, including oxidation of cysteine and methionine residues. Redox-dependent regulatory mechanisms have been identified for nearly every cellular process, but the major paradigm has been that cellular components are oxidized (damaged) by reactive oxygen species (ROS) in a relatively unspecific way, and then reduced (repaired) by designated reductases. While this scheme may work with cysteine, it cannot be ascribed to other residues, such as methionine, whose reaction with ROS is too slow to be biologically relevant. However, methionine is clearly oxidized in vivo and enzymes for its stereoselective reduction are present in all three domains of life. Here, we revisit the chemistry and biology of methionine oxidation, with emphasis on its generation by enzymes from the monooxygenase family. Particular attention is placed on MICALs, a recently discovered family of proteins that harbor an unusual flavin-monooxygenase domain with an NADPH-dependent methionine sulfoxidase activity. Based on structural and kinetic information we provide a rational framework to explain MICAL mechanism, inhibition, and regulation. Methionine residues that are targeted by MICALs are reduced back by methionine sulfoxide reductases, suggesting that reversible methionine oxidation may be a general mechanism analogous to the regulation by phosphorylation by kinases/phosphatases. The identification of new enzymes that catalyze the oxidation of methionine will open a new area of research at the forefront of redox signaling.
Isoform distinct time-, dose-, and castration-dependent alterations in flavin-containing monooxygenase expression in mouse liver after 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment
2010, Biochemical Pharmacology
Citation Excerpt :
Although our observed 1.7-fold increase in methionine S-oxidase activity was not large, it suggests an increase in FMO3 after TCDD exposure. Methionine S-oxidase activity has been previously observed in untreated male mouse liver [9] and is likely due to metabolism by FMO1 or FMO4, which are expressed in male mouse liver [6,27,50,53]. It is likely the observed 1.7-fold increase in activity is from the change in FMO3 level.
Flavin-containing monooxygenase (FMO) expression in male mouse liver is altered after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure or castration. Because TCDD is slowly eliminated from the body, we examined hepatic Fmo mRNA alterations for up to 32 days following 10 or 64 μg/kg TCDD exposure by oral gavage in male C57BL/6J mice. Fmo2 mRNA was significantly induced at 1, 4, and 8 days whereas Fmo3 mRNA was also induced at 32 days relative to controls. Fmo3 mRNA levels exhibited a dose-dependent increase at 4, 8, and 32 days after exposure; Fmo1, Fmo4, and Fmo5 mRNA did not exhibit clear trends. Because castration alone also increased Fmo2, Fmo3, and Fmo4 mRNA we examined the combined effects of castration and TCDD treatment on FMO expression. A greater than additive effect was observed with Fmo2 and Fmo3 mRNA expression. Fmo2 mRNA exhibited a 3–5-fold increase after castration or 10 μg/kg TCDD exposure by oral gavage, whereas an approximately 20-fold increase was observed between the sham-castrated control and castrated TCDD-treated mice. Similarly, treatment with 10 μg/kg TCDD alone increased Fmo3 mRNA 130- and 180-fold in the sham-castrated and castrated mice compared to their controls respectively, whereas, Fmo3 mRNA increased approximately 1900-fold between the sham control and castrated TCDD-treated mice. An increase in hepatic Fmo3 protein in TCDD-treated mice was observed by immunoblotting and assaying methionine S-oxidase activity. Collectively, these results provide evidence for isoform distinct time-, dose-, and castration-dependent effects of TCDD on FMO expression and suggest cross-talk between TCDD and testosterone signal transduction pathways.
Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney
2009, Toxicology Letters
Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm³/100 g to 0.6 cm³/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats.
Reduction of l-methionine selenoxide to seleno-l-methionine by endogenous thiols, ascorbic acid, or methimazole
2009, Biochemical Pharmacology
Citation Excerpt :
In the present study, the ability of the endogenous thiols l-cysteine, N-acetyl-l-cysteine and GSH, the FMO substrates methimazole (MIZ, 1-methyl-2-mercaptoimidazole) and l-methionine [18,19], and the antioxidant, ascorbic acid [20] to carry out the nonenzymatic reduction of MetSeO was examined to clarify the reactivity and/or selectivity of MetSeO towards these molecules and allow a better understanding of the mechanisms involved in SeMet-induced biological activities. MIZ was included because of its common use as a competitive inhibitor of FMO-mediated oxidations [19]. Ascorbic acid was previously shown to react with selenoxides under mild acidic conditions to give dehydroascorbic acid [21].
Seleno-l-methionine (SeMet) can be oxidized to l-methionine selenoxide (MetSeO) by flavin-containing monooxygenase 3 (FMO3) and rat liver microsomes in the presence of NADPH. MetSeO can be reduced by GSH to yield SeMet and GSSG. In the present study, the potential reduction of MetSeO to SeMet by other cellular components and antioxidants was investigated. Besides GSH, other thiols (l-cysteine, or N-acetyl-l-cysteine) and antioxidants (ascorbic acid and methimazole) also reduced MetSeO to SeMet. This reduction is unique to MetSeO since methionine sulfoxide was not reduced to methionine under similar conditions. The MetSeO reduction by thiols was instaneous and much faster than the reduction by ascorbic acid or methimazole. However, only one molar equivalent of ascorbic acid or methimazole was needed to complete the reduction, as opposed to two molar equivalents of thiols. Whereas the disulfides produced by the reactions of MetSeO with thiols are chemically stable, methimazole disulfide readily decomposed at pH 7.4, 37 °C to yield methimazole, methimazole-sulfenic acid, methimazole sulfinic acid, methimazole S-sulfonate, 1-methylimidazole (MI) and sulfite anion. Collectively, the results demonstrate reduction of MetSeO to SeMet by multiple endogenous thiols, ascorbic acid, and methimazole. Thus, oxidation of SeMet to MetSeO may result in depletion of endogenous thiols and antioxidant molecules. Furthermore, the novel reduction of MetSeO by methimazole provides clear evidence that methimazole should not be used as an alternative FMO substrate when studying FMO-mediated oxidation of SeMet.
Mammalian flavin-containing monooxygenases: Structure/function, genetic polymorphisms and role in drug metabolism
2005, Pharmacology and Therapeutics
Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a “soft-nucleophile”, usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics.
In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences.
The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration.
Methionine oxidation, α-synuclein and Parkinson's disease
2005, Biochimica et Biophysica Acta - Proteins and Proteomics
The aggregation of normally soluble α-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson's disease. Oxidative stress is believed to be a contributing factor in this disorder. Because it lacks Trp and Cys residues, mild oxidation of α-synuclein in vitro with hydrogen peroxide selectively converts all four methionine residues to the corresponding sulfoxides. Both oxidized and non-oxidized α-synucleins have similar unfolded conformations; however, the fibrillation of α-synuclein at physiological pH is completely inhibited by methionine oxidation. The inhibition results from stabilization of soluble oligomers of Met-oxidized α-synuclein. Furthermore, the Met-oxidized protein also inhibits fibrillation of unmodified α-synuclein. The degree of inhibition of fibrillation by Met-oxidized α-synuclein is proportional to the number of oxidized methionines. However, the presence of metals can completely overcome the inhibition of fibrillation of the Met-oxidized α-synuclein. Since oligomers of aggregated α-synuclein may be cytotoxic, these findings indicate that both oxidative stress and environmental metal pollution could play an important role in the aggregation of α-synuclein, and hence possibly Parkinson's disease. In addition, if the level of Met-oxidized α-synuclein was under the control of methionine sulfoxide reductase (Msr), then this could also be factor in the disease.

View all citing articles on Scopus

^☆: This research was supported by Grant DK44295 (A.A.E.) from NIDDK, National Institutes of Health, EPA graduate research fellowship (S.L.R.), and NIEHS Institutional Grant T32 ES07015 (P.J.S.)

²: Current address: G. D. Searle and Co., Skokie, IL 60077.

³: To whom reprint requests should be addressed at University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706. Fax: (608) 263-3926.

View full text

Regular ArticleCharacterization of the MethionineS-Oxidase Activity of Rat Liver and Kidney Microsomes: Immunochemical and Kinetic Evidence for FMO3 Being the Major Catalyst☆

Abstract

Regular Article
Characterization of the MethionineS-Oxidase Activity of Rat Liver and Kidney Microsomes: Immunochemical and Kinetic Evidence for FMO3 Being the Major Catalyst☆