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Adnan A Elfarra - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Biochemical pharmacology, 2009
    Co-Authors: Rachel M. Novick, Chad M. Vezina, Adnan A Elfarra
    Abstract:

    Abstract 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.

  • Differential Localization of Flavin-Containing Monooxygenase (FMO) Isoforms 1, 3, and 4 in Rat Liver and Kidney and Evidence for Expression of FMO4 in Mouse, Rat, and Human Liver and Kidney Microsomes
    The Journal of pharmacology and experimental therapeutics, 2009
    Co-Authors: Rachel M. Novick, Ann Mitzey, Mark S. Brownfield, Adnan A Elfarra
    Abstract:

    Flavin-containing monooxygenases (FMOs) play significant roles in the metabolism of drugs and endogenous or foreign compounds. In this study, the regional distribution of FMO isoforms 1, 3, and 4 was investigated in male Sprague-Dawley rat liver and kidney using immunohistochemistry (IHC). Rabbit polyclonal antibodies to rat FMO1 and FMO4, developed using anti-peptide technology, and commercial anti-human FMO3 antibody were used; specificities of the antibodies were verified using Western blotting, immunoprecipitation, and IHC. In liver, the highest immunoreactivity for FMO1 and FMO3 was detected in the perivenous region, and immunoreactivity decreased in intensity toward the periportal region. In contrast, FMO4 immunoreactivity was detected with the opposite lobular distribution. In the kidney, the highest immunoreactivity for FMO1, -3, and -4 was detected in the distal tubules. FMO1 and FMO4 immunoreactivity was also detected in the proximal tubules with strong staining in the brush borders, whereas less FMO3 immunoreactivity was detected in the proximal tubules. Immunoreactivity for FMO3 and FMO4 was detected in the collecting tubules in the renal medulla and the glomerulus, whereas little FMO1 immunoreactivity was detected in these regions. The FMO1 antibody did not react with human liver or kidney microsomes. However, the FMO4 antibody reacted with male and female mouse and human tissues. These data provided a compelling visual demonstration of the isoform-specific localization patterns of FMO1, -3, and -4 in the rat liver and kidney and the first evidence for expression of FMO4 at the protein level in mouse and human liver and kidney microsomes.

  • Oxidative metabolism of seleno-L-methionine to L-methionine selenoxide by flavin-containing monooxygenases.
    Chemical research in toxicology, 2006
    Co-Authors: Renee J Krause, Sharon L. Ripp, Steven C. Glocke, Anna Rita Sicuri, Adnan A Elfarra
    Abstract:

    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.

  • Potential roles of flavin-containing monooxygenases in sulfoxidation reactions of l-methionine, N-acetyl-l-methionine and peptides containing l-methionine.
    Biochimica et biophysica acta, 2004
    Co-Authors: Adnan A Elfarra, Renee J Krause
    Abstract:

    Abstract Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-dependent oxidation of a large number of sulfur-, selenium-, and nitrogen-containing compounds. Five active isoforms (FMO1–5) have been identified and shown to be differently expressed in various mammalian tissues. Previous work from this laboratory has shown l -methionine to be S-oxidized by rat, rabbit and human FMO1–4, with FMO3 exhibiting the highest stereoselectivity for the formation of the d-diastereomer of methionine sulfoxide. In this report, we describe new studies aimed at determining if N-acetyl- l -methionine and peptides containing l -methionine can be substrates for FMOs. Experiments were carried out using either rabbit liver microsomes or human cDNA-expressed FMOs. The results show that while N-acetyl- l -methionine and peptides with a modified methionine amino group may not function as substrates for FMOs, peptides containing a free N-terminal methionine may act as FMO substrates. With human cDNA-expressed FMO1, FMO3, and FMO5, both FMO1 and FMO3 exhibited activity with the active peptides whereas FMO5 was inactive. With FMO3, the activity measured with methionine was similar (1 mM) or higher (5 mM) than the activity measured with H-Met-Val-OH and H-Met-Phe-OH. With FMO1, H-Met-Phe-OH and methionine exhibited similar activities whereas activity with H-Met-Val-OH was much lower. Collectively, the results show that FMOs can oxidize peptides containing a free N-terminal methionine. Thus, the role of FMOs in the oxidation of methionine in larger peptides or proteins warrants further investigation.

  • human kidney flavin containing monooxygenases and their potential roles in cysteine s conjugate metabolism and nephrotoxicity
    Journal of Pharmacology and Experimental Therapeutics, 2003
    Co-Authors: Renee J Krause, Lawrence H Lash, Adnan A Elfarra
    Abstract:

    The potential roles of human hepatic and renal flavin-containing monooxygenases (FMOs) in the metabolism of the cysteine S -conjugates S -allyl cysteine (SAC) and S -(1,2-dichlorovinyl)-l-cysteine (DCVC) were investigated. Incubations of human cDNA-expressed FMO1, FMO3, FMO4, and FMO5 with SAC resulted in detection of SAC sulfoxide, with FMO3 exhibiting approximately 3-, 4-, and 10-fold higher activity than FMO1, FMO4, and FMO5, respectively. DCVC sulfoxide formation was only detected with FMO3 and was 59-fold lower than SAC sulfoxide formation. Incubations of human liver microsomes with SAC or DCVC resulted in detection of the corresponding sulfoxides and provided evidence for the involvement of FMO3. Incubations of SAC or DCVC with human kidney microsomes, however, led only to the detection of SAC sulfoxide. Immunoblots with monospecific antibodies to FMO1, FMO3, and FMO5 in kidney microsomes from 26 humans showed that the average expression levels for FMO1, FMO3, and FMO5 were 5.8 ± 2.3, 0.5 ± 0.4, and 2.4 ± 1.4 pmol/mg (means ± S.D.), respectively. Interestingly, African-American kidney samples ( n = 8) exhibited significantly higher FMO1 levels than Caucasian samples ( n = 17), whereas no difference in expression level between males and females was observed with any of the examined FMO isoforms. Collectively, the results provide evidence for the expression of three FMO isoforms in the human kidney and show that the contribution of renal FMOs in cysteine S -conjugate metabolism is likely to vary depending upon the cysteine S -conjugate and the relative expression levels of the active FMOs.

Richard M. Philpot - One of the best experts on this subject based on the ideXlab platform.

  • Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3
    Biochemical pharmacology, 1998
    Co-Authors: Dieter H. Lang, Richard M. Philpot, Catherine K. Yeung, Raimund M. Peter, Catherine Ibarra, Rodolfo Gasser, Kiyoshi Itagaki, Allan E. Rettie
    Abstract:

    In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.

  • Molecular Cloning, Sequence, and Expression of Mouse Flavin-Containing Monooxygenases 1 and 5 (FMO1 and FMO5)
    Journal of biochemical and molecular toxicology, 1998
    Co-Authors: Nathan J. Cherrington, Richard M. Philpot, J. Greg Falls, Patricia E. Levi, Randy L. Rose, Kieran M. Clements, Ernest Hodgson
    Abstract:

    Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5′-flanking region, and 413 in the 3′-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5′-flanking region, and 757 in the 3′-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83–94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83–84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward n -octylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 205–212, 1998

  • Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans
    Chemico-biological interactions, 1997
    Co-Authors: Lila H. Overby, Geraldine C Carver, Richard M. Philpot
    Abstract:

    Abstract Variable amounts of flavin-containing monooxygenase isoforms 3 and 5 (FMO3 and FMO5) are present in microsomal preparations from adult, male, human liver. Quantitation with monospecific antibodies and recombinant isoforms as standards showed levels of FMO3 and of FMO5 that ranged from 12.5 to 117 and 3.5 to 34 pmol/mg microsomal protein, respectively. The concentration of FMO3 was greater than that of FMO5 in all samples, but the ratio of FMO3 to FMO5 varied from 2:1 to 10:1. Human hepatic microsomal samples also showed variable activities for the S-oxidation of methimazole. This activity was associated totally with FMO3; no participation of FMO5 was apparent. This conclusion was supported by several lines of evidence: first, the catalytic efficiency of FMO3 with methimazole was found to be ∼5000 times greater than that of FMO5; second, the rate of metabolism showed a direct, quantitative relationship with FMO3 content; third, the plot of the relationship between metabolism and FMO3 content extrapolated close to the origin. A second reaction, the N-oxidation of ranitidine, exhibited a much higher K m with recombinant FMO3 than did methimazole (2 mM vs. 35 μ M). However, a direct relationship between this reaction and FMO3 content in human hepatic microsomal preparations was also apparent. This result shows that even with a high K m substrate, FMO3-catalyzed metabolism can account for the majority of the product formation with some drugs. Our findings demonstrate that the contribution of FMO isoforms to human hepatic drug metabolism can be assessed quantitatively on the basis of the characteristics of the enzymes expressed in Escherichia coli

  • Oxidation of cysteine S-conjugates by rabbit liver microsomes and cDNA-expressed flavin-containing mono-oxygenases: studies with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine.
    Molecular pharmacology, 1997
    Co-Authors: Sharon L. Ripp, Richard M. Philpot, Lila H. Overby, Adnan A Elfarra
    Abstract:

    Rabbit liver microsomes catalyzed the highly stereoselective, NADPH- and time-dependent S-oxidation ofS-benzyl-l-cysteine (SBC),S-allyl-l-cysteine (SAC),S-(1,2-dichlorovinyl)-l-cysteine (DCVC), andS-(1,2,2-trichlorovinyl)-l-cysteine (TCVC) to their respective sulfoxides. Methimazole, a flavin-containing mono-oxygenase (FMO) substrate, inhibited S-oxidation of all four conjugates. The cytochrome P450 inhibitor 1-benzylimidazole did not affect SAC, SBC, or DCVC S-oxidation but inhibited the S-oxidation of TCVC. Solubilization of microsomes also inhibited TCVC activity, whereas SBC, SAC, and DCVC activities were not affected. Because these results suggested that FMOs were the major catalysts of SBC, SAC, and DCVC sulfoxidations, the four conjugates were evaluated as substrates for cDNA-expressed rabbit FMO isoforms FMO1, FMO2, FMO3, and FMO5. At equimolar concentrations (10 mm), FMO1 S-oxidized SBC and SAC, but no sulfoxides were detected with DCVC or TCVC. FMO3S-oxidized all four conjugates.Km values determined with FMO3 were comparable to Km values from rabbit liver microsomes. S-Oxidation by FMO2 was detected only with SAC, and no sulfoxides were detected in incubations with FMO5. These results show that FMO isoforms can catalyze cysteine conjugate S-oxidation and that the specific isoform involved depends on the structure of the cysteine conjugate. The cysteine conjugates with more nucleophilic sulfur atoms, SAC and SBC, were much better FMO substrates than those having the less nucleophilic sulfur atoms DCVC and TCVC. The sulfoxides of TCVC and DCVC were reactive toward GSH, whereas the sulfoxides of SBC and SAC were not reactive. These results provide evidence for different chemical reactivities of these sulfoxides.

  • Characterization of the MethionineS-Oxidase Activity of Rat Liver and Kidney Microsomes: Immunochemical and Kinetic Evidence for FMO3 Being the Major Catalyst
    Archives of biochemistry and biophysics, 1996
    Co-Authors: Renee J Krause, Richard M. Philpot, Sharon L. Ripp, Lila H. Overby, Peter J. Sausen, Adnan A Elfarra
    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. TheVmax/Kmratios 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 methionineKmdetermined with the purified methionineS-oxidase was 3.4 mm, whereas the apparent methionineKmdetermined 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.

Allan E. Rettie - One of the best experts on this subject based on the ideXlab platform.

  • Prochiral sulfoxidation as a probe for flavin-containing monooxygenases.
    Methods in molecular biology (Clifton N.J.), 2006
    Co-Authors: Catherine K. Yeung, Allan E. Rettie
    Abstract:

    Asymmetric aryl alkyl sulfides (R-S-R') are metabolized by flavin-containing monooxygenase (FMO) and cytochrome P450 enzymes to enantiomerically enriched sulfoxide products (R-SO-R') that are readily analyzed with a host of commercially available chiral stationary phases. Prochiral sulfoxidation of probe compounds based on p-tolyl methyl sulfide is a particularly useful method for discriminating among FMO1, FMO3, and FMO5, because the stereochemistry of the resulting products is isoform dependent, but apparently species independent. If studies are performed with crude tissue microsomal preparations, the cytochrome P450 component must be quenched to unmask catalysis specifically by the FMO component of the tissue. This chapter details experimental protocols for stereochemical analysis of sulfoxides generated from methyl, ethyl, n-propyl, and n-butyl p-tolyl sulfide by purified FMO isoforms and tissue microsomal preparations.

  • Human Hepatic Flavin-Containing Monooxygenases 1 (FMO1) and 3 (FMO3) Developmental Expression
    Pediatric Research, 2002
    Co-Authors: Sevasti B Koukouritaki, Allan E. Rettie, Catherine K. Yeung, Pippa Simpson, Ronald N Hines
    Abstract:

    The flavin-containing monooxygenases (FMOs) are important for the metabolism of numerous therapeutics and toxicants. Six mammalian FMO genes ( FMO1–6 ) have been identified, each exhibiting developmental and tissue- and species-specific expression patterns. Previous studies demonstrated that human hepatic FMO1 is restricted to the fetus whereas FMO3 is the major adult isoform. These studies failed to describe temporal expression patterns, the precise timing of the FMO1/FMO3 switch, or potential control mechanisms. To address these questions, FMO1 and FMO3 were quantified in microsomal fractions from 240 human liver samples representing ages from 8 wk gestation to 18 y using Western blotting. FMO1 expression was highest in the embryo (8–15 wk gestation; 7.8 ± 5.3 pmol/mg protein). Low levels of FMO3 expression also were detectable in the embryo, but not in the fetus. FMO1 suppression occurred within 3 d postpartum in a process tightly coupled to birth, but not gestational age. The onset of FMO3 expression was highly variable, with most individuals failing to express this isoform during the neonatal period. FMO3 was detectable in most individuals by 1-2 y of age and was expressed at intermediate levels until 11 y (12.7 ± 8.0 pmol/mg protein). These data suggest that birth is necessary, but not sufficient for the onset of FMO3 expression. A gender-independent increase in FMO3 expression was observed from 11 to 18 y of age (26.9 ± 8.6 pmol/mg protein). Finally, 2- to 20-fold interindividual variation in FMO1 and FMO3 protein levels were observed, depending on the age bracket.

  • Role of human flavin-containing monooxygenases in the sulfoxidation of [14C]aldicarb
    Pesticide Biochemistry and Physiology, 2002
    Co-Authors: Daniel Schlenk, John R. Cashman, Cathy Yeung, Xiaofei Zhang, Allan E. Rettie
    Abstract:

    Abstract Biotransformation of the carbamate insecticide aldicarb to aldicarb sulfoxide has been shown to significantly increase its anticholinesterase activity. However, it is unclear what enzymes are responsible for this bioactivation. In this study, 51 human liver microsome samples were prepared and [14C]aldicarb S-oxygenation was examined in vitro under saturating substrate incubations ( 200 μM ). NADPH-catalyzed S-oxygenation activities were highly variable and observed in only 12 of 51 samples with a mean value of 0.034±0.17 nmol/min/mg of microsomal protein. Coincubation with 0.1% lubrol to inhibit contributions of cytochrome P450 failed to alter S-oxygenation activities ( 0.036±0.26 pmol/min/mg ). Benzydamine N-oxygenation was also examined and was 2.57±1.66 nmol/min/mg and observed in all samples. Flavin-containing monooxygenase (FMO)3 content was determined in all samples and showed a significant correlation with benzydamine N-oxygenation (r2=0.41), but FMO3 did not correlate with [14C]aldicarb S-oxygenation. Incubations of [14C]aldicarb with five human recombinant flavin-monooxygenase isoforms indicated catalysis by FMO1≫FMO3 with no activity detected from FMO2, FMO4, or FMO5. These results indicated limited hepatic biotransformation of [14C]aldicarb in human liver microsomes, but suggests other tissues may be more important such as fetal liver, the intestine or kidney that possess significant levels of FMO1.

  • Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes.
    Biochemical pharmacology, 2000
    Co-Authors: Mitchell A. Hamman, Barbara D. Haehner‐daniels, Steven A. Wrighton, Allan E. Rettie, Stephen D. Hall
    Abstract:

    The stereoselective sulfoxidation of the pharmacologically active metabolite of sulindac, sulindac sulfide, was characterized in human liver, kidney, and cDNA-expressed enzymes. Kinetic parameter estimates (pH = 7.4) for sulindac sulfoxide formation in human liver microsomes (N = 4) for R- and S-sulindac sulfoxide were Vmax = 1.5 ± 0.50 nmol/min/mg, Km = 15 ± 5.1 μM; and Vmax = 1.1 ± 0.36 nmol/min/mg, Km = 16 ± 6.1 μM, respectively. Kidney microsomes (N = 3) produced parameter estimates (pH = 7.4) of Vmax = 0.9 ± 0.29 nmol/min/mg, Km = 15 ± 2.9 μM; Vmax = 0.5 ± 0.21 nmol/min/mg, Km = 22 ± 1.9 μM for R- and S-sulindac sulfoxide, respectively. In human liver and flavin-containing monooxygenase 3 (FMO3) the Vmax for R-sulindac sulfoxide increased 60–70% at pH = 8.5, but for S-sulindac sulfoxide was unchanged. In fourteen liver microsomal preparations, significant correlations occurred between R-sulindac sulfoxide formation and either immunoquantified FMO or nicotine N-oxidation (r = 0.88 and 0.83; P < 0.01). The R- and S-sulindac sulfoxide formation rate also correlated significantly (r = 0.85 and 0.75; P < 0.01) with immunoquantified FMO in thirteen kidney microsomal samples. Mild heat deactivation of microsomes reduced activity by 30–60%, and a loss in stereoselectivity was observed. Methimazole was a potent and nonstereoselective inhibitor of sulfoxidation in liver and kidney microsomes. n-Octylamine and membrane solubilization with lubrol were potent and selective inhibitors of S-sulindac sulfoxide formation. cDNA-expressed CYPs failed to appreciably sulfoxidate sulindac sulfide, and CYP inhibitors were ineffective in suppressing catalytic activity. Purified mini-pig liver FMO1, rabbit lung FMO2, and human cDNA-expressed FMO3 efficiently oxidized sulindac sulfide with a high degree of stereoselectivity towards the R-isomer, but FMO5 lacked catalytic activity. The biotransformation of the sulfide to the sulfoxide is catalyzed predominately by FMOs and may prove to be useful in characterizing FMO activity.

  • In vitro evaluation of potential in vivo probes for human flavin-containing monooxygenase (FMO): metabolism of benzydamine and caffeine by FMO and P450 isoforms.
    British journal of clinical pharmacology, 2000
    Co-Authors: Dieter H. Lang, Allan E. Rettie
    Abstract:

    Aims To determine the FMO and P450 isoform selectivity for metabolism of benzydamine and caffeine, two potential in vivo probes for human FMO. Methods Metabolic incubations were conducted at physiological pH using substrate concentrations of 0.01–10 m m with either recombinant human FMOs, P450s or human liver microsomes serving as the enzyme source. Products of caffeine and benzydamine metabolism were analysed by reversed-phase h.p.l.c. with u.v. and fluorescence detection. Results CYP1A2, but none of the human FMOs, catalysed metabolism of caffeine. In contrast, benzydamine was a substrate for human FMO1, FMO3, FMO4 and FMO5. Apparent Km values for benzydamine N-oxygenation were 60 ± 8 µm, 80 ± 8 µm, > 3 m m and > 2 m m, for FMO1, FMO3, FMO4 and FMO5, respectively. The corresponding Vmax values were 46 ± 2 min−1, 36 ± 2 min−1, 

Andrea Mattevi - One of the best experts on this subject based on the ideXlab platform.

  • Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs
    Nature Structural & Molecular Biology, 2020
    Co-Authors: Callum R. Nicoll, Gautier Bailleul, Filippo Fiorentini, María Laura Mascotti, Marco W. Fraaije, Andrea Mattevi
    Abstract:

    Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-Å resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate–histidine switch is speculated to induce the distinctive Baeyer–Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins. Ancestral reconstruction leads to characterization and crystallization of three ancient mammalian flavin-containing monooxygenases, offering insights into their mechanisms of membrane binding, catalytic activity and substrate selection.

  • Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs.
    Nature structural & molecular biology, 2019
    Co-Authors: Callum R. Nicoll, Gautier Bailleul, Filippo Fiorentini, María Laura Mascotti, Marco W. Fraaije, Andrea Mattevi
    Abstract:

    Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-A resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate-histidine switch is speculated to induce the distinctive Baeyer-Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins.

  • Baeyer-Villiger Monooxygenase FMO5 as Entry Point in Drug Metabolism
    ACS chemical biology, 2017
    Co-Authors: Filippo Fiorentini, Marco W. Fraaije, Elvira Romero, Kurt Faber, Mã©lanie Hall, Andrea Mattevi
    Abstract:

    Flavin-containing monooxygenases (FMOs) are emerging as effective players in oxidative drug metabolism. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating molecules containing soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer–Villiger monooxygenase. With the aim of evaluating such an alternative entry point in the metabolism of active pharmaceutical ingredients, we selected and tested drug molecules bearing a carbonyl group on an aliphatic chain. Nabumetone and pentoxifylline, two widely used pharmaceuticals, were thereby demonstrated to be efficiently oxidized in vitro by FMO5 to the corresponding acetate esters with high selectivity. The proposed pathways explain the formation of a predominant plasma metabolite of pentoxifylline as well as the crucial transformation of the pro-drug nabumetone into the pharmacologically active compound. Using the recombinant enzyme, the ...

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  • Endogenous Roles of Mammalian Flavin-Containing Monooxygenases
    Catalysts, 2019
    Co-Authors: Ian Phillips, Elizabeth A. Shephard
    Abstract:

    Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of numerous foreign chemicals. This review considers the roles of FMOs in the metabolism of endogenous substrates and in physiological processes, and focuses on FMOs of human and mouse. Tyramine, phenethylamine, trimethylamine, cysteamine, methionine, lipoic acid and lipoamide have been identified as endogenous or dietary-derived substrates of FMOs in vitro. However, with the exception of trimethylamine, the role of FMOs in the metabolism of these compounds in vivo is unclear. The use, as experimental models, of knockout-mouse lines deficient in various Fmo genes has revealed previously unsuspected roles for FMOs in endogenous metabolic processes. FMO1 has been identified as a novel regulator of energy balance that acts to promote metabolic efficiency, and also as being involved in the biosynthesis of taurine, by catalyzing the S-oxygenation of hypotaurine. FMO5 has been identified as a regulator of metabolic ageing and glucose homeostasis that apparently acts by sensing or responding to gut bacteria. Thus, FMOs do not function only as xenobiotic-metabolizing enzymes and there is a risk that exposure to drugs and environmental chemicals that are substrates or inducers of FMOs would perturb the endogenous functions of these enzymes.

  • Effect of Flavin-Containing Monooxygenase Genotype, Mouse Strain, and Gender on Trimethylamine N-oxide Production, Plasma Cholesterol Concentration, and an Index of Atherosclerosis.
    Drug metabolism and disposition: the biological fate of chemicals, 2017
    Co-Authors: Sunil Veeravalli, Ian Phillips, Kersti Karu, Flora Scott, Diede Fennema, Elizabeth A. Shephard
    Abstract:

    The objectives of the study were to determine the contribution, in mice, of members of the flavin-containing monooxygenase (FMO) family to the production of trimethylamine (TMA) N-oxide (TMAO), a potential proatherogenic molecule, and whether under normal dietary conditions differences in TMAO production were associated with changes in plasma cholesterol concentration or with an index of atherosclerosis (Als). Concentrations of urinary TMA and TMAO and plasma cholesterol were measured in 10-week-old male and female C57BL/6J and CD-1 mice and in mouse lines deficient in various Fmo genes (Fmo1−/−, 2−/−, 4−/−, and FMO5−/−). In female mice most TMA N-oxygenation was catalyzed by FMO3, but in both genders 11%–12% of TMA was converted to TMAO by FMO1. Gender-, Fmo genotype-, and strain-related differences in TMAO production were accompanied by opposite effects on plasma cholesterol concentration. Plasma cholesterol was negatively, but weakly, correlated with TMAO production and urinary TMAO concentration. Fmo genotype had no effect on Als. There was no correlation between Als and either TMAO production or urinary TMAO concentration. Our results indicate that under normal dietary conditions TMAO does not increase plasma cholesterol or act as a proatherogenic molecule.

  • Identification of Flavin-Containing Monooxygenase 5 (FMO5) as a Regulator of Glucose Homeostasis and a Potential Sensor of Gut Bacteria.
    Drug metabolism and disposition: the biological fate of chemicals, 2017
    Co-Authors: Flora Scott, Ian Phillips, Sunil Veeravalli, Diede Fennema, Sandra G. Gonzalez Malagon, Brett A. O’brien, C.r. Coveney, Elizabeth A. Shephard
    Abstract:

    We have previously identified flavin-containing monooxygenase 5 (FMO5) as a regulator of metabolic aging. The aim of the present study was to investigate the role of FMO5 in glucose homeostasis and the impact of diet and gut flora on the phenotype of mice in which the FMO5 gene has been disrupted (FMO5−/− mice). In comparison with wild-type (WT) counterparts, FMO5−/− mice are resistant to age-related changes in glucose homeostasis and maintain the higher glucose tolerance and insulin sensitivity characteristic of young animals. When fed a high-fat diet, they are protected against weight gain and reduction of insulin sensitivity. The phenotype of FMO5−/− mice is independent of diet and the gut microbiome and is determined solely by the host genotype. FMO5−/− mice have metabolic characteristics similar to those of germ-free mice, indicating that FMO5 plays a role in sensing or responding to gut bacteria. In WT mice, FMO5 is present in the mucosal epithelium of the gastrointestinal tract where it is induced in response to a high-fat diet. In comparison with WT mice, FMO5−/− mice have fewer colonic goblet cells, and they differ in the production of the colonic hormone resistin-like molecule β. FMO5−/− mice have lower concentrations of tumor necrosis factor α in plasma and of complement component 3 in epididymal white adipose tissue, indicative of improved inflammatory tone. Our results implicate FMO5 as a regulator of body weight and of glucose disposal and insulin sensitivity and, thus, identify FMO5 as a potential novel therapeutic target for obesity and insulin resistance.

  • metabolism and pharmacokinetics of the anti tuberculosis drug ethionamide in a flavin containing monooxygenase null mouse
    Pharmaceuticals, 2012
    Co-Authors: Amy L Palmer, Sharon K Krueger, Ian Phillips, Elizabeth A. Shephard, Virginia L Leykam, Andrew Larkin, David E Williams
    Abstract:

    Multiple drug resistance (MDR) in Mycobacterium tuberculosis (mTB), the causative agent for tuberculosis (TB), has led to increased use of second-line drugs, including ethionamide (ETA). ETA is a prodrug bioactivated by mycobacterial and mammalian flavin-containing monooxygenases (FMOs). FMO2 is the major isoform in the lungs of most mammals, including primates. In humans a polymorphism exists in the expression of FMO2. FMO2.2 (truncated, inactive) protein is produced by the common allele, while the ancestral allele, encoding active FMO2.1, has been documented only in individuals of African and Hispanic origin, at an incidence of up to 50% and 7%, respectively. We hypothesized that FMO2 variability in TB-infected individuals would yield differences in concentrations and ratios of ETA prodrug and metabolites. In this study we assessed the impact of the FMO2 genetic polymorphism on the pharmacokinetics of ETA after administration of a single oral dose of ETA (125 mg/kg) to wild type and triple Fmo1/2/4-null mice, measuring levels of prodrug vs. metabolites in plasma collected from 0 to 3.5 h post-gavage. All mice metabolized ETA to ETA S-oxide (ETASO) and 2-ethyl-4-amidopyridine (ETAA). Wild type mice had higher plasma concentrations of metabolites than of parent compound (p = 0.001). In contrast, Fmo1/2/4-null mice had higher plasma concentrations of parent compound than of metabolites (p = 0.0001). Thus, the human FMO2 genotype could impact the therapeutic efficacy and/or toxicity of ETA.

  • human flavin containing monooxygenase 2 1 catalyzes oxygenation of the antitubercular drugs thiacetazone and ethionamide
    Drug Metabolism and Disposition, 2009
    Co-Authors: Asvi A Francois, Ian Phillips, Clinton R Nishida, Paul Ortiz R De Montellano, Elizabeth A. Shephard
    Abstract:

    The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the KM values for the four enzyme-catalyzed reactions are similar, kcat and, consequently, kcat/KM (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.