Human FMO3 Research Articles

Quinuclidine N-Oxygenation Mediated by Flavin-Containing Monooxygenases 1 and 3 in Kidney and Liver Microsomes from Humans, Monkeys, Dogs, and Pigs.

Flavin-containing monooxygenases (FMOs) are a family of enzymes that are involved in the oxygenation of heteroatom-containing molecules. In humans, FMO3 is the major hepatic form, whereas FMO1 is predominant in the kidneys. FMO1 and FMO3 have also been identified in monkeys, dogs, and pigs. The predicted contribution of human FMO3 to drug candidate N-oxygenation could be estimated using the classic base dissociation constants of the N-containing moiety. A basic quinuclidine moiety was found in natural quinine and medicinal products. Consequently, N-oxygenation of quinuclidine was evaluated using liver and kidney microsomes from humans, monkeys, dogs, and pigs as well as recombinant FMO1, FMO3, and FMO5 enzymes. Experiments using simple reversed-phase liquid chromatography with fluorescence monitoring revealed that recombinant FMO1 mediated quinuclidine N-oxygenation with a high capacity in humans. Moreover, recombinant FMO1, FMO3, and/or FMO5 in monkeys, dogs, and pigs exhibited relatively broad substrate specificity toward quinuclidine N-oxygenation. Kinetic analysis showed that human FMO1 efficiently, and pig FMO1 moderately, mediated quinuclidine N-oxygenation with high capacity, which is consistent with the reported findings for larger substrates readily accepted by pig FMO1 but excluded by human FMO1. In contrast, human FMO3-mediated quinuclidine N-oxygenation was slower than that of the typical FMO3 substrate trimethylamine. These results suggest that some species differences exist in terms of FMO-mediated quinuclidine N-oxygenation in humans and some animal models (monkeys, dogs, and minipigs); however, the potential for quinuclidine, which has a simple chemical structure, to be inhibited clinically by co-administered drugs should be relatively low, especially in human livers. SIGNIFICANCE STATEMENT: The high capacity of human flavin-containing monooxygenase (FMO) 1 to mediate quinuclidine N-oxygenation, a basic moiety in natural products and medicines, was demonstrated by simple reversed-phase liquid chromatography using fluorescence monitoring. The substrate specificity of FMO1 and FMO3 toward quinuclidine N-oxygenation in monkeys, dogs, and pigs was suggested to be relatively broad. Human FMO3-mediated quinuclidine N-oxygenation was slower than trimethylamine N-oxygenation. The likelihood of quinuclidine, with its simple chemical structure, being clinically inhibited by co-administered drugs is relatively low.

Molecular and functional characterization of flavin-containing monooxygenases (FMO1–6) in tree shrews

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1–5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1–6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85–90 % and 81–89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1–6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1–6, which are orthologous to human FMOs (FMO1–5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver.

Molecular and functional characterization of flavin-containing monooxygenases in pigs, dogs, and cats

Flavin-containing monooxygenases (FMOs) are drug-oxygenating enzymes that are present in the human genome as FMO1-5 and FMO6P. Among pig, dog, and cat FMOs, pig and dog FMO1 and FMO3 have been partly characterized, but other FMOs have not been systematically identified. In this study, orthologous FMO cDNAs were isolated from pig, dog, and cat livers and evaluated by sequence and phylogenetic analyses, tissue expression, and catalytic function. The amino acid sequences of pig, dog, and cat FMO1-5 shared high sequence identities (83–89%) with human FMO1-5 and were closely clustered in a phylogenetic tree. The gene structure and genomic organization of FMO1-5 were conserved across these species. Dog and pig FMO6P contained insertions of 1 and 83 bases, respectively, and are possibly pseudogenes similar to human FMO6P. Among the tissue types analyzed, pig FMO1 mRNA was abundant in liver, kidney, and lung; dog FMO3, FMO2, and FMO5 mRNAs were abundant in liver, lung, and kidney, respectively; cat FMO1 and FMO3 mRNAs were abundant in kidney and liver, respectively. Recombinant pig and dog FMO1-5 and cat FMO1-6 all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfoxide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by pig FMO1, dog FMO3, and cat FMO3. Cat FMO6 was also active toward trimethylamine. These results suggest some similarities in the drug-metabolizing capabilities of FMO3 in dogs, cats, and humans and that dog and cat FMO3 generally have molecular and functional characteristics similar to human FMO3, being the major FMO in human liver.

Human flavin-containing monooxygenase 1 and its long-sought hydroperoxyflavin intermediate

Out of the five isoforms of human flavin-containing monooxygenase (hFMO), FMO1 and FMO3 are the most relevant to Phase I drug metabolism. They are involved in the oxygenation of xenobiotics including drugs and pesticides using NADPH and FAD as cofactors. Majority of the characterization of these enzymes has involved hFMO3, where intermediates of its catalytic cycle have been described. On the other hand, research efforts have so far failed in capturing the same key intermediate that is responsible for the monooxygenation activity of hFMO1. In this work we demonstrate spectrophotometrically the formation of a highly stable C4a-hydroperoxyflavin intermediate of hFMO1 upon reduction by NADPH and in the presence of O2. The measured half-life of this flavin intermediate revealed it to be stable and not fully re-oxidized even after 30 min at 15 °C in the absence of substrate, the highest stability ever observed for a human FMO. In addition, the uncoupling reactions of hFMO1 show that this enzyme is <1% uncoupled in the presence of substrate, forming small amounts of H2O2 with no observable superoxide as confirmed by EPR spin trapping experiments. This behaviour is different from hFMO3, that is shown to form both H2O2 and superoxide anion radical as a result of ∼50% uncoupling. These data are consistent with the higher stability of the hFMO1 intermediate in comparison to hFMO3. Taken together, these data demonstrate the different behaviours of these two closely related enzymes with consequences for drug metabolism as well as possible toxicity due to reactive oxygen species.

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2’-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Role of human flavin-containing monooxygenase (FMO) 5 in the metabolism of nabumetone: Baeyer–Villiger oxidation in the activation of the intermediate metabolite, 3-hydroxy nabumetone, to the active metabolite, 6-methoxy-2-naphthylacetic acid in vitro

Nabumetone (NAB) is a non-steroidal anti-inflammatory drug used clinically, and its biotransformation includes the major active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA). One of the key intermediates between NAB and 6-MNA may be 3-hydroxy nabumetone (3-OH-NAB). The aim of the present study was to investigate the role of flavin-containing monooxygenase (FMO) isoform 5 in the formation of 6-MNA from 3-OH-NAB. To elucidate the biotransformation of 3-OH-NAB to 6-MNA, an authentic standard of 3-OH-NAB was synthesised and used as a substrate in an incubation with human liver samples or recombinant enzymes. The formation of 3-OH-NAB was observed after the incubation of NAB with various cytochrome P450 (CYP) isoforms. However, 6-MNA itself was rarely detected from NAB and 3-OH-NAB. Further experiments revealed a 6-MNA peak derived from 3-OH-NAB in human hepatocytes. 6-MNA was also detected in the extract obtained from 3-OH-NAB by a combined incubation of recombinant human FMO5 and human liver S9. We herein demonstrated that the reaction involves carbon-carbon cleavage catalyzed by the Baeyer–Villiger oxidation (BVO) of a carbonyl compound, the BVO substrate, such as a ketol, by FMO5. Further in vitro inhibition experiments showed that multiple non-CYP enzymes are involved in the formation of 6-MNA from 3-OH-NAB.

Flavin-Containing Monooxygenase 1 Catalyzes the Production of Taurine from Hypotaurine.

Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part, this is due to the incorrect assignment, in 1962, of the enzyme as an NAD-dependent hypotaurine dehydrogenase. For more than 55 years, the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase (FMO) 1. Metabolite analysis of the urine of Fmo1-null mice by 1H NMR spectroscopy revealed a buildup of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that human FMO1 catalyzes the conversion of hypotaurine to taurine, utilizing either NADPH or NADH as cofactor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biologic processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, antioxidant and anti-inflammatory functions, and the pathogenesis of obesity and skeletal muscle disorders. SIGNIFICANCE STATEMENT: The identity of the enzyme that catalyzes the biosynthesis of taurine from hypotaurine has remained elusive. Here we show, both in vivo and in vitro, that flavin-containing monooxygenase 1 catalyzes the oxygenation of hypotaurine to produce taurine.

Drug metabolite synthesis by immobilized human FMO3 and whole cell catalysts

BackgroundSufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro.ResultIn this work, using benzydamine as a model drug, two easy-to-perform approaches (whole cell catalysis and enzyme immobilization) were investigated for the synthesis of FMO3-generated drug metabolites. The whole cell catalysis was carried out by using cell suspensions of E. coli JM109 harboring FMO3 and E. coli BL21 harboring GDH (glucose dehydrogenase), giving 1.2 g/L benzydamine N-oxide within 9 h under the optimized conditions. While for another approach, two HisTrap HP columns respectively carrying His6-GDH and His6-FMO3 were connected in series used for the biocatalysis. In this case, 0.47 g/L benzydamine N-oxide was generated within 2.5 h under the optimized conditions. In addition, FMO3 immobilization at the C-terminal (membrane anchor region) significantly improved its enzymatic thermostability by more than 10 times. Moreover, the high efficiency of these two biocatalytic approaches was also confirmed by the N-oxidation of tamoxifen.ConclusionsThe results presented in this work provides new possibilities for the drug-metabolizing enzymes-mediated biocatalysis.

A direct time-based ITC approach for substrate turnover measurements demonstrated on human FMO3.

Transient binding events are a challenging issue in enzymology. Here we demostrate a time-based ITC approach to human flavin-containing monooxygenase 3, an important drug metabolising enzyme. We measure kinetic constants and we demonstrate how this approach can be exploited for measuring the inhibiton of the conversion of the key substrate trimethylamine into trimethylamine N-oxide.

Cope elimination mediated by human FMO1 leads to formation of reactive Michael Acceptor intermediate

Cope elimination mediated by human FMO1 leads to formation of reactive Michael Acceptor intermediate

Binding of methimazole and NADP(H) to human FMO3: In vitro and in silico studies

Human flavin-containing monooxygenase isoform 3 (hFMO3) is an important hepatic drug-metabolizing enzyme, catalyzing the monooxygenation of nucleophilic heteroatom-containing xenobiotics. Based on the structure of bacterial FMO, it is proposed that a conserved asparagine is involved in both NADP(H) and substrate binding. In order to explore the role of this amino acid in hFMO3, two mutants were constructed. In the case of N61Q, increasing the steric hindrance above the flavin N5-C4a causes poor NADP(H) binding, destabilizing the catalytic FAD intermediate, whereas the introduction of a negatively charged residue, N61D, interferes mainly with catalytic intermediate formation and its stability. To better understand the substrate-enzyme interaction, in vitro as well as in silico experiments were carried out with methimazole as substrate. Methimazole is a high-affinity substrate of hFMO3 and can competitively suppress the metabolism of other compounds. Our results demonstrate that methimazole Pi-stacks above the isoalloxazine ring of FAD in hFMO3, in a similar way to indole binding to the bacterial FMO. However, for hFMO3 indole is found to act as a non-substrate competitive inhibitor. Finally, understanding the binding mode of methimazole and indole could be advantageous for development of hFMO3 inhibitors, currently investigated as a possible treatment strategy for atherosclerosis.

Metabolism and Disposition of a Novel Selective α7 Neuronal Acetylcholine Receptor Agonist ABT-126 in Humans: Characterization of the Major Roles for Flavin-Containing Monooxygenases and UDP-Glucuronosyl Transferase 1A4 and 2B10 in Catalysis.

Mass balance, metabolism, and excretion of ABT-126, an α7 neuronal acetylcholine receptor agonist, were characterized in healthy male subjects (n = 4) after a single 100-mg (100 μCi) oral dose. The total recovery of the administered radioactivity was 94.0% (±2.09%), with 81.5% (±10.2%) in urine and 12.4% (±9.3%) in feces. Metabolite profiling indicated that ABT-126 had been extensively metabolized, with 6.6% of the dose remaining as unchanged parent drug in urine. Parent drug accounted for 12.2% of the administered radioactivity in feces. The primary metabolic transformations of ABT-126 involved aza-adamantane N-oxidation (M1, 50.3% in urine) and aza-adamantane N-glucuronidation (M11, 19.9% in urine). M1 and M11 were also major circulating metabolites, accounting for 32.6% and 36.6% of the drug-related material in plasma, respectively. These results demonstrated that ABT-126 is eliminated primarily by hepatic metabolism, followed by urinary excretion. Enzymatic studies suggested that M1 formation is mediated primarily by human liver flavin-containing monooxygenase (FMO)3 and, to a lesser extent, by human kidney FMO1; M11 is generated mainly by human uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A4, whereas UGT 2B10 also contributes to ABT-126 glucuronidation. Species-dependent formation of M11 was observed in hepatocytes; M11 was formed in human and monkey hepatocytes, but not in rat and dog hepatocytes, suggesting that monkeys constitute an appropriate model for predicting the fate of compounds undergoing significant N-glucuronidation. M1 and M11 are not expected to have clinically relevant on- or off-target pharmacologic activities. In summary, this study characterized ABT-126 metabolites in the circulation and excreta and the primary elimination pathways of ABT-126 in humans.

Inactivation mechanism of N61S mutant of human FMO3 towards trimethylamine

Human flavin-containing monooxygenase 3 (hFMO3) catalyses the oxygenation of a wide variety of compounds including drugs as well as dietary compounds. It is the major hepatic enzyme involved in the production of the N-oxide of trimethylamine (TMAO) and clinical studies have uncovered a striking correlation between plasma TMAO concentration and cardiovascular disease. Certain mutations within the hFMO3 gene cause defective trimethylamine (TMA) N-oxygenation leading to trimethylaminuria (TMAU) also known as fish-odour syndrome. In this paper, the inactivation mechanism of a TMAU-causing polymorphic variant, N61S, is investigated. Transient kinetic experiments show that this variant has a > 170-fold lower NADPH binding affinity than the wild type. Thermodynamic and spectroscopic experiments reveal that the poor NADP+ binding affinity accelerates the C4a-hydroperoxyFAD intermediate decay, responsible for an unfavourable oxygen transfer to the substrate. Steady-state kinetic experiments show significantly decreased N61S catalytic activity towards other substrates; methimazole, benzydamine and tamoxifen. The in vitro data are corroborated by in silico data where compared to the wild type enzyme, a hydrogen bond required for the stabilisation of the flavin intermediate is lacking. Taken together, the data presented reveal the molecular basis for the loss of function observed in N61S mutant.

Marmoset Flavin-Containing Monooxygenase 3 in the Liver Is a Major Benzydamine and Sulindac Sulfide Oxygenase.

Common marmosets (Callithrix jacchus) are potentially primate models for preclinical drug metabolism studies because there are similarities in the molecular characteristics of cytochrome P450 enzymes between this species and humans. However, characterization of non-cytochrome P450 enzymes has not been clarified in marmosets. Here, we report characterization of flavin-containing monooxygenases FMO1-FMO5 identified in marmoset tissues. Marmoset FMO forms shared high amino acid sequence identities (93%-95%) and phylogenetic closeness with human homologous FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in the liver and kidneys among five marmoset tissues examined, where FMO3 protein was detected by immunoblotting. FMO inhibition assays using preheated tissue microsomes indicated that benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the marmoset liver was mainly catalyzed by FMO3, the major hepatic FMO. Marmoset FMO3 protein heterologously expressed in Escherichia coli effectively catalyzed benzydamine N-oxygenation and sulindac sulfide S-oxygenation comparable to marmoset liver microsomes. These results indicate that the FMO3 enzyme expressed in marmoset livers mainly metabolizes benzydamine and sulindac sulfide (typical human FMO substrates), suggesting its importance for FMO-dependent drug metabolism in marmosets.

Bioelectrochemical profiling of two common polymorphic variants of human FMO3 in presence of graphene oxide

Genetic variation of phase I drug metabolising enzymes has been shown to greatly influence inter-individual reaction to pharmacological treatments. Among these enzymes, human flavin-containing monooxygenase 3 (hFMO3) plays a crucial role and understanding its pharmacogenetics is fundamental for the prediction of individual drug response and the efficacy of therapy. In this work the altered drug metabolism of two common polymorphic variants of hFMO3 (E158K and E308G) are studied by using an electrochemical platform modified with graphene oxide (GO). Electrochemistry was used to characterise the properties of these two engineered and purified hFMO3 variants followed by electrocatalysis experiments in the presence of three different hFMO3 substrates benzydamine, tamoxifen and sulindac sulfide. HPLC quantification of the electrochemically produced metabolites showed that E158K mutation leads to an impairment of N-oxygenation activity while E308G mutation enhances the same activity.Results demonstrate that electrocatalysis on GO modified glassy carbon electrodes provides a fast and reliable method for measuring kinetic parameters of hFMO3 polymorphic variants. This method can be considered suitable for deciphering metabolic implications of polymorphisms that might lead to adjustment of drug dosages depending on the individual’s genetic makeup, a step closer to the development of personalised medicine.

What is the contribution of human FMO3 in the N-oxygenation of selected therapeutic drugs and drugs of abuse?

Little is known about the role of flavin-containing monooxygenases (FMOs) in the metabolism of xenobiotics. FMO3 is the isoform in adult human liver with the highest impact on drug metabolism. The aim of the presented study was to elucidate the contribution of human FMO3 to the N-oxygenation of selected therapeutic drugs and drugs of abuse (DOAs). Its contribution to the in vivo hepatic net clearance of the N-oxygenation products was calculated by application of an extended relative activity factor (RAF) approach to differentiate from contribution of cytochrome P450 (CYP) isoforms. FMO3 and CYP substrates were identified using pooled human liver microsomes after heat inactivation and chemical inhibition, or single enzyme incubations. Kinetic parameters were subsequently determined using recombinant human enzymes and mass spectrometric analysis via authentic reference standards or simple peak areas of the products divided by those of the internal standard. FMO3 was identified as enzyme mainly responsible for the formation of N,N-diallyltryptamine N-oxide and methamphetamine hydroxylamine (>80% contribution for both). A contribution of 50 and 30% was calculated for the formation of N,N-dimethyltryptamine N-oxide and methoxypiperamide N-oxide, respectively. However, FMO3 contributed with less than 5% to the formation of 3-bromomethcathinone hydroxylamine, amitriptyline N-oxide, and clozapine N-oxide. There was no significant difference in the contributions when using calibrations with reference metabolite standards or peak area ratio calculations. The successful application of a modified RAF approach including FMO3 proved the importance of FMO3 in the N-oxygenation of DOAs in human metabolism.

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.5-49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM.

Biocatalytic Characterization of Human FMO5: Unearthing Baeyer-Villiger Reactions in Humans.

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism.

Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.

Regulation of flavin-containing mono-oxygenase (Fmo3) gene expression by steroids in mice and humans.

Abstract Flavin-containing mono-oxygenases (FMOs) are a family of microsomal chemical- and drug-metabolizing enzymes. FMO3 is a major FMO form in adult mouse and human liver. FMO3 mutations have been associated with the incidence and severity of trimethylaminuria (TMAU), a metabolic disorder characterized by the inability of the affected individual to metabolize the odorous trimethylamine to its non-odorous N-oxide. In addition to this primary genetic form, there are other forms of TMAU that support the hypothesis that FMO3 activity may be modulated by steroid hormones. To understand the molecular mechanism involved in the regulation of Fmo3 gene expression by steroid hormones, we performed this study in an in vitro cellular system, mouse liver cells, and on the human FMO3 gene. Dexamethasone, 5α-dihydrotestosterone, thyroid hormone, and progesterone had no effect on the accumulation of Fmo3 mRNA. The use of increased concentration of theophylline inhibited estrogen receptor α (ERα)-mediated transcription of Fmo3 mRNA. 17β-Estradiol inhibited Fmo3 mRNA accumulation. The use of ICI 164,384 abolished the inhibitory effect induced by estrogen. Gel-shift analyses showed a binding in the 5' region of the Fmo3 gene. This binding was abrogated by an excess of a cDNA containing an estrogen-responsive element. An estrogen-binding site was also present in the first intron of the human gene, as demonstrated by the gel-shift assay. Supershift experiments confirmed the binding of ERα in both mouse and human samples. Furthermore, chromatin immunoprecipitation assay confirmed the binding of ERα in the promoter region of mouse Fmo3 and in the first intron of the human FMO3 gene. Thus, 17β-estradiol plays a fundamental role in the regulation of Fmo3 gene transcription.