Evidence of the existence of structurally distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the rabbit

The flavin-containing monooxygenase has been purified from rabbit liver and lung microsomes. SDS-PAGE analysis shows that both enzyme forms migrate as a single band with an apparent Mr of 59000. The NH2-terminus of both forms is blocked. The liver oxidase contains a lower percentage of glutamine/glutamate and a greater amount of phenylalanine than does the lung flavoprotein. Polyclonal antibodies to a 14-amino-acid peptide obtained after CNBr cleavage of the liver oxidase cross-react with the microsomal and purified liver enzyme, but do not recognize the lung oxidase. HPLC profiles of tryptic digests of the liver and lung enzymes exhibit different patterns. Sequence alignment of selected peptides from the liver and lung oxidases reveals aberrant residues within homologous segments. These findings are interpreted to mean that both enzymes represent distinct gene products.     

The flavin-containing monooxygenase in mouse lung: Evidence for expression of multiple forms

The flavin-containing monooxygenase (FMO) was purified from mouse lung microsomes. On SDS-PAGE, the purified enzyme separated as two bands, a major band of 58,000 daltons and a minor band of 59,000 daltons. Antibodies to mouse liver FMO cross-reacted with both bands in the purified preparations, whereas antibodies to rabbit lung FMO cross-reacted only with the major band. In microsomal preparations the major band was recognized by both antibodies, but neither antibody detected the minor band in microsomes. A cDNA encoding the pig liver FMO hybridized with mRNA isolated from mouse liver, kidney, and lung, whereas cDNA encoding the rabbit lung FMO hybridized only with mouse lung and kidney mRNA. Thermal stability studies showed that the FMO preparation purified from mouse lung consisted of a heat-stable and a heat-labile component. The heat-labile component of lung FMO was inhibited competitively by imipramine, whereas the heat-stable component was insensitive to the presence of imipramine. Immunoprecipitation of purified mouse lung FMO with anti-rabbit lung FMO completely removed the protein band reactive to anti-rabbit lung FMO while leaving reactivity to anti-liver FMO. The catalytic and immunochemical differences seen between FMO from rabbit lung and mouse lung appear to result from the expression of at least two forms of FMO in the mouse lung, one similar to the rabbit pulmonary form and one similar to the major mouse liver form of FMO.     

Functional activity of the mouse flavin-containing monooxygenase forms 1, 3, and 5

Three functional mouse flavin-containing monooxygenases (mFMOs) (i.e., mFMO1, mFMO3, and mFMO5) have been reported to be the major FMOs present in mouse liver. To examine the biochemical features of these enzymes, recombinant enzymes were expressed as maltose-binding protein fusion proteins (i.e., MBP-mFMO1, MBP-mFMO3, and MBP-mFMO5) in Escherichia coli and isolated and purified with affinity chromatography. The substrate specificity of these three mouse hepatic FMO enzymes were examined using a variety of substrates, including mercaptoimidazole, trimethylamine, S-methyl esonarimod, and an analog thereof, and a series of 10-(N,N-dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazine analogs. The kinetic parameters of the three mouse FMOs for these substrates were compared in an attempt to explore substrate structure--function relationships specific for each mFMO. Utilizing a common phenothiazine substrate for all three enzymes, we compared the pH dependence for the recombinant enzymes under similar conditions. In addition, thermal stability for mFMO1, mFMO3, and mFMO5 enzymes was examined in the presence and absence of NADPH. The results revealed unique features for mFMO5, suggesting possible impact on the functional significance of this abundantly expressed FMO5 isoform in both human and mouse liver.     

Redox regulation of yeast flavin-containing monooxygenase

The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione. The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment. We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential. The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER. Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation. Cys 353 is the principal residue in the redox-sensitive switch. We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO. The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory.     

Covalent structure of liver microsomal flavin-containing monooxygenase form 1

Liver microsomal, flavin-containing monooxygenases catalyze NADPH- and oxygen-dependent oxidation of a wide variety of antipsychotic and narcotic drugs. Two forms of these enzymes have been isolated and partially characterized (Ozols, J. (1989) Biochem. Biophys. Res. Commun. 163, 49-55). The amino acid sequence of form 1 is presented here. Sequence determination has been achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The NH2 terminus of form 1 oxygenase is blocked. Partial acid hydrolysis of the blocked peptides removed acetyl groups and permitted their analysis by Edman degradation. Form 1 monooxygenase contains 536 residues. A peptide of 32 residues at the COOH terminus of the protein could not be sequenced in a gas-phase or pulsed liquid-phase sequenator, due to its extreme hydrophobicity. Covalent coupling of this peptide to an aryl amine membrane by means of carbodiimide, followed by automated solid-phase sequencing, established the order of 30 amino acid residues. The hydrophobic segment at the COOH terminus presumably functions to anchor the monooxygenase to the microsomal membrane. The amino acid sequence of form 1 monooxygenase, despite overlapping substrate specificity, is not related to the cytochrome P-450 superfamily. Comparison of the sequence of form 1 oxygenase with other known sequences, except for some short segments similar to those in the bacterial flavin-containing monooxygenases, did not reveal significant sequence similarities that would suggest a structural or evolutionary relationship.     

Asymmetric metabolic N-oxidation of N-ethyl-N-methylaniline by purified flavin-containing monooxygenase

The prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is known to be metabolically N-oxygenated in vitro with microsomal preparations. This biotransformation is thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Microsomal N-oxygenation of EMA is known to be stereoselective and varies between species. In order to further characterise this metabolic transformation, we have examined the in vitro metabolism of EMA using purified porcine hepatic FMO. Following incubation of EMA with purified FMO, EMA N-oxide, the only metabolite detected, was found to be produced stereoselectively [ratio (?)-(S):(+)-(R), ca. 4:1]. The enantiomeric ratio of the N-oxide product did not change markedly with respect to time, enzyme or substrate concentration. Determination of the kinetics of formation of the N-oxide indicated a single affinity for the prochiral substrate with differential rates of formation of the enantiomers. The extent of EMA N-oxide formation was shown to be affected by activators and inhibitors of FMO and pH, but its stereoselectively was unaltered. © 1994 Wiley-Liss, Inc.     

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.     

C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility

Flavin-containing monooxygenases (FMO) are membrane-associated enzymes contributing to oxidative metabolism of drugs and other chemicals. There are no known structures similar enough to FMO to provide accurate insights into the structural basis for differences in metabolism observed among FMOs. To develop an FMO amenable to crystallization, we introduced mutations into rabbit FMO2 (rF2) to increase solubility, decrease aggregation, and simplify isolation. Alterations included removal of 26 AA (Delta26) from the carboxyl-terminus, His(6)-fusion to the amino-terminus and a double Ser substitution designed to reduce local hydrophobicity. Only Delta26 FMO variants retained normal activity, increased the yield of cytosolic rF2 and decreased protein aggregation. Delta26 constructs increased rF2 in cytosol in low (from 2 to 13%), and high salt (from 24 to 62%) conditions. His-fusion proteins, while active and useful for purification, did not affect solubility. Delta26 variants should prove useful for identifying conditions suitable for production of an FMO crystal.     

Evidence for complex formation between rabbit lung flavin-containing monooxygenase and calreticulin

Rabbit lung flavin-containing monooxygenase (FMO, EC 1.14.13.8) was denatured, reduced, carboxymethylated, digested with endoproteinase Glu-C or trypsin, and subjected to mass spectrometric analysis. The amino acid sequences of selected peptides were determined by tandem mass spectrometry. Over 90% of rabbit lung FMO was mapped by liquid secondary ion mass spectrometry (LSIMS). The FMO N-terminal amino acid was found to be N-acetylated, and the N-terminal 23 amino acid peptide contained an FAD binding domain consisting of Gly-X-Gly-X-X-Gly. Another peptide was found to contain a NADP+ binding domain consisting of Gly-X-Gly-X-X-Ala. The mapped and/or sequenced peptides were found to be completely consistent with the peptide sequence deduced from the cDNA data and the previously published gas-phase sequencing data. Further mass spectrometry and protein analytical work unambiguously showed that rabbit lung FMO existed in tight association with a calcium-binding protein, calreticulin. Over 68% of rabbit lung calreticulin was mapped by LSIMS. Tandem mass spectrometric and gas-phase sequencing studies provided direct evidence for the identification of the N-terminal and other rabbit lung calreticulin-derived peptide sequences that were identical to other previously reported calreticulins. The complexation of calreticulin to rabbit lung FMO could account for some of the unusual physical properties of this FMO enzyme form.     

The rabbit pulmonary monooxygenase system: characteristics and activities of two forms of pulmonary cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450 have been characterized spectrally and their activities in reconstituted monooxygenase systems investigated. The presence of both microsomal phospholipids and sodium cholate was required to obtain optimum activity. Only one of the cytochromes (I) was active in the N-demethylation of benzphetamine and the O-deethylation of 7-ethoxycoumarin. However, cytochrome II was 20% more active than cytochrome I in the metabolism of benzo[a]pyrene. The profile of the metabolites formed from benzo[a]pyrene indicated that metabolism at the 9 and 10 positions was insignificant in the case of cytochrome I but represented about 40% of the metabolites produced by cytochrome II. The two forms of the cytochrome are present in pulmonary microsomes in approximately equal amounts.     

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.     

Oxidative metabolism of xenobiotics during pregnancy: Significance of microsomal flavin-containing monooxygenase

Pregnancy related changes in oxidative metabolism of model substrates were examined in CD₁ mice. As compared to nonpregnant females, a significant decrease in the hepatic microsomal aminopyrine-but not in dimethylaniline-N-demethylase activity was observed in pregnant mice. The rates of microsomal flavin-containing monooxygenase-catalyzed N-oxidation of dimethylaniline remained relatively unchanged during pregnancy in the liver, lung, kidney, and uterus. In contrast to this, N-oxidase activity of placental microsomes was increased nearly 5-fold when measured at day 12 and 18 of gestation.     

Microsomal oxidation of thiobenzamide. A photometric assay for the flavin-containing monooxygenase

The hepatotoxin thiobenzamide is S-oxidized by the microsomal flavin-containing monooxygenase (MFMO)¹ in liver, lung, and kidney of rabbit, mouse and rat. Its oxidation is accompanied by a large spectral shift which can be used as the basis of a simple convenient photometric assay for the MFMO system.     

Spectrophotometric measurement of flavin-containing monooxygenase activity in freshly isolated rat hepatocytes and their cultures.

The determination of the mixed function flavin-containing monooxygenase activity in rat liver and in hepatocytes and their cultures by spectrophotometric measurement of the oxygenation of methimazole is complicated by an inhibition caused by some of the reagents used during this method. Optimal conditions were determined for measuring this enzyme activity in microsomal preparations of rat liver and its hepatocytes. Optimal flavin-containing monooxygenase activities were obtained for measurements performed in a 0.25 M N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-EDTA buffer at pH 8.7 and at a methimazole concentration of 2 mM. Data are also presented which show that no interferences caused by either cytochrome P450-dependent enzymes or by the reduction of methimazole disulfide by glutathione have to be taken into account when determining methimazole oxygenation. Finally, the above assay was also used to study flavin-containing monooxygenase activity in primary monolayer cultures of hepatocytes for 6 days.     

Mutation, polymorphism and perspectives for the future of human flavin-containing monooxygenase 3

Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent monooxygenation of soft-nucleophilic nitrogen, sulfur, and phosphorous atoms contained within various drugs, pesticides, and xenobiotics. Flavin-containing monooxygenase 3 (FMO3) is responsible for the majority of FMO-mediated xenobiotic metabolism in the adult human liver. Mutations in the FMO3 gene can result in defective trimethylamine (TMA) N-oxygenation, which gives rise to the disorder known as trimethylaminuria (TMAU) or "fish-odour syndrome". To date 18 mutations of FMO3 gene have been reported that cause TMAU, and polymorphic variants of the gene have also been identified. Interindividual variability in the expression of FMO3 may affect drug and foreign chemical metabolism in the liver and other tissues. It is important therefore to study how base sequence variation of the FMO3 gene might affect the ability of individuals and different ethnic population groups to deal with the variety of environmental chemicals and pharmaceutical products that are substrates for FMO3.     

Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.     

Multiplicity of liver microsomal flavin-containing monooxygenase in the guinea pig: its purification and characterization

Two distinct forms (FMO-I and FMO-II) of flavin-containing monooxygenase were purified from the liver microsomes of guinea pig. The minimum molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 54,000 for FMO-I and 56,000 for FMO-II, respectively. Tryptic digestion of these enzymes gave different electrophoretic patterns, suggesting that FMO-I and -II have distinct amino acid sequences. The amino terminal sequence of FMO-II could not be estimated probably due to its blocking while that of FMO-I was determined to be highly homologous to the rabbit liver flavin-containing monooxygenase (J. Ozols, 1989, Biochem. Biophys. Res. Commun. 163, 49-55). Absorption maxima of FMO-I and -II were recorded at 368 and 440 nm and 381 and 456 nm, respectively. Molar ratios of FAD to both of these apoenzymes were shown to be one to one. Substrate specificity of FMO-I and -II was determined using 15 compounds as the substrate. The results showed two enzymes that exhibited overlapped but different specificity toward these substrates although FMO-I had lower activity than did FMO-II with all compounds except thiobenzamide. Of particular interest, only FMO-II showed considerably high activities for primary amines, n-octylamine, and n-decylamine. Immunoglobulin G raised against FMO-II could recognize FMO-I as well as FMO-II, but the reactivity of FMO-I toward the antibody was obviously lower than that of FMO-II. Electrophoresis followed by immunostaining revealed that microsomes of lung, kidney, urinary bladder, testis, and spleen contain the same protein as FMO-II and/or FMO-I. Only lung was shown to have an additional isozyme of FAD-monooxygenase with a molecular weight apparently higher than those of FMO-I and -II. These results strongly suggest that at least two forms of flavin-containing monooxygenases distinct from the lung-type isozyme are expressed in liver of guinea pigs.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Reactions of the 4a-hydroperoxide of liver microsomal flavin-containing monooxygenase with nucleophilic and electrophilic substrates

Liver microsomal flavin-containing monooxygenase (MFMO) has been shown to exhibit a stable 4a-flavin hydroperoxide intermediate in the absence of oxygenatable substrate (Poulsen, L. L., and Ziegler, D. M. (1979) J. Biol. Chem. 254, 6449-6455; Beaty, N. B., and Ballou, D. P. (1981) J. Biol. Chem. 256, 4619-4625). The reaction of this intermediate with an assortment of substrates was studied by stopped flow techniques. The first observed spectral change is a small blue shift in the absorbance peak of the 4a-flavin intermediate. The rate of this spectral change is dependent on the concentration of the substrate. This small spectral change is succeeded by a large increase in the absorbance at 450 nm. The rate of appearance of oxidized flavin is independent of substrate concentration but does increase at higher pH. Steady state turnover rates also greater at higher pH, consistent with earlier observations that the formation of oxidized flavin is rate determining in catalysis. Upon oxygenation by MFMO, thiobenzamide and iodide each undergo a spectral change which is dependent on substrate concentration. The spectral changes corresponding to oxygenation of these substrates occur at the same rates as do the initial small spectral changes contributed by the flavin chromophore as observed with all substrates. However, no substrate tested to date shows any effect on the rate of formation of oxidized flavin. Previous work has shown MFMO to catalyze the oxygenation of a variety of nitrogen- and sulfur-containing hydrophobic compounds. Two new classes of compounds are shown here to be substrates for this enzyme. The nucleophilic anions, iodide and thiocyanate, catalyze the decomposition of the 4a-flavin hydroperoxide. Organic boronic acids (e.g. phenylboronic acid and butylboronic acid) also appear to be oxygenated with no striking differences in kinetic characteristics from those of nucleophilic substrates. These organic boronic acids are classic electrophiles and suggest that like peracids, the 4a-flavin hydroperoxide is capable of oxygenating both nucleophiles and electrophiles (Lee, J. B., and Uff, B. C. (1967) Quart. Rev. 21, 429-457).