Flavin-containing monooxygenase activity can be inhibited by nitric oxide-mediated S-nitrosylation

Nitric oxide (NO) modifies the functions of a variety of proteins containing cysteine thiols or transition-metal centers, particularly by S-nitrosylation. In inflamed liver, NO is overproduced and hepatic drug-metabolizing enzymes, the flavin-containing monooxygenases (FMOs) and cytochrome P450s (CYPs), are suppressed. However, the NO-related mechanisms underlying the loss of these activities are not well understood, particularly for FMOs. In this study, we suggest that FMO3, the major FMO in human liver, is modified post-translationally by NO. This hypothesis is based on the imbalance observed between the decrease in FMO3 expression (40.7% of controls) and FMO3-specific ranitidine N-oxidation activity (15.1%), and on the partial or complete reversibility of FMO inhibition by sulfhydryl-reducing regents such as DTT (effective on both S-S and S-NO adducts) and ascorbate (effective on S-NO only). Furthermore, NO donors (SNP, SNAP, and Sin-1), including the pure NO donor DEA/NO, directly suppressed in vitro FMO activity (N- or S-oxidation of ranitidine, trimethylamine, and thiobenzamide) in human liver microsomal proteins and recombinant human FMO3. These activities were restored completely after treatment with DTT or ascorbate. These results suggest that NO-mediated S-nitrosylation is involved in the rigorous inhibition of FMO activity in vitro and in vivo, resulting in the suppression of FMO-based drug metabolism or detoxification.     

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.     

Flavin-containing monooxygenase activity in camel tissues: comparison with rat and human liver enzymes

We previously reported the occurrence of multiple forms of drug metabolizing enzymes in camel tissues. In this study, we demonstrated for the first time, flavin-containing monooxygenase (FMO)-dependent metabolism of two model substrates methimazole (MEM) and N,N'-dimethylaniline (DMA) by camel liver, kidney, brain and intestine. FMO-catalyzed metabolism in the microsomes of camel tissues was independent of cytochrome P450 (CYP) activity and exhibited a pH and temperature dependence characteristic of FMO enzymes. Use of inhibitors of CYP activities, SKF525A, octylamine or antibody against NADPH-P450 reductase, did not significantly alter the FMO-dependent substrate metabolism. Using MEM as a model substrate for FMO activity, we show that camel liver has an activity similar to that in rat and human livers. MEM metabolism in extrahepatic tissues in camels was significantly lower (60%-80%) than that in liver. Our results suggest occurrence of FMO in camel tissues, with catalytic properties similar to those in rat and human livers. These results may help in better understanding the effects of pharmacologically and toxicologically active compounds administered to camels.     

A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1-FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.     

Differential metabolism of 4-n- and 4-tert-octylphenols in perfused rat liver

Octylphenols, widely used in a variety of detergents and plastics, are known to exhibit estrogenicity in vivo. The details of their metabolism are needed to better understand the endocrine disruptions. We have previously shown that alkylphenols, having short alkyl chains, are glucuronidated and readily excreted into the bile from the liver, while 4-n-nonylphenol, having longer alkyl chains, remains as the alkylphenol's glucuronide in the tissue. This study elucidated the dependence of the metabolism on the shape of the alkyl chains by comparing 4-n-octylphenol and 4-tert-octylphenols in a perfused rat liver. Both octylphenols were highly glucuronidated by the liver microsomal fractions. The Vmax value of 4-tert-octylphenol glucuronidation was twice as high as that of 4-n-octylphenol in the liver microsomes. On the other hand, the Km values, being measures of enzymatic activity against these chemicals, were similar. 4-n-Octylphenol and 4-tert-octylphenol were both glucuronidated by a UDP-glucuronosyltransferase isoform, UGT2B1, expressed in the liver. In the liver perfusion, almost all of the 4-n-octylphenol perfused was metabolized directly to the glucuronide, whereas a portion of 4-tert-octylphenol was hydroxylated and then glucuronidated. The glucuronide of 4-n-octylphenol accumulated in the liver tissue in the same manner as 4-n-nonylphenol, but 4-tert-octylphenol and the hydroxylated metabolites were excreted readily into the bile. Only a small amount of 4-n-octylphenol-glucuronide and glucuronides of 4-tert-octylphenol and its hydroxylated metabolites could be excreted into the bile of Eisai hyperbilirubinemic rats (EHBR). These animals are deficient in xenobiotic conjugate transporter, multidrug resistance-associated protein (MRP-2), indicating that the glucuronides of both octylphenols are transported by MRP-2. These results indicate that the differences in metabolism of these octylphenols are due to the shape of their alkyl chains, suggesting that the estrogenic activities of not only the parent chemicals but also these metabolites must be taken into consideration.     

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH₄) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH₄ and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure–function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C₂–C₄ alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis—this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min^–1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C₂–C₄ alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.     

The action of zymosan on octanoate transport and metabolism in the isolated perfused rat liver

The effects of zymosan on transport, distribution, and metabolism of octanoate in the perfused rat liver were investigated using the multiple‐indicator dilution technique. Livers were perfused with 300 µM octanoate in the absence or in the presence of 100 µg/mL zymosan. Tracer amounts of [1‐¹⁴C]octanoate, [³H] water, and [¹³¹I]albumin were injected into the portal vein, and the effluent perfusate was fractionated. The normalized dilution curves were analyzed by means of a space‐distributed variable transit time model. Zymosan decreased the space into which octanoate undergoes flow‐limited distribution, possibly the first cellular exchanging pool represented by plasma membranes and their adjacencies. However, the rate of transfer of octanoate from the plasma membrane into the rest of the cell was not modified as indicated by the similar values of the influx rates and also the net uptake of octanoate per unit of accessible cellular volume. However, when referred to the wet weight of the liver, the net uptake of octanoate was 37.5% reduced, a value corresponding to the diminution of the cellular accessible space. It can be concluded that an exclusion of a fraction of the liver parenchyma from the microcirculation is the main mechanism by which zymosan reduces the metabolism of exogenous octanoate. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:155–165, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20269     

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.     

Direct methylation from mouse plasma and from liver and brain homogenates

The analysis of fatty acid composition of plasma and tissue is important as a method for studying lipid nutrition. We investigated the possibility of direct methylation of fatty acids by BF(3)-methanol from plasma and from liver and brain homogenates without lipid extraction. There were no ghost peaks in the chromatogram produced by the direct methylation method. The 18:0 percentages were significantly higher in the direct methylation method than in the lipid extraction method. There were not remarkable differences in fatty acid composition in the direct methylation and methylation after lyophilization methods. Furthermore, the recovery ratio of the internal standard in the direct methylation method was higher than that in the lipid extraction method. The difference of fatty acid composition with lipid extraction may be caused by the change of lipid class extraction. Therefore, the direct methylation method without lipid extraction is the most suitable for determining fatty acid composition in plasma and tissue.     

Baeyer-Villiger单加氧酶非保守Hinge影响酶的催化活性和立体选择性

Baeyer-Villiger单加氧酶是一种重要的生物催化剂,可用于合成一系列有价值的酯和内酯化合物。通过序列比对和晶体结构分析推测连接NADPH结构域和FAD结构域的一段非保守Hinge可能在酶对底物识别和催化氧化过程中扮演着重要角色。在以环己酮单加氧酶为模型的研究中发现,对该Hinge结构进行同源序列替换得到的突变体几乎完全丧失了催化活性,证明了其整体水平的重要性。丙氨酸扫描突变揭示其中一些位点对酶的功能有显著影响:K153位点的改变使酶的活性下降,立体选择性却更优化;L143位点的改变对酶的活性影响较小,却降低了立体选择性;L144位点的改变则同时大幅度削弱酶的活性和立体选择性。将同样的方法运用在苯丙酮单加氧酶中,我们得到了相似的结论,证明这些位点的重要功能在Baeyer-Villiger单加氧酶家族中有一定的普遍性。这一研究增进了对Baeyer-Villiger单加氧酶的结构与功能关系的认识,有助于底物结合口袋的精确描述和Baeyer-Villiger单加氧酶催化图景的进一步细化,对未来相关的理性设计和定向改造研究提供了借鉴。     

Tri- and tetramethylhydroquinone as electron donors for ammonia monooxygenase in whole cells of Nitrosomonas europaea

Abstract Ten redox reagents have been tested as electron donors to ammonia monooxygenase in whole cells of Nitrosomonas europaea . Positive results were obtained with tri- and tetramethylhydroquinone. An earlier study showed that phenol was converted into hydroquinone by the monooxygenase. Cells were therefore incubated with trimethylphenol, to see if its hydroxylation to trimethylhydroquinone would lead to a self-sufficient conversion of trimethylphenol into trimethylquinone. No trimethylquinone could be detected. The maximal rates of propene epoxidation obtained with tri-and tetramethylhydroquinone were 1.8 and 4.6 μmol · h⁻¹· mg protein⁻¹, respectively.     

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.     

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [³H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.     

Role of alpha-tocopherol in cadmium-induced oxidative stress in Wistar rat's blood, liver and brain

Cadmium (Cd) a highly toxic metal is considered to be a multitarget toxicant, and it accumulates principally in the liver and kidney after absorption. In vivo studies of mouse and rat liver have shown that apoptosis plays a primary role in Cd-induced hepatotoxicity. However, the detailed mechanisms by which toxic metals such as Cd produce their effects are still largely unknown. The present study aimed at investigating the consequences of exposure to Cd, alpha-tocopherol and their combination on stress biochemical parameters (lipoperoxidation and protein carbonyls levels). Male albino Wistar rats (1 month old) were treated intravenously with cadmium (2 mg CdCl(2)/kg body weight/day), and alpha-tocopherol (100 mg/kg body weight/day), or with alpha-tocopherol+Cd (100 mg Vit E/kg body weight, 2 mg CdCl(2)/kg). The lipoperoxidation was measured by the thiobarbituric acid reactive substances (TBARS) method and oxidatively generated damage to proteins by determining carbonyl (DNPH) levels. Among the hematological parameters measured the haematocrit value and haemoglobin concentration were significantly decreased in the blood of Cd-treated rats. A significant increase was observed in the level of malondialdehyde (MDA) and protein carbonyls in the cadmium exposed group compared to control group (p<0.001), and these values were decreased after administration of alpha-tocopherol (group 4). The activity of lactate dehydrogenase in rat liver and brain showed a significant increase as compared to that found in the control group and significant decrease of catalase and superoxide dismutase activities. In the liver of the Cd-treated group the contents of reduced glutathione were decreased. Our results suggest that cadmium induces an oxidation of cellular lipids and proteins and that administration of alpha-tocopherol can reduce Cd-induced oxidative stress and improve the glutathione level together with other biochemical parameters.     

Distribution of peptidyl-glycine alpha-amidating monooxygenase immunoreactivity in the brain,pituitary and islet organ of the anglerfish (Lophius americanus)

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is an enzyme that catalyzes conversion of glycine-extended peptides to alpha-amidated bioactive peptides. Two peptides that are processed at their carboxyl-termini by this enzyme are neuropeptide Y and anglerfish peptide Y, both of which possess a C-terminal glycine that is used as a substrate for amidation. Results from previous reports have demonstrated that neuropeptide Y-like and anglerfish peptide Y-like immunoreactivities are present in the brain of anglerfish (Lophius americanus). Furthermore, neuropeptide Y-like peptides, namely anglerfish peptide Y and anglerfish peptide YG (the homologues of pancreatic polypeptide) are present in the islet organ of this species. Neuropeptide Y has also been localized in the anterior, intermediated and posterior lobes of the pituitary gland in a variety of species. In order to learn more about the distribution of the enzyme responsible for alpha amidation of these peptides in the brain and pituitary and to specifically investigate the relationship of this enzyme to peptide synthesizing endocrine cells of the anglerfish islet, we performed an immunohistochemical study using several antisera generated against different peptide sequences of the enzyme. PAM antisera labeled cells in the islet organ, pituitary and brain, and fibers in the brain and pituitary gland. The PAM staining pattern in the brain was remarkably similar to the distribution of neuropeptide Y immunoreactivity reported previously. Clusters of cells adjacent to vessels in the anterior pituitary displayed punctate PAM immunoreactivity while varicose fibers were observed in the pituitary stalk and neurohypophysis. Endocrine cells of the islet organ were differentially labeled with different PAM antisera. Comparison of the staining patterns of insulin, glucagon, and anglerfish peptide Y in the islet organ to PAM immunoreactivity suggests a distribution of forms of PAM enzyme in insulin and anglerfish peptide Y-containing cells, but no overlap with glucagon-producing cells. The results also indicate that PAM immunoreactivity is widely distributed in the brain, pituitary and islet organ of anglerfish in cells that contain peptides that require presence of a C-terminal glycine for amidation.