Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl₃. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K_m for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.     

A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways

The aerobic and anaerobic degradation of trimethylamine by a newly isolated denitrifying bacterium from an enrichment culture with trimethylamine inoculated with activated sludge was studied. Based on 16S rDNA analysis, this strain was identified as a Paracoccus sp. The isolate, strain T231, aerobically degraded trimethylamine, dimethylamine and methylamine and released a stoichiometric amount of ammonium ion into the culture fluid as a metabolic product, indicating that these methylated amines were completely degraded to formaldehyde and ammonia. The strain degraded trimethylamine also under denitrifying conditions and consumed a stoichiometric amount of nitrate, demonstrating that complete degradation of trimethylamine was coupled with nitrate reduction. Cell-free extract prepared from cells grown aerobically on trimethylamine exhibited activities of trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase, dimethylamine mono-oxygenase, and methylamine mono-oxygenase. Cell-free extract from cells grown anaerobically on trimethylamine and nitrate exhibited activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase. These results indicate that strain T231 had two different pathways for aerobic and anaerobic degradation of trimethylamine. This is a new feature for trimethylamine metabolism in denitrifying bacteria.     

Adenosine 3′:5′-cyclic monophosphate in young and senescent human fibroblasts during growth and stationary phase in vitro. Effects of prostaglandin E1 and of adrenaline

1. A mono-oxygenase, which oxidizes trimethylamine and other tertiary amines bearing methyl or ethyl groups, was partially purified sixfold from Pseudomonas aminovorans grown on trimethylamine as sole carbon source. 2. The preferred electron donor was NADPH. The enzyme had a pH optimum of 8.0-9.4 for trimethylamine oxidation, and 8.8-9.2 for dimethylamine oxidation. 3. The oxidation product of trimethylamine was shown to be trimethylamine N-oxide. Other tertiary amines were probably also converted into N-oxides. 4. The enzyme also oxidized secondary amines. 5. The oxidation of trimethylamine was only slightly inhibited by CO and not at all by KCN or proadifen hydrochloride (SKF 525-A), but was inhibited by trimethylsulphonium chloride, tetramethylammonium chloride, 2,4-dichloro-6-phenylphenoxyethylamine (Lilly 53325) and its NN-diethyl derivative (Lilly 18947). 6. The oxidation of dimethylamine showed a similar response to inhibitors and a parallel loss in activity on heating at 35 degrees C. 7. The activities of the trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase and the secondary-amine mono-oxygenase increased severalfold during adaptation of succinate-grown bacteria to growth on trimethylamine, and the trimethylamine mono-oxygenase was the first enzyme to show an increase in activity. It is concluded that all three enzymes are involved in growth on trimethylamine by this organism.     

Assimilatory reduction of trimethylamine N-oxide in the yeast Sporopachydermia cereana

Summary Washed microsomal preparations (100 000 xg sediment) from the yeast Sporopachydermia cereana that had been grown on trimethylamine N-oxide as sole nitrogen source catalysed the NAD(P)H-dependent reduction of trimethylamine N-oxide to trimethylamine. Under anaerobic conditions, this was the sole reaction product, but under aerobic conditions only small amounts of trimethylamine accumulated, most being further metabolized to methylamine and formaldehyde (no detectable dimenthylamine accumulated due to its rapid turnover). In the absence of NAD(P)H, no formation of amines or formaldehyde from trimethylamine N-oxide was detected. The trimethylamine N-oxide reductase activity was inhibited by quinacrine, Cu²⁺ ions, triethylamine N-oxide (apparent K _i 0.43 mM) and dimethyl sulphoxide (K _i 0.94 mM). Chlorate and nitrate failed to inhibit the enzyme. The K _m for trimethylamine N-oxide was 29 M. Triethylamine N-oxide was also reduced by the microsomal preparation with the formation of acetaldehyde, and this reduction was sensitive to the same inhibitors as trimethylamine N-oxide, suggesting that both amine oxides are metabolized by the same enzyme(s). It is concluded that trimethylamine N-oxide is metabolized in this yeast via an NAD(P)H-dependent reductase.Abbreviations TMAO triemthylamine N-oxide     

A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells

A biosensor system based on the difference in the oxygen uptake response of two microbial electrodes was developed to monitor trimethylamine (TMA). The first electrode, constructed using Pseudomonas aminovorans grown on TMA, was sensitive to TMA, trimethylamine N-oxide (TMAO), dimethylamine (DMA) and monomethylamine (MMA). The second electrode responding to TMAO, DMA and MMA was prepared using Ps. aminovorans grown on TMAO. The difference in oxygen uptake was linearly related to the TMA concentration in the range of 5-26 microM. The minimum detectable level was 2.6 microM and the relative standard deviation was determined to be 14% for 16 repeated analyses. When operated and stored at 30 degrees C, the response of the system was stable for only 2 days. However, when the biosensor system was operated at 30 degrees C but stored overnight at 4 degrees C, the system was stable up to 20 days. The biosensor system was applicable for the determination of TMA in fish tissue extracts and the results compared well with those determined by HPLC.     

Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions.

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.     

The reaction of trimethylamine dehydrogenase with trimethylamine

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.     

Enzymological aspects of the pathways for trimethylamine oxidation and C1 assimilation of obligate methylotrophs and restricted facultative methylotrophs.

Extracts of trimethylamine-grown W6A and W3A1 (type M restricted facultative methylotrophs) contain trimethylamine dehydrogenase whereas similar extracts of Bacillus PM6 and Bacillus S2A1 (type L restricted facultative methylotrophs) contain trimethylamine mono-oxygenase and trimethylamine N-oxide demethylase but no trimethylamine dehydrogenase. Extracts of the restricted facultatives and of the obligate methylotroph C2A1 contain hexulose phosphate synthase-hexulose phosphate isomerase activity; hydroxypyruvate reductase was not detected. Neither the restricted facultatives nor the obligates 4B6 and C2A1 contain all the enzymes of the hexulose phosphate cycle of formaldehyde assimilation as originally proposed by Kemp & Quayle (1967). Organisms PM6 and S2A1 lack transaldolase and use a modified cycle involving sedoheptulose 1,7-diphosphate and sedoheptulose diphosphatase. The obligates 4B6 and C2A1, and the type M organisms W6A and W3A1, use a different modification of the assimilatory hexulose phosphate cycle involving the Entner-Doudoroff-pathway enzymes phosphogluconate dehydratase and phospho-2-keto-3-deoxygluconate aldolase. The lack of fructose diphosphate aldolase and hexose diphosphatase in these organisms may be a partial explanation of their restricted growth-substrate range. Enzymological evidence suggests that all the obligates and the restricted facultatives use a dissimilatory hexulose phosphate cycle to accomplish the complete oxidation of formaldehyde to CO2 and water.     

Adaptation of Pseudomonas sp. strain 7-6 to quaternary ammonium compounds and their degradation via dual pathways

Pseudomonas sp. strain 7-6, isolated from active sludge obtained from a wastewater facility, utilized a quaternary ammonium surfactant, n-dodecyltrimethylammonium chloride (DTAC), as its sole carbon, nitrogen, and energy source. When initially grown in the presence of 10 mM DTAC medium, the isolate was unable to degrade DTAC. The strain was cultivated in gradually increasing concentrations of the surfactant until continuous exposure led to high tolerance and biodegradation of the compound. Based on the identification of five metabolites by gas chromatography-mass spectrometry analysis, two possible pathways for DTAC metabolism were proposed. In pathway 1, DTAC is converted to lauric acid via n-dodecanal with the release of trimethylamine; in pathway 2, DTAC is converted to lauric acid via n-dodecyldimethylamine and then n-dodecanal with the release of dimethylamine. Among the identified metabolites, the strain precultivated on DTAC medium could utilize n-dodecanal and lauric acid as sole carbon sources and trimethylamine and dimethylamine as sole nitrogen sources, but it could not efficiently utilize n-dodecyldimethylamine. These results indicated pathway 1 is the main pathway for the degradation of DTAC.     

Mechanism of biosynthesis of trimethylamine oxide from choline in the teleost tilapia, Oreochromis niloticus, under freshwater conditions

The mechanism of biosynthesis of trimethylamine oxide (TMAO) from dietary precursors in the teleost tilapia (Oreochromis niloticus) was investigated. Diets supplemented with quaternary ammonium choline, glycine betaine, carnitine or phosphatidylcholine were administered and significant increases in TMAO levels in the muscle were only observed with choline. [Methyl-14C] and [1,2-14C] cholines were given through dietary and intraperitoneal injection routes, but 14C-TMAO was detected only in fish with dietary administration of [methyl-14C] choline. Dietary treatment with [15N] choline resulted in the formation of [15N] TMAO in the muscle. The incorporation of radioactivity into TMAO was also observed both following dietary administration and intraperitoneal injection of [14C] trimethylamine (TMA). When choline was introduced into the isolated intestine, marked increases in TMA levels occurred. These increases were significantly suppressed in the presence of penicillin. [14C]-TMA derived from [methyl-14C] choline was detected in the cavity of the isolated intestine. The introduction of [15N] choline into the intestinal cavity resulted in the formation of [15N] TMA. TMA mono-oxygenase activities were detected in the liver and kidney. We conclude that tilapia possess the ability to produce TMAO from choline, which is related to intestinal microorganisms and tissue mono-oxygenase under freshwater conditions.     

The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry

We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added. p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(III) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 mumol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Oxidation of dimethylamine and trimethylamine in methazotrophic yeasts by microsomal mono-oxygenases sensitive to carbon monoxide

Whole cells of Candida boidinii grown on di- or tri-methylamine as sole nitrogen source readily oxidized both amines. The oxidation was potently inhibited by carbon monoxide. Cell-free extracts required the presence of 20 μM FAD before mono-oxygenase activity with both amines could be demonstrated. NADH was a better electron donor than NADPH. Activity was present in cells grown on secondary and tertiary amines but not on primary amines, and was detected in a number of different yeasts. Enzyme activity could be sedimented at 187 000 x g, and was associated with NADPH-cytochrome c reductase activity. It is thus probably microsomal. Activity was inhibited by cyanide, mercaptoethanol, carbon monoxide and proadifen hydrochloride (SKF 525-A).     

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon-and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.Abbreviations HPS hexulose-6-phosphate synthase - RuMP ribulose monophosphate - DMA dimethylamine - TMA trimethylamine - TMA-NO trimethylamine-N-oxide - ICL isocitrate lyase - GS glutamine synthetase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOGAT glutamate synthase     

Fishy taint in chicken eggs is associated with a substitution within a conserved motif of the FMO3 gene

Fishy odor of urine and other secretions is a characteristic of trimethylaminuria in humans, resulting from loss-of-function mutations in the flavin-containing mono-oxygenase isoform FMO3. A similar phenotype exists in cattle, in which a nonsense mutation in the bovine orthologue causes fishy off-flavor in cow's milk. The fishy odor is caused by an elevated level of excreted odorous trimethylamine (TMA), due to deficient oxidation of TMA. We report the mapping of a similar disorder (fishy taint of eggs) and the chicken FMO3 gene to chicken chromosome 8. The only nonsynonymous mutation identified in the chicken FMO3 gene (T329S) changes an evolutionarily highly conserved amino acid and is associated with elevated levels of TMA and fishy taint in the egg yolk in several chicken lines. No differences in the expression of FMO3 were found among individuals with different associated genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The results support the importance and function of the evolutionarily conserved motif FATGY, which has been speculated to be a substrate recognition pocket of N-hydroxylating siderophore enzymes and flavin-containing mono-oxygenases.     

Adaptation of Pseudomonas sp. Strain 7-6 to Quaternary Ammonium Compounds and Their Degradation via Dual Pathways

Pseudomonas sp. strain 7-6, isolated from active sludge obtained from a wastewater facility, utilized a quaternary ammonium surfactant, n-dodecyltrimethylammonium chloride (DTAC), as its sole carbon, nitrogen, and energy source. When initially grown in the presence of 10 mM DTAC medium, the isolate was unable to degrade DTAC. The strain was cultivated in gradually increasing concentrations of the surfactant until continuous exposure led to high tolerance and biodegradation of the compound. Based on the identification of five metabolites by gas chromatography-mass spectrometry analysis, two possible pathways for DTAC metabolism were proposed. In pathway 1, DTAC is converted to lauric acid via n-dodecanal with the release of trimethylamine; in pathway 2, DTAC is converted to lauric acid via n-dodecyldimethylamine and then n-dodecanal with the release of dimethylamine. Among the identified metabolites, the strain precultivated on DTAC medium could utilize n-dodecanal and lauric acid as sole carbon sources and trimethylamine and dimethylamine as sole nitrogen sources, but it could not efficiently utilize n-dodecyldimethylamine. These results indicated pathway 1 is the main pathway for the degradation of DTAC.     

Anaerobic growth of halophilic archaeobacteria by reduction of dimethysulfoxide and trimethylamine N-oxide

Abstract Most representatives of the halophilic arachaeobacterial genera Halobacterium, Haloarcula and Haloferax tested were able to reduce dimethylsulfoxide (DMSO) to dimethylsulfide (DMS) and trimethylamine N -oxide (TMAO) to trimethylamine (TMA) under (semi)anaerobic conditions. In most cases the reduction of DMSO and TMAO was accompanied by an increase in cell yield. The ability to reduce DMSO or TMAO was not correlated to reduced DMSO or TMAO was not correlated with the ability to reduce nitrate to nitrite. Anaerobic respiration with DMSO and TMAO as electron acceptor supplies the halophilic archeobacteria with an additional mode of energy generation in the absence of molecular oxygen.     

Arabinan degrading enzymes from Aspergillus nidulans: Induction and purification

Abstract The expression and distribution of ferric reductase activity was examined in Shewanella putrefaciens MR-1. Formate-dependent ferric reductase was not detected in aerobically grown cells but was readily detectable in anaerobically grown cells. Ferric reductase activity was found exclusively in the membrane fractions, with 54–56% in the outer membrane. In contrast, the majority of formate dehydrogenase was in the soluble fraction with lesser amounts associated with the various membrane fractions. Outer membrane ferric reductase activity was markedly inhibited by p -chloromercuriphenylsulfonate, 2-heptyl-4-hydroxyquinolone- N -oxide, and antimycin A, but was unaffected by the presence of alternate electron acceptors (nitrate, nitrite, fumarate, and trimethylamine N -oxide). Both formate and NADH served as electron donors for ferric reductase; activity with l -lactate or NADPH was poor. The addition of FMN markedly stimulated formate- and NADH-dependent ferric reductase.     

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.     

Enzymatic characterization of the polynuclear aromatic hydrocarbons activating rat-liver preparations used in the mutagenicity test of Ames

Stability studies were performed on the mono-oxygenase system involved, in particular, in the activation of polynuclear aromatic hydrocarbons (PAHs) present in rat-liver preparations used in the Ames mutagenicity test. The results indicated a good stability of the spectral response of the cytochrome-P-450 system, but a much lower stability of its enzymatic activities measured with various substrates, thus showing the inadequacy of the spectral response to characterize the PAH mono-oxygenase activity of the preparations. Epoxide hydrolase activity was found to be stable. Various mono-oxygenase activities were measured in preparations induced with phenobarbital, 3-methylcholanthrene or Aroclor 1254. The activities of two enzymes, benzo[a]pyrene hydroxylase and ethoxyresorufin-O-dealkylase, were found suitable to characterize the capacity of the preparations to metabolize PAH to mutagens. The efficiency of the same preparations to promote the mutagenicity of benzo[a]pyrene and aflatoxin B1 in the Ames test was determined. There was an excellent general correlation between the efficiencies for mutagenic activation of the preparations and the two enzymatic activities mentioned above. Determination of ethoxyresorufin-O-dealkylase (or benzo[a]pyrene hydroxylase) and benzo[a]pyrene 4,5-oxide hydrolase activities is proposed for characterizing the rat-liver preparations used in the Ames test.