Inhibition by trimethylamine of methylamine oxidation by Paracoccus denitrificans and bacterium W3A1

Trimethylamine, a common substrate for methylotrophic growth, specifically inhibited methylamine-dependent respiration by Paracoccus denitrificans and bacterium W3A1. These effects were caused by the specific inhibition by trimethylamine of the periplasmic quinoprotein methylamine dehydrogenase. Steady-state kinetic analysis of the effect of trimethylamine on methylamine oxidation by methylamine dehydrogenase indicated that the inhibition was a mixed type. Apparent Ki values for trimethylamine of 1.1 mM and 4.7 mM, respectively, were obtained for the P. denitrificans and bacterium W3A1 enzymes. Methylamine-dependent oxygen consumption by each bacterium was inhibited either by preincubation of cells with trimethylamine prior to the addition of substrate or by addition of trimethylamine to actively respiring cells. Formate-dependent respiration was not inhibited by trimethylamine. A scheme is proposed which describes a regulatory role for trimethylamine in the metabolism and dissimilation of methylamine by methylotrophic bacteria.     

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.     

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.     

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.     

Trimethylamine oxidation in liver tissue is not catalyzed by a molybdenum cofactor-dependent enzyme

The oxidation of trimethylamine to trimethylamine N-oxide in animals is catalyzed by an enzyme which has not yet been fully characterized. The discovery that a bacterial enzyme catalyzing the reverse reaction, the reduction of trimethylamine N-oxide to trimethylamine, utilizes the molybdenum cofactor to carry out this function raised the possibility that trimethylamine oxidation may also be dependent on this cofactor. It was found, however, that liver tissue from tungsten-treated rats contained normal levels of trimethylamine oxidase. In addition, analysis of a urine sample from a patient with trimethylamine oxidase deficiency revealed the presence of normal levels of urothione, the degradation product of the molybdenum cofactor. These results suggest that trimethylamine oxidase is not a molybdoenzyme and that oxidation of trimethylamine proceeds by a mechanism which differs considerably from a simple reversal of trimethylamine N-oxide reduction.     

Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine

Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water. All strains were in alpha-Proteobacteria. Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria. Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium. Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase. The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.     

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.     

Enzymes metabolizing dimethylamine, trimethylamine and trimethylamine N-oxide in the yeast Sporopachydermia cereana grown on amines as sole nitrogen source

Abstract Sporopachydermia cereana , an ascosporogenous yeast, grew on dimethylamine, trimethylamine or trimethylamine N -oxide as sole nitrogen sources and produced mono-oxygenases for dimethylamine and trimethylamine that were significantly more stable than the corresponding enzymes found in Candida utilis . No trimethylamine mono-oxygenase activity was found in S. cereana grown on dimethylamine. In cells grown on trimethylamine N -oxide (but not on the other nitrogen sources), evidence for an enzyme metabolizing the N -oxide, possibly an aldolase, but more probably a reductase was obtained. All these activities showed a similar requirement for the presence of FAD or FMN in the extract buffer during isolation to retain activity. Amine mono-oxygenase activities showed a similar sensitivity to inhibitors, including proadifen hydrochloride and carbon monoxide as the corresponding enzymes in C. utilis . The trimethylamine N -oxide-dependent oxidation of NADH was more sensitive to inhibition by EDTA, N -ethylmaleimide and β-phenylethylamine than the mono-oxygenases, and less sensitive to KCN, and activity was significantly higher with NADPH than was observed with the 2 mono-oxygenases.     

Conversion of choline methyl groups through trimethylamine into methane in the rumen.

1. Choline methyl groups were rapidly metabolized to trimethylamine by rumen micro-organisms. 2. Trimethylamine was further metabolized to methane, but this system was more easily saturated by an excess of substrate, so that trimethylamine accumulated in the rumen of the fed animal. 3. Although trimethylamine was the only intermediate isolated in the conversion of the methyl groups of choline into methane, methylamine also served as a substrate for methane production. 4. The methyl group of methionine was also converted into methane by rumen fluid, but the methyl groups of carnitine were not.     

The fish odour syndrome: biochemical,familial, and clinical aspects.

OBJECTIVES--To study the biochemical, familial, and clinical features of the fish odour syndrome among subjects with suspected body malodour. DESIGN--Subjects who responded to a newspaper article were screened for the fish odour syndrome by interview and biochemical tests. Families of subjects with the syndrome were tested if possible. SETTING--St Mary''s Hospital, London, and some interviews at subjects'' homes. SUBJECTS--187 subjects (28 males) with suspected body malodour, of whom 156 (19 males) underwent biochemical tests. Five families of six of the subjects with the fish odour syndrome agreed to further tests. MAIN OUTCOME MEASURES--Amounts of trimethylamine and trimethylamine N-oxide in urine collected over 24 hours under normal dietary conditions and for eight hours after oral challenge with 600 mg trimethylamine. RESULTS--The fish odour syndrome was diagnosed in 11 subjects: the percentage of total trimethylamine excreted in their urine samples that was oxidised to trimethylamine N-oxide was < 55% under normal dietary conditions and < 25% after oral challenge with trimethylamine (in normal subjects > 80% of trimethylamine was N-oxidised). Parents of six of the subjects with the syndrome were tested: all showed impaired N-oxidation of excreted trimethylamine (< 80%) after oral challenge, indicating that they were heterozygous carriers of the allele for the syndrome. The syndrome was associated with various psychosocial reactions including clinical depression. CONCLUSIONS--The fish odour syndrome can be inherited in an autosomal recessive fashion. It should be considered as a possible causative factor in patients complaining of body malodour.     

Selective extraction of β-blockers from biological fluids by column-switching high-performance liquid chromatography using an internal-surface phenylboronic acid precolumn

A column-switching HPLC method using an internal-surface phenylboronic acid precolumn for the selective extraction of β-blockers from biological fluids has been developed. Filtered urine and plasma samples (50 μl) were injected onto the precolumn equilibrated with methanol-0.05 M disodium hydrogenphosphate (5:95, v/v). After the precolumn had been washed breifly, the selectively retained β-blockers were eluted with methol-0.05 M phosphate buffer (pH 2.0) and transferred to a reversed-phase analytical column, on which they were then separated. Even after exposure to at least 160 injections of non-treated urine and plasma samples, the retention efficiency of the precolumn was maintained with no increase in back pressure. Quantitative recoveries and good reproducibility were demonstrated with pindolol.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735.

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Efficacy of 1,4-diaminobutane (putrescine) in a food-based synthetic attractant for capture of Mediterranean and Mexican fruit flies (Diptera: Tephritidae)

Field trials were conducted in Guatemala to evaluate the importance of 1,4 diaminobutane (putrescine) in traps baited with ammonium acetate, trimethylamine, and putrescine. For the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), there were no differences in percentage of females captured in coffee and citrus or in percentage of males captured in citrus in traps with ammonium acetate and trimethylamine lures (females in coffee, 26.4 +/- 6.27%; females in citrus, 35.7 +/- 5.35%; males in citrus, 37.7 +/- 7.48%) versus ammonium acetate, trimethylamine, and putrescine lures (females in coffee, 36.6 +/- 9.64%; females in citrus, 41.1 +/- 5.18%; males in citrus, 37.1 +/- 6.09%). Percentage of males captured in coffee was reduced significantly when putrescine was not used with the ammonium acetate and trimethylamine (39.9 +/- 4.34 versus 31.6 +/- 5.29%). Lower percentages were captured in traps baited with ammonium acetate and putrescine, and the lowest percentages were captured in traps baited with putrescine and trimethylamine. When population level as indicated by capture in traps baited with ammonium acetate, trimethylamine, and putrescine was considered, a higher percentage of C. capitata males were captured in traps baited with all three components when one or more flies per trap per day were captured in coffee, and a higher percentage of females were captured when less than one fly per trap per day was captured in citrus. Percentage of the Mexican fruit fly, Anastrepha ludens (Loew), captured was significantly higher in traps baited with ammonium acetate and putrescine and significantly lower in traps baited putrescine and trimethylamine than in all other treatments. Results indicate that putrescine may be deleted when monitoring established populations of C. capitata but should be used in traps used to monitor A. ludens or to detect new infestations of C. capitata.     

Regulation by carbon source of enzyme expression and slime production in bacterium W3A1.

Slime production by bacterium W3A1 was greatly enhanced during growth on methanol and, to a lesser extent, during growth on trimethylamine. Of the major dehydrogenases synthesized, trimethylamine and methylamine dehydrogenases were induced to different levels by certain carbon sources, while methanol dehydrogenase was expressed during growth on all carbon sources.     

Detection of oestrous-related odour in bovine ( Bos taurus) saliva: bioassay of identified compounds

The present study was designed to identify the volatile constituents across the oestrous cycle of bovine in order to detect oestrous-specific chemical signal. The bovine saliva was extracted with diethyl ether (1 : 1 ratio, v/v) and analysed by gas chromatography-linked mass spectrometry. Numerous compounds were identified during oestrous cycle of bovine saliva. Among these, the compounds, namely, trimethylamine, acetic acid, phenol 4-propyl, pentanoic acid and propionic acid were specific to oestrous stage. The behaviour assay revealed that the compound, trimethylamine, is involved in attracting the male animal. The result concludes that the trimethylamine is considered as a putative oestrous-specific salivary chemo-signal in the bovine.     

Oxidation of trimethylamine to the level of formaldehyde by Methanosarcina barkeri is dependent on the protonmotive force

The conversion of trimethylamine to methane, carbon dioxide and ammonia as catalyzed by cell suspensions of Methanosarcina barkeri was coupled to the generation of a protonmotive force and to the synthesis of ATP. Methanogenesis as well as ATP formation and protonmotive force generation was abolished by the uncoupler tetrachloro-salicylanilide (TCS). Inhibition of methane formation was reversed by addition of formaldehyde, which was predominantly oxidized to carbon dioxide, whereas trimethylamine was predominantly reduced to methane and ammonia under these conditions. Cell extracts of M. barkeri were unable to convert trimethylamine to methane, carbon dioxide and ammonia independent from the presence or absence of ATP.     

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).     

Electron transfer flavoprotein from Methylophilus methylotrophus: properties, comparison with other electron transfer flavoproteins, and regulation of expression by carbon source.

When grown on methylated amines as a carbon source, Methylophilus methylotrophus synthesizes an electron transfer flavoprotein (ETF) which is the natural electron acceptor of trimethylamine dehydrogenase. It is composed of two dissimilar subunits of 38,000 and 42,000 daltons and 1 mol of flavin adenine dinucleotide. It was reduced by trimethylamine dehydrogenase to a stable anionic semiquinone form, which could not be converted, either enzymatically or chemically, to the fully reduced dihydroquinone. This ETF exhibited spectral properties which were nearly identical to ETFs from bacterium W3A1, Paracoccus denitrificans, and pig liver mitochondria. M. methylotrophus ETF cross-reacted immunologically and enzymatically with the ETF of bacterium W3A1 but not with the other two ETFs. In M. methylotrophus and bacterium W3A1, ETF and trimethylamine dehydrogenase were each expressed during growth on trimethylamine and were each absent during growth on methanol.