The effect of the osmolyte trimethylamine N-oxide on the stability of the prion protein at low pH

A study of the effect of trimethylamine N-oxide on the stability of two recombinant forms of the prion protein PrP, an ovine full-length and a human truncated form, is here reported. Both thermal denaturation and denaturation at room temperature were analyzed at pH values above and below the pKa of trimethylamine N-oxide, which is close to 4.7. Surprisingly, results showed that not only is trimethylamine N-oxide able to decrease PrP thermal stability at low pH but it also acts as a strong denaturant at room temperature. Likely, this destabilization is due to the capability of the cationic form of trimethylamine N-oxide to interact with the protein backbone as well as to weaken electrostatic interactions which are important for PrP fold. These results constitute the first experimental measurement of the effect of trimethylamine N-oxide on PrP stability and provide an unambiguous proof of the destabilizing effect of this osmolyte on PrP at low pH.     

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.     

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.     

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.     

Trimethylamine metabolism in obligate and facultative methylotrophs

1. Twelve bacterial isolates that grow with trimethylamine as sole source of carbon and energy were obtained in pure culture. All the isolates grow on methylamine, dimethylamine and trimethylamine. One isolate, bacterium 4B6, grows only on these methylamines whereas another isolate, bacterium C2A1, also grows on methanol but neither grows on methane; these two organisms are obligate methylotrophs. The other ten isolates grow on a variety of C(i) and other organic compounds and are therefore facultative methylotrophs. 2. Washed suspensions of the obligate methylotrophs bacteria 4B6 and C2A1, and of the facultative methylotrophs bacterium 5B1 and Pseudomonas 3A2, all grown on trimethylamine, oxidize trimethylamine, dimethylamine, formaldehyde and formate; only bacterium 5B1 and Ps. 3A2 oxidize trimethylamine N-oxide; only bacterium 4B6 does not oxidize methylamine. 3. Cell-free extracts of trimethylamine-grown bacteria 4B6 and C2A1 contain a trimethylamine dehydrogenase that requires phenazine methosulphate as primary hydrogen acceptor, and evidence is presented that this enzyme is important for the growth of bacterium 4B6 on trimethylamine. 4. Cell-free extracts of eight facultative methylotrophs, including bacterium 5B1 and Ps. 3A2, do not contain trimethylamine dehydrogenase but contain instead a trimethylamine monooxygenase and trimethylamine N-oxide demethylase. It is concluded that two different pathways for the oxidation of trimethylamine occur amongst the isolates.     

Functional protection of human haemoglobin against protein dissociation

The spectroscopic and functional properties of human adult haemoglobin are clearly disrupted by concentrations of urea > 0.4 M. This disruption of structure and function is completely obviated by the presence of 0.2 M trimethylamine N-oxide (TMAO). Spectroscopic data suggest that TMAO prevents urea-induced production of high-spin haem. Functional analysis shows that TMAO exerts its influence by counteracting urea-induced destabilisation of the T state of the haemoglobin protein. Further studies show, however, that TMAO is not able to exert any such stabilising influences in the presence of high concentrations of typical organic solvent denaturants.     

Anaerobic growth of Escherichia coli on formate by reduction of nitrate, fumarate, and trimethylamine N-oxide.

Anaerobic growth of E. coli, strain K-10, depending on formate oxidation by nitrate, fumarate, and trimethylamine N-oxide was followed in a medium containing peptone. The presence of formate and peptone was indispensable for growth with fumarate and trimethylamine N-oxide reduction. While there was no growth in the absence of acceptor, growth was observed in the absence of formate by nitrate reduction though not as much as under aerobic conditions. Per mole consumed formate equimolar succinate or trimethylamine was formed, but 1.2 mole of nitrate was produced, probably depending partly on peptone oxidation. The molar growth yield on formate was found to be 6.5, 7.6, and 7.0 g cells/mole depending on the reduction of nitrate, fumarate, and trimethylamine N-oxide, respectively, suggesting the formation of one mole ATP coupled to the anaerobic electron transfers from formate.     

Effects of organic solvents, methylamines, and urea on the affinity for Pi of the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. In a previous report (de Meis, L., Alves, E., and Martins, O.B. (1980) Biochemistry 19, 4252-4261), it was shown that organic solvent such as dimethyl sulfoxide and glycerol cause a decrease in the apparent Km for Pi. In this report it is shown that a similar effect is obtained with the methylamines glycine betaine and trimethylamine N-oxide. The apparent Km value for Pi in totally aqueous medium and in the presence of either 6.4 M glycerol, 1.4 M dimethyl sulfoxide, 0.4 M trimethylamine N-oxide, or 1 M glycine betaine were found to be respectively 2.85, 0.52, 0.52, 0.81, and 0.93 mM at pH 6.2 and greater than 10.0, 1.08, 2.53, 3.05, and 2.05 mM at pH 7.5. In contrast to the effect of methylamines, urea caused an increase in the apparent Km for Pi. When mixed in the appropriate concentration ratio, the effect of either organic solvent or methylamines is cancelled by urea.     

The periplasmic TorT protein is required for trimethylamine N-oxide reductase gene induction in Escherichia coli.

Expression of the Escherichia coli torCAD operon, which encodes the trimethylamine N-oxide reductase system, is regulated by the presence of trimethylamine N-oxide through the action of the TorR response regulator. We have identified an additional gene, torT, located just downstream from the torR gene, which is necessary for torCAD structural operon expression. Insertion within the torT gene dramatically reduced the expression of a torA'-'lacZ fusion, while presence of the gene in trans restored the wild-type phenotype. Overproduction of TorR in a torT strain resulted in partial constitutive expression of the torA'-'lacZ fusion, suggesting that TorR acts downstream from TorT. The torT gene codes for a 35.7-kDa periplasmic protein which presents some homology with the periplasmic ribose-binding protein of E. coli. We discuss the possible role of TorT as an inducer-binding protein involved in signal transduction of the tor regulatory pathway.     

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.     

A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.     

Inductions of superoxide dismutases in Escherichia coli under anaerobic conditions. Accumulation of an inactive form of the manganese enzyme

Escherichia coli growing anaerobically respond to NO3- with a 3-fold induction of the iron-containing superoxide dismutase. Mutants lacking nitrate reductase do not show this response. Anaerobically grown cells also contain an inactive form of the manganese-containing superoxide dismutase (MnSOD) which can be activated by addition of Mn(II) salts in the presence of acidic guanidinium chloride, followed by dialysis against neutral buffer. Direct addition of Mn(II) to a neutral solution of the inactive MnSOD does not impart activity. This inactive MnSOD thus behaves as would the apoenzyme or the enzyme bearing a metal other than Mn(II) at its active sites. Terminal electron acceptors, such as NO3- or trimethylamine N-oxide, increase the amount of inactive MnSOD produced by anaerobic E. coli. Paraquat, which is itself ineffective in this regard, markedly augments the effect of these terminal electron acceptors. It appears that flow of electrons to sinks such as NO3- or trimethylamine N-oxide, facilitated by paraquat, is sufficient to elicit biosynthesis of the MnSOD protein and that O2- is not needed for this process. Yet, oxygenation and concomitant O2- production do appear important for the insertion of manganese into the growing MnSOD polypeptide, possibly because O-2 oxidizes Mn(II) to Mn(III), and the latter is the valence state most effective in combining with the apoenzyme.     

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.     

The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N-oxide, against chemical denaturant was investigated by NMR spin-relaxation measurements and model-free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond-nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N-oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by (1)H-(15)N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.     

Functional analysis of the twin-arginine translocation pathway in Sodalis glossinidius, a bacterial symbiont of the tsetse fly

This study demonstrates a functional twin-arginine (Tat) translocation pathway present in the tsetse fly symbiont Sodalis glossinidius and its potential to export active heterologous proteins to the periplasm. Functionality was demonstrated using green fluorescent protein (GFP) fused to the Tat signal peptide of Escherichia coli trimethylamine N-oxide reductase (TorA).     

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.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

Formation and breakdown of glycine betaine and trimethylamine in hypersaline environments

Glycine betaine is accumulated as a compatible solute in many photosynthetic and non-photosynthetic bacteria — the last being unable to synthesize the compound - and thus large pools of betaine can be expected to be present in hypersaline environments. A variety of aerobic and anaerobic microorganisms degrade betaine to among other products trimethylamine and methylamine, in a number of different pathways. Curiously, very few of these betaine breakdown processes have yet been identified in hypersaline environments. Trimethylamine can also be formed by bacterial reduction of trimethylamine N-oxide (also by extremely halophilic archaeobacteria). Degradation of trimethylamine in hypersaline environments by halophilic methanogenic bacteria is relatively well documented, and leads to the formation of methane, carbon dioxide and ammonia.