Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide.

Investigations of protein-solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid-dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 M). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid-inactivated porcine heart lactate dehydrogenase reflects a non-native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non-native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid-dissociated porcine heart lactate dehydrogenase as well as other viscosity-dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.     

Preventing misfolding of the prion protein by trimethylamine N-oxide

Transmissible spongiform encephalopathies are a class of fatal neurodegenerative diseases linked to the prion protein. The prion protein normally exists in a soluble, globular state (PrP(C)) that appears to participate in copper metabolism in the central nervous system and/or signal transduction. Infection or disease occurs when an alternatively folded form of the prion protein (PrP(Sc)) converts soluble and predominantly alpha-helical PrP(C) into aggregates rich in beta-structure. The structurally disordered N-terminus adopts beta-structure upon conversion to PrP(Sc) at low pH. Chemical chaperones, such as trimethylamine N-oxide (TMAO), can prevent formation of PrP(Sc) in scrapie-infected mouse neuroblastoma cells [Tatzelt, J., et al. (1996) EMBO J. 15, 6363-6373]. To explore the mechanism of TMAO protection of PrP(C) at the atomic level, molecular dynamics simulations were performed under conditions normally leading to conversion (low pH) with and without 1 M TMAO. In PrP(C) simulations at low pH, the helix content drops and the N-terminus is brought into the small native beta-sheet, yielding a PrP(Sc)-like state. Addition of 1 M TMAO leads to a decreased radius of gyration, a greater number of protein-protein hydrogen bonds, and a greater number of tertiary contacts due to the N-terminus forming an Omega-loop and packing against the structured core of the protein, not due to an increase in the level of extended structure as with the PrP(C) to PrP(Sc) simulation. In simulations beginning with the "PrP(Sc)-like" structure (derived from PrP(C) simulated at low pH in pure water) in 1 M TMAO, similar structural reorganization at the N-terminus occurred, disrupting the extended sheet. The mechanism of protection by TMAO appears to be exclusionary in nature, consistent with previous theoretical and experimental studies. The TMAO-induced N-terminal conformational change prevents residues that are important in the conversion of PrP(C) to PrP(Sc) from assuming extended sheet structure at low pH.     

Measurement of trimethylamine and trimethylamine N-oxide independently in urine by fast atom bombardment mass spectrometry

We report a method based upon fast atom bombardment mass spectrometry (FAB-MS) and stable isotope dilution techniques for the measurement of urinary trimethylamine (TMA) and trimethylamine N-oxide (TMAOx). TMA is extracted from urine that was spiked with (15)N-labeled TMA. The extracted TMA isotopomers are quaternized with trideuteromethyl iodide and analyzed in FAB-MS with hexaethylene glycol as matrix. TMAOx is measured by evaporation of another sample of the urine spiked with (15)N-labeled TMAOx on the FAB probe and analyzed as for the TMA. The method allows the ready and simple distinguishing of controls and patients with TMAuria, and is useful in monitoring patients with the disorder. We give examples of its use in determining normal control ranges for these metabolites and in evaluating patients.     

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.     

Reduction of trimethylamine N-oxide by Escherichia coli as anaerobic respiration.

E. coli was found to grow anaerobically on lactate in the presence of trimethylamine N-oxide (TMANO), reducing it to trimethylamine. Anaerobic growth on glucose was promoted in the presence of TMANO. When a culture grown in complex medium was transferred to defined medium, growth on glucose and ammonia took place in the presence of TMANO after consumption of complex nutrients introduced with the preculture, in contrast to growth in nitrate respiration. The amounts of ethanol, succinate, and lactate among the fermentation products were decreased and that of acetate was increased in the presence of TMANO. Formate generation was much reduced at pH 7.4, whereas stoichiometric formation of formate was observed in the absence of TMANO. Cells grown anaerobically in the presence of TMANO had a higher activity of amine N-oxide reductase than cells grown under other conditions. The content of cytochrome-558 was elevated in the presence of TMANO during growth in complex medium. Cytochrome c-552 found in cells grown in diluted complex medium or defined medium in the presence of TMANO was oxidized by TMANO in cell extracts. The molar growth yield on glucose was higher in the presence of TMANO than in its absence and lower than that in the presence of nitrate.     

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.     

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.     

Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte,trimethylamine N-oxide

The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations. When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days. When it was incubated with 2 M NaCl or 3 M KCl, however, it gradually regained activity. To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M. The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl. TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK. Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.     

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-N-oxide counteracts urea effects on rabbit muscle lactate dehydrogenase function: a test of the counteraction hypothesis.

Trimethylamine-N-oxide (TMAO) in the cells of sharks and rays is believed to counteract the deleterious effects of the high intracellular concentrations of urea in these animals. It has been hypothesized that TMAO has the generic ability to counteract the effects of urea on protein structure and function, regardless of whether that protein actually evolved in the presence of these two solutes. Rabbit muscle lactate dehydrogenase (LDH) did not evolve in the presence of either solute, and it is used here to test the validity of the counteraction hypothesis. With pyruvate as substrate, results show that its Km and the combined Km of pyruvate and NADH are increased by urea, decreased by TMAO, and in 1:1 and 2:1 mixtures of urea:TMAO the Km values are essentially equivalent to the Km values obtained in the absence of the two solutes. In contrast, values of k(cat) and the Km for NADH as a substrate are unperturbed by urea, TMAO, or urea:TMAO mixtures. All of these effects are consistent with TMAO counteraction of the effects of urea on LDH kinetic parameters, supporting the premise that counteraction is a property of the solvent system and is independent of the evolutionary history of the protein.     

Use of dietary phytochemicals for inhibition of trimethylamine N-oxide formation

Trimethylamine-N-oxide (TMAO) has been reported as a risk factor for atherosclerosis development, as well as for other cardiovascular disease (CVD) pathologies. The objective of this review is to provide a useful summary on the use of phytochemicals as TMAO-reducing agents. This review discusses the main mechanisms by which TMAO promotes CVD, including the modulation of lipid and bile acid metabolism, and the promotion of endothelial dysfunction and oxidative stress. Current knowledge on the available strategies to reduce TMAO formation are discussed, highlighting the effect and potential of phytochemicals. Overall, phytochemicals (i.e., phenolic compounds or glucosinolates) reduce TMAO formation by modulating gut microbiota composition and/or function, inhibiting host's capacity to metabolize TMA to TMAO, or a combination of both. Perspectives for design of future studies involving phytochemicals as TMAO-reducing agents are discussed. Overall, the information provided by this review outlines the current state of the art of the role of phytochemicals as TMAO reducing agents, providing valuable insight to further advance in this field of study.     

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     

Suppression of intestinal microbiota-dependent production of pro-atherogenic trimethylamine N-oxide by shifting L-carnitine microbial degradation

Aims

Trimethylamine-N-oxide (TMAO) is produced in host liver from trimethylamine (TMA). TMAO and TMA share common dietary quaternary amine precursors, carnitine and choline, which are metabolized by the intestinal microbiota. TMAO recently has been linked to the pathogenesis of atherosclerosis and severity of cardiovascular diseases. We examined the effects of anti-atherosclerotic compound meldonium, an aza-analogue of carnitine bioprecursor gamma-butyrobetaine (GBB), on the availability of TMA and TMAO.

Main methods

Wistar rats received L-carnitine, GBB or choline alone or in combination with meldonium. Plasma, urine and rat small intestine perfusate samples were assayed for L-carnitine, GBB, choline and TMAO using UPLC-MS/MS. Meldonium effects on TMA production by intestinal bacteria from L-carnitine and choline were tested.

Key findings

Treatment with meldonium significantly decreased intestinal microbiota-dependent production of TMA/TMAO from L-carnitine, but not from choline. 24 hours after the administration of meldonium, the urinary excretion of TMAO was 3.6 times lower in the combination group than in the L-carnitine-alone group. In addition, the administration of meldonium together with L-carnitine significantly increased GBB concentration in blood plasma and in isolated rat small intestine perfusate. Meldonium did not influence bacterial growth and bacterial uptake of L-carnitine, but TMA production by the intestinal microbiota bacteria K. pneumoniae was significantly decreased.

Significance

We have shown for the first time that TMA/TMAO production from quaternary amines could be decreased by targeting bacterial TMA-production. In addition, the production of pro-atherogenic TMAO can be suppressed by shifting the microbial degradation pattern of supplemental/dietary quaternary amines.     

Phosphorylation of lactate dehydrogenase by ATP

Evidence is presented indicating that phosphorylation of porcine muscle lactate dehydrogenase by [gamma-32P] ATP occurs at carboxyl residues of the protein. The phosphoenzyme complex was moderately stable at pH 6.8 and 25 degrees C, with a half-life of 3.5 h. In the presence of NADH rapid dephosphorylation occurred. Formation of an abortive complex with NAD-pyruvate also caused hydrolysis of the phosphoenzyme. The phosphorylated lactate dehydrogenase was shown to serve as a phosphate donor for phosphorylation of ADP.     

Microcoulometric analysis of trimethylamine dehydrogenase.

Trimethylamine dehydrogenase, which contains one covalently bound 6-S-cysteinyl-FMN and one Fe4S4 cluster per subunit of molecular mass 83,000 Da, was purified to homogeneity from the methylotrophic bacterium W3A1. Microcoulometry at pH 7 in 50 mM-Mops buffer containing 0.1 mM-EDTA and 0.1 M-KCl revealed that the native enzyme required the addition of 3 reducing equivalents per subunit for complete reduction. In contrast, under identical conditions the phenylhydrazine-inhibited enzyme required the addition of 0.9 reducing equivalent per subunit with a midpoint potential of +110 mV. Least-squares analysis of the microcoulometric data obtained for the native enzyme, assuming uptake of 1 electron by Fe4S4 and 2 electrons by FMN, indicated midpoint potentials of +44 mV and +36 mV for the FMN/FMN.- and FMN.-/FMNH2 couples respectively and +102 mV for reduction of the Fe4S4 cluster.     

Gut microbes impact stroke severity via the trimethylamine N-oxide pathway

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