Mechanistic aspects of the tyrosinase oxidation of hydroquinone

Contradictory reports on the behaviour of hydroquinone as a tyrosinase substrate are reconciled in terms of the ability of the initially formed ortho-quinone to tautomerise to the thermodynamically more stable para-quinone isomer. Oxidation of phenols by native tyrosinase requires activation by in situ formation of a catechol formed via an enzyme generated ortho-quinone. In the special case of hydroquinone, catechol formation is precluded by rapid tautomerisation of the ortho-quinone precursor to catechol formation.     

Global Metabolic Response of Enterococcus faecalis to Oxygen

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and ¹³C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.     

Oxidation of N-β-alanyldopamine by insect cuticles and its role in cuticular sclerotization

The oxidation products formed when various types of insect cuticle were incubated with N-β-alanyldopamine (NBAD) have been studied by means of reversed phase high performance liquid chromatography, and compared to the corresponding products obtained when N-acetyldopamine (NADA) was incubated with the cuticles. The results indicate that NBAD is oxidized to o-quinone and quinone methide derivatives. In contrast, NADA can be oxidized by some cuticles not only to o-quinone and quinone methide derivatives, but it can also be desaturated to α,β-dehydro-N-acetyldopamine, a probable intermediate in β-sclerotization. Some implications for in vivo sclerotization are discussed.     

Effect of Phloroglucinol and Resorcinol on the Clingstone Peach Polyphenol Oxidase-catalyzed Oxidation of 4-Methylcatechol

Phloroglucinol and resorcinol are not substrates for clingstone peach (Prunus persica) polyphenol oxidase, but they react with 4-methyl-o-quinone, produced either enzymatically or nonenzymatically, to give an intense red or red-brown color with a maximal absorption at about 470 nanometers. Several colored products were isolated from an ethyl acetate extract of the reaction by two-dimensional thin layer chromatography. Based on thin layer chromatographic and spectral studies of the enzymatic and nonenzymatic reactions, polyphenol oxidase does not play a role in the reaction between 4-methyl-o-quinone and phloroglucinol, resorcinol, d-catechin, or orcinol. In such reactions, the function of polyphenol oxidase is the formation of 4-methyl-o-quinone which then reacts nonenzymatically with the above phenols. Activation energies of both enzymatic and nonenzymatic reactions were determined.     

Regiospecificity of Two Multicomponent Monooxygenases from Pseudomonas stutzeri OX1: Molecular Basis for Catabolic Adaptation of This Microorganism to Methylated Aromatic Compounds

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.     

Regulation of mitochondrial FoF1ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977 bp deletion of mitochondrial DNA

Sirt3, a mitochondrial NAD⁺-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F_oF₁ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F_oF₁ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F_oF₁ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F_oF₁ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F_oF₁ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ? 85% of mtDNA with 4977 bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F_oF₁ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5–10 μM of menadione impaired the Sirt3-mediated deacetylation and activation on F_oF₁ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F_oF₁ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.     

Effect of quercetin and its conjugated metabolite on the hydrogen peroxide-induced intracellular production of reactive oxygen species in mouse fibroblasts

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.     

Chicoric Acid Is an Antioxidant Molecule That Stimulates AMP Kinase Pathway in L6 Myotubes and Extends Lifespan in Caenorhabditis elegans

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5–100 μM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.     

Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K⁺ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z_g values and a less negative ΔG_o, indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased ΔG_o, indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.     

Mutation of Glutamic Acid 103 of Toluene o-Xylene Monooxygenase as a Means To Control the Catabolic Efficiency of a Recombinant Upper Pathway for Degradation of Methylated Aromatic Compounds

Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.     

Impact of Hydrogen Peroxide on the Activity, Structure, and Conformational Stability of the Oxidized Protein Repair Enzyme Methionine Sulfoxide Reductase A

The oxidized protein repair methionine sulfoxide reductase (Msr) system has been implicated in aging, in longevity, and in the protection against oxidative stress. This system is made of two different enzymes (MsrA and MsrB) that catalyze the reduction of the two diastereoisomers S- and R-methionine sulfoxide back to methionine within proteins, respectively. Due to its role in cellular protection against oxidative stress that is believed to originate from its reactive oxygen species scavenging ability in combination with exposed methionine at the surface of proteins, the susceptibility of MsrA to hydrogen-peroxide-mediated oxidative inactivation has been analyzed. This study is particularly relevant to the oxidized protein repair function of MsrA in both fighting against oxidized protein formation and being exposed to oxidative stress situations. The enzymatic properties of MsrA indeed rely on the activation of the catalytic cysteine to the thiolate anion form that is potentially susceptible to oxidation by hydrogen peroxide. The residual activity and the redox status of the catalytic cysteine were monitored before and after treatment. These experiments showed that the enzyme is only inactivated by high doses of hydrogen peroxide. Although no significant structural modification was detected by near- and far-UV circular dichroism, the conformational stability of oxidized MsrA was decreased as compared to that of native MsrA, making it more prone to degradation by the 20S proteasome. Decreased conformational stability of oxidized MsrA may therefore be considered as a key factor for determining its increased susceptibility to degradation by the proteasome, hence avoiding its intracellular accumulation upon oxidative stress.     

Degradation of Quercetin and Luteolin by Eubacterium ramulus

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by ¹H and ¹³C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.     

Isolation and characterization of a methionine adduct of DOPA o-quinone

The o-quinone of DOPA, an important intermediate implicated in many biological processes, has been found to react with methionine. The product has been isolated and studied, and tentative structure has been assigned.     

Mechanistic analyses of the suppression of amyloid β42 aggregation by apomorphine

(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid β-protein (Aβ42), a causative agent of Alzheimer’s disease (AD). Although the inhibition of Aβ42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC–MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aβ42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aβ42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. ¹H-¹⁵N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aβ42 monomer. These regions form intermolecular β-sheets in Aβ42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aβ42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 to investigate whether 3 associates with Aβ42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aβ42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the β-sheet of Aβ42 nuclei, thereby suppressing further aggregation.     

A rapid stimulation of phosphatidylinositol metabolism in rabbit leukocytes by pseudomonal leukocidin

The time course experiments of ³²P_i-labelling and breakdown of phospholipids in rabbit leukocytes exposed to leukocidin from Pseudomonas aeruginosa suggested that the initial action of this toxin was to stimulate phosphatidic acid production, presumably by causing a rapid metabolic change of phosphatidylinositol (PI response) correlating with phosphatidylinositol-specific phospholipase C and 1,2-diacylglycerol kinase. It appears that a rapid formation of phosphatidic acid and degradation of polyphosphoinositides in leukocytes treated with the toxin might be related a Ca²⁺-movement from extra- and intracellular spaces, resuling in the activation of Ca²⁺-dependent enzymes involved in the leukocidic process.     

Bupivacaine uncouples the mitochondrial oxidative phosphorylation,inhibits respiratory chain complexes I and III and enhances ROS production: Results of a study on cell cultures

This study aimed to validate, in situ, proposed mechanisms of bupivacaine citotoxicity pointing to impairment of the mitochondrial oxidative metabolism. High resolution oxymetry, carried out on a panel of cell cultures, revealed a dual dose- and time-dependent effect of bupivacaine consisting of uncoupling of the mtΔμ_H+-controlled respiratory rates in a cyclosporine A-insensitive manner and further inhibition of the respiratory rates. Intriguingly, a relatively small decrease on the mtΔΨ (about 20 mV) was sufficient to account for both the bupivacaine- and the FCCP-mediated impairment of the oxidative phosphorylation coupling thereby supporting a common protonophoric mechanism of action. The bupivacaine-induced depression of the cell respiration related to specific inhibition of complexes I and III and accompanied with production of reactive oxygen species. Importantly, inhibition of the respiratory chain complexes was prevented by antioxidant treatment and reversed following removal of the anaesthetic thereby suggesting an oxidant-mediated feed-back mechanism reinforcing the primary inhibitory action of the anaesthetic.     

Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO^?), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO^?. Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO^? or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases.     

Suppressive effect of quercetin on acute stress-induced hypothalamic-pituitary-adrenal axis response in Wistar rats

The flavonoid quercetin is considered to have beneficial effects on human health. We recently have shown that quercetin-enriched foods reduced the duration of immobility time in a rat forced swimming test, indicating that dietary quercetin is promising as an antidepressant-like factor, whereas its mechanism of action is poorly understood. The aim of this study is to investigate the effects of quercetin on water immersion-restraint (WIR), stress-induced hypothalamic-pituitary-adrenal (HPA) axis activation, which is a major component of stress response and plays an important role in the pathology of depression. Quercetin administration to rats significantly suppressed WIR stress-induced increase of plasma corticosterone and adrenocorticotropic hormone levels as well as the mRNA expression of corticotropin-releasing factor (CRF) in the hypothalamic region. In addition, quercetin modulated the DNA binding activities of glucocorticoid receptor and phosphorylated cyclic adenosine 3′,5′-monophosphate (cAMP) response element binding protein as well as the phosphorylation of extracellular signal-regulated kinase 1/2 in the hypothalamic region, all of which are known to regulate the expression of CRF mRNA. Taken together, these results suggest that dietary quercetin attenuates the HPA axis activation by the suppression of the CRF mRNA expression.