Ergothioneine-Sodium Hyaluronate Dressing: A Promising Approach for Protecting against Radiation-Induced Skin Injury.

Radiotherapy commonly causes damage to healthy tissues, particularly radiation-induced skin injury (RISI) that affects a significant majority of patients undergoing radiotherapy. Effective treatments for RISI are lacking. This study focuses on the pathogenesis of RISI, which primarily involves oxidative stress. Excessive reactive oxygen species (ROS) generation during radiation induces damage to biological macromolecules, triggering oxidative stress and inflammation. To address this, ergothioneine (EGT), a natural and biocompatibile thiol compound with excellent antioxidant activity, is explored as a potential radiation-protective agent. By utilizing its specific transport and absorption in the skin tissue, as well as its efficient and stable clearance of radiation-induced "ROS storm", EGT is combined with sodium hyaluronate (NaHA) to develop a novel radiation protective dressing suitable for the skin. This EGT-NaHA dressing demonstrates an effective ability to scavenge free radicals and reduce oxidative stress in vitro and in vivo, reducing cellular apoptosis and inflammation. These results demonstrate the protective properties of EGT against RISI, with far-reaching implications for research and development in the field of radioprotection.

Structural insights into a novel nonheme iron-dependent oxygenase in selenoneine biosynthesis.

Selenoneine (SEN) is a natural histidine derivative with radical-scavenging activity and shows higher antioxidant potential than its sulfur-containing isolog ergothioneine (EGT). Recently, the SEN biosynthetic pathway in Variovorax paradoxus was reported. Resembling EGT biosynthesis, the committed step of SEN synthesis is catalyzed by a nonheme Fe-dependent oxygenase termed SenA. This enzyme catalyzes oxidative carbon­selenium (C-Se) bond formation to conjugate N-α-trimethyl histidine (TMH) and selenosugar to yield selenoxide; the process parallels the EGT biosynthetic route, in which sulfoxide synthases known as EgtB members catalyze the conjugation of TMH and cysteine or γ-glutamylcysteine to afford sulfoxides. Here, we report the crystal structures of SenA and its complex with TMH and thioglucose (SGlc), an analog of selenoglucose (SeGlc) at high resolution. The overall structure of SenA adopts the archetypical fold of EgtB, which comprises a DinB-like domain and an FGE-like domain. While the TMH-binding site is highly conserved to that of EgtB, a various substrate-enzyme interaction network in the selenosugar-binding site of SenA features a number of water-mediated hydrogen bonds. The obtained structural information is beneficial for understanding the mechanism of SenA-mediated C-Se bond formation.

Isotope-labeled ergothioneine clarifies the mechanism of reaction with singlet oxygen.

Recently we have uncovered a non-enzymatic multi-step cycle for the regeneration of ergothioneine (ET), after reaction with noxious singlet oxygen (1O2), by glutathione (GSH). When living cells were loaded with ET labeled with deuterium and N-15 atoms (D5-ET) and exposed to light in the presence of a photosensitizer, no loss of deuterium at position 5 of the imidazole ring was observed, in contradiction to our previous mechanistic proposal. Therefore, it was necessary to reexamine the in vitro products of ET and 1O2 by liquid chromatography coupled to high resolution mass spectrometry. Pure 1O2 was generated by thermolysis at 37 °C of the endoperoxide DHPNO2. The use of D5-ET enabled us to revise and extend the reaction scheme. On the main pathway, 1O2 attacks the imidazole ring, and the hydroperoxide intermediates are reduced rapidly by ET or GSH via different mechanisms. The intramolecular water elimination from the 5-hydroperoxide described previously is slower and not a part of the cycle. On another side path, 1O2 attacks the sulfur of ET to form a sulfine (S-oxide). The reduction of the sulfine also allows for the complete regeneration of ET. Experiments with methanol instead of water as solvent revealed that, in the absence of GSH, ET was attacked 6 times more frequently at the ring than at the sulfur. In the presence of 1 mM GSH or higher, both side paths were abandoned. ET efficiently captures 1O2 with its ring and can then be regenerated to a large extent by GSH, without enzyme involvement.

Inhibitors of Mycobacterium tuberculosis EgtD target both substrate binding sites to limit hercynine production.

Ergothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.

A novel antioxidant ergothioneine PET radioligand for in vivo imaging applications.

Ergothioneine (ERGO) is a rare amino acid mostly found in fungi, including mushrooms, with recognized antioxidant activity to protect tissues from damage by reactive oxygen species (ROS) components. Prior to this publication, the biodistribution of ERGO has been performed solely in vitro using extracted tissues. The aim of this study was to develop a feasible chemistry for the synthesis of an ERGO PET radioligand, [11C]ERGO, to facilitate in vivo study. The radioligand probe was synthesized with identical structure to ERGO by employing an orthogonal protection/deprotection approach. [11C]methylation of the precursor was performed via [11C]CH3OTf to provide [11C]ERGO radioligand. The [11C]ERGO was isolated by RP-HPLC with a molar activity of 690 TBq/mmol. To demonstrate the biodistribution of the radioligand, we administered approximately 37 MBq/0.1 mL in 5XFAD mice, a mouse model of Alzheimer's disease via the tail vein. The distribution of ERGO in the brain was monitored using 90-min dynamic PET scans. The delivery and specific retention of [11C]ERGO in an LPS-mediated neuroinflammation mouse model was also demonstrated. For the pharmacokinetic study, the concentration of the compound in the serum started to decrease 10 min after injection while starting to distribute in other peripheral tissues. In particular, a significant amount of the compound was found in the eyes and small intestine. The radioligand was also distributed in several regions of the brain of 5XFAD mice, and the signal remained strong 30 min post-injection. This is the first time the biodistribution of this antioxidant and rare amino acid has been demonstrated in a preclinical mouse model in a highly sensitive and non-invasive manner.

A proposed antioxidation mechanism of ergothioneine based on the chemically derived oxidation product hercynine and further decomposition products.

Ergothioneine (ERGO), a thiohistidine betaine, exists in various fungi, plants, and animals. Humans take in ERGO from their diet. ERGO is a strong biological antioxidant, but there are only a limited number of reports about its redox mechanism. The purpose of this study was to clarify the oxidation mechanism of ERGO. Reactions of ERGO with chemical oxidants were performed. The oxidation products of ERGO were analyzed by nuclear magnetic resonance and liquid chromatography-mass spectrometry (LC-MS). The major product of oxidation of ERGO by hydrogen peroxide in physiological conditions was identified as hercynine (histidine betaine). One molecule of ERGO was able to reduce 2 molecules of hydrogen peroxide. Hercynine was found to react with the more potent oxidant hypochlorite. One unstable decomposition product was detected by LC-MS. As a result, a mechanism of oxidation of ERGO, and hence its physiological antioxidant activity, was developed.

Oral Administration of the Food-Derived Hydrophilic Antioxidant Ergothioneine Enhances Object Recognition Memory in Mice.

BACKGROUND: The enhancement of learning and memory through food-derived ingredients is of great interest to healthy individuals as well as those with diseases. Ergothioneine (ERGO) is a hydrophilic antioxidant highly contained in edible golden oyster mushrooms (Pleurotus cornucopiae var. citrinopileatus), and systemically absorbed by its specific transporter, carnitine/organic cation transporter OCTN1/SLC22A4. OBJECTIVE: This study aims to examine the possible enhancement of object recognition memory by oral administration of ERGO in normal mice. METHODS: Novel object recognition test, spatial recognition test, LC-MS/MS, Golgi staining, neuronal culture, western blotting, immunocytochemistry, and quantitative RT-PCR were utilized. RESULT: After oral administration of ERGO (at a dose of 1-50 mg/kg) three times per week for two weeks in ICR mice, the novel object recognition test revealed a longer exploration time for the novel object than for the familiar object. After oral administration of ERGO, the spatial recognition test also revealed a longer exploration time for the spatially moved object than the unmoved one in mice fed ERGO-free diet. The discrimination index was significantly higher in the ERGO-treated group than the control in both behavioral tests. ERGO administration led to an increase in its concentration in the plasma and hippocampus. The systemic concentration reached was relevant to those found in humans after oral ERGO administration. Golgi staining revealed that ERGO administration increased the number of matured spines in the hippocampus. Exposure of cultured hippocampal neurons to ERGO elevated the expression of the synapse formation marker, synapsin I. This elevation of synapsin I was inhibited by the tropomyosin receptor kinase inhibitor, K252a. Treatment with ERGO also increased the expression of neurotrophin-3 and -5, and phosphorylated mammalian target of rapamycin in hippocampal neurons. CONCLUSION: Oral intake of ERGO may enhance object recognition memory at its plasma concentration achievable in humans, and this enhancement effect could occur, at least in part, through the promotion of neuronal maturation in the hippocampus.

Successful biosynthesis of natural antioxidant ergothioneine in Saccharomyces cerevisiae required only two genes from Grifola frondosa.

BACKGROUND: Ergothioneine (EGT) has a unique antioxidant ability and diverse beneficial effects on human health. But the content of EGT is very low in its natural producing organisms such as Mycobacterium smegmatis and mushrooms. Therefore, it is necessary to highly efficient heterologous production of EGT in food-grade yeasts such as Saccharomyces cerevisiae. RESULTS: Two EGT biosynthetic genes were cloned from the mushroom Grifola frondosa and successfully heterologously expressed in Saccharomyces cerevisiae EC1118 strain in this study. By optimization of the fermentation conditions of the engineered strain S. cerevisiae EC1118, the 11.80 mg/L of EGT production was obtained. With daily addition of 1% glycerol to the culture medium in the fermentation process, the EGT production of the engineered strain S. cerevisiae EC1118 can reach up to 20.61 mg/L. CONCLUSION: A successful EGT de novo biosynthetic system of S. cerevisiae containing only two genes from mushroom Grifola frondosa was developed in this study. This system provides promising prospects for the large scales production of EGT for human health.

Examining the health effects and bioactive components in Agaricus bisporus mushrooms: a scoping review.

There is evidence from both in vitro and animal models that the consumption of edible mushrooms has beneficial effects on health. It is unclear whether similar effects exist in humans and which bioactive compounds are present. This review synthesises the evidence on the world's most commonly consumed mushroom, Agaricus bisporus to (i) examine its effect on human health outcomes; and (ii) determine the nutrient density of its bioactive compounds, which may explain their health effects. A systematic literature search was conducted on the consumption of A. bisporus, without date and study design limits. Bioactive compounds included ergosterol, ergothioneine, flavonoids, glucans and chitin. Two authors independently identified studies for inclusion and assessed methodological quality. Beneficial effects of A. bisporus on metabolic syndrome, immune function, gastrointestinal health and cancer, with the strongest evidence for the improvement in Vitamin D status in humans, were found. Ultraviolet B (UVB) exposed mushrooms may increase and maintain serum 25(OH)D levels to a similar degree as vitamin D supplements. A. bisporus contain beta-glucans, ergosterol, ergothioneine, vitamin D and an antioxidant compound usually reported as flavonoids; with varying concentrations depending on the type of mushroom, cooking method and duration, and UVB exposure. Further research is required to fully elucidate the bioactive compounds in mushrooms using vigorous analytical methods and expand the immunological markers being tested. To enable findings to be adopted into clinical practice and public health initiatives, replication of existing studies in different population groups is required to confirm the impact of A. bisporus on human health.

The Biosynthetic Pathway of Ergothioneine in Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

Ergothioneine is a natural 2-thiol-amidazole amino acid that plays an important role in inflammation, depression, and cardiovascular disease. Flammulina velutipes is a common basidiomycete mushroom rich in ergothioneine (EGT). However, the biosynthetic pathway of EGT in F. velutipes is still unclear. In this study, the F. velutipes ergothioneine biosynthetic gene 1 (Fvegtl), F. velutipes ergothioneine biosynthetic gene 2 (Fvegt2), and F. velutipes ergothioneine biosynthetic gene 3 (Fvegt3) were cloned and expressed, and the activities of the proteins encoded by these three genes (FvEgt1, F. velutipes ergothioneine biosynthase 1; FvEgt2, F. velutipes ergothioneine biosynthase 2; and FvEgt3, F. velutipes ergothioneine biosynthase 3) were identified. The results showed that FvEgtl not only has the function of methyltransferase, but also has the function of hercynlcysteineteine sulfoxide (Hersul) synthase, which can catalyze the production of Hersul from histidine and cysteine in F. velutipes. FvEgt2 and FvEgt3 are two functionally different cysteine desulfurase enzymes. Among them, FvEgt2 is a cysteine-cysteine desulfurase-which catalyzes the activation of the S-H bond on cysteine, while FvEgt3 is a pyridoxal phosphate (PLP)-dependent cysteine desulfurase responsible for catalyzing the production of ketimine complex. Our results show that FvEgt1/FvEgt2/FvEgt3 can simultaneously catalyze the production of EGT by histidine, cysteine, and pyridoxal phosphate. Collectively, the in vitro synthesis of EGT in the edible fungus F. velutipes was first achieved, which laid the foundation for the biological production of EGT.

A Study on the Antioxidant Properties and Stability of Ergothioneine from Culinary-Medicinal Mushrooms.

The naturally occurring amino acid ergothioneine (EGT) has excellent free radical scavenging ability, which was not different to ascorbic acid. The IC50 values for EGT scavenging hydrogen peroxide, hydroxyl radicals, and superoxide anions were 11.65 ± 0.31, 70.31 ± 1.59, and 160.44 ± 0.32 µg/mL, respectively. The EGT concentration in different species of mushrooms was significantly different (p < 0.05), but it was not significantly related to the ability of the mushrooms to scavenge reactive oxygen species (p > 0.05). After isolating EGT from mushrooms, we demonstrated that the antioxidant ability of EGT accounts for about 25% of the total antioxidant ability of the extract. We studied the stability of EGT and found that it has excellent light, thermal, and acid-base stability. However, the presence of Cu2+ decreased the concentration of EGT. Unlike EGT, the thermal stability of the EGT extracted from Pleurotus citrinopileatus (PEGT) was not as good as EGT, while long-term high-temperature heating caused a decrease in the concentration of PEGT. The results of our study provide a basis for further investigating EGT from mushrooms for research and development.

Selenocysteine as a Substrate, an Inhibitor and a Mechanistic Probe for Bacterial and Fungal Iron-Dependent Sulfoxide Synthases.

Sulfoxide synthases are non-heme iron enzymes that participate in the biosynthesis of thiohistidines, such as ergothioneine and ovothiol A. The sulfoxide synthase EgtB from Chloracidobacterium thermophilum (CthEgtB) catalyzes oxidative coupling between the side chains of N-α-trimethyl histidine (TMH) and cysteine (Cys) in a reaction that entails complete reduction of molecular oxygen, carbon-sulfur (C-S) and sulfur-oxygen (S-O) bond formation as well as carbon-hydrogen (C-H) bond cleavage. In this report, we show that CthEgtB and other bacterial sulfoxide synthases cannot efficiently accept selenocysteine (SeCys) as a substrate in place of cysteine. In contrast, the sulfoxide synthase from the filamentous fungus Chaetomium thermophilum (CthEgt1) catalyzes C-S and C-Se bond formation at almost equal efficiency. We discuss evidence suggesting that this functional difference between bacterial and fungal sulfoxide synthases emerges from different modes of oxygen activation.

Anti-oxidative Amino Acid L-ergothioneine Modulates the Tumor Microenvironment to Facilitate Adjuvant Vaccine Immunotherapy.

Cancer vaccines consist of a tumor-associated antigen (TAA) and adjuvant. These vaccines induce and activate proliferation of TAA-specific cytotoxic T lymphocytes (CTLs), suppressing tumor growth. The therapeutic efficacy of TAA-specific CTLs depends on the properties of tumor microenvironment. The environments make immunosuppressive by function of regulatory T cells and tumor-associated myeloid cells; thus, regulation of these cells is important for successful cancer immunotherapy. We report here that L-ergothioneine (EGT) with the adjuvant Toll-like receptor 2 (TLR2) ligand modulated suppressive microenvironments to be immune-enhancing. EGT did not augment DC-mediated CTL priming or affect CTL activation in draining lymph node and spleen. However, EGT decreased the immuno-suppressive function of tumor-associated macrophages (TAMs). TLR2 stimulation accompanied with EGT administration downregulated expression of PD-L1, CSF-1R, arginase-1, FAS ligand, and TRAIL in TAMs, reflecting reduction of CTL suppression. An anti-oxidative thiol-thione residue of EGT was essential to dampening CTL suppression. The effect was specific to the thiol-thione residue of EGT because no effect was observed with another anti-oxidant N-acetyl-L-cysteine (NAC). A CTL-suppressive environment made by TLR2 is relieved to be improved by the addition of EGT, which may ameliorate the efficacy of vaccine immunotherapy.

An Alternative Active Site Architecture for O2 Activation in the Ergothioneine Biosynthetic EgtB from Chloracidobacterium thermophilum.

Sulfoxide synthases are nonheme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-α-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the γ-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system, we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five subtypes, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.

Regeneration of ergothioneine after reaction with singlet oxygen.

Ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. Important antioxidants are typically regenerated from their oxidized products, to prevent the interceptors from being lost after a single chemical reaction with a reactive oxygen species. However, no mechanism for the complete regeneration of ET has yet been uncovered. Here we define a non-enzymatic multi-step cycle for the regeneration of ET after reaction with singlet oxygen (1O2). All reaction steps were verified by density functional theory computations. Four molecules of GSH are used per turn to detoxify 1O2 to water. Pure 1O2 was generated by thermolysis at 37 °C of the endoperoxide DHPNO2. Addition of 1 mM ET to 10 mM DHPNO2 and 10 mM GSH increased the production of oxidized GSH (GSSG), measured by LC-MS/MS, by a factor of 26 (water) and 28 (D2O), respectively. In the same assay, the ring of ET alone was able to drive the cycle at equal speed; thus, the zwitterionic amino acid backbone was not involved. Our data suggest that ET reacts at least 4-fold faster with 1O2 than ascorbic acid. ET must now be viewed as tightly linked with the GSH/GSSG redox couple. The necessary thiol foundation is present in all mammalian and vertebrate cells, and also in all species that generate ET, such as cyanobacteria, mycobacteria, and fungi. Regeneration provides a decisive advantage for ET over other reactive, but non-recoverable, compounds. Our findings substantiate the importance of ET for the eradication of noxious 1O2.

A comprehensive screening shows that ergothioneine is the most abundant antioxidant in the wild macrofungus Phylloporia ribis Ryvarden.

The polar and non-polar extracts from the authenticated wild mushroom Phylloporia ribis were separated by hydrophilic interaction liquid chromatography (HILIC) and by reverse phase (RP)-HPLC, respectively. A split valve separated the eluents into two fractions for free-radical scavenging analysis and for structural identification. Forty-six compounds showed scavenging activity of the stable-free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). The structures of 8 antioxidants (inosine, caffeic acid, ergothioneine, p-hydroxybenzoic acid, adenosine, 3,4-dihydroxybenzaldehyde, apigenin, and naringenin) are characterized by Mass Spectrometer. Among them, ergothioneine was the most abundant (>65%) and most active antioxidant in P. ribis.

Ergothioneine stands out from hercynine in the reaction with singlet oxygen: Resistance to glutathione and TRIS in the generation of specific products indicates high reactivity.

The candidate vitamin ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. However, the precise physiological role of ET is still unresolved. Here, we investigated in vitro the hypothesis that ET serves specifically to eradicate noxious singlet oxygen (1O2). Pure 1O2 was generated by thermolysis at 37°C of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide 1,4-endoperoxide (DHPNO2). Assays of DHPNO2 with ET or hercynine (= ET minus sulfur) at pH 7.4 were analyzed by LC-MS in full scan mode to detect products. Based on accurate mass and product ion scan data, several products were identified and then quantitated as a function of time by selected reaction monitoring. All products of hercynine contained, after a [4+2] cycloaddition of 1O2, a carbonyl at position 2 of the imidazole ring. By contrast, because of the doubly bonded sulfur, we infer from the products of ET as the initial intermediates a 4,5-dioxetane (after [2+2] cycloaddition) and hydroperoxides at position 4 and 5 (after Schenck ene reactions). The generation of single products from ET, but not from hercynine, was fully resistant to a large excess of tris(hydroxymethyl)aminomethane (TRIS) or glutathione (GSH). This suggests that 1O2 markedly favors ET over GSH (at least 50-fold) and TRIS (at least 250-fold) for the initial reaction. Loss of ET was almost abolished in 5mM GSH, but not in 25mM TRIS. Regeneration of ET seems feasible, since some ET products - by contrast to hercynine products - decomposed easily in the MS collision cell to become aromatic again.

Ergothioneine Antioxidant Function: From Chemistry to Cardiovascular Therapeutic Potential.

Ergothioneine (ESH), the betaine of 2-mercapto-L-histidine, is a water-soluble naturally occurring amino acid with antioxidant properties. ESH accumulates in several human and animal tissues up to millimolar concentration through its high affinity transporter, namely the organic cation transporter 1 (OCTN1). ESH, first isolated from the ergot fungus (Claviceps purpurea), is synthesized only by Actinomycetales and non-yeast-like fungi. Plants absorb ESH via symbiotic associations between their roots and soil fungi, whereas mammals acquire it solely from dietary sources. Numerous evidence demonstrated the antioxidant and cytoprotective effects of ESH, including protection against cardiovascular diseases, chronic inflammatory conditions, ultraviolet radiation damages, and neuronal injuries. Although more than a century after its discovery has gone by, our understanding on the in vivo ESH mechanism is limited and this compound still intrigues researchers. However, recent evidence about differences in chemical redox behavior between ESH and alkylthiols, such as cysteine and glutathione, has opened new perspectives on the role of ESH during oxidative damage. In this short review, we discuss the role of ESH in the complex machinery of the cellular antioxidant defense focusing on the current knowledge on its chemical mechanism of action in the protection against cardiovascular disease.

Theoretical Study of the Mechanism of the Nonheme Iron Enzyme EgtB.

EgtB is a nonheme iron enzyme catalyzing the C-S bond formation between γ-glutamyl cysteine (γGC) and N-α-trimethyl histidine (TMH) in the ergothioneine biosynthesis. Density functional calculations were performed to elucidate and delineate the reaction mechanism of this enzyme. Two different mechanisms were considered, depending on whether the sulfoxidation or the S-C bond formation takes place first. The calculations suggest that the S-O bond formation occurs first between the thiolate and the ferric superoxide, followed by homolytic O-O bond cleavage, very similar to the case of cysteine dioxygenase. Subsequently, proton transfer from a second-shell residue Tyr377 to the newly generated iron-oxo moiety takes place, which is followed by proton transfer from the TMH imidazole to Tyr377, facilitated by two crystallographically observed water molecules. Next, the S-C bond is formed between γGC and TMH, followed by proton transfer from the imidazole CH moiety to Tyr377, which was calculated to be the rate-limiting step for the whole reaction, with a barrier of 17.9 kcal/mol in the quintet state. The calculated barrier for the rate-limiting step agrees quite well with experimental kinetic data. Finally, this proton is transferred back to the imidazole nitrogen to form the product. The alternative thiyl radical attack mechanism has a very high barrier, being 25.8 kcal/mol, ruling out this possibility.

Administration of Pure Ergothioneine to Healthy Human Subjects: Uptake, Metabolism, and Effects on Biomarkers of Oxidative Damage and Inflammation.

AIM: We investigated the uptake and pharmacokinetics of l-ergothioneine (ET), a dietary thione with free radical scavenging and cytoprotective capabilities, after oral administration to humans, and its effect on biomarkers of oxidative damage and inflammation. RESULTS: After oral administration, ET is avidly absorbed and retained by the body with significant elevations in plasma and whole blood concentrations, and relatively low urinary excretion (<4% of administered ET). ET levels in whole blood were highly correlated to levels of hercynine and S-methyl-ergothioneine, suggesting that they may be metabolites. After ET administration, some decreasing trends were seen in biomarkers of oxidative damage and inflammation, including allantoin (urate oxidation), 8-hydroxy-2'-deoxyguanosine (DNA damage), 8-iso-PGF2α (lipid peroxidation), protein carbonylation, and C-reactive protein. However, most of the changes were non-significant. INNOVATION: This is the first study investigating the administration of pure ET to healthy human volunteers and monitoring its uptake and pharmacokinetics. This compound is rapidly gaining attention due to its unique properties, and this study lays the foundation for future human studies. CONCLUSION: The uptake and retention of ET by the body suggests an important physiological function. The decreasing trend of oxidative damage biomarkers is consistent with animal studies suggesting that ET may function as a major antioxidant but perhaps only under conditions of oxidative stress. Antioxid. Redox Signal. 26, 193-206.