Substrate binding and catalytic mechanism of the Se-glycosyltransferase SenB in the biosynthesis of selenoneine.

Selenium is an essential multifunctional trace element in diverse organisms. The only Se-glycosyltransferase identified that catalyzes the incorporation of selenium in selenoneine biosynthesis is SenB from Variovorax paradoxus. Although the biochemical function of SenB has been investigated, its substrate specificity, structure, and catalytic mechanism have not been elucidated. Here, we reveal that SenB exhibits sugar donor promiscuity and can utilize six UDP-sugars to generate selenosugars. We report crystal structures of SenB complexed with different UDP-sugars. The key elements N20/T23/E231 contribute to the sugar donor selectivity of SenB. A proposed catalytic mechanism is tested by structure-guided mutagenesis, revealing that SenB yields selenosugars by forming C-Se glycosidic bonds via spontaneous deprotonation and disrupting Se-P bonds by nucleophilic water attack, which is initiated by the critical residue K158. Furthermore, we functionally and structurally characterize two other Se-glycosyltransferases, CbSenB from Comamonadaceae bacterium and RsSenB from Ramlibacter sp., which also exhibit sugar donor promiscuity.

Bis(1-methylimidazol-2-yl) diselenide and its evaluation as a chemical radio-protector: role of kinetic rate constants for ROS scavenging and glutathione peroxidase like activity.

Bis(1-methylimidazol-2-yl) diselenide (MeImSe), a derivative of selenoneine, has been examined for bimolecular rate constants for scavenging of various radiolytically and non-radiolytically generated reactive oxygen species (ROS). Further, its potential to show glutathione peroxidase (GPx)-like activity and to protect in vitro models of DNA and lipid against radiation induced strand breakage and lipid peroxidation, respectively were studied. The results confirmed that MeImSe scavenged all major short-lived (hydroxyl radical) and long-lived (peroxyl radical, carbonate radical, nitrogen dioxide radical, hypochlorite and hydrogen peroxide) oxidants involved in the radiation toxicity either directly or through GPx-like catalytic mechanism. The rate constants of MeImSe for these oxidants were found to be comparable to analogous sulfur and selenium-based compounds. The enzyme kinetics study established that MeImSe took part in the GPx cycle through the reductive pathway. Further, MeImSe inhibited the radiation induced DNA strand cleavage and lipid peroxidation with half maximal inhibitory concentration (IC50) of ∼ 60 µM and ∼100 µM, respectively. Interestingly, MeImSe treatment in the above concentration range (>100 µM) did not show any significant toxicity in normal human lung fibroblast (WI26) cells. The balance between efficacy and toxicity of MeImSe as a chemical radioprotector was attributed to the formation of less reactive intermediates during its oxidation/reduction reactions as evidenced from NMR studies.HighlightsMeImSe, a derivative of selenoneine protects DNA and lipid from radiation damageMeImSe scavenges all major short- and long-lived oxidants involved in radiation toxicityRate constants of MeImSe for ROS scavenging determined by pulse radiolysis techniqueFirst organoselenium compound reported to scavenge nitrogen dioxide radicalMeImSe exhibits GPx-like activity through reductive pathway.

Ergothioneine and its congeners: anti-ageing mechanisms and pharmacophore biosynthesis.

Ergothioneine, Ovothiol, and Selenoneine are sulfur/selenium-containing histidine-derived natural products widely distributed across different organisms. They exhibit significant antioxidant properties, making them as potential lead compounds for promoting health. Increasing evidence suggests that Ergothioneine is positively correlated with healthy ageing and longevity. The mechanisms underlying Ergothioneine's regulation of the ageing process at cellular and molecular levels are beginning to be understood. In this review, we provide an in-depth and extensive coverage of the anti-ageing studies on Ergothioneine and discuss its possible intracellular targeting pathways. In addition, we highlight the recent efforts in elucidating the biosynthetic details for Ergothioneine, Ovothiol, and Selenoneine, with a particular focus on the study of their pharmacophore-forming enzymology.

Diphthamide - a conserved modification of eEF2 with clinical relevance.

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases.

Inositol hexaphosphate enhances chemotherapy by reversing senescence induced by persistently activated PERK and diphthamide modification of eEF2.

Oxaliplatin is an important initial chemotherapy benefiting advanced-stage colorectal cancer patients. Frustratingly, acquired oxaliplatin resistance always occurs after sequential chemotherapy with diverse antineoplastic drugs. Therefore, an exploration of the mechanism of oxaliplatin resistance formation in-depth is urgently needed. We generated oxaliplatin-resistant colorectal cancer models by four representative compounds, and RNA-seq revealed that oxaliplatin resistance was mainly the result of cells' response to stimulus. Moreover, we proved persistent stimulus-induced endoplasmic reticulum stress (ERs) and associated cellular senescence were the core causes of oxaliplatin resistance. In addition, we screened diverse phytochemicals for ER inhibitors in silico, identifying inositol hexaphosphate (IP6), whose strong binding was confirmed by surface plasmon resonance. Finally, we confirmed the ability of IP6 to reverse colorectal cancer chemoresistance and investigated the mechanism of IP6 in the inhibition of diphthamide modification of eukaryotic elongation factor 2 (eEF2) and PERK activation. Our study demonstrated that oxaliplatin resistance contributed to cell senescence induced by persistently activated PERK and diphthamide modification of eEF2 levels, which were specifically reversed by combination therapy with IP6.

Quantitation of phosphohistidine in proteins in a mammalian cell line by 31P NMR.

There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 µmol/g and 68 µmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells.

Biosynthesis of selenium-containing small molecules in diverse microorganisms.

Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids, via selenocysteine and 2-selenouridine, respectively1. However, despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.

Translational fidelity and growth of Arabidopsis require stress-sensitive diphthamide biosynthesis.

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.

Context-specific roles of diphthamide deficiency in hepatocellular carcinogenesis.

Diphthamide biosynthesis protein 1 (DPH1) is biochemically involved in the first step of diphthamide biosynthesis, a post-translational modification of eukaryotic elongation factor 2 (EEF2). Earlier studies showed that DPH1, also known as ovarian cancer-associated gene 1 (OVCA1), is involved in ovarian carcinogenesis. However, the role of DPH1 in hepatocellular carcinoma (HCC) remains unclear. To investigate the impact of DPH1 in hepatocellular carcinogenesis, we performed data mining from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset. We found that reduced DPH1 levels were associated with advanced stages and poor survival of patients with HCC. Also, we generated hepatocyte-specific Dph1-deficient mice and showed that diphthamide-deficient EEF2 resulted in a reduced translation elongation rate in the hepatocytes and led to mild liver damage with fatty accumulation. After N-diethylnitrosamine (DEN)-induced acute liver injury, p53-mediated pericentral hepatocyte death was increased, and compensatory proliferation was reduced in Dph1-deficient mice. Consistent with these effects, Dph1 deficiency decreased the incidence of DEN-induced pericentral-derived HCC and revealed a protective effect against p53 loss. In contrast, Dph1 deficiency combined with Trp53- or Trp53/Pten-deficient hepatocytes led to increased tumor loads associated with KRT19 (K19)-positive periportal-like cell expansion in mice. Further gene set enrichment analysis also revealed that HCC patients with lower levels of DPH1 and TP53 expression had enriched gene-sets related to the cell cycle and K19-upregulated HCC. Additionally, liver tumor organoids obtained from 6-month-old Pten/Trp53/Dph1-triple-mutant mice had a higher frequency of organoid re-initiation cells and higher proliferative index compared with those of the Pten/Trp53-double-mutant. Pten/Trp53/Dph1-triple-mutant liver tumor organoids showed expression of genes associated with stem/progenitor phenotypes, including Krt19 and Prominin-1 (Cd133) progenitor markers, combined with low hepatocyte-expressed fibrinogen genes. These findings indicate that diphthamide deficiency differentially regulates hepatocellular carcinogenesis, which inhibits pericentral hepatocyte-derived tumors and promotes periportal progenitor-associated liver tumors. © 2022 The Pathological Society of Great Britain and Ireland.

A journey from phosphotyrosine to phosphohistidine and beyond.

Protein phosphorylation is a reversible post-translational modification. Nine of the 20 natural amino acids in proteins can be phosphorylated, but most of what we know about the roles of protein phosphorylation has come from studies of serine, threonine, and tyrosine phosphorylation. Much less is understood about the phosphorylation of histidine, lysine, arginine, cysteine, aspartate, and glutamate, so-called non-canonical phosphorylations. Phosphohistidine (pHis) was discovered 60 years ago as a mitochondrial enzyme intermediate; since then, evidence for the existence of histidine kinases and phosphohistidine phosphatases has emerged, together with examples where protein function is regulated by reversible histidine phosphorylation. pHis is chemically unstable and has thus been challenging to study. However, the recent development of tools for studying pHis has accelerated our understanding of the multifaceted functions of histidine phosphorylation, revealing a large number of proteins that are phosphorylated on histidine and implicating pHis in a wide range of cellular processes.

Ergothioneine, Ovothiol A, and Selenoneine-Histidine-Derived, Biologically Significant, Trace Global Alkaloids.

The history, chemistry, biology, and biosynthesis of the globally occurring histidine-derived alkaloids ergothioneine (10), ovothiol A (11), and selenoneine (12) are reviewed comparatively and their significance to human well-being is discussed.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Copper-histidine therapy in an infant with novel splice-site variant in the ATP7A gene of Menkes disease: the first experience in South East Asia and literature review.

Menkes disease (MD) is an X linked recessive multi-systemic disorder of copper metabolism, resulting from an ATP7A gene mutation. We report a male infant aged 4 months who presented with kinky hair, hypopigmented skin, epilepsy and delayed development. Magnetic resonance imaging (MRI) of brain demonstrated multiple tortuosities of intracranial vessels and brain atrophy. Investigation had showed markedly decreased serum copper and ceruloplasmin. The novel c.2172+1G>T splice-site mutation in the ATP7A gene confirmed MD. He was treated with subcutaneous administration of locally prepared copper-histidine (Cu-His). Following the therapy, hair manifestation was restored and serum ceruloplasmin was normalised 1 month later. Despite the treatment, epilepsy, neurodevelopment and osteoporosis still progressed. He died from severe respiratory tract infection at the age of 9.5 months. These findings suggest that the benefit of Cu-His in our case is limited which might be related to severe presentations and degree of ATP7A mutation.

First Time Identification of Selenoneine in Seabirds and Its Potential Role in Mercury Detoxification.

Birds are principally exposed to selenium (Se) through their diet. In long-lived and top predator seabirds, such as the giant petrel, extremely high concentrations of Se are found. Selenium speciation in biota has aroused great interest in recent years; however, there is a lack of information about the chemical form of Se in (sea)birds. The majority of publications focus on the growth performance and antioxidant status in broilers in relation to Se dietary supplementation. The present work combines elemental and molecular mass spectrometry for the characterization of Se species in wild (sea)birds. A set of eight giant petrels (Macronectes sp.) with a broad age range from the Southern Ocean were studied. Selenoneine, a Se-analogue of ergothioneine, was identified for the first time in wild avian species. This novel Se-compound, previously reported in fish, constitutes the major Se species in the water-soluble fraction of all of the internal tissues and blood samples analyzed. The levels of selenoneine found in giant petrels are the highest reported in animal tissues until now, supporting the trophic transfer in the marine food web. The characterization of selenoneine in the brain, representing between 78 and 88% of the total Se, suggests a crucial role in the nervous system. The dramatic decrease of selenoneine (from 68 to 3%) with an increase of Hg concentrations in the liver strongly supports the hypothesis of its key role in Hg detoxification.

Investigating histidinylated highly branched poly(lysine) for siRNA delivery.

The temporary silencing of disease-associated genes utilising short interfering RNA (siRNA) is a potent and selective route for addressing a wide range of life limiting disorders. However, the few clinically approved siRNA therapies rely on lipid based formulations, which although potent, provide limited chemical space to tune the stability, efficacy and tissue selectivity. In this study, we investigated the role of molar mass and histidinylation for poly(lysine) based non-viral vectors, synthesised through a fully aqueous thermal condensation polymerisation. Formulation and in vitro studies revealed that higher molar mass derivatives yielded smaller polyplexes attributed to a greater affinity for siRNA at lower N/P ratios yielding greater transfection efficiency, albeit with some cytotoxicity. Histidinylation had a negligible effect on formulation size, yet imparted a moderate improvement in biocompatibility, but did not provide any meaningful improvement over silencing efficiency compared to non-histidinylated derivatives. This was attributed to a greater degree of cellular internalisation for non-histidinylated analogues, which was enhanced with the higher molar mass material.

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.

The many ways that nature has exploited the unusual structural and chemical properties of phosphohistidine for use in proteins.

Histidine phosphorylation is an important and ubiquitous post-translational modification. Histidine undergoes phosphorylation on either of the nitrogens in its imidazole side chain, giving rise to 1- and 3- phosphohistidine (pHis) isomers, each having a phosphoramidate linkage that is labile at high temperatures and low pH, in contrast with stable phosphomonoester protein modifications. While all organisms routinely use pHis as an enzyme intermediate, prokaryotes, lower eukaryotes and plants also use it for signal transduction. However, research to uncover additional roles for pHis in higher eukaryotes is still at a nascent stage. Since the discovery of pHis in 1962, progress in this field has been relatively slow, in part due to a lack of the tools and techniques necessary to study this labile modification. However, in the past ten years the development of phosphoproteomic techniques to detect phosphohistidine (pHis), and methods to synthesize stable pHis analogues, which enabled the development of anti-phosphohistidine (pHis) antibodies, have accelerated our understanding. Recent studies that employed anti-pHis antibodies and other advanced techniques have contributed to a rapid expansion in our knowledge of histidine phosphorylation. In this review, we examine the varied roles of pHis-containing proteins from a chemical and structural perspective, and present an overview of recent developments in pHis proteomics and antibody development.

Dietary Intake of Selenoneine Enhances Antioxidant Activity in the Muscles of the Amberjack Seriola dumerili Grown in Aquaculture.

The selenium (Se)-containing imidazole compound selenoneine (2-selenyl-Nα, Nα, Nα-trimethyl-L-histidine) is a strong scavenger of reactive oxygen species (ROS) in the blood and tissues of fish. Intravenous injection of selenoneine into yellowtail has been shown to delay changes in meat color and prevent met-myoglobin formation in red muscle. In this study, to determine whether selenoneine can improve stress tolerance and meat quality in fish, we examined the biological antioxidant functions of selenoneine in fish in vivo. Juvenile amberjack (Seriola dumerili) were cultured and fed a diet containing selenoneine for 9 weeks. Total Se and selenoneine concentrations increased in amberjack blood and muscles during the study period. We also measured the oxidative-redox potential (ORP) in fish muscle using an ORP electrode and found that muscle ORP and ROS levels were closely correlated with the Se concentration in blood and muscles. We conclude that dietary administration of selenoneine led to its accumulation in amberjack blood and muscles, resulting in reduced ORP and ROS levels in the muscles.

Diphthamide promotes TOR signaling by increasing the translation of proteins in the TORC1 pathway.

Diphthamide, a modification found only on translation elongation factor 2 (EF2), was proposed to suppress -1 frameshifting in translation. Although diphthamide is conserved among all eukaryotes, exactly what proteins are affected by diphthamide deletion is not clear in cells. Through genome-wide profiling for a potential -1 frameshifting site, we identified that the target of rapamycin complex 1 (TORC1)/mammalian TORC1 (mTORC1) signaling pathway is affected by deletion of diphthamide. Diphthamide deficiency in yeast suppresses the translation of TORC1-activating proteins Vam6 and Rtc1. Interestingly, TORC1 signaling also promotes diphthamide biosynthesis, suggesting that diphthamide forms a positive feedback loop to promote translation under nutrient-rich conditions. Our results provide an explanation for why diphthamide is evolutionarily conserved and why diphthamide deletion can cause severe developmental defects.

Histidine phosphorylation in metalloprotein binding sites.

Post-translational modifications (PTMs) are invaluable regulatory tools for the control of catalytic functionality, protein-protein interactions, and signaling pathways. Historically, the study of phosphorylation as a PTM has been focused on serine, threonine, and tyrosine residues. In contrast, the significance of mammalian histidine phosphorylation remains largely unexplored. This gap in knowledge regarding the molecular basis for histidine phosphorylation as a regulatory agent exists in part because of the relative instability of phosphorylated histidine as compared with phosphorylated serine, threonine and tyrosine. However, the unique metal binding abilities of histidine make it one of the most common metal coordinating ligands in nature, and it is interesting to consider how phosphorylation would change the metal coordinating ability of histidine, and consequently, the properties of the phosphorylated metalloprotein. In this review, we examine eleven metalloproteins that have been shown to undergo reversible histidine phosphorylation at or near their metal binding sites. These proteins are described with respect to their biological activity and structure, with a particular emphasis on how phosphohistidine may tune the primary coordination sphere and protein conformation. Furthermore, several common methods, challenges, and limitations of studying sensitive, high affinity metalloproteins are discussed.