Metal Cofactor Research Articles

UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor.

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

Dielectric barrier discharge plasma treatment affects stability, metal ion coordination, and enzyme activity of bacterial superoxide dismutases

AbstractA molecular‐level understanding of the effects of atmospheric‐pressure plasma on biological samples requires knowledge of the effects on proteins. Superoxide dismutases, which detoxify superoxide under oxidative stress conditions, play a key role in bacterial plasma resistance. Investigation of the impact of dielectric barrier discharge (DBD) treatment on purified superoxide dismutases SodA and SodB of Escherichia coli showed that DBD treatment caused a rapid protein degradation, with only 8% of protein remaining after 10 min. The affinity of SodA for the metal cofactor Mn2+ was reduced. Mass spectrometry, in conjunction with coupled‐cluster calculations, revealed that modifications of amino acid residues in the active site can explain the decreased metal affinity and a distortion of the coordination geometry responsible for the activity loss.

Establishment of an in vitro RNA polymerase transcription system: a new tool to study transcriptional activation in Borrelia burgdorferi

The Lyme disease spirochete Borrelia burgdorferi exhibits dramatic changes in gene expression as it transits between its tick vector and vertebrate host. A major hurdle to understanding the mechanisms underlying gene regulation in B. burgdorferi has been the lack of a functional assay to test how gene regulatory proteins and sigma factors interact with RNA polymerase to direct transcription. To gain mechanistic insight into transcriptional control in B. burgdorferi, and address sigma factor function and specificity, we developed an in vitro transcription assay using the B. burgdorferi RNA polymerase holoenzyme. We established reaction conditions for maximal RNA polymerase activity by optimizing pH, temperature, and the requirement for divalent metals. Using this assay system, we analyzed the promoter specificity of the housekeeping sigma factor RpoD to promoters encoding previously identified RpoD consensus sequences in B. burgdorferi. Collectively, this study established an in vitro transcription assay that revealed RpoD-dependent promoter selectivity by RNA polymerase and the requirement of specific metal cofactors for maximal RNA polymerase activity. The establishment of this functional assay will facilitate molecular and biochemical studies on how gene regulatory proteins and sigma factors exert control of gene expression in B. burgdorferi required for the completion of its enzootic cycle.

Co-delivery of doxorubicin and DNAzyme using ZnO@polydopamine core-shell nanocomposites for chemo/gene/photothermal therapy

Multi-modal nanomedicines that synergistically combine chemo-, gene-, and photothermal therapy have shown great potential for cancer treatment. In this study, a core-shell nanosystem-based on a zinc oxide (ZnO) nanocore and a polydopamine (PDA) shell was constructed to integrate chemo- (doxorubicin, DOX), gene- (DNAzyme, DZ), and photothermal (PDA layer) therapy in one system. Instead of small interfering RNAs, we employed DZ for tumor-related gene (survivin) regulation owing to its higher stability, biocompatibility, and predictable activity. DOX and amino-modified DZ were loaded onto the PDA shell via physisorption and covalent conjugation, respectively. Specifically, the ZnO nanocore was designed as a metal cofactor reservoir to release Zn2+ in response to intracellular stimuli, which triggered the activation of DZ for gene silencing after endocytosis into cells. Both in vitro and in vivo experiments demonstrated the enhanced anti-tumor efficacy of these multifunctional nanocomposites and highlighted the advantages of these nano-drug delivery systems to alleviate the side effects of DOX. This study provides a strategy for synergistic cancer therapy via chemo/gene/photothermal combination and offers a strategy to harness DZ as a gene-silencing tool for disease treatment in combination with other therapeutic modalities. Statement of SignificanceIn this work, we constructed a core-shell nanosystem containing a zinc oxide (ZnO) nanocore and a polydopamine (PDA) outer layer, which integrated chemo- (doxorubicin, DOX), gene- (DNAzyme, DZ), and photothermal (PDA layer) therapies for multimodal cancer therapy. Specifically, the ZnO core was incorporated to solve the key issue of DZ for gene silencing applications, which acted as the metal cofactor reservoir to release Zn2+ inside cells for effective DZ activation. In addition, the PDA shell could detoxify the ZnO by scavenging the reactive oxygen species produced by ZnO, thus increasing the biocompatibility of the nanocarrier. This work solves the key issue of DZ for RNAi-based applications, offers a platform to combine DZ with other therapeutic modalities, and also provides a smart strategy to achieve triggered activation of biocatalytic reactions for therapeutic applications.

Phylogenetic Analysis and In Vitro Bifunctional Nuclease Assay of Arabidopsis BBD1 and BBD2.

Nucleases are a very diverse group of enzymes that play important roles in many crucial physiological processes in plants. We previously reported that the highly conserved region (HCR), domain of unknown function 151 (DUF151) and UV responsive (UVR) domain-containing OmBBD is a novel nuclease that does not share homology with other well-studied plant nucleases. Here, we report that DUF151 domain-containing proteins are present in bacteria, archaea and only Viridiplantae kingdom of eukarya, but not in any other eukaryotes. Two Arabidopsis homologs of OmBBD, AtBBD1 and AtBBD2, shared 43.69% and 44.38% sequence identity and contained all three distinct domains of OmBBD. We confirmed that the recombinant MBP-AtBBD1 and MBP-AtBBD2 exhibited non-substrate-specific DNase and RNase activity, like OmBBD. We also found that a metal cofactor is not necessarily required for DNase activity of AtBBD1 and AtBBD2, but their activities were much enhanced in the presence of Mg2+ or Mn2+. Using a yeast two-hybrid assay, we found that AtBBD1 and AtBBD2 each form a homodimer but not a heterodimer and that the HCR domain is possibly crucial for dimerization.

Artificial Metalloenzymes: Recent Developments and Innovations in Bioinorganic Catalysis.

Cellular life is orchestrated by the biochemical components of cells that include nucleic acids, lipids, carbohydrates, proteins, and cofactors such as metabolites and metals, all of which coalesce and function synchronously within the cell. Metalloenzymes allow for such complex chemical processes, as they catalyze a myriad of biochemical reactions both efficiently and selectively, where the metal cofactor provides additional functionality to promote reactivity not readily achieved in their absence. While the past 60 years have yielded considerable insight on how enzymes catalyze these reactions, a need to engineer and develop artificial metalloenzymes has been driven not only by industrial and therapeutic needs, but also by innate human curiosity. The design of miniature enzymes, both rationally and through serendipity, using both organic and inorganic building blocks has been explored by many scientists over the years and significant progress has been made. Herein, recent developments over the past 5 years in areas that have not been recently reviewed are summarized, and prospects for future research in these areas are addressed.

Alteration of iron responsive gene expression in Arabidopsis glutaredoxin S17 loss of function plants with or without iron stress

ABSTRACT Iron (Fe) is a mineral nutrient and a metal cofactor essential for plants. Iron limitation can have detrimental effects on plant growth and development, while excess iron inside plant cells leads to oxidative damage. As a result, plants have evolved complex regulatory networks to respond to fluctuations in cellular iron concentrations. The mechanisms that regulate these responses however, are not fully understood. Heterologous expression of an Arabidopsis thaliana monothiol glutaredoxin S17 (GRXS17) suppresses the over-accumulation of iron in the Saccharomyces cerevisiae Grx3/Grx4 mutant and disruption of GRXS17 causes plant sensitivity to exogenous oxidants and iron deficiency stress. GRXS17 may act as an important regulator in the plant’s ability to respond to iron deficiency stress and maintain redox homeostasis. Here, we extend this investigation by analyzing iron-responsive gene expression of the Fer-like iron deficiency-induced transcription factor (FIT) network (FIT, IRT1, FRO1, and FRO2) and the bHLH transcription factor POPEYE (PYE) network (PYE, ZIF1, FRO3, NAS4, and BTS) in GRXS17 KO plants and wildtype controls grown under iron sufficiency and deficiency conditions. Our findings suggest that GRXS17 is required for tolerance to iron deficiency, and plays a negative regulatory role under conditions of iron sufficiency.

Insights on the Functions and Ecophysiological Relevance of the Diverse Carbonic Anhydrases in Microalgae.

Carbonic anhydrases (CAs) exist in all kingdoms of life. They are metalloenzymes, often containing zinc, that catalyze the interconversion of bicarbonate and carbon dioxide—a ubiquitous reaction involved in a variety of cellular processes. So far, eight classes of apparently evolutionary unrelated CAs that are present in a large diversity of living organisms have been described. In this review, we focus on the diversity of CAs and their roles in photosynthetic microalgae. We describe their essential role in carbon dioxide-concentrating mechanisms and photosynthesis, their regulation, as well as their less studied roles in non-photosynthetic processes. We also discuss the presence in some microalgae, especially diatoms, of cambialistic CAs (i.e., CAs that can replace Zn by Co, Cd, or Fe) and, more recently, a CA that uses Mn as a metal cofactor, with potential ecological relevance in aquatic environments where trace metal concentrations are low. There has been a recent explosion of knowledge about this well-known enzyme with exciting future opportunities to answer outstanding questions using a range of different approaches.

Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation.

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3′-OH RNA strands in the context of 3′-OH/5′-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

Genome-wide in silico analysis of SOD genes in common bean (Phaseolus vulgaris L.)

Superoxide dismutases (SODs) are a group of enzymes that play essential roles in catalyzing the dismutation of superoxide radicals to protect cells from oxidative damage caused by various adverse conditions. SODs are classified into three types based on their metal cofactors: Cu/ZnSOD, FeSOD, and MnSOD. This study presents the first genome-wide identification of SOD genes in Phaseolus vulgaris L. Eight SOD genes were identified in the genome of P. vulgaris L., including four Cu/ZnSODs (CSD), three FeSODs (FSD) and one MnSOD (MSD). The protein sequence ranged between 166 and 312 amino acids with 5–8 introns each. Most PvSOD genes are in chromosome 7. The syntenic analysis revealed that PvFSD1 and PvFSD2 genes were derived from segmental duplication. As expected, phylogenetic analyses grouped the PvSOD proteins into three classes based on their metal cofactors: copper/zinc (Cu/Zn)SOD, manganese (Mn)SOD, and iron (Fe)SOD. Expression pattern analysis using RNAseq data indicated that three of the PvSOD genes (PvMSD1, PvCSD3, and PvFSD1) were detected in majority of tissues and developmental stages. This study provides a comprehensive analysis about SOD genes in P. vulgaris L. and new resources for future studies on the functional analysis of PvSOD genes in this species of great agronomic importance.

Detecting reactive oxygen species in yeast at early growth points

Reactive Oxygen Species (ROS) at low levels can act as important signaling molecules; however, when there is a surplus of ROS this can lead to harmful and or lethal results for the cell. ROS are primarily generated in the mitochondria of cells. This development occurs in the electron transport chain (ETC) which is embedded in the inner membrane of the mitochondria. Production of the mitochondrial superoxide anion (a type of ROS), occurs at redox active prosthetic groups, or electron carriers, bound to ETC proteins where kinetic factors favor O2 to becoming the superoxide anion. These kinetic factors can include the inhibition of ETC by small molecules. If an electron interacts “early” with O2 then superoxide will form, which could ultimately result in peroxides or more toxic hydroxyl radicals (HO•). Both proteins and small molecules are used within cells to mediate ROS levels and maintain redox states within these mechanisms to ensure ROS levels do not reach damaging levels. The accumulation of too much ROS can prove to be harmful to the organism. Mutations and dysfunctional molecules can result from ROS reacting with both nuclear and mitochondrial DNA, proteins, and lipids; however, more recent research shows that low levels of ROS provide important signaling mechanisms. Our interest is in how metal cofactors are incorporated into the ETC protein complexes of yeast and how misincorporation or modulation of available metals, such as copper and iron, in yeast mitochondria leads to the production of ROS and how under these conditions ROS changes during yeast lifespan. This project is particularly interested in how ROS changes “early” in yeast lifespan, meaning timepoints before yeast cells complete stationary phase and begin to enter the death phase. To detect the ROS superoxide, yeast cultures were grown in rich media for one to three days and then stained with dyhydroethidum (DHE). DHE is a fluorescent indicator for cytosolic superoxide, and cells were assayed utilizing both a fluorescence plate reader and FACS. To enhance superoxide production cells were also treated with the ETC inhibitor Antimycin A (a Complex II inhibitor), and the control inhibitor Oligomycin A (an ATPase inhibitor). Our results indicate that we are able to detect cellular superoxide using the fluorescent dye DHE and that cells cultured in the presence of the inhibitor Antimycin A have higher DHE fluorescence values compared to cells cultured in the presence of Oligomycin A. Since Antimycin A inhibits Complex III in the ETC, these results are consistent with ROS being generated from this complex. Our current work is to culture cells in supplemental copper ranging from no treatment to 0.5 mM CuSO4, or bathocuproine disulphonate (BCS) a copper chelator. Ultimately, we intend to use DHE fluorescence to assess superoxide levels in multiple yeast strains to provide insight into how ROS changes during yeast lifespan.

Copper Modulation and Reactive Oxygen Species Production Across Yeast Lifespan

The mitochondrial electron transport chain (ETC) machinery is the primary site of superoxide formation, a reactive oxygen species (ROS) associated with premature cell aging. A primary defense against ROS in yeast (Saccharomyces cerevisiae) is the enzyme superoxide dismutase (Sod1p). This enzyme requires copper and zinc as metal cofactors in the conversion of superoxide to hydrogen peroxide, a less harmful ROS. How mitochondrial function contributes to yeast chronological lifespan (CLS) is particularly of interest, specifically how essential metals for mitochondrial function and their role in the production of ROS drive the aging process of yeast cells. Our aim is to determine the effect exogenous copper treatment on the CLS of yeast strains lacking Sod1p and its copper chaperone, Lys7p. Our results indicate that low levels of exogenous copper (0.25 mM copper sulfate or less in restricted nutrient media) extend yeast chronological lifespan, especially in cells lacking Lys7p. All cell types see a dramatic reduction in lifespan when exogenous copper levels are increased to 2.0 mM copper sulfate or higher. This indicates that a small increase in the amount of copper in the media is beneficial for yeast in these restricted media conditions. Our current studies are to assess protein levels by western blot and gene expression by qRT‐PCR of various components the yeast ETC machinery, specifically Cox2 and Cox4, to mechanistically understand how exogenous copper in a small dose is contributing to lifespan extension. ROS levels are also being assessed via fluorescent staining. By using biochemical dyes such as Dihydroethidium (DHE) for cytosolic ROS, and MitoSox and mitochondria superoxide production, we are able to monitor ROS production and show localization of these metabolic byproducts with both fluorescent microscopy and FACS at various points of the chronological lifespan yeast.

Biosynthetic Origin of Complex Terpenoid Mixtures by Multiproduct Enzymes, Metal Cofactors, and Substrate Isomers

Terpenoids form a substantial portion of chemical diversity in nature. The enormous terpenoid diversity of more than 80,000 compounds is supported by the multisubstrate and multiproduct nature of certain enzymes from the various terpene synthases and terpene cyclases. These highly versatile enzymes are not only able to accept multiple substrates in their active site, but also simultaneously catalyze multiple reactions to the resultant multiple products. Interestingly, apart from the substrates and catalytic mechanisms, multiple regulation factors are able to alter the product profile of multiproduct terpene synthases. Simple variations in cellular conditions by changes in metal cofactors, assay pH, temperature and substrate geometry lead to significant shifts in product profiles. Switch in substrate stereochemistry for multiproduct terpene synthases in some case shows enhanced biocatalysis and in others initiates even a novel cyclization cascade. Hence, organisms can get access to a greater chemodiversity and avoid the expensive process of developing new biocatalysts just by simple changes in the cellular environment. This possibility of modulating chemical diversity provides immobile plants in the same generation access to an enhanced chemical arsenal for defense and communication by simply altering cofactors, pH level, and temperature and substrate geometry.

Studying Electron Transfer Pathways in Oxidoreductases

Oxidoreductases containing transition metal ions are widespread in nature and are essential for living organisms. The copper-containing nitrite reductase (NirK) and the molybdenum-containing aldehyde oxidoreductase (Aor) are typical examples of oxidoreductases. Metal ions in these enzymes are present either as mononuclear centers or organized into clusters and accomplish two main roles. One of them is to be the active site where the substrate is converted into product, and the other one is to serve as electron transfer center. Both enzymes transiently bind the substrate and an external electron donor/acceptor in NirK/Aor, respectively, at distinct protein points for them to exchange the electrons involved in the redox reaction. Electron exchange occurs through a specific intra-protein chemical pathway that connects the different enzyme metal cofactors. Based on the two oxidoreductases presented here, we describe how the different actors involved in the intra-protein electron transfer process can be characterized and studied employing molecular biology, spectroscopic, electrochemical, and structural techniques.

Dysregulation of Magnesium Transport Protects Bacillus subtilis against Manganese and Cobalt Intoxication

Transition metals are essential for life but are toxic when in excess. Metal ion intoxication may result from the mismetallation of essential metal-dependent enzymes with a noncognate metal. To begin to identify enzymes and processes that are susceptible to mismetallation, we have selected for strains with increased resistance to Mn(II) and Co(II). In Bacillus subtilis, cells lacking the MntR metalloregulator are exquisitely sensitive to Mn(II) but can easily become resistant by acquiring mutations affecting the MntH Mn(II) importer. Using transposon mutagenesis, and starting with an mntR mntH strain, we recovered mariner insertions that inactivated the mpfA gene encoding a putative Mg(II) efflux system. Loss of MpfA leads to elevated intracellular Mg(II), increased sensitivity to high Mg(II), and reduced Mn(II) sensitivity. Consistently, we also recovered an insertion disrupting the mgtE riboswitch, which normally restricts expression of the major Mg(II) importer. These results suggest that Mn(II) intoxication results from disruption of a Mg(II)-dependent enzyme or process. Mutations that inactivate MpfA were also recovered in a selection for Co(II) resistance beginning with sensitized strains lacking the major Co(II) efflux pump, CzcD. Since both Mn(II) and Co(II) may mismetallate iron-dependent enzymes, we repeated the selections under conditions of iron depletion imposed by expression of the Listeria monocytogenes FrvA iron exporter. Under conditions of iron depletion, a wider variety of suppressor mutations were recovered, but they still point to a central role for Mg(II) in maintaining metal ion homeostasis.IMPORTANCE Cellular metal ion homeostasis is tightly regulated. When metal ion levels are imbalanced, or when one metal is at toxic levels, enzymes may bind to the wrong metal cofactor. Enzyme mismetallation can impair metabolism, lead to new and deleterious reactions, and cause cell death. Beginning with Bacillus subtilis strains genetically sensitized to metal intoxication through loss of efflux or by lowering intracellular iron, we identified mutations that suppress the deleterious effects of excess Mn(II) or Co(II). For both metals, mutations in mpfA, encoding a Mg(II) efflux pump, suppressed toxicity. These mutant strains have elevated intracellular Mg(II), suggesting that Mg(II)-dependent processes are very sensitive to disruption by transition metals.

Biochemical and structural characterization of the Holliday junction resolvase RuvC from Pseudomonas aeruginosa

The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5′-TTC-3′. PaRuvC uses Mg2+ as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0–9.0. Elevated temperatures (37–60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system.

The evolving redox chemistry and bioavailability of vanadium in deep time.

The incorporation of metal cofactors into protein active sites and/or active regions expanded the network of microbial metabolism during the Archean eon. The bioavailability of crucial metal cofactors is largely influenced by earth surface redox state, which impacted the timing of metabolic evolution. Vanadium (V) is a unique element in geo-bio-coevolution due to its complex redox chemistry and specific biological functions. Thus, the extent of microbial V utilization potentially represents an important link between the geo- and biospheres in deep time. In this study, we used geochemical modeling and network analysis to investigate the availability and chemical speciation of V in the environment, and the emergence and changing chemistry of V-containing minerals throughout earth history. The redox state of V shifted from a more reduced V(III) state in Archean aqueous geochemistry and mineralogy to more oxidized V(IV) and V(V) states in the Proterozoic and Phanerozoic. The weathering of vanadium sulfides, vanadium alkali metal minerals, and vanadium alkaline earth metal minerals were potential sources of V to the environment and microbial utilization. Community detection analysis of the expanding V mineral network indicates tectonic and redox influence on the distribution of V mineral-forming elements. In reducing environments, energetic drivers existed for V to potentially be involved in early nitrogen fixation, while in oxidizing environments vanadate ( ) could have acted as a metabolic electron acceptor and phosphate mimicking enzyme inhibitor. The coevolving chemical speciation and biological functions of V due to earth's changing surface redox conditions demonstrate the crucial links between the geosphere and biosphere in the evolution of metabolic electron transfer pathways and biogeochemical cycles from the Archean to Phanerozoic.

Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast

BackgroundCobalt is an important metal cofactor of many living cells. However, excessive cobalt is toxic and can cause cell death and even several diseases in humans. Saccharomyces cerevisiae is a useful tool for studying metal homeostasis and many of the genes and pathways are highly conserved in higher eukaryotes including humans. MethodsThe intracellular cobalt and reactive oxygen species (ROS) levels were measured by an atomic absorption spectrometer and DHE staining method, respectively. The expression of genes involved in scavenging oxidative stress was tested by qPCR method, while the expression of UPRE-lacZ report gene was analyzed via β-galactosidase activity assay. ResultsUsing a genome-scale genetic screen, 153 cobalt-sensitive and 37 cobalt-tolerant gene deletion mutants were identified from Saccharomyces cerevisiae. We showed that 101 of the cobalt-sensitive mutants accumulated higher intracellular cobalt compared to wild-type. The intracellular ROS levels in 112 of the mutants were induced by cobalt, which might be caused by the decreased expression of genes involved in scavenging oxidative stress in response to cobalt. Moreover, more than one-third of the cobalt-sensitive mutants were also sensitive to tunicamycin, and cobalt stress might induce the unfolded protein response (UPR) through serine/threonine kinase and endoribonuclease Ire1. ConclusionsThis study reinforced the fact that cobalt toxicity might be due to the high intracellular cobalt and ROS levels, and the endoplasmic reticulum stress responses induced by cobalt. General significanceElucidating the toxicity mechanisms of cobalt stress response will help reveal new routes for the treatment of the diseases induced by cobalt.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

Enzyme Mimicking Based on the Natural Melanin Particles from Human Hair.

SummaryNatural enzymes are mainly composed by the protein part and metallic cofactor part, both of which work cooperatively to achieve high catalytic activity. Here, natural melanin particles (NMPs) were extracted from human hair and further bound with metal ions to mimic natural enzymes. The different metal-bound NMPs (M-NMPs) exhibited different enzyme-like activities with great promise in diverse biomedical applications. It was found that Fe-bound NMPs (Fe-NMPs) showed outstanding peroxidase (POD)-like activity that possessed potential in antibacterial applications, and Mn-bound NMPs (Mn-NMPs) displayed catalase (CAT)-like activity with a remarkable radiotherapy sensitization effect in cancer therapy. Besides, Cu-bound NMPs (Cu-NMPs) could serve as combined POD, superoxide dismutase (SOD), and CAT alternatives, which exhibited prominent reactive oxygen species (ROS) scavenging ability, revealing great potential in anti-inflammation. The versatile enzyme-like activities of M-NMPs derived from hair might give extensive perspective for designing biomedical materials and provide a promising tool in solving biomedical problems.