ABSTRACT
Methyl-coenzyme M reductase (MCR) is the key enzyme in pathways for the formation and anaerobic oxidation of methane. As methane is a potent greenhouse gas and biofuel, investigations of MCR catalysis and maturation are of interest for the development of both methanogenesis inhibitors and natural gas conversion strategies. The activity of MCR is dependent on a unique, nickel-containing coenzyme F430, the most highly reduced tetrapyrrole found in nature. Coenzyme F430 is biosynthesized from sirohydrochlorin in four steps catalyzed by the CfbABCDE enzymes. Here, methods for the expression and purification of the coenzyme F430 biosynthesis enzymes are described along with conditions for the synthesis and purification of biosynthetic intermediates on the milligram scale from commercially available porphobilinogen.
Subject(s)
Bacterial Proteins , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Methane/metabolism , Nickel/metabolism , Nickel/chemistry , Metalloporphyrins , OxidoreductasesABSTRACT
RATIONAL: Nickel is one of humans' most prevalent triggers of allergic contact dermatitis. However, the underlying mechanisms of this allergy still need to be fully understood. One aspect that has yet to be explored is the direct impact of common metal allergens on the skin's metabolites and lipids composition. METHOD: Our study employed matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to analyze spatially resolved metabolic alterations induced by nickel exposure. Cross-sections of ex vivo porcine ear skin exposed to increasing nickel (II) ion concentrations (17-167 µg/cm2) were measured with an AP-SMALDI5 AF ion source coupled to Q Exactive HF Orbitrap mass spectrometer. Additionally, the penetration of nickel ions into the skin was observed through its pink complexation with dimethylglyoxime under light microscopy. RESULTS: For nickel ion concentrations up to 84 µg/cm2, most nickel ions were stopped within the stratum corneum, while only a very small proportion of nickel ions penetrated the viable epidermis and dermis. Stratum corneum locations with high nickel ion concentrations showed a decrease in arginine and ceramides. Furthermore, several phosphatidylcholine and sphingomyelin species were found to be downregulated in the viable epidermis and dermis due to the nickel exposure. CONCLUSION: Nickel penetrates at a trace level into the viable skin and induces severe metabolomic and lipidomic changes in the stratum corneum, epidermis, and dermis, indicating a change in the skin (barrier) function. These findings contribute to a deeper understanding of nickel-induced skin allergies and provide a solid foundation for further research.
Subject(s)
Nickel , Skin , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Swine , Nickel/analysis , Nickel/metabolism , Nickel/pharmacokinetics , Skin/metabolism , Skin/drug effects , Skin/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Ear , Skin Absorption/drug effectsABSTRACT
Although the production of carbon monoxide (CO) within the human body has been detected, only two CO-utilizing prokaryotes (CO utilizers) have been reported in the human gut. Therefore, the phylogenetic diversity of the human gut CO-utilizing prokaryotes remains unclear. Here, we unveiled more than a thousand representative genomes containing genes for putative nickel-containing CO dehydrogenase (pCODH), an essential enzyme for CO utilization. The taxonomy of genomes encoding pCODH was expanded to include 8 phyla, comprising 82 genera and 248 species. In contrast, putative molybdenum-containing CODH genes were not detected in the human gut microbial genomes. pCODH transcripts were detected in 97.3â% (n=110) of public metatranscriptome datasets derived from healthy human faeces, suggesting the ubiquitous presence of prokaryotes bearing transcriptionally active pCODH genes in the human gut. More than half of the pCODH-encoding genomes contain a set of genes for the autotrophic Wood-Ljungdahl pathway (WLP). However, 79â% of these genomes commonly lack a key gene for the WLP, which encodes the enzyme that synthesizes formate from CO2, suggesting that potential human gut CO-utilizing prokaryotes share a degenerated gene set for WLP. In the other half of the pCODH-encoding genomes, seven genes, including putative genes for flavin adenine dinucleotide-dependent NAD(P) oxidoreductase (FNOR), ABC transporter and Fe-hydrogenase, were found adjacent to the pCODH gene. None of the putative genes associated with CO-oxidizing respiratory machinery, such as energy-converting hydrogenase genes, were found in pCODH-encoding genomes. This suggests that the human gut CO utilization is not for CO removal, but potentially for fixation and/or biosynthesis, consistent with the harmless yet continuous production of CO in the human gut. Our findings reveal the diversity and distribution of prokaryotes with pCODH in the human gut microbiome, suggesting their potential contribution to microbial ecosystems in human gut environments.
Subject(s)
Aldehyde Oxidoreductases , Bacteria , Carbon Monoxide , Gastrointestinal Microbiome , Multienzyme Complexes , Nickel , Phylogeny , Humans , Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Gastrointestinal Microbiome/genetics , Nickel/metabolism , Carbon Monoxide/metabolism , Multienzyme Complexes/genetics , Bacteria/genetics , Bacteria/classification , Bacteria/enzymology , Bacteria/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
BACKGROUND: At lower concentrations copper (Cu), zinc (Zn) and nickel (Ni) are trace metals essential for some bacterial enzymes. At higher concentrations they might alter and inhibit microbial functioning in a bioreactor treating wastewater. We investigated the effect of incremental concentrations of Cu, Zn and Ni on the bacterial community structure and their metabolic functions by shotgun metagenomics. Metal concentrations reported in previous studies to inhibit bacterial metabolism were investigated. RESULTS: At 31.5 µM Cu, 112.4 µM Ni and 122.3 µM Zn, the most abundant bacteria were Achromobacter and Agrobacterium. When the metal concentration increased 2 or fivefold their abundance decreased and members of Delftia, Stenotrophomonas and Sphingomonas dominated. Although the heterotrophic metabolic functions based on the gene profile was not affected when the metal concentration increased, changes in the sulfur biogeochemical cycle were detected. Despite the large variations in the bacterial community structure when concentrations of Cu, Zn and Ni increased in the bioreactor, functional changes in carbon metabolism were small. CONCLUSIONS: Community richness and diversity replacement indexes decreased significantly with increased metal concentration. Delftia antagonized Pseudomonas and members of Xanthomonadaceae. The relative abundance of most bacterial genes remained unchanged despite a five-fold increase in the metal concentration, but that of some EPS genes required for exopolysaccharide synthesis, and those related to the reduction of nitrite to nitrous oxide decreased which may alter the bioreactor functioning.
Subject(s)
Bacteria , Biodiversity , Bioreactors , Copper , Metagenomics , Nickel , Zinc , Bioreactors/microbiology , Zinc/metabolism , Nickel/metabolism , Bacteria/genetics , Bacteria/classification , Bacteria/metabolism , Bacteria/isolation & purification , Copper/metabolism , Wastewater/microbiology , Wastewater/chemistryABSTRACT
Toxic and heavy metals cause direct and indirect damage to the environment and ultimately to humans. This study involved the isolation of indigenous bacteria from heavy metal-contaminated environments that have the ability to bioabsorb heavy metals such as cadmium, nickel, and lead. The bioabsorption process was optimized by varying parameters such as temperature, metal concentration, number of bacteria, pH, and more. The bacterial isolates were investigated in terms of morphology, biochemistry, and phylogeny, with 12 strains chosen in the initial stage and one strain chosen in the final stage. It should be remembered that the metal uptake capacity of all isolates was approximately calculated. A box and reactor were designed to house these optimized microorganisms. Based on biochemical, morphological, and molecular results, the isolated strain was found to be closely related to the Bacillus genus. In the first five steps of testing, the ideal pH for removing lead alone, lead with cadmium, lead with nickel, and lead ternary (with cadmium and nickel) by Bacillus bacteria was found to be 7, 6, 5.5, and 6.5, respectively. The absorption efficiencies for single lead (unary), lead together with nickel, cadmium (binary), and ternary (lead with cadmium and nickel) were found to be 0.36, 0.25, 0.22, and 0.21 mmol/g, respectively. The ideal temperature for lead removal was around 30 °C. The adsorption isotherm for each lead metal in different states was found to be similar to the Langmuir isotherm, indicating that the surface absorption process is a single-layer process. The kinetics of the process follow the second-order kinetic model. The amount of Bacillus bacteria biomass obtained during this process was approximately 1.5 g per liter.
Subject(s)
Biofilms , Bioreactors , Metals, Heavy , Metals, Heavy/metabolism , Bioreactors/microbiology , Biofilms/growth & development , Biodegradation, Environmental , Nickel/metabolism , Nickel/chemistry , Cadmium/metabolism , Bacillus/metabolism , Phylogeny , Hydrogen-Ion Concentration , Bacteria/metabolism , Adsorption , Lead/metabolism , TemperatureABSTRACT
In mitochondria, the detoxification of molar excess H2S as polysulfide proceeded via an oxidation process promoted by Cu/Zn containing superoxide dismutase (SOD1) enzyme, which has been very recently reported as the alternative enzyme for cytosolic H2S oxidation. Herein, we present Ni(II) complexes bearing the terminal SH group as a synthetic functional analogue for the sulfide oxidase function of SOD1. Synthesis, crystal structure and complete spectroscopic characterization of two sets of complexes, [NiLOMe/tBu(PPh3)] (2OMe/tBu) and tetraethyl salt of [NiLOMe/tBu(SH)]-1 (3OMe/tBu), were described (LOMe = (E)-2-methoxy-6-(((2-sulfidophenyl)imino)methyl)phenolate and LtBu = (E)-2,4-di-tert-butyl-6-(((2-sulfidophenyl)imino)methyl)phenolate). Under anaerobic conditions, 3OMe/tBu responded to a catalytic sulfur atom transfer (SAT) reaction with PPh3 to produce SPPh3. The SAT reaction was analyzed using detailed studies of 1H and 31P NMR spectra. Finally, the SAT reactivity pattern was compared with the same in the native enzyme of SOD1.
Subject(s)
Coordination Complexes , Nickel , Sulfur , Nickel/chemistry , Nickel/metabolism , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Coordination Complexes/chemical synthesis , Sulfur/chemistry , Sulfur/metabolism , Superoxide Dismutase-1/chemistry , Superoxide Dismutase-1/metabolism , Models, Molecular , Catalysis , Anaerobiosis , Superoxide Dismutase/metabolism , Superoxide Dismutase/chemistry , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolismABSTRACT
The innate immune system employs a variety of antimicrobial oxidants to control and kill host-associated bacteria. Hypothiocyanite/hypothiocyanous acid (-OSCN/HOSCN) is one such antimicrobial oxidant that is synthesized by lactoperoxidase, myeloperoxidase, and eosinophil peroxidase at sites throughout the human body. HOSCN has potent antibacterial activity while being largely non-toxic toward human cells. The molecular mechanisms by which bacteria sense and defend themselves against HOSCN have only recently begun to be elaborated, notably by the discovery of bacterial HOSCN reductase (RclA), an HOSCN-degrading enzyme widely conserved among bacteria that live on epithelial surfaces. In this paper, I show that Ni2+ sensitizes Escherichia coli to HOSCN by inhibiting glutathione reductase and that inorganic polyphosphate protects E. coli against this effect, probably by chelating Ni2+ ions. I also found that RclA is very sensitive to inhibition by Cu2+ and Zn2+, metals that are accumulated to high levels by innate immune cells, and that, surprisingly, thioredoxin and thioredoxin reductase are not involved in HOSCN stress resistance in E. coli. These results advance our understanding of the contribution of different oxidative stress responses and redox buffering pathways to HOSCN resistance in E. coli and illustrate important interactions between metal ions and the enzymes bacteria use to defend themselves against oxidative stress. IMPORTANCE: Hypothiocyanite (HOSCN) is an antimicrobial oxidant produced by the innate immune system. The molecular mechanisms by which host-associated bacteria defend themselves against HOSCN have only recently begun to be understood. The results in this paper are significant because they show that the low molecular weight thiol glutathione and enzyme glutathione reductase are critical components of the Escherichia coli HOSCN response, working by a mechanism distinct from that of the HOSCN-specific defenses provided by the RclA, RclB, and RclC proteins and that metal ions (including nickel, copper, and zinc) may impact the ability of bacteria to resist HOSCN by inhibiting specific defensive enzymes (e.g., glutathione reductase or RclA).
Subject(s)
Escherichia coli , Thiocyanates , Escherichia coli/genetics , Escherichia coli/drug effects , Escherichia coli/metabolism , Thiocyanates/pharmacology , Thiocyanates/metabolism , Nickel/pharmacology , Nickel/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Drug Resistance, Bacterial , Glutathione Reductase/metabolism , Glutathione Reductase/genetics , Anti-Bacterial Agents/pharmacology , Zinc/metabolism , Zinc/pharmacology , Copper/metabolism , Copper/pharmacologyABSTRACT
Acetyl coenzyme A synthase (ACS) catalyzes the formation and deconstruction of the key biological metabolite, acetyl coenzyme A (acetyl-CoA). The active site of ACS features a {NiNi} cluster bridged to a [Fe4S4]n+ cubane known as the A-cluster. The mechanism by which the A-cluster functions is debated, with few model complexes able to replicate the oxidation states, coordination features, or reactivity proposed in the catalytic cycle. In this work, we isolate the first bimetallic models of two hypothesized intermediates on the paramagnetic pathway of the ACS function. The heteroligated {Ni2+Ni1+} cluster, [K(12-crown-4)2][1], effectively replicates the coordination number and oxidation state of the proposed "Ared" state of the A-cluster. Addition of carbon monoxide to [1]- allows for isolation of a dinuclear {Ni2+Ni1+(CO)} complex, [K(12-crown-2)n][2] (n = 1-2), which bears similarity to the "ANiFeC" enzyme intermediate. Structural and electronic properties of each cluster are elucidated by X-ray diffraction, nuclear magnetic resonance, cyclic voltammetry, and UV/vis and electron paramagnetic resonance spectroscopies, which are supplemented by density functional theory (DFT) calculations. Calculations indicate that the pseudo-T-shaped geometry of the three-coordinate nickel in [1]- is more stable than the Y-conformation by 22 kcal mol-1, and that binding of CO to Ni1+ is barrierless and exergonic by 6 kcal mol-1. UV/vis absorption spectroscopy on [2]- in conjunction with time-dependent DFT calculations indicates that the square-planar nickel site is involved in electron transfer to the CO π*-orbital. Further, we demonstrate that [2]- promotes thioester synthesis in a reaction analogous to the production of acetyl coenzyme A by ACS.
Subject(s)
Nickel , Nickel/chemistry , Nickel/metabolism , Acetate-CoA Ligase/chemistry , Acetate-CoA Ligase/metabolism , Models, Molecular , Coordination Complexes/chemistry , Coordination Complexes/metabolism , Oxidation-Reduction , Acetyl Coenzyme A/metabolism , Acetyl Coenzyme A/chemistryABSTRACT
Biological environments present a complex array of metal-binding ligands. Metal-binding proteins have been the overwhelming focus of study because of their important and well-defined biological roles. Consequently, the presence of functional low molecular weight (LMW) metal-ligand complexes has been overlooked in terms of their roles in metallobiochemistry, particularly within cells. Recent studies in microbial systems have illuminated the different roles of L-histidine in nickel uptake, gene expression, and metalloenzyme maturation. In this focused critical review, these roles are surveyed in the context of the coordination chemistry of Ni(II) ions and the amino acid histidine, and the physico-chemical properties of nickel complexes of histidine. These complexes are fundamentally important to cellular metal homeostasis and further work is needed to fully define their contributions.
Subject(s)
Histidine , Nickel , Histidine/chemistry , Histidine/metabolism , Nickel/chemistry , Nickel/metabolism , Coordination Complexes/chemistry , Coordination Complexes/metabolismABSTRACT
The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni2+-dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.
Subject(s)
Hydrolases , Metformin , Nickel , Metformin/metabolism , Metformin/chemistry , Nickel/metabolism , Nickel/chemistry , Hydrolases/metabolism , Hydrolases/chemistry , Hydrolases/genetics , Crystallography, X-Ray , Hydrolysis , Substrate Specificity , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Models, MolecularABSTRACT
In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes.
Subject(s)
Oxidation-Reduction , Urate Oxidase , Uric Acid , Substrate Specificity , Urate Oxidase/chemistry , Urate Oxidase/metabolism , Uric Acid/chemistry , Uric Acid/metabolism , Uric Acid/urine , Ligands , Humans , Nickel/chemistry , Nickel/metabolism , Binding Sites , Catalytic Domain , Catalysis , Models, Molecular , X-Ray Absorption SpectroscopyABSTRACT
Nickel(Ni)-containing materials have been widely used in a wide range of medical applications, including orthopaedics. Despite their excellent properties, there is still a problem with the release of nickel ions into the patient's body, which can cause changes in the behaviour of surrounding cells and tissues. This study aims to evaluate the effects of Ni on bone cells with an emphasis on the determination of Ni localization in cellular compartments in time. For these purposes, one of the most suitable models for studying the effects induced by metal implants was used-the patient's osteoarthritic cells. Thanks to this it was possible to simulate the pathophysiological conditions in the patient's body, as well as to evaluate the response of the cells which come into direct contact with the material after the implantation of the joint replacement. The largest differences in cell viability, proliferation and cell cycle changes occurred between Ni 0.5 mM and 1 mM concentrations. Time-dependent localization of Ni in cells showed that there is a continuous transport of Ni ions between the nucleus and the cytoplasm, as well as between the cell and the environment. Moreover, osteoarthritic osteoblasts showed faster changes in concentration and ability to accumulate more Ni, especially in the nucleus, than physiological osteoblasts. The differences in Ni accumulation process explains the higher sensitivity of patient osteoblasts to Ni and may be crucial in further studies of implant-derived cytotoxic effects.
Subject(s)
Cell Proliferation , Cell Survival , Nickel , Osteoarthritis , Osteoblasts , Nickel/metabolism , Osteoblasts/metabolism , Osteoblasts/drug effects , Humans , Osteoarthritis/metabolism , Osteoarthritis/pathology , Cell Survival/drug effects , Cell Proliferation/drug effects , Ions/metabolism , Cell Cycle/drug effects , Cells, CulturedABSTRACT
BACKGROUND: Cable bacteria are filamentous members of the Desulfobulbaceae family that are capable of performing centimetrescale electron transport in marine and freshwater sediments. This longdistance electron transport is mediated by a network of parallel conductive fibres embedded in the cell envelope. This fibre network efficiently transports electrical currents along the entire length of the centimetrelong filament. Recent analyses show that these fibres consist of metalloproteins that harbour a novel nickelcontaining cofactor, which indicates that cable bacteria have evolved a unique form of biological electron transport. This nickeldependent conduction mechanism suggests that cable bacteria are strongly dependent on nickel as a biosynthetic resource. Here, we performed a comprehensive comparative genomic analysis of the genes linked to nickel homeostasis. We compared the genomeencoded adaptation to nickel of cable bacteria to related members of the Desulfobulbaceae family and other members of the Desulfobulbales order. RESULTS: Presently, four closed genomes are available for the monophyletic cable bacteria clade that consists of the genera Candidatus Electrothrix and Candidatus Electronema. To increase the phylogenomic coverage, we additionally generated two closed genomes of cable bacteria: Candidatus Electrothrix gigas strain HY106 and Candidatus Electrothrix antwerpensis strain GW34, which are the first closed genomes of their respective species. Nickel homeostasis genes were identified in a database of 38 cable bacteria genomes (including 6 closed genomes). Gene prevalence was compared to 19 genomes of related strains, residing within the Desulfobulbales order but outside of the cable bacteria clade, revealing several genomeencoded adaptations to nickel homeostasis in cable bacteria. Phylogenetic analysis indicates that nickel importers, nickelbinding enzymes and nickel chaperones of cable bacteria are affiliated to organisms outside the Desulfobulbaceae family, with several proteins showing affiliation to organisms outside of the Desulfobacterota phylum. Conspicuously, cable bacteria encode a unique periplasmic nickel export protein RcnA, which possesses a putative cytoplasmic histidinerich loop that has been largely expanded compared to RcnA homologs in other organisms. CONCLUSION: Cable bacteria genomes show a clear genetic adaptation for nickel utilization when compared to closely related genera. This fully aligns with the nickeldependent conduction mechanism that is uniquely found in cable bacteria.
Subject(s)
Genome, Bacterial , Genomics , Homeostasis , Nickel , Phylogeny , Nickel/metabolism , Deltaproteobacteria/genetics , Deltaproteobacteria/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric-type class II chelatase which is able to use not only Ni2+ as a physiological metal substrate, but also Co2+ as a nonphysiological substrate with higher activity than for Ni2+. The Ni/Co-chelatase function found in CfbA is also observed in SirB, a descendant, monomeric-type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni-chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni-chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni-chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni-binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni-chelatase reaction in a class II chelatase architecture.
Subject(s)
Nickel , Nickel/metabolism , Nickel/chemistry , Catalytic Domain/genetics , Histidine/metabolism , Histidine/chemistry , Histidine/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Models, Molecular , Lyases/metabolism , Lyases/chemistry , Lyases/geneticsABSTRACT
One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.
Subject(s)
Acetate-CoA Ligase , Carbon Monoxide , Catalytic Domain , Nickel , Nickel/metabolism , Nickel/chemistry , Acetate-CoA Ligase/metabolism , Acetate-CoA Ligase/genetics , Acetate-CoA Ligase/chemistry , Carbon Monoxide/metabolism , Carbon Monoxide/chemistry , Carbon Cycle , Anaerobiosis , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carbon Dioxide/metabolism , Carbon Dioxide/chemistryABSTRACT
The role of the carapace in the uptake and storage of newly accumulated metals was investigated in the green crab exposed to environmentally relevant concentrations of calcium ([Ca] = 389 mg L-1 or 9.7 mmol L-1), zinc ([Zn] = 82 µg L-1 or 1.25 µmol L-1), and nickel ([Ni] = 8.2 µg L-1 or 0.14 µmol L-1) in 12 °C seawater, using radio-tracers (45Ca, 65Zn, 63Ni). After 24-h exposure, carapace exhibited the highest concentration of newly accumulated Ca, whereas carapace and gills exhibited the highest concentrations of both newly accumulated Zn and Ni relative to other tissues. For all three metals, the carapace accounted for >85 % of the total body burden. Acute temperature changes (to 2 °C and 22 °C) revealed the highest overall temperature coefficient Q10 (2.15) for Ca uptake into the carapace, intermediate Q10 for Ni (1.87) and lowest Q10 (1.45) for Zn. New Ca uptake into the carapace continued linearly with time for 24 h, new Zn uptake gradually deviated from linearity, whereas Ni uptake reached a plateau by 6 h. Attachment of a rubber membrane to the dorsal carapace, thereby shielding about 20 % of the total crab surface area from the external water, eliminated both new Zn and Ni incorporation into the shielded carapace, whereas 36 % of new Ca incorporation persisted. When recently euthanized crabs were exposed, new Zn uptake into the carapace remained unchanged, whereas Ca and Ni uptake were reduced by 89 % and 71 %, respectively. We conclude that the carapace is a very important uptake and storage site for all three metals. All of the uptake of new Zn and new Ni, and most of the uptake of new Ca into this tissue comes directly from the external water. For Zn, the mechanism involves only physicochemical processes, whereas for Ca and Ni, life-dependent processes make the major contribution.
Subject(s)
Brachyura , Calcium , Nickel , Seawater , Water Pollutants, Chemical , Zinc , Animals , Brachyura/metabolism , Nickel/metabolism , Zinc/metabolism , Water Pollutants, Chemical/metabolism , Seawater/chemistry , Calcium/metabolism , Animal Shells/chemistry , Animal Shells/metabolism , Environmental MonitoringABSTRACT
This study investigates Ni phytoremediation and accumulation potential in the presence of salicylic acid (SA) (0, 50 and 200 µM) and jasmonic acid (JA) (0, 5 and 10 µM) in two populations of Alyssum inflatum under various nickel (Ni) doses (0, 100 and 400 µM). By measuring Ni levels in the shoots and roots, values of bioaccumulation coefficient (BAC), biological concentration factor (BCF) and translocation factor (TF) were calculated to quantify Ni accumulation and translocation between plant organs. Additionally, the amounts of histidine (His), citric acid (CA) and malic acid (MA) were explored. The results showed that plant dry weight (DW) [in shoot (29.8%, 8.74%) and in root (21.6%, 24.4%)] and chlorophyll [a (17.1%, 32.5%), b (10.1%, 30.9%)] declined in M and NM populations respectively, when exposed to Ni (400 µM). Conversely, the levels of MA [in shoot (37.0%, 32.0%) and in root (25.5%, 21.2%)], CA [in shoot (17.0%, 10.0%) and in root (47.9%, 37.2%)] and His [in shoot (by 1.59- and 1.34-fold) and in root (by 1.24- and 1.18-fold)] increased. Also, in the presence 400 µM Ni, the highest accumulation of Ni was observed in shoots of M (1392 µg/g DW) and NM (1382 µg/g DW). However, the application of SA and JA (especially in Ni 400 µM + SA 200 µM + JA 5 and 10 µM treatments) mitigated the harmful impact of Ni on physiological parameters. Also, a decreasing trend was observed in the contents of MA, CA, and His. The reduction of these compounds as important chelators of Ni caused a decrease in root-to-shoot Ni transfer and reducing accumulation in the shoots of both populations. The values of phytoremediation indices in both populations exposed to Ni (400 µM) were above one. In presence of the SA and JA, these indices showed a decreasing trend, although the values remained above one (BAC, BCF and TF > 1). Overall, the results indicated that SA and JA can reduce phytoremediation potential of the two populations through different mechanisms.
Subject(s)
Biodegradation, Environmental , Cyclopentanes , Nickel , Oxylipins , Plant Roots , Salicylic Acid , Oxylipins/metabolism , Oxylipins/pharmacology , Nickel/metabolism , Cyclopentanes/metabolism , Cyclopentanes/pharmacology , Salicylic Acid/metabolism , Plant Roots/metabolism , Plant Roots/drug effects , Soil Pollutants/metabolism , Plant Shoots/metabolism , Plant Shoots/drug effects , Brassicaceae/metabolism , BioaccumulationABSTRACT
Microbial Ni2+ homeostasis underpins the virulence of several clinical pathogens. Ni2+ is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization and persistence. Many microbes produce metallophores to sequester metals necessary for their metabolism and starve competing neighboring organisms. The fungal metallophore aspergillomarasmine A (AMA) shows narrow specificity for Zn2+, Ni2+, and Co2+. Here, we show that this specificity allows AMA to block the uptake of Ni2+ and attenuate bacterial Ni-dependent enzymes, offering a potential strategy for reducing virulence. Bacterial exposure to AMA perturbs H2 metabolism, ureolysis, struvite crystallization, and biofilm formation and shows efficacy in a Galleria mellonella animal infection model. The inhibition of Ni-dependent enzymes was aided by Zn2+, which complexes with AMA and competes with the native nickelophore for the uptake of Ni2+. Biochemical analyses demonstrated high-affinity binding of AMA-metal complexes to NikA, the periplasmic substrate-binding protein of the Ni2+ uptake system. Structural examination of NikA in complex with Ni-AMA revealed that the coordination geometry of Ni-AMA mimics the native ligand, Ni-(L-His)2, providing a structural basis for binding AMA-metal complexes. Structure-activity relationship studies of AMA identified regions of the molecule that improve NikA affinity and offer potential routes for further developing this compound as an anti-virulence agent.
Subject(s)
Bacterial Proteins , Nickel , Nickel/metabolism , Nickel/chemistry , Animals , Virulence/drug effects , Bacterial Proteins/metabolism , Biofilms/drug effects , Zinc/metabolism , Zinc/chemistry , Moths/microbiology , Urease/metabolism , Urease/antagonists & inhibitors , Biological TransportABSTRACT
The uptake of nickel (Ni) by Asteraceae/Cichorioideae species Cichorium intybus, Leontodon hispidus and Hieracium aurantiacum exposed to Ni (0.3 or 30 µM) over 14 days and subsequent changes of metabolites were compared in order to identify their phytoaccumulation potential. Hieracium contained the most Ni (194 and 1558 µg Ni/g DW at 30 µM Ni in shoots and roots) but had unchanged amount of antioxidants (vitamin C and thiols) in the shoots and an elevated amount in the roots, which may be the reason for the absence of visible damage. On the contrary, Leontodon reacted by a decrease in antioxidants to an excess of Ni, which can be related to enhanced oxidative stress (an increase in ROS and a decrease in nitric oxide detected by fluorescence microscopy). All roots were anatomically in the secondary state and Ni-induced cell wall thickening (i.e. lignin/suberin deposition) was most visible in Hieracium roots, which also contained 2-times more Ni than the other species. Among essential elements, mainly Fe accumulation was affected by Ni excess. The content of soluble phenols increased while organic acids (malic and citric) decreased sometimes extensively (up to 90%) in individual species. PCA analyses showed that especially ascorbic acid, thiols and phenols affect the separation in the shoots especially with regard to applied concentration of Ni, while these metabolites in the roots clearly separated the species (Cichorium from the others). The data show the highest tolerance to Ni in Hieracium, but the highest phytoaccumulation of Ni was found in Cichorium (626 µg Ni/plant or 122 µg Ni/shoot at a dose of 30 µM Ni).
Subject(s)
Antioxidants , Asteraceae , Nickel , Oxidative Stress , Plant Roots , Nickel/metabolism , Antioxidants/metabolism , Asteraceae/metabolism , Plant Roots/metabolism , Oxidative Stress/drug effects , Soil Pollutants/metabolism , Reactive Oxygen Species/metabolism , Ascorbic Acid/metabolism , Plant Shoots/metabolism , Sulfhydryl Compounds/metabolismABSTRACT
Phytoremediation, an eco-friendly approach for mitigating heavy metal contamination, is reliant on hyperaccumulators. This study focused on Leersia hexandra Swart, a known chromium (Cr) hyperaccumulator with demonstrated tolerance to multiple heavy metals. Our objective was to investigate its response to simultaneous Cr and nickel (Ni) stress over 12 days. Results from physiological experiments demonstrated a significant increase in the activities of antioxidant enzymes (APX, SOD, CAT) and glutathione (GSH) content under Cr and Ni stress, indicating enhanced antioxidant mechanisms. Transcriptome analysis revealed that stress resulted in the differential expression of 27 genes associated with antioxidant activity and metal binding, including APX, SOD, CAT, GSH, metallothionein (MT), and nicotinamide (NA). Among them, twenty differentially expressed genes (DEGs) related to GSH metabolic cycle were identified. Notably, GSTU6, GND1, and PGD were the top three related genes, showing upregulation with fold changes of 4.57, 6.07, and 3.76, respectively, indicating their crucial role in metal tolerance. The expression of selected DEGs was validated by quantitative real-time PCR, confirming the reliability of RNA-Seq data. Metabolomic analysis revealed changes in 1121 metabolites, with amino acids, flavonoids, and carbohydrates being the most affected. Furthermore, glucosinolate biosynthesis and amino acid biosynthesis pathways were represented in the KEGG pathway of differentially expressed metabolites (DEMs). This study provides insights into the tolerance mechanisms of L. hexandra under the co-stress of Cr and Ni, offering a new perspective for enhancing its remediation performance.