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1.
Biomolecules ; 11(12)2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34944503

RESUMO

This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese (TST, EC: 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC: 2.8.1.2) and cystathionine γ-lyase (CTH, EC: 4.4.1.1) in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line (NMuNG, ATCC: CRL-1636), mouse mammary gland tumor (4T1, ATCC: CRL-2539), mouse fibroblast (MEF, ATCC: SCRC-1008), mouse melanoma (B16-F1, ATCC: CRL-6323), human colorectal adenocarcinoma (Caco-2, ATCC: HTB-37), human embryonic kidney (HEK-293, ATCC: CRL-1573), human osteosarcoma (MG-63, ATCC: CRL-1427) and rat myocardium (H9c2, ATCC: CRL-1446). Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them.


Assuntos
Cistationina gama-Liase/metabolismo , Polimorfismo de Nucleotídeo Único , Sulfurtransferases/metabolismo , Tiossulfato Sulfurtransferase/metabolismo , Animais , Células CACO-2 , Linhagem Celular , Cistationina gama-Liase/genética , Cisteína/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Ratos , Enxofre/metabolismo , Sulfurtransferases/genética , Tiossulfato Sulfurtransferase/genética
2.
Elife ; 102021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34612205

RESUMO

Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.


Assuntos
Proteína de Transporte de Acila/genética , Ácidos Graxos/biossíntese , Ferro/metabolismo , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/genética , Enxofre/metabolismo , Proteína de Transporte de Acila/metabolismo , Biogênese de Organelas , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo
3.
Redox Biol ; 47: 102164, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34656823

RESUMO

Iron-sulfur (Fe-S) clusters are essential cofactors most commonly known for their role mediating electron transfer within the mitochondrial respiratory chain. The Fe-S cluster pathways that function within the respiratory complexes are highly conserved between bacteria and the mitochondria of eukaryotic cells. Within the electron transport chain, Fe-S clusters play a critical role in transporting electrons through Complexes I, II and III to cytochrome c, before subsequent transfer to molecular oxygen. Fe-S clusters are also among the binding sites of classical mitochondrial inhibitors, such as rotenone, and play an important role in the production of mitochondrial reactive oxygen species (ROS). Mitochondrial Fe-S clusters also play a critical role in the pathogenesis of disease. High levels of ROS produced at these sites can cause cell injury or death, however, when produced at low levels can serve as signaling molecules. For example, Ndufs2, a Complex I subunit containing an Fe-S center, N2, has recently been identified as a redox-sensitive oxygen sensor, mediating homeostatic oxygen-sensing in the pulmonary vasculature and carotid body. Fe-S clusters are emerging as transcriptionally-regulated mediators in disease and play a crucial role in normal physiology, offering potential new therapeutic targets for diseases including malaria, diabetes, and cancer.


Assuntos
Proteínas Ferro-Enxofre , Ferro , Biologia , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , Enxofre/metabolismo
4.
FASEB J ; 35(10): e21936, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547129

RESUMO

Lipopolysaccharide (LPS)-stimulated macrophages express an aconitate decarboxylase (IRG1, also called ACOD1), leading to accumulation of the endogenous metabolite itaconate. However, the precise mechanisms by which elevated itaconate levels alter macrophage function are not clear. Our hypothesis is itaconate affects macrophage function through some uncertain mechanism. Based on this, we established a transcriptional and proteomic signature of macrophages stimulated by itaconate and identified the pathways of IL-1ß secretion and altered iron metabolism. Consistently, the effect of IRG1 deficiency on IL-1ß secretion and iron metabolism was confirmed in IRG1 knockout THP-1 cell lines. Several common inhibitors and other compounds were used to examine the molecular mechanisms involved. Only cysteine and antioxidants (catechin hydrate) could inhibit caspase-1 activation and IL-1ß secretion in itaconate-stimulated macrophages. We further found that aconitase activity was decreased by itaconate stimulation. Our results demonstrate the counteracting effects of overexpression of mitochondrial aconitase (ACO2, a tricarboxylic acid cycle enzyme) or cytosolic aconitase (ACO1, an iron regulatory protein) on IL-1ß secretion and altered iron metabolism. Both enzyme activities were inhibited by itaconate because of iron-sulfur (Fe-S) cluster destruction. Our findings indicate that the immunoregulatory functions of IRG1 and itaconate in macrophages are stressful Fe-S cluster of aconitases disrupting and iron metabolism rebalancing.


Assuntos
Ferro/metabolismo , Macrófagos/metabolismo , Succinatos/metabolismo , Enxofre/metabolismo , Aconitato Hidratase/metabolismo , Carboxiliases/metabolismo , Linhagem Celular , Cisteína/metabolismo , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteoma/metabolismo , Proteômica
5.
Nat Commun ; 12(1): 4403, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285238

RESUMO

Sulfur cycling is ubiquitous in sedimentary environments, where it mediates organic carbon remineralization, impacting both local and global redox budgets, and leaving an imprint in pyrite sulfur isotope ratios (δ34Spyr). It is unclear to what extent stratigraphic δ34Spyr variations reflect local aspects of the depositional environment or microbial activity versus global sulfur-cycle variations. Here, we couple carbon-nitrogen-sulfur concentrations and stable isotopes to identify clear influences on δ34Spyr of local environmental changes along the Peru margin. Stratigraphically coherent glacial-interglacial δ34Spyr fluctuations (>30‰) were mediated by Oxygen Minimum Zone intensification/expansion and local enhancement of organic matter deposition. The higher resulting microbial sulfate reduction rates led to more effective drawdown and 34S-enrichment of residual porewater sulfate and sulfide produced from it, some of which is preserved in pyrite. We identify organic carbon loading as a major influence on δ34Spyr, adding to the growing body of evidence highlighting the local controls on these records.


Assuntos
Bactérias Anaeróbias/metabolismo , Sedimentos Geológicos/microbiologia , Ferro/metabolismo , Oxigênio/metabolismo , Sulfetos/metabolismo , Enxofre/metabolismo , Carbono/metabolismo , Ciclo do Carbono , Geografia , Sedimentos Geológicos/análise , Sedimentos Geológicos/química , Ferro/química , Oxirredução , Peru , Sulfetos/química , Isótopos de Enxofre/análise
6.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199378

RESUMO

Iron-sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe-S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron-sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe-2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)2 complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe-S cluster assembly.


Assuntos
Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/genética , Ferro/metabolismo , Enxofre/metabolismo , Liases de Carbono-Enxofre/genética , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Ligação Proteica/genética
7.
J Toxicol Sci ; 46(7): 341-344, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34193771

RESUMO

Reactive sulfur species (RSS) include biological persulfide molecules that protect cells against oxidative stress and heavy metal toxicity. Vascular endothelial cells regulate blood coagulation and fibrinolytic activity, and prevent vascular disorders such as atherosclerosis. We hypothesized that RSS protect vascular endothelial cells not only from nonspecific cell damage but also from specific functional damage through regulation of specific cell functions. In the present study, cultured bovine aortic endothelial cells were treated with sodium trisulfide, a sulfane sulfur donor, and both [3H]thymidine incorporation and effects on cell cycle were analyzed. These results suggest that RSS stimulate vascular endothelial cell proliferation. RSS may reduce the functional cytotoxicity of antiproliferative agents.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Enxofre/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Bovinos
8.
PLoS One ; 16(7): e0253714, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34260590

RESUMO

Co-digestion of organic biomass mixed with inorganic amendments could have an impact on composting dynamics. Various studies highlighted fertilizers' role as an additive to lesser the nitrogen loss, while some studies focused on the addition of fertilizers to enhance the efficiency. The changes in carbon, nitrogen components, and humic substances during the organic-inorganic co-compost process were seldom studied. Clarifying these changes might help improve the production process and compost nutrients contents. Thus, this study's purpose is to investigate the effects of inorganic amendments on compost characteristics, compost temperature, biochemical methane production (BMP), and nutritional contents. The inorganic phosphorous (P), sulfur (S), and sulfur solubilizing agent (SSA) were added to Farmyard manure (FYM) mixed with biodegradable waste (BW), including wheat straw, corn stalks, and green lawn waste. The P and S amended treatments were carried out into two sets, with and without SSA. The mixed feedstocks were added in the insulated RBC composting pit (15 x 15 x 10 feet). The compost material's moisture content was maintained 50-65% during the entire composting process for optimum waste digestion i.e., the moisture content (MC) of FYM was 82.7% and for BW ranged 8.8-10.2%, while the C/N ratio was found 10.5 for FYM, 74.5 for wheat straw, 83.5 for corn stalks, and 84.8 for lawn waste. At the condition of compost maturity, the inorganic amendments have no significant effect on composted material's moisture content. The maximum organic matter of 69.7% and C/N ratio of 44.6 was measured in T1. On the 6th day of composting, the temperature reached to thermophilic range (>45 oC) in all the treatments due to aeration of compost increased microbial activities and waste decomposition rate and decreased gradually to mesophilic range (35-45 oC) because the supply of high-energy compounds becomes exhausted. The highest temperature was reached in T4 (58 oC) and lowest in CT (47 oC). The significantly maximum methane of 8.95 m3 and biogas burning was 818 minutes in CT, followed by T1 and T4. The results of this study revealed that P enriched compost is a feasible and sustainable way to overcome P deficiency in the soil as well as in plants and best way to use low-grade P and organic waste material.


Assuntos
Biomassa , Compostagem/métodos , Esterco , Carbono/metabolismo , Estudos de Viabilidade , Metano/metabolismo , Nitrogênio/metabolismo , Fosfatos/metabolismo , Enxofre/metabolismo
9.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281196

RESUMO

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ferredoxinas/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/imunologia , Resistência à Doença , Ferredoxinas/imunologia , Ferredoxinas/metabolismo , Glucosinolatos/genética , Glucosinolatos/imunologia , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Família Multigênica , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Estresse Fisiológico/genética , Enxofre/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-34270399

RESUMO

A novel extreme thermophilic and piezophilic chemoorganoheterotrophic archaeon, strain EXT12cT, was isolated from a hydrothermal chimney sample collected at a depth of 2496 m at the East Pacific Rise 9° N. Cells were strictly anaerobic, motile cocci. The strain grew at NaCl concentrations ranging from 1 to 5 % (w/v; optimum, 2.0%), from pH 6.0 to 7.5 (optimum, pH 6.5-7.0), at temperatures between 60 and 95 °C (optimum, 80-85 °C), and at pressures from 0.1 to at least 50 MPa (optimum, 30 MPa). Strain EXT12cT grew chemoorganoheterotrophically on complex proteinaceous substrates. Its growth was highly stimulated by the presence of elemental sulphur or l-cystine, which were reduced to hydrogen sulfide. The DNA G+C content was 54.58 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain EXT12cT falls into the genus Thermococcus and is most closely related to Thermococcus nautili strain 30-1T. Overall genome relatedness index analyses (average nucleotide identity scores and in silico DNA-DNA hybridizations) showed a sufficient genomic distance between the new genome and the ones of the Thermococcus type strains for the delineation of a new species. On the basis of genotypic and phenotypic data, strain EXT12cT is considered to represent a novel species, for which the name Thermococcus henrietii sp. nov. is proposed, with the type strain EXT12cT (=UBOCC M-2417T=DSM 111004T).


Assuntos
Fontes Hidrotermais/microbiologia , Filogenia , Água do Mar/microbiologia , Thermococcus/classificação , Composição de Bases , DNA Arqueal/genética , Temperatura Alta , Hibridização de Ácido Nucleico , Oceano Pacífico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Enxofre/metabolismo , Thermococcus/isolamento & purificação
11.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203823

RESUMO

There are two main types of bacterial photosynthesis: oxygenic (cyanobacteria) and anoxygenic (sulfur and non-sulfur phototrophs). Molecular mechanisms of photosynthesis in the phototrophic microorganisms can differ and depend on their location and pigments in the cells. This paper describes bacteria capable of molecular oxidizing hydrogen sulfide, specifically the families Chromatiaceae and Chlorobiaceae, also known as purple and green sulfur bacteria in the process of anoxygenic photosynthesis. Further, it analyzes certain important physiological processes, especially those which are characteristic for these bacterial families. Primarily, the molecular metabolism of sulfur, which oxidizes hydrogen sulfide to elementary molecular sulfur, as well as photosynthetic processes taking place inside of cells are presented. Particular attention is paid to the description of the molecular structure of the photosynthetic apparatus in these two families of phototrophs. Moreover, some of their molecular biotechnological perspectives are discussed.


Assuntos
Chlorobi/genética , Chlorobi/fisiologia , Chromatiaceae/genética , Chromatiaceae/fisiologia , Processos Fototróficos/genética , Anaerobiose , Chlorobi/classificação , Chromatiaceae/classificação , Filogenia , Enxofre/metabolismo
12.
Appl Environ Microbiol ; 87(17): e0079421, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34190607

RESUMO

Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and the Calvin-Benson-Bassham cycle (CBB) in "Candidatus Endoriftia persephonae," the autotrophic sulfur-oxidizing bacterial endosymbiont from the giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than that by the CBB. R. pachyptila was incubated under in situ conditions in high-pressure aquaria under low (28 to 40 µmol · h-1) or high (180 to 276 µmol · h-1) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under the two conditions were capable of similar rates of CO2 fixation. Activities of the rCAC enzyme ATP-dependent citrate lyase (ACL) and the CBB enzyme 1,3-bisphosphate carboxylase/oxygenase (RubisCO) did not differ between the two conditions, although transcript abundances for ATP-dependent citrate lyase were 4- to 5-fold higher under low-sulfide conditions. δ13C values of internal dissolved inorganic carbon (DIC) pools were varied and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ13C values of lipids fell between those collected for organisms using either the rCAC or the CBB exclusively. These observations are consistent with cooccurring activities of the rCAC and the CBB in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from "omics" studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila was maintained and monitored in high-pressure aquaria prior to measuring its CO2 fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and the CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO2-fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.


Assuntos
Gammaproteobacteria/fisiologia , Poliquetos/microbiologia , Simbiose , Animais , Processos Autotróficos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/isolamento & purificação , Fontes Hidrotermais/microbiologia , Fontes Hidrotermais/parasitologia , Fotossíntese , Poliquetos/fisiologia , Sulfetos/metabolismo , Enxofre/metabolismo
13.
Science ; 373(6551): 236-241, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34083449

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.


Assuntos
Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Óxidos N-Cíclicos/farmacologia , Ferro/metabolismo , SARS-CoV-2/efeitos dos fármacos , Enxofre/metabolismo , Motivos de Aminoácidos , Animais , Antivirais/farmacologia , Sítios de Ligação , Domínio Catalítico , Chlorocebus aethiops , Coenzimas/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/farmacologia , Ferro/química , Domínios Proteicos , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Marcadores de Spin , Enxofre/química , Células Vero , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Zinco/metabolismo
14.
J Bacteriol ; 203(17): e0011721, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34124941

RESUMO

Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), and the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2 dehydrogenase), protoheme (AhbABCD), cytochrome c (Cyt c) (CcmCF), and iron storage/detoxification (Bfr, FtrA, and IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. The results indicate several prevalent but nonuniversal features, including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold, as well as its siroheme (and F430) derivatives. However, several early-diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited equivalent prevalences of [Fe-S] cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities, while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved. IMPORTANCE Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for cofactor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group. Variation in these characters was generally congruent with the phylogenetic placement of these genomes, indicating that variation in Fe/S usage and deployment has contributed to the diversification and ecology of these organisms. However, incongruency was observed among the distribution of cofactor biosynthesis pathways and known protein destinations for those cofactors, suggesting auxotrophy or yet-to-be-discovered pathways for cofactor biosynthesis.


Assuntos
Alcanos/metabolismo , Archaea/classificação , Archaea/metabolismo , Coenzimas/metabolismo , Ferro/metabolismo , Metano/metabolismo , Enxofre/metabolismo , Archaea/genética , Archaea/isolamento & purificação , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Processos Autotróficos , Vias Biossintéticas , Cisteína/metabolismo , Compostos Férricos/metabolismo , Heme/análogos & derivados , Heme/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Filogenia
15.
Nat Commun ; 12(1): 3503, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108477

RESUMO

Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate (dsrA, dsrC/tusE, soxC, soxD and soxYZ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations.


Assuntos
Bacteriófagos/metabolismo , Ecossistema , Enxofre/metabolismo , Motivos de Aminoácidos , Bacteriófagos/classificação , Bacteriófagos/genética , Caudovirales/classificação , Caudovirales/genética , Caudovirales/metabolismo , Metabolismo Energético , Microbiologia Ambiental , Genes Virais/genética , Variação Genética , Genoma Viral/genética , Metagenômica , Oxirredução , Filogenia , Domínios Proteicos , Tiossulfatos/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
16.
Sci Rep ; 11(1): 13226, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168171

RESUMO

Lignosulfonate (LS) is a by-product obtained during sulfite pulping process and is commonly used as a growth enhancer in plant growth. However, the underlying growth promoting mechanism of LS on shoot growth remains largely unknown. Hence, this study was undertaken to determine the potential application of eco-friendly ion-chelated LS complex [sodium LS (NaLS) and calcium LS (CaLS)] to enhance recalcitrant indica rice MR 219 shoot growth and to elucidate its underlying growth promoting mechanisms. In this study, the shoot apex of MR 219 rice was grown on Murashige and Skoog medium supplemented with different ion chelated LS complex (NaLS and CaLS) at 100, 200, 300 and 400 mg/L The NaLS was shown to be a better shoot growth enhancer as compared to CaLS, with optimum concentration of 300 mg/L. Subsequent comparative proteomic analysis revealed an increase of photosynthesis-related proteins [photosystem II (PSII) CP43 reaction center protein, photosystem I (PSI) iron-sulfur center, PSII CP47 reaction center protein, PSII protein D1], ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), carbohydrate metabolism-related proteins (glyceraldehyde-3-phosphate dehydrogenase 3, fructose-bisphosphate aldolase) and stress regulator proteins (peptide methionine sulfoxide reductase A4, delta-1-pyrroline-5-carboxylate synthase 1) abundance in NaLS-treated rice as compared to the control (MSO). Consistent with proteins detected, a significant increase in biochemical analyses involved in photosynthetic activities, carbohydrate metabolism and protein biosynthesis such as total chlorophyll, rubisco activity, total sugar and total protein contents were observed in NaLS-treated rice. This implies that NaLS plays a role in empowering photosynthesis activities that led to plant growth enhancement. In addition, the increased in abundance of stress regulator proteins were consistent with low levels of peroxidase activity, malondialdehyde content and phenylalanine ammonia lyase activity observed in NaLS-treated rice. These results suggest that NaLS plays a role in modulating cellular homeostasis to provide a conducive cellular environment for plant growth. Taken together, NaLS improved shoot growth of recalcitrant MR 219 rice by upregulation of photosynthetic activities and reduction of ROS accumulation leading to better plant growth.


Assuntos
Lignina/análogos & derivados , Oryza/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sódio/farmacologia , Antioxidantes/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Clorofila/metabolismo , Lignina/farmacologia , Oryza/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/metabolismo , Proteômica/métodos , Ribulose-Bifosfato Carboxilase/metabolismo , Enxofre/metabolismo
17.
Appl Environ Microbiol ; 87(17): e0069821, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34160273

RESUMO

Hypersaline microbial mats are dense microbial ecosystems capable of performing complete element cycling and are considered analogs of early Earth and hypothetical extraterrestrial ecosystems. We studied the functionality and limits of key biogeochemical processes, such as photosynthesis, aerobic respiration, and sulfur cycling, in salt crust-covered microbial mats from a tidal flat at the coast of Oman. We measured light, oxygen, and sulfide microprofiles as well as sulfate reduction rates at salt saturation and in flood conditions and determined fine-scale stratification of pigments, biomass, and microbial taxa in the resident microbial community. The salt crust did not protect the mats against irradiation or evaporation. Although some oxygen production was measurable at salinities of ≤30% (wt/vol) in situ, at saturation-level salinity (40%), oxygenic photosynthesis was completely inhibited and only resumed 2 days after reducing the porewater salinity to 12%. Aerobic respiration and active sulfur cycling occurred at low rates under salt saturation and increased strongly upon salinity reduction. Apart from high relative abundances of Chloroflexi, photoheterotrophic Alphaproteobacteria, Bacteroidetes, and Archaea, the mat contained a distinct layer harboring filamentous Cyanobacteria, which is unusual for such high salinities. Our results show that the diverse microbial community inhabiting this salt flat mat ultimately depends on periodic salt dilution to be self-sustaining and is rather adapted to merely survive salt saturation than to thrive under the salt crust. IMPORTANCE Due to their abilities to survive intense radiation and low water availability, hypersaline microbial mats are often suggested to be analogs of potential extraterrestrial life. However, even the limitations imposed on microbial processes by saturation-level salinity found on Earth have rarely been studied in situ. While abundance and diversity of microbial life in salt-saturated environments are well documented, most of our knowledge on process limitations stems from culture-based studies, few in situ studies, and theoretical calculations. In particular, oxygenic photosynthesis has barely been explored beyond 5 M NaCl (28% wt/vol). By applying a variety of biogeochemical and molecular methods, we show that despite abundance of photoautotrophic microorganisms, oxygenic photosynthesis is inhibited in salt-crust-covered microbial mats at saturation salinities, while rates of other energy generation processes are decreased several-fold. Hence, the complete element cycling required for self-sustaining microbial communities only occurs at lower salt concentrations.


Assuntos
Archaea/metabolismo , Bactérias/isolamento & purificação , Sedimentos Geológicos/microbiologia , Cloreto de Sódio/metabolismo , Archaea/classificação , Archaea/genética , Archaea/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Sedimentos Geológicos/análise , Microbiota , Oxigênio/análise , Oxigênio/metabolismo , Fotossíntese , Filogenia , Cloreto de Sódio/análise , Enxofre/análise , Enxofre/metabolismo
18.
Biochemistry ; 60(20): 1569-1572, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33938220

RESUMO

IscU serves as a scaffold for the de novo assembly of a [2Fe-2S] cluster prior to its delivery to recipient protein. It has also been proposed that on one dimer of bacterial IscU, two [2Fe-2S] clusters can be converted into a single [4Fe-4S] cluster. However, lack of structural information about the dimeric state of IscU has hindered our understanding of the underlying mechanisms. In this study, we determine the X-ray crystal structure of IscU from the thermophilic archaeon Methanothrix thermoacetophila and demonstrate a dimer structure of IscU in which two [2Fe-2S] clusters are facing each other in close proximity at the dimer interface. Our structure also reveals for the first time that Asp40 serves as a fourth ligand to the [2Fe-2S] cluster with three Cys ligands in each monomer, consistent with previous spectroscopic data. We confirm by EPR spectroscopic analysis that in solution two adjacent [2Fe-2S] clusters in the wild-type dimer are converted to a [4Fe-4S] cluster via reductive coupling. Furthermore, we find that the H106A substitution abolishes the reductive conversion to the [4Fe-4S] cluster without structural alteration, suggesting that His106 is functionally involved in this process. Overall, these findings provide a structural explanation for the assembly and conversion of Fe-S clusters on IscU and highlight a dynamic process that advances via association and dissociation of the IscU dimer.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Methanosarcinaceae/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas de Escherichia coli/fisiologia , Ferro/metabolismo , Proteínas Ferro-Enxofre/fisiologia , Relação Estrutura-Atividade , Enxofre/metabolismo
19.
J Sci Food Agric ; 101(15): 6595-6601, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33973247

RESUMO

BACKGROUND: Soybeans (Glycine max) are a major protein source both for humans and non-ruminant livestock; however, the usability of soybean protein is limited by the concentration of the essential sulfur (S)-containing amino acids methionine and cysteine (MET+CYS). Traditional efforts to improve protein quality in soybeans have largely been focused on plant breeding but soil S fertility may also influence seed MET+CYS concentration. Crop S deficiencies are increasingly common due to soil depletion by high yields and reduced atmospheric deposition. We report on a survey of commercial soybean fields and two replicated split-plot field experiments in the mid-Atlantic region, USA. The experimental treatments were two levels (0 or 100 kg S ha-1 ) of broadcast gypsum (CaSO4 ) and two levels (0 or 11 kg-S ha-1 ) of foliar Epsom salt (MgSO4 ) applied to two soybean cultivars. The objective was to assess the variability of, and effect of, S fertilization on S and MET+CYS concentrations in soybean seeds. RESULTS: Sulfur ranged from 2.35 to 3.54 mg g-1 and MET+CYS ranged from 5.5 to 9.2 mg g-1 protein in seeds from commercial fields surveyed. Sulfur application increased seed MET+CYS concentration 1.3 to twofold in two replicated field experiments. Overall, MET+CYS concentration in protein ranged from 3.9 to 12.8 mg g-1 and was linearly predicted (R2  = 0.65) by seed S. CONCLUSIONS: Soybean seed S and MET+CYS concentrations vary widely. We show that field-scale S application can greatly enhance soybean MET+CYS content and therefore protein quality. © 2021 Society of Chemical Industry.


Assuntos
Cisteína/metabolismo , Metionina/metabolismo , Solo/química , Soja/metabolismo , Enxofre/análise , Cisteína/análise , Metionina/análise , Sementes/química , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Soja/química , Soja/crescimento & desenvolvimento , Enxofre/metabolismo
20.
J Biol Chem ; 297(1): 100823, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34029591

RESUMO

Bacterial two-component flavin-dependent monooxygenases cleave the stable C-S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS-) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C-S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS- closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD's functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Multimerização Proteica , Pseudomonas fluorescens/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Mononucleotídeo de Flavina/metabolismo , Mesilatos/metabolismo , Modelos Moleculares , Especificidade por Substrato , Enxofre/metabolismo
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