ABSTRACT
Metamonads are a diverse group of heterotrophic microbial eukaryotes adapted to living in hypoxic environments. All metamonads but one harbour metabolically altered 'mitochondrion-related organelles' (MROs) with reduced functions, however the degree of reduction varies. Here, we generate high-quality draft genomes, transcriptomes, and predicted proteomes for five recently discovered free-living metamonads. Phylogenomic analyses placed these organisms in a group we name the 'BaSk' (Barthelonids+Skoliomonads) clade, a deeply branching sister group to the Fornicata, a phylum that includes parasitic and free-living flagellates. Bioinformatic analyses of gene models shows that these organisms are predicted to have extremely reduced MRO proteomes in comparison to other free-living metamonads. Loss of the mitochondrial iron-sulfur cluster assembly system in some organisms in this group appears to be linked to the acquisition in their common ancestral lineage of a SUF-like minimal system Fe/S cluster pathway by lateral gene transfer. One of the isolates, Skoliomonas litria, appears to have lost all other known MRO pathways. No proteins were confidently assigned to the predicted MRO proteome of this organism suggesting that the organelle has been lost. The extreme mitochondrial reduction observed within this free-living anaerobic protistan clade demonstrates that mitochondrial functions may be completely lost even in free-living organisms.
Subject(s)
Mitochondria , Phylogeny , Proteome , Mitochondria/metabolism , Mitochondria/genetics , Proteome/metabolism , Proteome/genetics , Transcriptome , Eukaryota/genetics , Eukaryota/metabolism , Eukaryota/classification , Gene Transfer, Horizontal , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/geneticsABSTRACT
BACKGROUND: Glutaric aciduria type II (GA2) is a rare genetic disorder inherited in an autosomal recessive manner. Double dosage mutations in GA2 corresponding genes, ETFDH, ETFA, and ETFB, lead to defects in the catabolism of fatty acids, and amino acids lead to broad-spectrum phenotypes, including muscle weakness, developmental delay, and seizures. product of these three genes have crucial role in transferring electrons to the electron transport chain (ETC), but are not directly involve in ETC complexes. METHODS: Here, by using exome sequencing, the cause of periodic cryptic gastrointestinal complications in a 19-year-old girl was resolved after years of diagnostic odyssey. Protein modeling for the novel variant served as another line of validation for it. RESULTS: Exome Sequencing (ES) identified two variants in ETFDH: ETFDH:c.926T>G and ETFDH:c.1141G>C. These variants are likely contributing to the crisis in this case. To the best of our knowledge at the time of writing this manuscript, variant ETFDH:c.926T>G is reported here for the first time. Clinical manifestations of the case and pathological analysis are in consistent with molecular findings. Protein modeling provided another line of evidence proving the pathogenicity of the novel variant. ETFDH:c.926T>G is reported here for the first time in relation to the causation GA2. CONCLUSION: Given the milder symptoms in this case, a review of GA2 cases caused by compound heterozygous mutations was conducted, highlighting the range of symptoms observed in these patients, from mild fatigue to more severe outcomes. The results underscore the importance of comprehensive genetic analysis in elucidating the spectrum of clinical presentations in GA2 and guiding personalized treatment strategies.
Subject(s)
Electron-Transferring Flavoproteins , Heterozygote , Iron-Sulfur Proteins , Oxidoreductases Acting on CH-NH Group Donors , Humans , Female , Electron-Transferring Flavoproteins/genetics , Iron-Sulfur Proteins/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Young Adult , Brain Diseases, Metabolic/genetics , Brain Diseases, Metabolic/pathology , Brain Diseases, Metabolic/diagnosis , Mutation , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/genetics , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/pathologyABSTRACT
OBJECTIVE: To explore the clinical characteristics and genetic variants in three children with late-onset Multiple acyl-Coenzyme A dehydrogenase deficiency (MADD type â ¢). METHODS: Clinical data of three children diagnosed with late-onset MADD at the Children's Hospital Affiliated to Zhengzhou University between March 2020 and March 2022 were retrospectively analyzed. All children were subjected to whole exome sequencing (WES), and candidate variants were verified by Sanger sequencing. All children had received improved metabolic therapy and followed up for 1 ~ 3 years. RESULTS: The children had included 2 males and 1 female, and aged from 2 months to 11 years and 7 months. Child 1 had intermittent vomiting, child 2 had weakness in lower limbs, while child 3 had no symptom except abnormal neonatal screening. Tandem mass spectrometry of the three children showed elevation of multiple acylcarnitines with short, medium and long chains. Children 1 and 2 showed increased glutaric acid and multiple dicarboxylic acids by urine Gas chromatography-mass spectrometry (GC-MS) analysis. All children were found to harbor compound heterozygous variants of the ETFDH gene, including a paternal c.1211T>C (p.M404T) and a maternal c.488-22T>G variant in child 1, a paternal c.1717C>T (p.Q573X) and a maternal c.250G>A (p.A84T) variant in child 2, and a paternal c.1285+1G>A and maternal c.629A>G (p.S210N) variant in child 3. As for the treatment, high-dose vitamin B2, levocarnitine and coenzyme Q10 were given to improve the metabolism, in addition with a low fat, hypoproteinic and high carbohydrate diet. All children showed a stable condition with normal growth and development during the follow-up. CONCLUSION: The compound heterozygous variants of the ETFDH gene probably underlay the muscle weakness, remittent vomiting, elevated short, medium, and long chain acylcarnitine, as well as elevated glutaric acid and various dicarboxylic acids in the three children with type â ¢ MADD.
Subject(s)
Electron-Transferring Flavoproteins , Multiple Acyl Coenzyme A Dehydrogenase Deficiency , Humans , Male , Female , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/genetics , Infant , Child , Child, Preschool , Electron-Transferring Flavoproteins/genetics , Mutation , Retrospective Studies , Carnitine/analogs & derivatives , Carnitine/blood , Iron-Sulfur Proteins/genetics , Exome Sequencing , Oxidoreductases Acting on CH-NH Group Donors/genetics , Genetic VariationABSTRACT
Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.
Subject(s)
Iron-Sulfur Proteins , Humans , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Male , Female , Neuromuscular Diseases/genetics , Neuromuscular Diseases/enzymology , Neuromuscular Diseases/metabolism , Neuromuscular Diseases/pathology , Child , Cell Nucleus/metabolism , Cell Nucleus/enzymology , Cell Nucleus/genetics , Cytoplasm/metabolism , Cytoplasm/enzymology , MetallochaperonesABSTRACT
Maintaining the integrity of the genome is fundamental to living organisms. To this end, nature developed several mechanisms to find and promptly repair DNA lesions. Among them, base excision repair (BER) enzymes evolved to efficiently carry out this task. Notably, the mechanisms allowing these proteins to search for, detect, and fix DNA damage on a biologically relevant time scale still remain partially unclear. By taking MutY, a BER enzyme implied in the repair of the 8-oxoguanine-adenine mismatches, as a model system, we shed some light on the repair mechanism through a theoretical-computational approach. First, we estimated the effect of the oxidation state of the MutY iron-sulfur cluster on the protein-DNA binding. Then, the redox thermodynamics of both the protein cluster and DNA nucleobases are calculated. Finally, the charge migration kinetics along the double strand bound to the enzyme has been evaluated. The rationalization of our results indicates that the search for DNA lesions is essentially dictated by the redox chemistry of the species involved, i.e., the iron-sulfur redox cofactor and the DNA bound to the enzyme.
Subject(s)
DNA Glycosylases , DNA Repair , Oxidation-Reduction , DNA Glycosylases/metabolism , DNA Glycosylases/chemistry , DNA Repair/physiology , DNA/metabolism , DNA/chemistry , Kinetics , DNA Damage , Thermodynamics , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/geneticsABSTRACT
The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.
Subject(s)
Bacterial Proteins , Enterococcus faecium , Iron-Sulfur Proteins , Enterococcus faecium/genetics , Enterococcus faecium/metabolism , Enterococcus faecium/growth & development , Animals , Mice , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Anaerobiosis , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Gastrointestinal Tract/microbiology , Gastrointestinal Microbiome , Gram-Positive Bacterial Infections/microbiology , Humans , DNA Transposable Elements , Carbohydrate Metabolism , Female , AcetyltransferasesABSTRACT
All sulfur transfer pathways have generally a l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In Escherichia coli, the housekeeping l-cysteine desulfurase IscS has several interaction partners, which bind at different sites of the protein. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron-sulfur (Fe-S) cluster assembly have been mapped, in addition to TusA, which is required for molybdenum cofactor biosynthesis and mnm5s2U34 tRNA modifications, and ThiI, which is involved in thiamine biosynthesis and s4U8 tRNA modifications. Previous studies predicted that the sulfur acceptor proteins bind to IscS one at a time. E. coli TusA has, however, been suggested to be involved in Fe-S cluster assembly, as fewer Fe-S clusters were detected in a ∆tusA mutant. The basis for this reduction in Fe-S cluster content is unknown. In this work, we investigated the role of TusA in iron-sulfur cluster assembly and iron homeostasis. We show that the absence of TusA reduces the translation of fur, thereby leading to pleiotropic cellular effects, which we dissect in detail in this study.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The ferric uptake regulator plays a major role in controlling the expression of iron homeostasis genes in bacteria. We show that a ∆tusA mutant is impaired in the assembly of Fe-S clusters and accumulates iron. TusA, therefore, reduces fur mRNA translation leading to pleiotropic cellular effects.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Homeostasis , Iron-Sulfur Proteins , Iron , Repressor Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Iron/metabolism , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Carbon-Sulfur Lyases/metabolism , Carbon-Sulfur Lyases/genetics , Gene Expression Regulation, Bacterial , Sulfur/metabolism , Protein Biosynthesis , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Pteridines/metabolism , Molybdenum CofactorsABSTRACT
Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.
Subject(s)
Archaea , Hydrogen , Hydrogenase , Phylogeny , Archaea/genetics , Archaea/enzymology , Archaeal Proteins/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Genome, Archaeal , Hydrogen/metabolism , Hydrogenase/metabolism , Hydrogenase/genetics , Hydrogenase/chemistry , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/chemistry , Models, Molecular , Protein Structure, TertiaryABSTRACT
Magnetic sense, or termed magnetoreception, has evolved in a broad range of taxa within the animal kingdom to facilitate orientation and navigation. MagRs, highly conserved A-type iron-sulfur proteins, are widely distributed across all phyla and play essential roles in both magnetoreception and iron-sulfur cluster biogenesis. However, the evolutionary origins and functional diversification of MagRs from their prokaryotic ancestor remain unclear. In this study, MagR sequences from 131 species, ranging from bacteria to humans, were selected for analysis, with 23 representative sequences covering species from prokaryotes to Mollusca, Arthropoda, Osteichthyes, Reptilia, Aves, and mammals chosen for protein expression and purification. Biochemical studies revealed a gradual increase in total iron content in MagRs during evolution. Three types of MagRs were identified, each with distinct iron and/or iron-sulfur cluster binding capacity and protein stability, indicating continuous expansion of the functional roles of MagRs during speciation and evolution. This evolutionary biochemical study provides valuable insights into how evolution shapes the physical and chemical properties of biological molecules such as MagRs and how these properties influence the evolutionary trajectories of MagRs.
Subject(s)
Iron-Sulfur Proteins , Animals , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Biological Evolution , Evolution, Molecular , Phylogeny , Iron/metabolismABSTRACT
Aspergillus fumigatus is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including A. fumigatus. [2Fe-2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both A. fumigatus homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various Aspergillus species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe-2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.
Subject(s)
Aspergillus fumigatus , Cytosol , Fungal Proteins , Iron , Mitochondria , Aspergillus fumigatus/metabolism , Aspergillus fumigatus/genetics , Fungal Proteins/metabolism , Fungal Proteins/genetics , Cytosol/metabolism , Mitochondria/metabolism , Iron/metabolism , Adaptation, Physiological , Cell Nucleus/metabolism , Protein Transport , Proteomics/methods , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Gene Expression Regulation, Fungal , AcetylationABSTRACT
Glutaric aciduria type II (GAII) is a heterogeneous genetic disorder affecting mitochondrial fatty acid, amino acid and choline oxidation. Clinical manifestations vary across the lifespan and onset may occur at any time from the early neonatal period to advanced adulthood. Historically, some patients, in particular those with late onset disease, have experienced significant benefit from riboflavin supplementation. GAII has been considered an autosomal recessive condition caused by pathogenic variants in the gene encoding electron-transfer flavoprotein ubiquinone-oxidoreductase (ETFDH) or in the genes encoding electron-transfer flavoprotein subunits A and B (ETFA and ETFB respectively). Variants in genes involved in riboflavin metabolism have also been reported. However, in some patients, molecular analysis has failed to reveal diagnostic molecular results. In this study, we report the outcome of molecular analysis in 28 Australian patients across the lifespan, 10 paediatric and 18 adult, who had a diagnosis of glutaric aciduria type II based on both clinical and biochemical parameters. Whole genome sequencing was performed on 26 of the patients and two neonatal onset patients had targeted sequencing of candidate genes. The two patients who had targeted sequencing had biallelic pathogenic variants (in ETFA and ETFDH). None of the 26 patients whose whole genome was sequenced had biallelic variants in any of the primary candidate genes. Interestingly, nine of these patients (34.6%) had a monoallelic pathogenic or likely pathogenic variant in a single primary candidate gene and one patient (3.9%) had a monoallelic pathogenic or likely pathogenic variant in two separate genes within the same pathway. The frequencies of the damaging variants within ETFDH and FAD transporter gene SLC25A32 were significantly higher than expected when compared to the corresponding allele frequencies in the general population. The remaining 16 patients (61.5%) had no pathogenic or likely pathogenic variants in the candidate genes. Ten (56%) of the 18 adult patients were taking the selective serotonin reuptake inhibitor antidepressant sertraline, which has been shown to produce a GAII phenotype, and another two adults (11%) were taking a serotonin-norepinephrine reuptake inhibitor antidepressant, venlafaxine or duloxetine, which have a mechanism of action overlapping that of sertraline. Riboflavin deficiency can also mimic both the clinical and biochemical phenotype of GAII. Several patients on these antidepressants showed an initial response to riboflavin but then that response waned. These results suggest that the GAII phenotype can result from a complex interaction between monoallelic variants and the cellular environment. Whole genome or targeted gene panel analysis may not provide a clear molecular diagnosis.
Subject(s)
Electron-Transferring Flavoproteins , Multiple Acyl Coenzyme A Dehydrogenase Deficiency , Humans , Female , Male , Child , Adult , Child, Preschool , Electron-Transferring Flavoproteins/genetics , Adolescent , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/genetics , Queensland , Riboflavin/therapeutic use , Young Adult , Infant , Iron-Sulfur Proteins/genetics , Cohort Studies , Oxidoreductases Acting on CH-NH Group Donors/genetics , Infant, Newborn , Mutation , Whole Genome SequencingABSTRACT
Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.
Subject(s)
Organelles , Organelles/metabolism , Mitochondria/metabolism , Eukaryota/metabolism , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/geneticsABSTRACT
Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.
Subject(s)
Iron-Sulfur Proteins , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Methanosarcina/enzymology , Methanosarcina/genetics , Ferredoxins/metabolism , Ferredoxins/chemistry , Ferredoxins/genetics , Oxidation-Reduction , Models, Molecular , Thioredoxins/metabolism , Thioredoxins/chemistry , Thioredoxins/genetics , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Oxidoreductases/genetics , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxin-Disulfide Reductase/chemistry , Thioredoxin-Disulfide Reductase/genetics , Archaeal Proteins/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Electron Spin Resonance SpectroscopyABSTRACT
Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in Zymomonas mobilis, the substrates of IspG and IspH accumulate in cells in response to O2, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in Escherichia coli that even under anaerobic conditions, Z. mobilis IspG and IspH are not as functional as their E. coli counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of Z. mobilis IspG and IspH since no improvement in viability was observed in E. coli expressing the Z. mobilis SUF pathway or having increased expression of the E. coli SUF pathway. Complementation of single and double mutants with various combinations of Z. mobilis and E. coli IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, Z. mobilis IspH conferred an O2-sensitive growth defect to E. coli that could be partially rescued by co-expression of Z. mobilis IspG. In vitro analysis showed O2 sensitivity of the [4Fe-4S] cluster of both Z. mobilis IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in Z. mobilis IspH function under both aerobic and anaerobic conditions. IMPORTANCE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.
Subject(s)
Bacterial Proteins , Erythritol , Escherichia coli , Zymomonas , Zymomonas/metabolism , Zymomonas/enzymology , Zymomonas/genetics , Erythritol/metabolism , Erythritol/analogs & derivatives , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli/enzymology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Terpenes/metabolism , OxidoreductasesABSTRACT
AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial ironsulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and ironsulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.
Subject(s)
Aconitate Hydratase , Ferroptosis , Mice, Knockout , Myocytes, Cardiac , Phenylenediamines , Sirtuin 3 , Animals , Sirtuin 3/metabolism , Sirtuin 3/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Aconitate Hydratase/metabolism , Ferroptosis/drug effects , Mice , Acetylation , Phenylenediamines/pharmacology , Mitochondria/metabolism , Mitochondria/drug effects , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Iron/metabolism , Frataxin , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Mitochondria, Heart/metabolism , Mitochondria, Heart/drug effects , Iron-Binding Proteins/metabolism , Iron-Binding Proteins/genetics , Heart Failure/metabolism , Heart Failure/genetics , Cytosol/metabolism , CyclohexylaminesABSTRACT
Iron-sulfur (Fe-S) clusters are ubiquitous and are necessary to sustain basic life processes. The intracellular Fe-S clusters do not form spontaneously and many proteins are required for their biosynthesis and delivery. The bacterial P-loop NTPase family protein ApbC participates in Fe-S cluster assembly and transfers the cluster into apoproteins, with the Walker A motif and CxxC motif being essential for functionality of ApbC in Fe-S protein biogenesis. However, the structural basis underlying the ApbC activity and the motifs' role remains unclear. Here, we report the crystal structure of Escherichia coli ApbC at 2.8 Å resolution. The dimeric structure is in a W shape and the active site is located in the 2-fold center. The function of the motifs can be annotated by structural analyses. ApbC has an additional N-terminal domain that differs from other P-loop NTPases, possibly conferring its inherent specificity in vivo.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Iron-Sulfur Proteins , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Models, Molecular , Protein Conformation , Protein MultimerizationABSTRACT
Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.
Subject(s)
Beauveria , Electron-Transferring Flavoproteins , Beauveria/pathogenicity , Beauveria/genetics , Beauveria/enzymology , Electron-Transferring Flavoproteins/genetics , Electron-Transferring Flavoproteins/metabolism , Virulence , Fungal Proteins/genetics , Fungal Proteins/metabolism , Spores, Fungal/growth & development , Oxidoreductases/metabolism , Oxidoreductases/genetics , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Oxidoreductases Acting on CH-NH Group DonorsABSTRACT
Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how biochemical-genetic approaches using A. vinelandii to study nitrogen fixation led to the formulation of the "scaffold hypothesis" for the assembly of both simple and complex [Fe-S] clusters associated with biological nitrogen fixation. These studies also led to the discovery of a parallel, but genetically distinct, pathway for maturation of [Fe-S] proteins that support central metabolic processes.
Subject(s)
Azotobacter vinelandii , Bacterial Proteins , Iron-Sulfur Proteins , Nitrogen Fixation , Azotobacter vinelandii/metabolism , Azotobacter vinelandii/genetics , Iron-Sulfur Proteins/metabolism , Iron-Sulfur Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/geneticsABSTRACT
Pa0665 in Pseudomonas aeruginosa shares homologous sequences with that of the essential A-type iron-sulfur (Fe-S) cluster insertion protein ErpA in Escherichia coli. However, its essentiality in P. aeruginosa and its complementation with E. coli erpA has not been experimentally examined. To fulfill this task, we constructed plasmid-based ts-mutant Δpa0665/pTS-pa0665 using a three-step protocol. The mutant displayed growth defects at 42 °C, which were complemented by expressing ec.erpA. Microscopic observations indicated a petite cell phenotype for Δpa0665/pTS-pa0665 at 42 °C, correlated with the downregulation of the oprG gene. RNA sequencing revealed significant transcriptional changes in genes associated with the oxidative phosphorylation (OXPHOS) system, aligning with reduced ATP levels in Δpa0665/pTS-pa0665 under 42 °C. Additionally, the ts-mutant showed heightened sensitivity to H2O2 at 42 °C. Overall, our study demonstrates the essential role of pa0665 for OXPHOS function and is complemented by ec.erpA. We propose that the plasmid-based ts-allele is useful for genetic analysis of essential genes of interest in P. aeruginosa.
Subject(s)
Bacterial Proteins , Oxidative Phosphorylation , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mutation , Escherichia coli/genetics , Escherichia coli/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Gene Expression Regulation, BacterialABSTRACT
Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.