Key Enzyme Complex Research Articles

Growth response of Bacillus coagulans on TiO2 nanoparticles by RNA-Seq analysis of the customary probiotic strain

The phosphoenolpyruvate sugar phosphotransferase systems in bacteria are complex enzyme systems carrying out various important functions like detection, phosphorylation and transport of several sugar substrates including mono and disaccharides, amino sugars, etc. This key enzyme complex prevents the efflux of sugar substrates by phosphorylation and prepares them for energy production and metabolism. In this study, a high throughput RNA sequencing method was used to compare the gene expression in Bacillus coagulans treated with nano titanium dioxide (TiO2) rutile. Out of 3175 genes, 499 were differentially expressed. Gene Ontology analysis revealed 239 significant genes. In the biological process category, we found the phosphoenolpyruvate-dependent sugar phosphotransferase systems are the dominant ones and are involved in the uptake of specific carbohydrate sources. In our previous study, we showed increased growth of bacteria in presence of nano TiO2 anatase with a sharp rise in ATP concentrations. We know that, among metal oxide-based nanoparticles, TiO2 is placed at the topmost position in the agri-food sector based on its use and their reactivity with human beneficial gut flora, commonly known as probiotics, becoming a major concern. Hence, the interaction of TiO2 rutile nanoparticles on Bacillus coagulans was studied thoroughly at the transcriptomics level; with depth.

Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration.

Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N-acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases.

A tunable metabolic valve for precise growth control and increased product formation in Pseudomonas putida

Metabolic engineering of microorganisms aims to design strains capable of producing valuable compounds under relevant industrial conditions and in an economically competitive manner. From this perspective, and beyond the need for a catalyst, biomass is essentially a cost-intensive, abundant by-product of a microbial conversion. Yet, few broadly applicable strategies focus on the optimal balance between product and biomass formation. Here, we present a genetic control module that can be used to precisely modulate growth of the industrial bacterial chassis Pseudomonas putida KT2440. The strategy is based on the controllable expression of the key metabolic enzyme complex pyruvate dehydrogenase (PDH) which functions as a metabolic valve. By tuning the PDH activity, we accurately controlled biomass formation, resulting in six distinct growth rates with parallel overproduction of excess pyruvate. We deployed this strategy to identify optimal growth patterns that improved the production yield of 2-ketoisovalerate and lycopene by 2.5- and 1.38-fold, respectively. This ability to dynamically steer fluxes to balance growth and production substantially enhances the potential of this remarkable microbial chassis for a wide range of industrial applications.

Refining the Role of Pyruvate Dehydrogenase Kinases in Glioblastoma Development.

Simple SummaryFinding new treatment strategies is an urgent need in cancer medicine, especially for the fast-growing and aggressive primary brain tumor glioblastoma (GB). Patients with GB face a median survival of about 15 months, despite tumor resection and radio-chemotherapy. In this study, we targeted cancer cell metabolism, specifically the pyruvate dehydrogenase (PDH) regulators PDH kinases (PDHK), to disturb GB progression. Indeed, PDHK1 and PDHK2 were found to be highly expressed in GB tissue cohorts and presented different expression patterns in distinct areas of the tumor. We used patient-derived stem-like cells to knockout PDHK1 or PDHK2, and dichloroacetate (DCA) to inhibit all PDHK activity. DCA showed massive in vitro effects on proliferation and invasion but no in vivo benefit as monotherapy when injected into GB-bearing mice. Finally, mice implanted with PDHK KO cells presented a higher survival rate than the control group; this effect was enhanced by performing cranial irradiation.Glioblastoma (GB) are the most frequent brain cancers. Aggressive growth and limited treatment options induce a median survival of 12–15 months. In addition to highly proliferative and invasive properties, GB cells show cancer-associated metabolic characteristics such as increased aerobic glycolysis. Pyruvate dehydrogenase (PDH) is a key enzyme complex at the crossroads between lactic fermentation and oxidative pathways, finely regulated by PDH kinases (PDHKs). PDHKs are often overexpressed in cancer cells to facilitate high glycolytic flux. We hypothesized that targeting PDHKs, by disturbing cancer metabolic homeostasis, would alter GB progression and render cells vulnerable to additional cancer treatment. Using patient databases, distinct expression patterns of PDHK1 and PDHK2 in GB tissues were obvious. To disturb protumoral glycolysis, we modulated PDH activity through the genetic or pharmacological inhibition of PDHK in patient-derived stem-like spheroids. Striking effects of PDHKs inhibition using dichloroacetate were observed in vitro on cell morphology and metabolism, resulting in increased intracellular ROS levels and decreased proliferation and invasion. In vivo findings confirmed a reduction in tumor size and better survival of mice implanted with PDHK1 and PDHK2 knockout cells. Adding a radiotherapeutic protocol further resulted in a reduction in tumor size and improved mouse survival in our model.

A Lipoate-Protein Ligase Is Required for De Novo Lipoyl-Protein Biosynthesis in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner. In the hyperthermophilic archaeon Thermococcus kodakarensis, TK1234 and TK1908 are predicted to encode the N- and C-terminal regions of Lpl, respectively. TK1908 and TK1234 recombinant proteins form a heterodimer and together displayed significant ligase activity toward octanoate in addition to lipoate when a chemically synthesized octapeptide was used as the acceptor. The proteins also displayed activity toward other fatty acids, indicating broad fatty acid specificity. On the other hand, lipoyl synthase from T. kodakarensis only recognized octanoyl-peptide as a substrate. Examination of individual proteins indicated that the TK1908 protein alone was able to catalyze the ligase reaction although with a much lower activity. Gene disruption of TK1908 led to lipoate/serine auxotrophy, whereas TK1234 gene deletion did not. Acyl carrier protein homologs are not found on the archaeal genomes, and the TK1908/TK1234 protein complex did not utilize octanoyl-CoA, raising the possibility that the substrate of the ligase reaction is octanoic acid itself. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results imply that in T. kodakarensis, the TK1908 and TK1234 proteins function in de novo lipoyl-protein biosynthesis. IMPORTANCE Based on previous studies in bacteria and eukaryotes, lipoate-protein ligases (Lpls) have been considered to be involved exclusively in lipoate salvage. The genetic analyses in this study on the lipoate-protein ligase in T. kodakarensis, however, suggest otherwise and that the enzyme is additionally involved in de novo protein lipoylation. We also provide biochemical evidence that the lipoate-protein ligase displays broad substrate specificity and is capable of ligating acyl groups of various chain-lengths to the peptide substrate. We show that this apparent ambiguity in Lpl is resolved by the strict substrate specificity of the lipoyl synthase LipS in this organism, which only recognizes octanoyl-peptide. The results provide relevant physiological insight into archaeal protein lipoylation.

Arabidopsis ACYL-ACTIVATING ENZYME 9 (AAE9) encoding an isobutyl-CoA synthetase is a key factor connecting branched-chain amino acid catabolism with iso-branched wax biosynthesis.

Iso-branched wax compounds are well known in plants, but their biosynthetic pathways are still mostly unknown. It has been speculated that branched waxes are derived from branched-chain amino acid (BCAA) catabolism, but the evidence for this is very limited. Gas chromatography-flame ionisation detection (GC-FID) analysis revealed that mutations in two subunits of the branched-chain ketoacid dehydrogenase (BCKDH) complex, a key enzyme complex in the degradation of BCAAs, significantly decreased the amounts of branched wax compounds, indicating that BCAA degradation may be integral to the synthesis of iso-branched wax. Substrate feeding studies further revealed that the metabolic precursor of iso-branched wax compounds is isobutyric acid (iBA), which is derived from valine degradation in Arabidopsis. We also isolated a novel mutant and found that its branched wax deficient phenotype could not be rescued by iBA. Map-based cloning together with complementation analysis revealed that mutation in ACYL-ACTIVATING ENZYME 9 (AAE9) is responsible for this phenotype. Genetic and enzyme activity analysis demonstrated that AAE9 is located downstream of the BCAA degradation pathway, and that it activates iBA to isobutyryl-CoA for use on branched wax synthesis. Taken together, our study demonstrates that AAE9 is a key factor connecting BCAA catabolism with branched wax biosynthesis.

Hexaconazole exposure ravages biosynthesis pathway of steroid hormones: revealed by molecular dynamics and interaction.

Widespread application of hexaconazole for agriculture purpose poses a threat to human health by disrupting normal endocrine homeostasis. To avoid adverse health effects on human, it is crucial to identify the effects of hexaconazole on key enzymes responsible for steroidal hormone synthesis. In view of this, present study was conducted to investigate the interaction mechanisms of hexaconazole with key enzymes in comparison with their food drug administration (FDA) approved inhibitor by molecular docking and molecular dynamics simulations. Results indicate that hexaconazole contacts with the active site of the key enzymes required for steroidal hormonal synthesis. Results pertaining to root-mean-square deviation, root-mean-square calculation, radius of gyration, hydrogen bonding and solvent accessible surface area exhibited that the interaction pattern and stability of interaction of hexaconazole was similar to enzyme specific inhibitor. In addition, ligand and enzyme complex interaction energy of hexaconazole was almost similar to key enzyme and FDA-approved enzyme specific inhibitor complex. This study offers a molecular level of understanding of hexaconazole with different enzymes required for steroidal hormonal synthesis. Findings of the study clearly suggest that hexaconazole has efficacy to stably interact with various enzyme required to progress the pathway of hormonal synthesis. If incessant exposure of hexaconazole occurs during agricultural work it may lead to ravage hormonal synthesis or potent endocrine disruption. The result of binding energy and complex interaction energy is depicted in the graphical abstract.

Tuning Gene Expression by Phosphate in the Methanogenic Archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

Role of succinate dehydrogenase deficiency and oncometabolites in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. At the molecular level, GISTs can be categorized into two groups based on the causative oncogenic mutations. Approximately 85% of GISTs are caused by gain-of-function mutations in the tyrosine kinase receptor KIT or platelet-derived growth factor receptor alpha (PDGFRA). The remaining GISTs, referred to as wild-type (WT) GISTs, are often deficient in succinate dehydrogenase complex (SDH), a key metabolic enzyme complex in the tricarboxylic acid (TCA) cycle and electron transport chain. SDH deficiency leads to the accumulation of succinate, a metabolite produced by the TCA cycle. Succinate inhibits α-ketoglutarate-dependent dioxygenase family enzymes, which comprise approximately 60 members and regulate key aspects of tumorigenesis such as DNA and histone demethylation, hypoxia responses, and m6A mRNA modification. For this reason, succinate and metabolites with similar structures, such as D-2-hydroxyglutarate and fumarate, are considered oncometabolites. In this article, we review recent advances in the understanding of how metabolic enzyme mutations and oncometabolites drive human cancer with an emphasis on SDH mutations and succinate in WT GISTs.

Selective linkage of mitochondrial enzymes to intracellular calcium stores differs between human-induced pluripotent stem cells, neural stem cells, and neurons.

Mitochondria and releasable endoplasmic reticulum (ER) calcium modulate neuronal calcium signaling, and both change in Alzheimer's disease (AD). The releasable calcium stores in the ER are exaggerated in fibroblasts from AD patients and in multiple models of AD. The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme complex, is diminished in brains from AD patients, and can be plausibly linked to plaques and tangles. Our previous studies in cell lines and mouse neurons demonstrate that reductions in KGDHC increase the ER releasable calcium stores. The goal of these studies was to test whether the relationship was true in human iPSC-derived neurons. Inhibition of KGDHC for one or 24hr increased the ER releasable calcium store in human neurons by 69% and 144%, respectively. The effect was mitochondrial enzyme specific because inhibiting the pyruvate dehydrogenase complex, another key mitochondrial enzyme complex, diminished the ER releasable calcium stores. The link of KGDHC to ER releasable calcium stores was cell type specific as the interaction was not present in iPSC or neural stem cells. Thus, these studies in human neurons verify a link between KGDHC and releasable ER calcium stores, and support the use of human neurons to examine mechanisms and potential therapies for AD.

Assembly of the [4Fe-4S] cluster of NFU1 requires the coordinated donation of two [2Fe-2S] clusters from the scaffold proteins, ISCU2 and ISCA1.

NFU1, a late-acting iron-sulfur (Fe-S) cluster carrier protein, has a key role in the pathogenesis of the disease, multiple mitochondrial dysfunctions syndrome. In this work, using genetic and biochemical approaches, we identified the initial scaffold protein, mitochondrial ISCU (ISCU2) and the secondary carrier, ISCA1, as the direct donors of Fe-S clusters to mitochondrial NFU1, which appears to dimerize and reductively mediate the formation of a bridging [4Fe-4S] cluster, aided by ferredoxin 2. By monitoring the abundance of target proteins that acquire their Fe-S clusters from NFU1, we characterized the effects of several novel pathogenic NFU1 mutations. We observed that NFU1 directly interacts with each of the Fe-S cluster scaffold proteins known to ligate [2Fe-2S] clusters, ISCU2 and ISCA1, and we mapped the site of interaction to a conserved hydrophobic patch of residues situated at the end of the C-terminal alpha-helix of NFU1. Furthermore, we showed that NFU1 lost its ability to acquire its Fe-S cluster when mutagenized at the identified site of interaction with ISCU2 and ISCA1, which thereby adversely affected biochemical functions of proteins that are thought to acquire their Fe-S clusters directly from NFU1, such as lipoic acid synthase, which supports the Fe-S-dependent process of lipoylation of components of multiple key enzyme complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and the glycine cleavage complex.

Cellular Mechanisms of Metabolic Remodeling During Fluid Sheer Stress-Induced Metastasis

Abstract Objectives Fluid sheer stress (FSS) is a physical stimuli of circulating tumor cells responsible for development of and progression to cancer. FSS is reported to enhance chemoresistance and proliferation in breast cancer cells. However, cellular mechanisms explaining how FSS contributes to the metastatic phenotype of breast cancer cell are less known. Chemoresistance is highly dependent upon active transport systems, and cell division and growth require ATP. In this study, we hypothesize that FSS contributes to mitochondrial remodeling and leads to alterations in energy metabolism which favor metastasis. Methods MDA-MB-231 human breast cancer cells were exposed to fluid sheer stress (FSS). MDA-MB-231 cells were then grown in culture media for 24 h, and intracellular energy (ATP) and abundance of ATP synthase were analyzed. Results FSS significantly increases intracellular ATP in MDA-MB-231 breast cancer cells. Interestingly, MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP indicating remodeling and decreased reliance on mitochondrial energy metabolism. We then quantified the abundance of ATP synthase, the key enzyme complex that produces mitochondrial ATP. FSS significantly decreased protein levels of the c-subunit of ATP synthase. Conclusions Our data show that FSS causes metabolic remodeling of mitochondria-dependent ATP production. We suggest that the c-subunit of ATP synthase is an important target of FSS-mediated metastasis. Strategies to enhance the abundance or activity of the c-subunit may prevent metabolic remodeling-associated with metastasis in FSS-exposed circulating cancer cells. Funding Sources Alabama Life Research Institute (ALRI) 14,565.

Modeling the Dynamics of Pigment Content in Cells of Dunaliella Salina Teod. Unicellular Alga at the Stage of Carotenogenesis

The work is focused on modeling of chlorophyll and carotenoids content dynamics in the in cells of the unicellular algae D. salina, living in salt water, at carotenogenesis induction phase. A mathematical model of pigments content in microalgae cells, which experience excess of light energy and the limit of nutrient medium mineral components, is proposed. The model is based on assumption, that observed rate of variation in pigment concentration is an algebraic sum of the rates of synthesis, photodestruction and partial recovery of photo-oxidized pigments. The rate of secondary carotenoids synthesis does not depend on external conditions and is determined by the quantity of key enzyme complex and its turnover rate. The rate of secondary carotenoids and chlorophyll photodestruction depends on the effective light intensity and is proportional to the amount of absorbed photosynthetically active radiation energy. The verification of the derived equations was conducted in the course of D. salina cultivation at the carotenogenesis stage. The specific rate of chlorophyll a photodestruction was determined, which resulted in 0.12 days–1. The secondary carotenoids concentration increases up to the maximum value, which is determined by the ratio of synthesis and photodestruction specific rates, as well as the maximum culture density. Under conditions of natural light in the Sevastopol region, the maximum concentration of carotenoids was 18.33 mg/l or 0.73 g/m2.

Modeling of photosynthesis light curves by linear splines

The paper presents an example of the linear splines use to describe the photosynthesis light curves for microalgae culture. The main mathematical models of the relationship between photosynthesis rate and light are listed. Based on the previously formulated basic principles of modeling microalgae photobiosynthesis, a mathematical model is proposed that describes the dependence of the assimilation number of chlorophyll a on the value of the light flux by linear splines. The advantage of the proposed approach is a clear definition of the point of change of the limiting factor. It is shown that light-limited photosynthesis rate is determined not only by external irradiation, but also by the concentration of chlorophyll a. The light-saturated rate depends on the amount of a key enzyme complex, which limits the rate of energy exchange reactions in the cell. Verification of the proposed model on the example of the diatom microalgae Skeletonema costatum was carried out. It is shown that the higher the degree of cell adaptation to high irradiation, the better the photosynthesis curve is described by linear splines. If S. costatum cells are adapted to low irradiation, deviations of experimental data from the idealized broken line are observed, which are caused by changes in the pigment composition. When the experimental data are normalized, the cell adaptation factor is reduced, all points are described by a single broken line, which indicates the universality of the proposed approach.

Lysine-14 acetylation of histone H3 in chromatin confers resistance to the deacetylase and demethylase activities of an epigenetic silencing complex.

The core CoREST complex (LHC) contains histone deacetylase HDAC1 and histone demethylase LSD1 held together by the scaffold protein CoREST. Here, we analyze the purified LHC with modified peptide and reconstituted semisynthetic mononucleosome substrates. LHC demethylase activity toward methyl-Lys4 in histone H3 is strongly inhibited by H3 Lys14 acetylation, and this appears to be an intrinsic property of the LSD1 subunit. Moreover, the deacetylase selectivity of LHC unexpectedly shows a marked preference for H3 acetyl-Lys9 versus acetyl-Lys14 in nucleosome substrates but this selectivity is lost with isolated acetyl-Lys H3 protein. This diminished activity of LHC to Lys-14 deacetylation in nucleosomes is not merely due to steric accessibility based on the pattern of sensitivity of the LHC enzymatic complex to hydroxamic acid-mediated inhibition. Overall, these studies have revealed how a single Lys modification can confer a composite of resistance in chromatin to a key epigenetic enzyme complex involved in gene silencing.

Electron Transfer to Nitrogenase in Different Genomic and Metabolic Backgrounds.

Nitrogenase catalyzes the reduction of dinitrogen (N2) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.

Succinate Dehydrogenase B (SDHB) Immunohistochemistry for the Evaluation of Muscle Biopsies.

Succinate dehydrogenase (SDH) is a key mitochondrial enzyme complex composed of 4 subunits. SDH histochemistry is routinely utilized in the assessment of muscle biopsies to reveal underlying pathology such as subsarcolemmal mitochondrial aggregates. In this study, we evaluated the utility of succinate dehydrogenase B (SDHB) immunohistochemistry (IHC) in 27 muscle biopsies, including 13 mitochondrial myopathies (MMs), 9 inflammatory myopathies, and 5 controls. SDHB IHC was performed on formalin-fixed, paraffin-embedded tissue sections with a mouse monoclonal antibody (Abcam 21A11AE7) in parallel with histochemical SDH stains on a fresh-frozen tissue. In all muscle biopsies, SDHB IHC exhibited granular immunoreactivity and highlighted the dark type 1 and lighter type 2 staining pattern observed by histochemistry. In all cases of MM, SDHB IHC showed subsarcolemmal granular aggregates involving the entire periphery of the fibers that were more distinct than those seen by SDH histochemistry. In 3 extraocular muscle biopsies, SDHB immunoreactive speckles of various sizes were distributed throughout the entire sarcoplasm that were more prominent than those seen on SDH histochemistry. Subsarcolemmal and cytoplasmic granular aggregates seen on SDHB IHC correlated with mitochondrial pathology on electron microscopy. In cases of inflammatory myopathy, there was diffuse sarcoplasmic SDHB immunoreactivity in degenerating fibers, but no evidence of subsarcolemmal aggregates. This study demonstrates that SDHB IHC is highly sensitive and specific in the identification of MM. The automation, reproducibility, and cost efficiency of SDHB IHC offer advantages over the labor-intensive histochemical method requiring frozen sections. As this technique is performed on formalin-fixed, paraffin-embedded tissues, it can be easily applied for retrospective studies.

Exploratory plasma proteomic analysis in a randomized crossover trial of aspirin among healthy men and women.

Long-term use of aspirin is associated with lower risk of colorectal cancer and other cancers; however, the mechanism of chemopreventive effect of aspirin is not fully understood. Animal studies suggest that COX-2, NFκB signaling and Wnt/β-catenin pathways may play a role, but no clinical trials have systematically evaluated the biological response to aspirin in healthy humans. Using a high-density antibody array, we assessed the difference in plasma protein levels after 60 days of regular dose aspirin (325 mg/day) compared to placebo in a randomized double-blinded crossover trial of 44 healthy non-smoking men and women, aged 21–45 years. The plasma proteome was analyzed on an antibody microarray with ~3,300 full-length antibodies, printed in triplicate. Moderated paired t-tests were performed on individual antibodies, and gene-set analyses were performed based on KEGG and GO pathways. Among the 3,000 antibodies analyzed, statistically significant differences in plasma protein levels were observed for nine antibodies after adjusting for false discoveries (FDR adjusted p-value<0.1). The most significant protein was succinate dehydrogenase subunit C (SDHC), a key enzyme complex of the mitochondrial tricarboxylic acid (TCA) cycle. The other statistically significant proteins (NR2F1, MSI1, MYH1, FOXO1, KHDRBS3, NFKBIE, LYZ and IKZF1) are involved in multiple pathways, including DNA base-pair repair, inflammation and oncogenic pathways. None of the 258 KEGG and 1,139 GO pathways was found to be statistically significant after FDR adjustment. This study suggests several chemopreventive mechanisms of aspirin in humans, which have previously been reported to play a role in anti- or pro-carcinogenesis in cell systems; however, larger, confirmatory studies are needed.

Combining ancestral sequence reconstruction with protein design to identify an interface hotspot in a key metabolic enzyme complex.

It is important to identify hotspot residues that determine protein-protein interactions in interfaces of macromolecular complexes. We have applied a combination of ancestral sequence reconstruction and protein design to identify hotspots within imidazole glycerol phosphate synthase (ImGPS). ImGPS is a key metabolic enzyme complex, which links histidine and de novo purine biosynthesis and consists of the cyclase subunit HisF and the glutaminase subunit HisH. Initial fluorescence titration experiments showed that HisH from Zymomonas mobilis (zmHisH) binds with high affinity to the reconstructed HisF from the last universal common ancestor (LUCA-HisF) but not to HisF from Pyrobaculum arsenaticum (paHisF), which differ by 103 residues. Subsequent titration experiments with a reconstructed evolutionary intermediate linking LUCA-HisF and paHisF and inspection of the subunit interface of a contemporary ImGPS allowed us to narrow down the differences crucial for zmHisH binding to nine amino acids of HisF. Homology modeling and in silico mutagenesis studies suggested that at most two of these nine HisF residues are crucial for zmHisH binding. These computational results were verified by experimental site-directed mutagenesis, which finally enabled us to pinpoint a single amino acid residue in HisF that is decisive for high-affinity binding of zmHisH. Our work shows that the identification of protein interface hotspots can be very efficient when reconstructed proteins with different binding properties are included in the analysis. Proteins 2017; 85:312-321. © 2016 Wiley Periodicals, Inc.