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Enzymes of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily catalyze the reversible oxidation of 2-oxoacids to acyl-coenzyme A esters and carbon dioxide (CO 2 )using ferredoxin or flavodoxin as the redox partner. Although members of the family share primary sequence identity, a variety of domain and subunit arrangements are known. Here, we characterize the structure of a four-subunit family member: the pyruvate:ferredoxin oxidoreductase (PFOR) from the methane producing archaeon Methanosarcina acetivorans (MaPFOR). The 1.92 Å resolution crystal structure of MaPFOR shows a protein fold like those of single- or two-subunit PFORs that function in 2-oxoacid oxidation, including the location of the requisite thiamine pyrophosphate (TPP), and three [4Fe-4S] clusters. Of note, MaPFOR typically functions in the CO 2 reductive direction, and structural comparisons to the pyruvate oxidizing PFORs show subtle differences in several regions of catalytical relevance. These studies provide a framework that may shed light on the biochemical mechanisms used to facilitate reductive pyruvate synthesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pyruvate:quinone oxidoreductase (PQO) is a flavin-containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO 2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate-medium. A similar pattern with about 30-fold higher specific PQO activities was observed in C. glutamicum with plasmid-bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post-transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h -1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate-medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein -1 ) and turnover number (k cat : 440 vs. 78 s -1 , respectively), suggesting post-translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C-terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C-terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C-terminus for enzyme activation and lipid binding., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The effectiveness of metronidazole against the tetraploid intestinal parasite Giardia lamblia is dependent on its activation/inactivation within the cytoplasm. There are several activating enzymes, including pyruvate ferredoxin reductase (PFOR) and nitroreductase (NR) 1 which metabolize metronidazole into toxic forms, while NR2 on the other hand inactivates it. Metronidazole treatment failures have been increasing rapidly over the last decade, indicating genetic resistance mechanisms. Analyzing genetic variation in the PFOR and NR genes in susceptible and refractory Giardia isolates may help identify potential markers of resistance. Full length PFOR1, PFOR2, NR1 and NR2 genes from clinical culturable isolates and non-cultured clinical Giardia assemblage B samples were cloned, sequenced and single nucleotide variants (SNVs) were analyzed to assess genetic diversity and alleles. A similar ratio of amino acid changing SNVs per gene length was found for the NRs; 4.2% for NR1 and 6.4% for NR2, while the PFOR1 and PFOR2 genes had less variability with a ratio of 1.1% and 1.6%, respectively. One of the samples from a refractory case had a nonsense mutation which caused a truncated NR1 gene in one out of six alleles. Further, we found three NR2 alleles with frameshift mutations, possibly causing a truncated protein in two susceptible isolates. One of these isolates was homozygous for the affected NR2 allele. Three nsSNVs with potential for affecting protein function were found in the ferredoxin domain of the PFOR2 gene. The considerable variation and discovery of mutations possibly causing dysfunctional NR proteins in clinical Giardia assemblage B isolates, reveal a potential for genetic link to metronidazole susceptibility and resistance., Competing Interests: Declaration of competing interest No conflict of interest has been reported by any of the authors or funding institutions., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd 2- ) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg -1 ) and ferredoxin (Fd; 27.2 U·mg -1 ) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein -1 , consistent with the presence of three predicted [4Fe-4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Trichomoniasis, is the most prevalent non-viral sexually transmitted disease worldwide. Although metronidazole (MDZ) is the recommended treatment, several strains of the parasite are resistant to MDZ, and new treatments are required. Curcumin (CUR) is a polyphenol with anti-inflammatory, antioxidant and antiparasitic properties. In this study, we evaluated the effects of CUR on two biochemical targets: on proteolytic activity and hydrogenosomal metabolism in Trichomonas vaginalis . We also investigated the role of CUR on pro-inflammatory responses induced in RAW 264.7 phagocytic cells by parasite proteinases on pro-inflammatory mediators such as the nitric oxide (NO), tumor necrosis factor α (TNFα), interleukin-1beta (IL-1β), chaperone heat shock protein 70 (Hsp70) and glucocorticoid receptor (mGR). CUR inhibited the growth of T. vaginalis trophozoites, with an IC 50 value between 117 ± 7 μM and 173 ± 15 μM, depending on the culture phase. CUR increased pyruvate:ferredoxin oxidoreductase (PfoD), hydrogenosomal enzyme expression and inhibited the proteolytic activity of parasite proteinases. CUR also inhibited NO production and decreased the expression of pro-inflammatory mediators in macrophages. The findings demonstrate the potential usefulness of CUR as an antiparasitic and anti-inflammatory treatment for trichomoniasis. It could be used to control the disease and mitigate the associated immunopathogenic effects.

Two variants of the enzyme family pyruvate:ferredoxin oxidoreductase (PFOR), derived from the anaerobic sulfate-reducing bacterium Desulfovibrio africanus and the extremophilic crenarchaeon Sulfolobus acidocaldarius, respectively, were evaluated for their capacity to fixate CO 2 in vitro. PFOR reversibly catalyzes the conversion of acetyl-CoA and CO 2 to pyruvate using ferredoxin as redox partner. The oxidative decarboxylation of pyruvate is thermodynamically strongly favored, and most previous studies only considered the oxidative direction of the enzyme. To assay the pyruvate synthase function of PFOR during reductive carboxylation of acetyl-CoA is more challenging and requires to maintain the reaction far from equilibrium. For this purpose, a biochemical assay was established where low-potential electrons were introduced by photochemical reduction of EDTA/deazaflavin and the generated pyruvate was trapped by chemical derivatization with semicarbazide. The product of CO 2 fixation could be detected as pyruvate semicarbazone by HPLC-MS. In a combinatorial approach, both PFORs were tested with ferredoxins from different sources. The pyruvate semicarbazone product could be detected with low-potential ferredoxins of the green sulfur bacterium Chlorobium tepidum and of S. acidocaldarius whereas CO 2 fixation was not supported by the native ferredoxin of D. africanus. Methylviologen as an artificial electron carrier also allowed CO 2 fixation. For both enzymes, the results are the first demonstration of CO 2 fixation in vitro. Both enzymes exhibited high stability in the presence of oxygen during purification and storage. In conclusion, the employed PFOR enzymes in combination with non-native ferredoxin cofactors might be promising candidates for further incorporation in biocatalytic CO 2 conversion. ENZYMES: EC1.2.7.1. Pyruvate:Ferredoxin Oxidoreductase., (© 2019 Federation of European Biochemical Societies.)

The genus Mesotoga, the only described mesophilic Thermotogae lineage, is common in mesothermic anaerobic hydrocarbon-rich environments. Besides mesophily, Mesotoga displays lineage-specific phenotypes, such as no or little H 2 production and dependence on sulfur-compound reduction, which may influence its ecological role. We used comparative genomics of 18 Mesotoga strains (pairwise 16S rRNA identity >99%) and a transcriptome of M. prima to investigate how life at moderate temperatures affects phylogeography and to interrogate the genomic features of its lineage-specific metabolism. We propose that Mesotoga accomplish H 2 oxidation and thiosulfate reduction using a sulfide dehydrogenase and a hydrogenase-complex and that a pyruvate:ferredoxin oxidoreductase acquired from Clostridia is responsible for oxidizing acetate. Phylogenetic analysis revealed three distinct Mesotoga lineages (89.6%-99.9% average nucleotide identity [ANI] within lineages, 79.3%-87.6% ANI between lineages) having different geographic distribution patterns and high levels of intra-lineage recombination but little geneflow between lineages. Including data from metagenomes, phylogeographic patterns suggest that geographical separation historically has been more important for Mesotoga than hyperthermophilic Thermotoga and we hypothesize that distribution of Mesotoga is constrained by their anaerobic lifestyle. Our data also suggest that recent anthropogenic activities and environments (e.g., wastewater treatment, oil exploration) have expanded Mesotoga habitats and dispersal capabilities., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Mitochondria are not just the powerhouses of the cell; these 'end of function' organelles are crucial components of cellular physiology and influence many central metabolic and signaling pathways that support complex multicellular life. Not surprisingly, these organelles play vital roles in adaptations for extreme survival strategies including hibernation and freeze tolerance, both of which are united by requirements for a strong reduction and reprioritization of metabolic processes. To facilitate metabolic rate depression, adaptations of all aspects of mitochondrial function are required, including; energetics, physiology, abundance, gene regulation, and enzymatic controls. This review discusses these factors with a focus on the stress-specific nature of mitochondrial genes and transcriptional regulators, and processes including apoptosis and chaperone protein responses. We also analyze the regulation of glutamate dehydrogenase and pyruvate dehydrogenase, central mitochondrial enzymes involved in coordinating the shifts in metabolic fuel use associated with extreme survival strategies. Finally, an emphasis is given to the novel mitochondrial research areas of microRNAs, peptides, epigenetics, and gaseous mediators and their potential roles in facilitating hypometabolism. © 2018 IUBMB Life, 70(12):1260-1266, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Purpose: The gastrointestinal tract is home to thousands of commensal bacterial species. Therefore, competition for nutrients is paramount for successful bacterial pathogen invasion of intestinal ecosystems. The human pathogen Vibrio cholerae, the causative agent of the severe diarrhoeal disease, cholera, is able to colonize the small intestine, which is protected by mucus. However, it is unclear which metabolic pathways or nutrients V. cholerae utilizes during intestinal colonization and growth., Methodology: In this study, we investigated the effect of various metabolic key genes, including those involved in the gluconeogenesis pathway, on V. cholerae physiology and in vivo colonization., Results: We found that gluconeogenesis is important for infant mouse colonization. Growth assays showed that mutations in the key components of gluconeogenesis pathway, PpsA and PckA, lead to a growth defect in a minimal medium supplemented with mucin as a carbon source. Furthermore, the ppsA/pckA mutants colonized poorly in the adult mouse intestine, particularly when more gut commensal flora are present., Conclusion: Gluconeogenesis biosynthesis is important for the successful colonization of V. cholerae in a niche that is full of competing microbiota.

Background: Blastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children., Methods: Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity., Results: The 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower., Conclusions: Although the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs.

Mesaconate, a branched unsaturated dicarboxylic acid, has drawn great interest because of its versatile applications. In this work, we optimized the fermentation efficiency of Escherichia coli to produce mesaconate from glucose. We first drove the carbon flux to 2-ketoglutarate by overexpressing genes involved in TCA precursor pathway and anaplerotic pathways. Then, to increase the pool of phosphoenolpyruvate (PEP), an upstream precursor for 2-ketoglutarate, the phosphotransferase system (PTS) of E. coli was inactivated by deleting glucose PTS permease and the import of glucose was altered by overexpressing galactose/H + symporter GalP. Further, production optimization was achieved by deleting a class I fumarase (FumA) to block the hydration of mesaconate. Finally, we overexpressed PEP synthase (PpsA) to increase the availability of phosphoenolpyruvate and accelerate the production of mesaconate. These genetic modifications led to mesaconate production with a titer of 23.1 g L -1 and a yield of 0.46 g g -1 glucose (64% of the theoretical maximum). This work demonstrates the possibility of engineering a highly efficient bacteria strain that converts glucose into mesaconate with promising titer, rate, and yield.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a microbial enzyme that uses thiamine pyrophosphate (TPP), three [4Fe-4S] clusters, and coenzyme A (CoA) in the reversible oxidation of pyruvate to generate acetyl-CoA and carbon dioxide. The two electrons that are generated as a result of pyruvate decarboxylation are used in the reduction of low potential ferredoxins, which provide reducing equivalents for central metabolism, including the Wood-Ljungdahl pathway. PFOR is a member of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily, which plays major roles in both microbial redox reactions and carbon dioxide fixation. Here, we present a set of crystallographic snapshots of the best-studied member of this superfamily, the PFOR from Moorella thermoacetica ( Mt PFOR). These snapshots include the native structure, those of lactyl-TPP and acetyl-TPP reaction intermediates, and the first of an OFOR with CoA bound. These structural data reveal the binding site of CoA as domain III, the function of which in OFORs was previously unknown, and establish sequence motifs for CoA binding in the OFOR superfamily. Mt PFOR structures further show that domain III undergoes a conformational change upon CoA binding that seals off the active site and positions the thiolate of CoA directly adjacent to the TPP cofactor. These structural findings provide a molecular basis for the experimental observation that CoA binding accelerates catalysis by 10 5 -fold., Competing Interests: The authors declare no conflict of interest.

We describe the development of an optimized glycolytic flux biosensor and its application in detecting altered flux in a production strain and in a mutant library. The glycolytic flux biosensor is based on the Cra-regulated ppsA promoter of E. coli controlling fluorescent protein synthesis. We validated the glycolytic flux dependency of the biosensor in a range of different carbon sources in six different E. coli strains and during mevalonate production. Furthermore, we studied the flux-altering effects of genome-wide single gene knock-outs in E. coli in a multiplex FlowSeq experiment. From a library consisting of 2126 knock-out mutants, we identified 3 mutants with high-flux and 95 mutants with low-flux phenotypes that did not have severe growth defects. This approach can improve our understanding of glycolytic flux regulation improving metabolic models and engineering efforts., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Clostridium pasteurianum as an emerging new microbial cell factory can produce both n-butanol (BuOH) and 1,3-propanediol (1,3-PDO), and the pattern of product formation changes significantly with the composition of the culture medium. Among others iron content in the medium was shown to strongly affect the products selectivity. However, the mechanism behind this metabolic regulation is still unclear. For a better understanding of such metabolic regulation and for process optimization, we carried out fermentation experiments under either iron excess or iron limitation conditions, and performed metabolic, stoichiometric and proteomic analyses., Results: 1,3-PDO is most effectively produced under iron limited condition (Fe-), whereas 1,3-PDO and BuOH were both produced under iron rich condition (Fe+). With increased iron availability the BuOH/1,3-PDO ratio increased significantly from 0.27 mol/mol (at Fe-) to 1.4 mol/mol (at Fe+). Additionally, hydrogen production was enhanced significantly under Fe+ condition. Proteomic analysis revealed differentiated expression of many proteins including several ones of the central carbon metabolic pathway. Among others, pyruvate: ferredoxin oxidoreductase, hydrogenases, and several electron transfer flavoproteins was found to be strongly up-regulated under Fe+ condition, pointing to their strong involvement in the regeneration of the oxidized form of ferredoxin, and consequently their influences on the product selectivity in C. pasteurianum. Of particular significance is the finding that H 2 formation in C. pasteurianum is coupled to the ferredoxin-dependent butyryl-CoA dehydrogenase catalyzed reaction, which significantly affects the redox balance and thus the product selectivity., Conclusions: The metabolic, stoichiometric and proteomic results clearly show the key roles of hydrogenases and ferredoxins dependent reactions in determining the internal redox balance and hence product selectivity. Not only the NADH pool but also the regulation of the ferredoxin pool could explain such product variation under different iron conditions.

Shikimic acid is an important metabolic intermediate with various applications. This paper presents a novel control strategy for the construction of shikimic acid producing strains, without completely blocking the aromatic amino acid biosynthesis pathways. Growth phase-dependent expression and gene deletion was performed to regulate the aroK gene expression in the shikimic acid producing Escherichia coli strain, SK4/rpsM. In this strain, the aroL and aroK genes were deleted, and the aroB, aroG*, ppsA, and tktA genes were overexpressed. The relative amount of shikimic acid that accumulated in SK4/rpsM was 1.28-fold higher than that in SK4/pLac. Furthermore, a novel shikimic acid production pathway, combining the expression of the dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) enzyme from woody plants, was constructed in E. coli strains. The results demonstrated that a growth phase-dependent control of the aroK gene leads to higher SA accumulation (5.33 g/L) in SK5/pSK6. This novel design can achieve higher shikimic acid production by using the same amount of medium used by the current methods and can also be widely used for modifying other metabolic pathways.

Escherichia coli cells require RNase E, encoded by the essential gene rne, to propagate. The growth properties on different carbon sources of E. coli cells undergoing suppression of RNase E production suggested that reduction in RNase E is associated with decreased expression of phosphoenolpyruvate synthetase (PpsA), which converts pyruvate to phosphoenolpyruvate during gluconeogenesis. Western blotting and genetic complementation confirmed the role of RNase E in PpsA expression. Adventitious ppsA overexpression from a multicopy plasmid was sufficient to restore colony formation of ∆rne E. coli on minimal media containing glycerol or succinate as the sole carbon source. Complementation of ∆rne by ppsA overproduction was observed during growth on solid media but was only partial, and bacteria showed slowed cell division and grew as filamentous chains. We found that restoration of colony-forming ability by ppsA complementation occurred independent of the presence of endogenous RNase G or second-site suppressors of RNase E essentiality. Our investigations demonstrate the role of phosphoryl transfer catalyzable by PpsA as a determinant of RNase E essentiality in E. coli.

Unlabelled: The aim of this study was to investigate the effect of iron (Fe) availability on butyrate production in the complex bacterial ecosystem of the human gut. Hence, different Fe availabilities were mimicked in an in vitro colonic fermentation model (the polyfermenter intestinal model called PolyFermS) inoculated with immobilized gut microbiota from a child and in batch cultures of the butyrate producer Roseburia intestinalis. Shifts in the microbial community (16S rRNA sequencing and quantitative PCR), metabolic activity (high-performance liquid chromatography), and expression of genes involved in butyrate production were assessed. In the PolyFermS, moderate Fe deficiency resulted in a 1.4-fold increase in butyrate production and a 5-fold increase in butyryl-coenzyme A (CoA):acetate CoA-transferase gene expression, while very strong Fe deficiency significantly decreased butyrate concentrations and butyrate-producing bacteria compared with the results under normal Fe conditions. Batch cultures of R. intestinalis grown in a low-Fe environment preferentially produced lactate and had reduced butyrate and hydrogen production, in parallel with upregulation of the lactate dehydrogenase gene and downregulation of the pyruvate:ferredoxin-oxidoreductase gene. In contrast, under high-Fe conditions, R. intestinalis cultures showed enhanced butyrate and hydrogen production, along with increased expression of the corresponding genes, compared with the results under normal-Fe conditions. Our data reveal the strong regulatory effect of Fe on gut microbiota butyrate producers and on the concentrations of butyrate, which contributes to the maintenance of host gut health., Importance: Fe deficiency is one of the most common nutritional deficiencies worldwide and can be corrected by Fe supplementation. In this in vitro study, we show that environmental Fe concentrations in a continuous gut fermentation model closely mimicking a child's gut microbiota strongly affect the composition of the gut microbiome and its metabolic activity, particularly butyrate production. The differential expression of genes involved in the butyrate production pathway under different Fe conditions and the enzyme cofactor role of Fe explain the observed modulation of butyrate production. Our data reveal that the level of dietary Fe reaching the colon affects the microbiome, and its essential function of providing the host with beneficial butyrate., (Copyright © 2015 Dostal et al.)

Unlabelled: Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr(+) strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr(+) strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri., Importance: The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

Introduction: Giardia intestinalis is the most important and common diarrhea-causing parasitic protozoa worldwide with growing clinical relevance in public health. There are many documented cases of G. intestinalis resistance to metronidazole (MZ). Pyruvate: ferredoxin oxidoreductase (PFOR), the membrane-localized enzyme, plays a key role in the development of resistance to drugs. The aim of the present study was to evaluate the difference in the levels of PFOR gene expression between MZ-resistant and MZ-susceptible strains of G. intestinatlis., Methodology: From 159 samples with G. intestinalis cysts, 48 strains were successfully cultivated. Using specific pair primers, PFOR gene expressions were estimated in different groups of Giardia. The polymerase chain reaction (PCR) data were analyzed with Bayesian analysis of qRT-PCR data using MCMC.qpcr package, with relative expression software tool (REST) and quantitative PCR CopyCount web source., Results: In the group of Giardia with minimum inhibitory concentration (MIC) of 6.3 µM, the level of PFOR gene expression was downregulated and compared with controls, differed by 1.5 to 2.8 times. At the same time, there was no significant difference in PFOR gene expression between the control (susceptible) group and the group with MIC of 3.2 µM., Conclusions: Though there is association between PFOR gene expression and metronidazole resistance of Giardia intestinalis, the level of PFOR gene expression cannot be a strong genetic marker to predict level of resistance to metronidazole based on MICs.

Pyruvate ferredoxin oxidoreductase is the crucial enzyme that involves in bioethanol synthesis pathway of Clostridium thermocellum. It is an ethanologenic organism but has been investigated less on its enzyme structure. The amino acid sequence of Pyruvate ferredoxin oxidoreductase was derived from UNIPROT and the screened crystal structure was taken as the template for homology modeling using MODELLER 9V11. The model was loop refined and was validated using RMSD, ProSA and PROCHECK. The docking and per residue interaction studies were carried out to elucidate the interaction energies of amino acid residues with pyruvate. To enhance the binding of pyruvate with the enzyme, mutation studies were carried out by replacing Thr31 as it had a less interaction energy. Out of 10 mutants, T31N, T31Q and T31G were selected using potential energy and the residual energy calculations. Five nanoseconds explicit MD simulations were run for apo, wild type and mutants T31N, T31Q and T31G using Desmond. RMSD, RMSF, distance plots and H-bonds analysis proved T31G to be a favorable mutant for binding of pyruvate. Thus, modeling PFOR would help in profound understanding of its structural clefts and mutation studies would aid in improving the enzyme efficiency.

Ruminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO2, and 2.6 H2 when growing in batch culture and to 2 acetate, 2 CO2, and 4 H2 when growing in continuous culture in syntrophic association with H2-consuming microorganisms that keep the H2 partial pressure low. The organism uses NAD(+) and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO2, NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H2. Of all the enzymes involved, only the enzyme catalyzing the formation of H2 from NADH remained unknown. Here, we report that R. albus 7 grown in batch culture on glucose contained, besides a ferredoxin-dependent [FeFe]-hydrogenase (HydA2), a ferredoxin- and NAD-dependent electron-bifurcating [FeFe]-hydrogenase (HydABC) that couples the endergonic formation of H2 from NADH to the exergonic formation of H2 from reduced ferredoxin. Interestingly, hydA2 is adjacent to the hydS gene, which is predicted to encode an [FeFe]-hydrogenase with a C-terminal PAS domain. We showed that hydS and hydA2 are part of a larger transcriptional unit also harboring putative genes for a bifunctional acetaldehyde/ethanol dehydrogenase (Aad), serine/threonine protein kinase, serine/threonine protein phosphatase, and a redox-sensing transcriptional repressor. Since HydA2 and Aad are required only when R. albus grows at high H2 partial pressures, HydS could be a H2-sensing [FeFe]-hydrogenase involved in the regulation of their biosynthesis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H2-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H2 uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H2 yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH3, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β -keto acids.

In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H(2)) as one of several fermentation products. H(2) is generated mostly by the [Fe-Fe]-hydrogenase HYDA1, which uses plant type ferredoxin PETF/FDX1 (PETF) as an electron donor. Dark fermentation of the alga is mainly of the mixed acid type, because formate, ethanol, and acetate are generated by a pyruvate:formate lyase pathway similar to Escherichia coli. However, C. reinhardtii also possesses the pyruvate:ferredoxin oxidoreductase PFR1, which, like pyruvate:formate lyase and HYDA1, is localized in the chloroplast. PFR1 has long been suggested to be responsible for the low but significant H(2) accumulation in the dark because the catalytic mechanism of pyruvate:ferredoxin oxidoreductase involves the reduction of ferredoxin. With the aim of proving the biochemical feasibility of the postulated reaction, we have heterologously expressed the PFR1 gene in E. coli. Purified recombinant PFR1 is able to transfer electrons from pyruvate to HYDA1, using the ferredoxins PETF and FDX2 as electron carriers. The high reactivity of PFR1 toward oxaloacetate indicates that in vivo, fermentation might also be coupled to an anaerobically active glyoxylate cycle. Our results suggest that C. reinhardtii employs a clostridial type H(2) production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H(2) evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA.

Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min⁻¹ mg⁻¹ protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1-FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent K(m) values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist.

Intracellular precursor supply is a critical factor for amino acid productivity. In the present study, ppsA and tktA genes were overexpressed in genetically engineered Escherichia coli to enhance the availability of two precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate. The engineered strain, TRTH0709 carrying pSV709, produced 35.9 g/L tryptophan from glucose after 40 h in fed-batch cultivation. The two genes were inserted, independently or together, into a low-copy-number expression vector (pSTV28) and transferred to TRTH0709. Fed-batch fermentations at high cell densities of the recombination strains revealed that overexpression of the ppsA gene alone does not significantly increase tryptophan yield. On the other hand, overexpression of the tktA gene, alone or with the ppsA gene, could further improve tryptophan yield to a final tryptophan titer of 37.9 and 40.2 g/L, respectively. These results represent a 5.6% and 11.9% enhancement over the titer achieved by TRTH0709. No evident genetic modifications leading to growth impairment were observed.

Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.

We discovered the phenyllactate (PLA)-producing fungal strain Wickerhamia fluorescens TK1 and purified phenylpyruvate reductase (PPR) from fungal cell-free extracts. The PPR used both NADPH and NADH as cofactors with more preference for the former. The enzyme reaction as well as the fungal culture produced optically active d-PLA. The gene for the PPR (pprA) was cloned and expressed in Escherichia coli cells. Purified preparations of both native and recombinant PPR used hydroxyphenylpyruvate, glyoxylate and hydroxypyruvate as substrates but not pyruvate, oxaloacetate or benzoylformate. The predicted PPR protein had sequence similarity to proteins in the d-isomer-specific 2-hydroxyacid dehydrogenase family. Phylogenetic analyses indicated that the predicted PPR protein together with fungal predicted proteins constitutes a novel group of glyoxylate/hydroxypyruvate reductases. The fungus efficiently converted phenylalanine and phenylpyruvate to d-PLA. These compounds up-regulated the transcription of pprA, suggesting that it plays a role in fungal phenylalanine metabolism., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: E. coli B (BL21), unlike E.coli K-12 (JM109) is insensitive to glucose concentration and, therefore, grows faster and produces less acetate than E. coli K-12, especially when growing to high cell densities at high glucose concentration. By performing genomic analysis, it was demonstrated that the cause of this difference in sensitivity to the glucose concentration is the result of the differences in the central carbon metabolism activity. We hypothesized that the global transcription regulator Cra (FruR) is constitutively expressed in E. coli B and may be responsible for the different behaviour of the two strains. To investigate this possibility and better understand the function of Cra in the two strains, cra - negative E. coli B (BL21) and E. coli K-12 (JM109) were prepared and their growth behaviour and gene expression at high glucose were evaluated using microarray and real-time PCR., Results: The deletion of the cra gene in E. coli B (BL21) minimally affected the growth and maximal acetate accumulation, while the deletion of the same gene in E.coli K-12 (JM109) caused the cells to stop growing as soon as acetate concentration reached 6.6 g/L and the media conductivity reached 21 mS/cm. ppsA (gluconeogenesis gene), aceBA (the glyoxylate shunt genes) and poxB (the acetate producing gene) were down-regulated in both strains, while acs (acetate uptake gene) was down-regulated only in E.coli B (BL21). These transcriptional differences had little effect on acetate and pyruvate production. Additionally, it was found that the lower growth of E. coli K-12 (JM109) strain was the result of transcription inhibition of the osmoprotectant producing bet operon (betABT)., Conclusions: The transcriptional changes caused by the deletion of cra gene did not affect the activity of the central carbon metabolism, suggesting that Cra does not act alone; rather it interacts with other pleiotropic regulators to create a network of metabolic effects. An unexpected outcome of this work is the finding that cra deletion caused transcription inhibition of the bet operon in E. coli K-12 (JM109) but did not affect this operon transcription in E. coli B (BL21). This property, together with the insensitivity to high glucose concentrations, makes this the E. coli B (BL21) strain more resistant to environmental changes.

It is becoming increasingly clear that the so-called remnant organelles of microaerophilic unicellular eukaryotes, hydrogenosomes and mitosomes, are significantly reduced versions of mitochondria. They normally lack most of the classic mitochondrial attributes, such as an electron transport chain and a genome. While hydrogenosomes generate energy by substrate-level phosphorylation along a hydrogen-producing fermentation pathway, involving iron-sulfur-cluster-containing enzymes pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, whether mitosomes participate in ATP synthesis is currently unknown. Both enzymes were recently described in the mitosome-bearing diplomonad Giardia intestinalis, also shown to produce molecular hydrogen. As published data show that giardial PFO is a membrane-associated enzyme, it could be suspected that PFO and hydrogenase operate in the mitosome, in which case the latter would by definition be a hydrogenosome. Using antibodies against recombinant enzymes of G. intestinalis, it was shown by Western blot analysis of subcellular fractions and by confocal immunofluorescence microscopy of whole cells that neither PFO nor hydrogenase localize to the mitosome, but are mostly found in the cytosol. The giardial mitosome is known to play a role in iron-sulfur cluster assembly and to contain chaperones Cpn60 and mtHsp70, which assist, in particular, in protein import. In mitochondria, transmembrane potential is essential for this complex process. Using MitoTracker Red and organelle-specific antibodies, transmembrane potential could be detected in the Trichomonas vaginalis hydrogenosome, but not in the G. intestinalis mitosome. These results provide further evidence that the Giardia mitosome is one of the most highly reduced mitochondrial homologues.

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

Protists that live under low-oxygen conditions often lack conventional mitochondria and instead possess mitochondrion-related organelles (MROs) with distinct biochemical functions. Studies of mostly parasitic organisms have suggested that these organelles could be classified into two general types: hydrogenosomes and mitosomes. Hydrogenosomes, found in parabasalids, anaerobic chytrid fungi, and ciliates, metabolize pyruvate anaerobically to generate ATP, acetate, CO(2), and hydrogen gas, employing enzymes not typically associated with mitochondria. Mitosomes that have been studied have no apparent role in energy metabolism. Recent investigations of free-living anaerobic protists have revealed a diversity of MROs with a wider array of metabolic properties that defy a simple functional classification. Here we describe an expressed sequence tag (EST) survey and ultrastructural investigation of the anaerobic heteroloboseid amoeba Sawyeria marylandensis aimed at understanding the properties of its MROs. This organism expresses typical anaerobic energy metabolic enzymes, such as pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenase, and associated hydrogenase maturases with apparent organelle-targeting peptides, indicating that its MRO likely functions as a hydrogenosome. We also identified 38 genes encoding canonical mitochondrial proteins in S. marylandensis, many of which possess putative targeting peptides and are phylogenetically related to putative mitochondrial proteins of its heteroloboseid relative Naegleria gruberi. Several of these proteins, such as a branched-chain alpha keto acid dehydrogenase, likely function in pathways that have not been previously associated with the well-studied hydrogenosomes of parabasalids. Finally, morphological reconstructions based on transmission electron microscopy indicate that the S. marylandensis MROs form novel cup-like structures within the cells. Overall, these data suggest that Sawyeria marylandensis possesses a hydrogenosome of mitochondrial origin with a novel combination of biochemical and structural properties.

Protists that live in low oxygen conditions often oxidize pyruvate to acetate via anaerobic ATP-generating pathways. Key enzymes that commonly occur in these pathways are pyruvate:ferredoxin oxidoreductase (PFO) and [FeFe]-hydrogenase (H(2)ase) as well as the associated [FeFe]-H(2)ase maturase proteins HydE, HydF, and HydG. Determining the origins of these proteins in eukaryotes is of key importance to understanding the origins of anaerobic energy metabolism in microbial eukaryotes. We conducted a comprehensive search for genes encoding these proteins in available whole genomes and expressed sequence tag data from diverse eukaryotes. Our analyses of the presence/absence of eukaryotic PFO, [FeFe]-H(2)ase, and H(2)ase maturase sequences across eukaryotic diversity reveal orthologs of these proteins encoded in the genomes of a variety of protists previously not known to contain them. Our phylogenetic analyses revealed: 1) extensive lateral gene transfers of both PFO and [FeFe]-H(2)ase in eubacteria, 2) decreased support for the monophyly of eukaryote PFO domains, and 3) that eukaryotic [FeFe]-H(2)ases are not monophyletic. Although there are few eukaryote [FeFe]-H(2)ase maturase orthologs characterized, phylogenies of these proteins do recover eukaryote monophyly, although a consistent eubacterial sister group for eukaryotic homologs could not be determined. An exhaustive search for these five genes in diverse genomes from two representative eubacterial groups, the Clostridiales and the alpha-proteobacteria, shows that although these enzymes are nearly universally present within the former group, they are very rare in the latter. No alpha-proteobacterial genome sequenced to date encodes all five proteins. Molecular phylogenies and the extremely restricted distribution of PFO, [FeFe]-H(2)ases, and their associated maturases within the alpha-proteobacteria do not support a mitochondrial origin for these enzymes in eukaryotes. However, the unexpected prevalence of PFO, pyruvate:NADP oxidoreductase, [FeFe]-H(2)ase, and the maturase proteins encoded in genomes of diverse eukaryotes indicates that these enzymes have an important role in the evolution of microbial eukaryote energy metabolism.

Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thiamine pyrophosphate-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA and CO(2). The thermophilic, obligate chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates CO(2) via the reductive tricarboxylic acid cycle. In this cycle, POR acts as pyruvate synthase catalyzing the reverse reaction (i.e. reductive carboxylation of acetyl-CoA) to form pyruvate. The pyruvate synthesis reaction catalyzed by POR is an energetically unfavorable reaction and requires a strong reductant. Moreover, the reducing equivalents must be supplied via its physiological electron mediator, a small iron-sulfur protein ferredoxin. Therefore, the reaction is difficult to demonstrate in vitro and the reaction mechanism has been poorly understood. In the present study, we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxoglutarate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently low-potential electrons to reduce ferredoxin, to drive the energy-demanding pyruvate synthesis by POR. We demonstrate that H. thermophilus POR catalyzes pyruvate synthesis from acetyl-CoA and CO(2), confirming the operation of the reductive tricarboxylic acid cycle in this bacterium. We also measured the electron paramagnetic resonance spectra of the POR intermediates in both the forward and reverse reactions, and demonstrate the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical in both reactions. The reaction mechanism of the reductive carboxylation of acetyl-CoA is also discussed.

Ss-LrpB is an Lrp-like transcriptional regulator from Sulfolobus solfataricus. Previously, in vitro binding of Ss-LrpB to the control region of its own gene has been extensively studied. However, nothing was known about the physiological role of this regulator yet. Here, using the knowledge of the DNA-binding sequence specificity of Ss-LrpB, several potential binding sites were predicted in silico in promoter regions of genes located adjacently to the Ss-lrpB gene. These genes include an operon encoding a pyruvate ferredoxin oxidoreductase (porDAB) and two genes encoding putative permeases. In vitro protein-DNA interaction studies allowed the identification of the Ss-LrpB binding sites in the cognate control regions. Intriguingly, the binding site organization in the por operator is identical to that in the Ss-lrpB control region. An Ss-lrpB gene disruption mutant was constructed and the gene expression of the above-mentioned targets in this mutant was analysed by qRT-PCR and compared with isogenic wild type. Our data demonstrate that in vivo Ss-LrpB acts as an activator at the promoters of the three predicted targets. Based on these results, it appears that not all regulators belonging to the archaeal Lrp family perform a function related to the amino acid metabolism, unlike the bacterial Lrp-like regulators.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens but currently no effective vaccine is available. Our previous study showed that certain C. perfringens secreted proteins when administered intramuscularly protected chickens against experimental infection. In the current study, genes encoding three C. perfringens proteins: fructose-biphosphate-aldolase (FBA), pyruvate:ferredoxin-oxidoreductase (PFOR) and hypothetical protein (HP), were cloned into an avirulent Salmonella enterica sv. typhimurium vaccine vector. Broiler chickens immunized orally with recombinant Salmonella expressing FBA or HP proteins were significantly protected against NE challenge. Immunized birds developed serum and mucosal antibodies to both clostridial and Salmonella antigens. This study showed the oral immunizing ability of two C. perfringens antigens against NE in broiler chickens through an attenuated Salmonella vaccine vector.

Oxidative decarboxylation of pyruvate forming acetyl-coenzyme A is a crucial step in many metabolic pathways. In most anaerobes, this reaction is carried out by pyruvate-ferredoxin oxidoreductase (PFOR), an enzyme normally oxygen sensitive except in Desulfovibrio africanus (Da), where it shows an abnormally high oxygen stability. Using site-directed mutagenesis, we have specified a disulfide bond-dependent protective mechanism against oxidative conditions in Da PFOR. Our data demonstrated that the two cysteine residues forming the only disulfide bond in the as-isolated PFOR are crucial for the stability of the enzyme in oxidative conditions. A methionine residue located in the environment of the proximal [4Fe-4S] cluster was also found to be essential for this protective mechanism. In vivo analysis demonstrated unambiguously that PFOR in Da cells as well as two other Desulfovibrio species was efficiently protected against oxidative stress. Importantly, a less active but stable Da PFOR in oxidized cells rapidly reactivated when returned to anaerobic medium. Our work demonstrates the existence of an elegant disulfide bond-dependent reversible mechanism, found in the Desulfovibrio species to protect one of the key enzymes implicated in the central metabolism of these strict anaerobes. This new mechanism could be considered as an adaptation strategy used by sulfate-reducing bacteria to cope with temporary oxidative conditions and to maintain an active dormancy.

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.

Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k(cat) of 122 s(-1), and an apparent K(m) of 14 muM for menadione and 26 muM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO(2), and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO(2) fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO(2)-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO(2) fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.

Classical ideas for early eukaryotic evolution often posited a period of anaerobic evolution producing a nucleated phagocytic cell to engulf the mitochondrial endosymbiont, whose presence allowed the host to colonize emerging aerobic environments. This idea was given credence by the existence of contemporary anaerobic eukaryotes that were thought to primitively lack mitochondria, thus providing examples of the type of host cell needed. However, the groups key to this hypothesis have now been shown to contain previously overlooked mitochondrial homologues called hydrogenosomes or mitosomes; organelles that share common ancestry with mitochondria but which do not carry out aerobic respiration. Mapping these data on the unfolding eukaryotic tree reveals that secondary adaptation to anaerobic habitats is a reoccurring theme among eukaryotes. The apparent ubiquity of mitochondrial homologues bears testament to the importance of the mitochondrial endosymbiosis, perhaps as a founding event, in eukaryotic evolution. Comparative study of different mitochondrial homologues is needed to determine their fundamental importance for contemporary eukaryotic cells.

A genotyping method has been developed to distinguish each Trichomonas vaginalis isolate and has provided the first genome mapping studies of this protist with an estimated 180Mb genome. The technique was developed using high molecular weight DNA prepared from five laboratory isolates from Australia and USA and 20 clinical isolates from South Africa. Inhibition of the notorious T. vaginalis endogenous nucleases by addition of potent inhibitors was essential to the success of this study. Chromosomal DNA larger than 2.2Mb was macrorestricted to a minimum segment size of approximately 50kb, separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate generated a unique pattern that was distinguished by each of the probes. Four single copy gene probes (fd, hmp35, ibp39 and pfoD) were identified but probes which identified several bands (pfoB and alpha-scs) per isolate were most informative for genotyping. The pyruvate:ferredoxin oxidoreductase B gene probe identified two to seven copies of pfoB (or its closely related homologue pfoA) per genome in different isolates and is an obvious candidate probe to identify epidemiological linkage between infections by this genotyping method. Cleavage of the genomes into smaller fragments failed to distinguish isolates from diverse locations indicating the proximal regions of genes are conserved.

Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.