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Sample records for pyrococcus furiosus ferredoxin

  1. Crystallization of [Fe4S3]-ferredoxin from the hyperthermophile archaeon pyrococcus furiosus

    DEFF Research Database (Denmark)

    Nielsen, Michael Ericsson Skovbo; Harris, Pernille; Christensen, Hans Erik Mølager

    2003-01-01

    Recombinant Pyrococcus furiosus ferredoxin with a [Fe3S4]-cluster was crystallized through steps of optimization and X-ray diffraction data were collected from several crystal forms. Flat plate-like crystals were grown by hanging-drop vapour diffusion. The precipitant used was 30% PEG 400; the p...

  2. Accurate Computation of Reduction Potentials of 4Fe−4S Clusters Indicates a Carboxylate Shift in Pyrococcus furiosus Ferredoxin

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Ooi, Bee Lean; Christensen, Hans Erik Mølager

    2007-01-01

    This work describes the computation and accurate reproduction of subtle shifts in reduction potentials for two mutants of the iron-sulfur protein Pyrococcus furiosus ferredoxin. The computational models involved only first-sphere ligands and differed with respect to one ligand, either acetate (as...

  3. In situ STM imaging and direct electrochemistry of Pyrococcus furiosus ferredoxin assembled on thiolate-modified Au(111) surfaces

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Christensen, Hans Erik Mølager; Ooi, Bee Lean

    2004-01-01

    We have addressed here electron transfer (ET) of Pyrococcus furiosus ferredoxin (PfFd, 7.5 kDa) in both homogeneous solution using edge plane graphite (EPG) electrodes and in the adsorbed state by electrochemistry on surface-modified single-crystal Au(111) electrodes, This has been supported...... surface modified by the same functional group monolayer and to address diffusionless direct electrochemistry, as well as surface microstructures of the protein monolayer. PfFd molecules were found to assemble on either mercaptopropionic acid (MPA) or cysteine-modified Au(111) surfaces in stable monolayers...

  4. The 1.5 resolution structure of the [Fe4S3]-ferredoxin from the hyperthermiphilic archaeon Pyrococcus furiosus

    DEFF Research Database (Denmark)

    Nielsen, Michael Ericsson Skovbo; Harris, Pernille; Ooi, Bee Lean;

    2004-01-01

    contains a double-conformation disulfide bond existing in a left-handed and a right-handed spiral conformation. The crystal packing reveals a beta-sheet interaction, which supports the suggestion that P. furiosus ferredoxin is a functional dimer. The extraordinary thermostability of P. furiosus ferredoxin...

  5. Electronic, Magnetic, and Redox Properties of [MFe(3)S(4)] Clusters (M = Cd, Cu, Cr) in Pyrococcus furiosus Ferredoxin.

    Science.gov (United States)

    Staples, Christopher R.; Dhawan, Ish K.; Finnegan, Michael G.; Dwinell, Derek A.; Zhou, Zhi Hao; Huang, Heshu; Verhagen, Marc F. J. M.; Adams, Michael W. W.; Johnson, Michael K.

    1997-12-01

    The ground- and excited-state properties of heterometallic [CuFe(3)S(4)](2+,+), [CdFe(3)S(4)](2+,+), and [CrFe(3)S(4)](2+,+) cubane clusters assembled in Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR and variable-temperature/variable-field magnetic circular dichroism (MCD) studies. The results indicate Cd(2+) incorporation into [Fe(3)S(4)](0,-) cluster fragments to yield S = 2 [CdFe(3)S(4)](2+) and S = (5)/(2) [CdFe(3)S(4)](+) clusters and Cu(+) incorporation into [Fe(3)S(4)](+,0) cluster fragments to yield S = (1)/(2) [CuFe(3)S(4)](2+) and S = 2 [CuFe(3)S(4)](+) clusters. This is the first report of the preparation of cubane type [CrFe(3)S(4)](2+,+) clusters, and the combination of EPR and MCD results indicates S = 0 and S = (3)/(2) ground states for the oxidized and reduced forms, respectively. Midpoint potentials for the [CdFe(3)S(4)](2+,+), [CrFe(3)S(4)](2+,+), and [CuFe(3)S(4)](2+,+) couples, E(m) = -470 +/- 15, -440 +/- 10, and +190 +/- 10 mV (vs NHE), respectively, were determined by EPR-monitored redox titrations or direct electrochemistry at a glassy carbon electrode. The trends in redox potential, ground-state spin, and electron delocalization of [MFe(3)S(4)](2+,+) clusters in P. furiosus ferredoxin are discussed as a function of heterometal (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Tl).

  6. Pyrococcus furiosus strains and methods of using same

    Energy Technology Data Exchange (ETDEWEB)

    Lipscomb, Gina L; Farkas, Joel Andrew; Adams, Michael W. W.; Westpheling, Janet

    2015-01-06

    Provided herein are methods for transforming a Pyrococcus furiosus with a polynucleotide. In one embodiment, the method includes contacting a P. furiosus with a polynucleotide under conditions suitable for uptake of the polynucleotide by the P. furiosus, and identifying transformants at a frequency of, for instance, at least 10.sup.3 transformants per microgram DNA. Also provided are isolated Pyrococcus furiosus having the characteristics of Pyrococcus furiosus COM1, and plasmids that include an origin of replication that functions in a Pyrococcus furiosus. The plasmid is stable in a recipient P. furiosus without selection for more than 100 generations and is structurally unchanged after replication in P. furiosus for more than 100 generations.

  7. The tungsten metallome of Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M.; Pinkse, M.; Bol, E.; Krijgen, G.; Wolterbeek, H.; Verhaert, P.D.E.M.; Hagedoorn, P.L.; Hagen, W.R.

    2009-01-01

    The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native–native 2D-PAGE with the radioactive tungsten isotope W-187 (t1/2 = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 mM, of

  8. The tungsten metallome of Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M.; Pinkse, M.W.H.; Bol, E.; Krijger, G.C.; Wolterbeek, H.T.; Verhaert, P.; Hagedoorn, P.L.; Hagen, W.R.

    2009-01-01

    The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope W-187 (t(1/2) = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 mu M

  9. Dynamics of the [4Fe-4S] cluster in Pyrococcus furiosus D14C ferredoxin via nuclear resonance vibrational and resonance Raman spectroscopies, force field simulations, and density functional theory calculations.

    Science.gov (United States)

    Mitra, Devrani; Pelmenschikov, Vladimir; Guo, Yisong; Case, David A; Wang, Hongxin; Dong, Weibing; Tan, Ming-Liang; Ichiye, Toshiko; Jenney, Francis E; Adams, Michael W W; Yoda, Yoshitaka; Zhao, Jiyong; Cramer, Stephen P

    2011-06-14

    We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ∼400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.

  10. Dynamics of the [4Fe-4S] Cluster in Pyrococcus furiosus D14C Ferredoxin via Nuclear Resonance Vibrational and Resonance Raman Spectroscopies, Force Field Simulations, and Density Functional Theory Calculations

    Science.gov (United States)

    Mitra, Devrani; Pelmenschikov, Vladimir; Guo, Yisong; Case, David A.; Wang, Hongxin; Dong, Weibing; Tan, Ming-Liang; Ichiye, Toshiko; Jenney, Francis E.; Adams, Michael W. W.; Yoda, Yoshitaka; Zhao, Jiyong; Cramer, Stephen P.

    2011-01-01

    We have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal mode assignments, we recorded the NRVS of D14C ferredoxin samples with 36S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains: (Ph4P)2[Fe4S4Cl4]. Several distinct regions of NRVS intensity are identified, ranging from `protein' and torsional modes below 100 cm−1, through bending and breathing modes near 150 cm−1, to strong bands from Fe-S stretching modes between 250 cm−1 and ~400 cm−1. The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The 57Fe partial vibrational densities of states (PVDOS) for the oxidized samples were interpreted by normal mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe4S4]2+/1+ redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins. PMID:21500788

  11. Molybdenum incorporation in tungsten aldehyde oxidoreductase enzymes from Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M; Bevers, L.E.; Pinkse, M.W.H.; Krijger, G.C.; Wolterbeek, H.T.; Verhaert, P.D.E.M.; Hagen, W.R.; Hagedoorn, P.L.

    2010-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is no

  12. Molybdenum incorporation in tungsten aldehyde oxidoreductase enzymes from Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M; Bevers, L.E.; Pinkse, M.W.H.; Krijger, G.C.; Wolterbeek, H.T.; Verhaert, P.D.E.M.; Hagen, W.R.; Hagedoorn, P.L.

    2010-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is no

  13. Enzymology and bioenergetics of the glycolytic pathway of pyrococcus furiosus

    NARCIS (Netherlands)

    Tuininga, J.E.

    2003-01-01

    During growth ondi- and polysaccharides, the hyperthermophilic Archaeon Pyrococcusfuriosus uses a modified Embden-Meyerhof pathway

  14. A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei

    Directory of Open Access Journals (Sweden)

    Wirojne Kanoksilapatham

    2004-01-01

    Full Text Available Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 °C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S°, H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10–100 µM sodium tungstate in S°-free medium. However,P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcusfuriosus and P. woesei share three closely related families of insertion sequences (ISs. A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.

  15. Oligosaccharide synthesis by the hyperthermostable b-glucosidase from Pyrococcus furiosus: kinetics and modelling

    NARCIS (Netherlands)

    Bruins, M.E.; Strubel, M.; Lieshout, van J.F.T.; Janssen, A.E.M.; Boom, R.M.

    2003-01-01

    Oligosaccharides can be synthesised from monosaccharides or disaccharides, using glycosidases as a catalyst. To investigate the potential of this synthesis with beta-glycosidase from Pyrococcus furiosus we determined kinetic parameters for substrate conversion and product formation from cellobiose,

  16. Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration

    NARCIS (Netherlands)

    Mertens, R.; Greiner, L.; Ban, van den E.C.D.; Haaker, H.B.C.M.; Liese, A.

    2003-01-01

    The soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzy

  17. Production and Application of a Soluble Hydrogenase from Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Chang-Hao Wu

    2015-01-01

    Full Text Available Hydrogen gas is a potential renewable alternative energy carrier that could be used in the future to help supplement humanity’s growing energy needs. Unfortunately, current industrial methods for hydrogen production are expensive or environmentally unfriendly. In recent years research has focused on biological mechanisms for hydrogen production and specifically on hydrogenases, the enzyme responsible for catalyzing the reduction of protons to generate hydrogen. In particular, a better understanding of this enzyme might allow us to generate hydrogen that does not use expensive metals, such as platinum, as catalysts. The soluble hydrogenase I (SHI from the hyperthermophile Pyrococcus furiosus, a member of the euryarchaeota, has been studied extensively and used in various biotechnological applications. This review summarizes the strategies used in engineering and characterizing three different forms of SHI and the properties of the recombinant enzymes. SHI has also been used in in vitro systems for hydrogen production and NADPH generation and these systems are also discussed.

  18. Exploring the reductive capacity of Pyrococcus furiosus. The reduction of carboxylic acids and pyridine nucleotides

    NARCIS (Netherlands)

    Ban, van den E.C.D.

    2001-01-01

    This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon Pyrococcus furiosus . The research was focused on the biocatalytic reduction of carboxylic acids.Reductions of carboxylic acids are interes

  19. Identification and molecular characterization of a novel type of alpha-galactosidase from Pyrococcus furiosus

    NARCIS (Netherlands)

    Lieshout, van J.F.T.; Verhees, C.H.; Ettema, T.J.G.; Sar, van der S.; Imamura, H.; Matsuzawa, H.; Oost, van der J.; Vos, de W.M.

    2003-01-01

    An -galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first -galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other -galactosidases. Highest homology

  20. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA

    NARCIS (Netherlands)

    Swarts, D.C.; Hegge, J.W.; Hinojo, Ismael; Shiimori, Masami; Ellis, Michael A.; Dumrongkulraksa, Justin; Terns, Rebecca M.; Terns, Michael P.; Oost, Van Der John

    2015-01-01

    Functions of prokaryotic Argonautes (pAgo) have long remained elusive. Recently, Argonautes of the bacteria Rhodobacter sphaeroides and Thermus thermophilus were demonstrated to be involved in host defense. The Argonaute of the archaeon Pyrococcus furiosus (PfAgo) belongs to a different branch in

  1. Exploring the reductive capacity of Pyrococcus furiosus : the reduction of carboxylic acids and pyridine nucleotides

    NARCIS (Netherlands)

    Ban, van den E.C.D.

    2001-01-01

    This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon Pyrococcus furiosus . The research was focused on the biocatalytic reduction of carboxylic

  2. Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Machielsen, M.P.; Oost, van der J.

    2006-01-01

    The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of ¿ 155 kDa.

  3. Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase

    NARCIS (Netherlands)

    Berrisford, J.M.; Hounslow, A.M.; Akerboom, A.P.; Hagen, W.R.; Brouns, S.J.J.; Oost, van der J.; Murray, I.A.; Blackburn, G.M.; Waltho, J.P.; Rice, D.W.; Baker, P.J.

    2006-01-01

    The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPG

  4. Identification and molecular characterization of a novel type of alpha-galactosidase from Pyrococcus furiosus

    NARCIS (Netherlands)

    Lieshout, van J.F.T.; Verhees, C.H.; Ettema, T.J.G.; Sar, van der S.; Imamura, H.; Matsuzawa, H.; Oost, van der J.; Vos, de W.M.

    2003-01-01

    An -galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first -galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other -galactosidases. Highest homology

  5. Crystal structure of Pyrococcus furiosus phosphoglucose isomerase: Implications for substrate binding and catalysis

    NARCIS (Netherlands)

    Berrisford, J.M.; Akerboom, A.P.; Turnbull, A.P.; Geus, de D.; Sedelnikova, S.E.; Staton, I.; McLeod, C.W.; Verhees, C.H.; Oost, van der J.; Rice, D.W.; Baker, P.J.

    2003-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between D-fructose 6-phosphate and D-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-Angstrom

  6. Adsorption of an endoglucanase from the hyperthermophilic Pyrococcus furiosus of hydrophobic (polysterene) and hydrophilic (silica) surfaces increases protein heat stability

    NARCIS (Netherlands)

    Koutsopoulos, S.; Oost, van der J.; Norde, W.

    2004-01-01

    The interaction of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus with two types of surfaces, that is, hydrophobic polystyrene and hydrophilic silica, was investigated, and the adsorption isotherms were determined. The adsorbed hyperthermostable enzyme did not undergo

  7. Adsorption of an endoglucanase from the hyperthermophilic Pyrococcus furiosus on hydrophobic (polystyrene) and hydrophilic (silica) surfaces increases protein heat stability

    NARCIS (Netherlands)

    Koutsopoulos, S.; van der Oost, J.; Norde, Willem

    2004-01-01

    The interaction of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus with two types of surfaces, that is, hydrophobic polystyrene and hydrophilic silica, was investigated, and the adsorption isotherms were determined. The adsorbed hyperthermostable enzyme did not undergo

  8. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

    Directory of Open Access Journals (Sweden)

    Hui eYuan

    2015-09-01

    Full Text Available Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of Pyrococcus furiosus proteins at whole genome level, we constructed expression plasmids of each Pyrococcus furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3pLysS. In summary, this recombinant expression library of Pyrococcus furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.

  9. Exploring the reductive capacity of Pyrococcus furiosus. The reduction of carboxylic acids and pyridine nucleotides

    OpenAIRE

    Ban, van den, A.W.

    2001-01-01

    This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon Pyrococcus furiosus . The research was focused on the biocatalytic reduction of carboxylic acids.Reductions of carboxylic acids are interesting reactions, since the generated products, aldehydes and alcohols, are potentially applicable in the fine-chemical industry. However, the reduction of carboxylic acids to the corresponding aldehydes is a thermodynamicall...

  10. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Gerrit Jan Schut

    2016-01-01

    Full Text Available Carbon monoxide (CO is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a carbon monoxide dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally-relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.

  11. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

    Science.gov (United States)

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-01

    Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms. PMID:26858706

  12. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria

    2015-03-13

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption by the ferritin. Thermostable ferritin proved to be an excellent system for rapid phosphate and arsenate removal from aqueous solutions down to residual concentrations at the picomolar level. These very low concentrations make thermostable ferritin a potential tool to considerably mitigate industrial biofouling by phosphate limitation or to remove arsenate from drinking water.

  13. A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology.

    Science.gov (United States)

    Lewis, Derrick L; Notey, Jaspreet S; Chandrayan, Sanjeev K; Loder, Andrew J; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2015-03-01

    A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

  14. Increased susceptibility of ß-glucosidase from the hyperthermophile Pyrococcus furiosus to thermal inactivation at higher pressures

    NARCIS (Netherlands)

    Bruins, M.E.; Meersman, F.; Janssen, A.E.M.; Heremans, K.; Boom, R.M.

    2009-01-01

    The stability of ß-glucosidase from the hyperthermophile Pyrococcus furiosus was studied as a function of pressure, temperature and pH. The conformational stability was monitored using FTIR spectroscopy, and the functional enzyme stability was monitored by inactivation studies. The enzyme proved to

  15. Biochemical evidence for the presence of two α-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, Sonja M.; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of α-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports tr

  16. Hydrolysis of isoflavone glycosides by a thermostable β-glucosidase from Pyrococcus furiosus.

    Science.gov (United States)

    Yeom, Soo-Jin; Kim, Bi-Na; Kim, Yeong-Su; Oh, Deok-Kun

    2012-02-15

    The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.

  17. Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

    Science.gov (United States)

    2012-04-26

    12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS prolidase, organophosphate , OP nerve agent, thermostable enzyme, Pyrococcus furiosus...Singh, 2009). Annually, there are an estimated 3 million poisonings and 300,000 human deaths owing to OP compounds (Singh, 2009). There is a need... organophosphate anhydrolase/prolidase. Biochemistry, 49, 547-59. Wang, Q., Sun, M., Zhang, H. & Huang, C. 1998. Purification and properties of soman

  18. Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcus furiosus

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    Rahman M. Mizanur

    2008-01-01

    Full Text Available Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535 from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml–1 glycogen concentration was 52 U mg–1 and 31 U mg–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min–1 µg–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.

  19. Mutational analyses of the enzymes involved in the metabolism of hydrogen by the hyperthermophilic archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Gerrit J Schut

    2012-05-01

    Full Text Available Pyrococcus furiosus grows optimally near 100°C by fermenting carbohydrates to produce hydrogen (H2 or, if elemental sulfur (S0, is present hydrogen sulfide instead. It contains two cytoplasmic hydrogenases, SHI and SHII, that use NADP(H as an electron carrier, and a membrane bound hydrogenase (MBH, that utilizes the redox protein ferredoxin. We previously constructed deletion strains lacking SHI and/or SHII and showed that they exhibited no obvious phenotype. This study has now been extended to include biochemical analyses and growth studies using the ΔSHI and ΔSHII deletion strains together with strains lacking a functional MBH (ΔMbhL. Hydrogenase activities in cytoplasmic extracts of ΔSHII and the parent strain were similar but were much lower (<10% in the ΔSHI strain, and no activity was detected in the ΔSHIΔSHII double deletion strain, indicating that SHI is responsible for most of the cytoplasmic hydrogenase activity. In contrast, the ΔmbhL strain showed no growth in the absence of S0, confirming the hypothesis that, in the absence of S0, MBH is the only enzyme that can dispose of reductant (as H2 generated during sugar oxidation. The deletion strain devoid of all three hydrogenases also grew only in the presence of S0 and did not produce any detectable H2. When grown in the presence of limiting S0, both H2S and H2 were produced by the parent and ΔSHI/ΔSHII strains. A significant amount of H2 was also produced by the ΔmbhL strain, showing that SHI can produce H2 from NADPH in vivo, although this does not enable significant growth of ΔmbhL in the absence of S0. We propose that the physiological function of SHI is to recycle H2 and provide a link between external H2 and the intracellular pool of NADPH needed for biosynthesis. This likely has a distinct energetic advantage in the environment, but it is clearly not required for growth of the organism under the usual laboratory conditions. The function of SHII, however, remains

  20. Differential signal transduction via TrmB, a sugar sensing transcriptional repressor of Pyrococcus furiosus.

    Science.gov (United States)

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-06-01

    TrmB is a transcriptional repressor of the hyperthermophilic archaeon Pyrococcus furiosus serving at least two operons. TrmB represses genes encoding an ABC transporter for trehalose and maltose (the TM system) with trehalose and maltose as inducers. TrmB also represses genes encoding another ABC transporter for maltodextrins (the MD system) with maltotriose and sucrose as inducers. Here we report that glucose which was also bound by TrmB acted as a corepressor (causing stronger repression) for both the TM and the MD system. Binding of glucose by TrmB was increased in the presence of TM promoter DNA. Maltose which acted as inducer for the TM system acted as a corepressor for the MD system intensifying repression. We propose that the differential conformational changes of TrmB in response to binding the different sugars governs the ability of TrmB to interact with the promoter region and represents a simple mechanism for selecting the usage of one carbon source over the other, reminiscent of catabolite repression in bacteria.

  1. Coupled TLC and MALDI-TOF/MS Analyses of the Lipid Extract of the Hyperthermophilic Archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Simona Lobasso

    2012-01-01

    Full Text Available The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers and the remaining part by caldarchaeol lipids (tetraethers. The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

  2. N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

    Science.gov (United States)

    Chang, Michelle M; Imperiali, Barbara; Eichler, Jerry; Guan, Ziqiang

    2015-01-01

    In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

  3. N-Linked Glycans Are Assembled on Highly Reduced Dolichol Phosphate Carriers in the Hyperthermophilic Archaea Pyrococcus furiosus.

    Directory of Open Access Journals (Sweden)

    Michelle M Chang

    Full Text Available In all three domains of life, N-glycosylation begins with the assembly of glycans on phosphorylated polyisoprenoid carriers. Like eukaryotes, archaea also utilize phosphorylated dolichol for this role, yet whereas the assembled oligosaccharide is transferred to target proteins from dolichol pyrophosphate in eukaryotes, archaeal N-linked glycans characterized to date are derived from a dolichol monophosphate carrier, apart from a single example. In this study, glycan-charged dolichol phosphate from the hyperthermophile Pyrococcus furiosus was identified and structurally characterized. Normal and reverse phase liquid chromatography-electrospray ionization mass spectrometry revealed the existence of dolichol phosphate charged with the heptasaccharide recently described in in vitro studies of N-glycosylation on this species. As with other described archaeal dolichol phosphates, the α- and ω-terminal isoprene subunits of the P. furiosus lipid are saturated, in contrast to eukaryal phosphodolichols that present only a saturated α-position isoprene subunit. Interestingly, an additional 1-4 of the 12-14 isoprene subunits comprising P. furiosus dolichol phosphate are saturated, making this lipid not only the longest archaeal dolichol phosphate described to date but also the most highly saturated.

  4. Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.

    Science.gov (United States)

    Tanabe, Maiko; Ishino, Sonoko; Ishino, Yoshizumi; Nishida, Hirokazu

    2014-01-21

    The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

  5. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Meagher, Martin; Enemark, Eric J.

    2016-06-22

    The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

  6. Crystal structure and nucleic acid-binding activity of the CRISPR-associated protein Csx1 of Pyrococcus furiosus.

    Science.gov (United States)

    Kim, Young Kwan; Kim, Yeon-Gil; Oh, Byung-Ha

    2013-02-01

    In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C-terminal domain of PF1127 has a unique β-hairpin structure that protrudes out of an α-helix and contains several positively charged residues. We demonstrate that PF1127 binds double-stranded DNA and RNA and that this activity requires an intact β-hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β-hairpin structure and exhibits no DNA-binding properties under the same experimental conditions. Notably, the C-terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N-terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid-binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Copyright © 2012 Wiley Periodicals, Inc.

  7. Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

    Science.gov (United States)

    Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi

    2003-05-15

    We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

  8. Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri.

    Science.gov (United States)

    Voorhorst, W G; Warner, A; de Vos, W M; Siezen, R J

    1997-08-01

    The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate-binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.

  9. X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus, and its cys-free mutant.

    Science.gov (United States)

    Tanaka, H; Chinami, M; Mizushima, T; Ogasahara, K; Ota, M; Tsukihara, T; Yutani, K

    2001-07-01

    In order to elucidate the mechanism of the thermostability of proteins from hyperthermophiles, X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus (PfPCP), and its mutant protein with Ser substituted at Cys142 and Cys188 were determined at 2.2 and 2.7 A resolution, respectively. The obtained structures were compared with those previously reported for pyrrolidone carboxyl peptidases from a hyperthermophilie, Thermococcus litoralis (TlPCP), and from a mesophile, Bacillus amyloliquefaciens (BaPCP). The PfPCP structure is a tetramer of four identical subunits similar to that of the TlPCP and BaPCP. The largest structural changes among the three PCPs were detected in the C-terminal protrusion, which interacts with that of another subunit. A comparison of the three structures indicated that the high stability of PfPCP is caused by increases in hydrophobic interactions and hydrogen bonds, the formation of an intersubunit ion-pair network, and improvement to an ideal conformation. On the basis of the structures of the three proteins, it can be concluded that PfPCP does not have any special factors responsible for its extremely high stability and that the conformational structure of PfPCP is superior in its combination of positive and negative stabilizing factors compared with BaPCP.

  10. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  11. Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity

    Directory of Open Access Journals (Sweden)

    Fanghua Wang

    2016-05-01

    Full Text Available Glycerophosphodiester phosphodiesterases (GDPD are enzymes which degrade various glycerophosphodiesters to produce glycerol-3-phosphate and the corresponding alcohol moiety. Apart from this, a very interesting finding is that this enzyme could be used in the degradation of toxic organophosphorus esters, which has resulted in much attention on the biochemical and application research of GDPDs. In the present study, a novel GDPD from Pyrococcus furiosus DSM 3638 (pfGDPD was successfully expressed in Escherichia coli and biochemically characterized. This enzyme hydrolyzed bis(p-nitrophenyl phosphate, one substrate analogue of organophosphorus diester, with an optimal reaction temperature 55 °C and pH 8.5. The activity of pfGDPD was strongly dependent on existing of bivalent cations. It was strongly stimulated by Mn2+ ions, next was Co2+ and Ni2+ ions. Further investigations were conducted on its substrate selectivity towards different phospholipids. The results indicated that except of glycerophosphorylcholine (GPC, this enzyme also possessed lysophospholipase D activity toward both sn1-lysophosphatidylcholine (1-LPC and sn2-lysophosphatidylcholine (2-LPC. Higher activity was found for 1-LPC than 2-LPC; however, no hydrolytic activity was found for phosphatidylcholine (PC. Molecular docking based on the 3D-modeled structure of pfGDPD was conducted in order to provide a structural foundation for the substrate selectivity.

  12. Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.

    OpenAIRE

    Hayashi, I; Morikawa, K; ISHINO, Y.

    1999-01-01

    Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purifie...

  13. Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.

    OpenAIRE

    I. Hayashi; Morikawa, K.; Ishino, Y

    1999-01-01

    Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purifie...

  14. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [University of Georgia; W. W. Adams, Michael

    2014-01-07

    Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

  15. Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable beta-glycosylhydrolase CelB from Pyrococcus Furiosus.

    Science.gov (United States)

    Lang, Markus; Kamrat, Thomas; Nidetzky, Bernd

    2006-12-20

    The synthesis of glycosides by enzymatic transglycosylation is a kinetically controlled reaction performed in the context of a non-favorable thermodynamic equilibrium. An unreactive organic cosolvent which increases the selectivity of the enzyme for glycosyl transfer to the acceptor nucleophile compared with water (Ksel) could improve maximum product yield. Here we report on the effect of the ionic liquid 1,3-dimethylimidazoliummethylsulfate on hydrolase and transferase activities of the hyperthermostable beta-glycosidase CelB from the archaeon Pyrococcus furiosus. CelB retained full catalytic efficiency for lactose hydrolysis at 80 degrees C in a 50% (by vol.) solution of ionic liquid in sodium citrate buffer, pH 5.5. It was inactive but not irreversibly denatured at 70% ionic liquid. Using lactose (0.15 M) as galactosyl donor, values of Ksel for a representative series of eight acceptor alcohols were determined in kinetic assays at 80 degrees C and found to increase between 1.3-fold (D-xylose) and 3.1-fold (glycerol) in 45% ionic liquid. Enhancement of Ksel was dependent on ionic liquid concentration and higher than expected from the decrease in water activity caused by the cosolvent. Experimental molar ratios of D-glucose and D-galactose produced during enzymatic conversion of lactose (75-150 mM) in the presence of D-xylose (0.5 M) or glycerol (0.5 M) showed excellent agreement with predictions based on Ksel values and confirm a significant, yet moderate effect of 45% ionic liquid on increasing the yield of D-galactoside product, by < or = 10%.

  16. Structural basis of thermal stability of the tungsten cofactor synthesis protein MoaB from Pyrococcus furiosus.

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    Nastassia Havarushka

    Full Text Available Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15 °C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 °C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.

  17. The role of TrmB and TrmB-like transcriptional regulators for sugar transport and metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Lee, Sung-Jae; Surma, Melanie; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2008-09-01

    TrmB of Pyrococcus furiosus was discovered as the trehalose/maltose-specific repressor for the genes encoding the trehalose/maltose high-affinity ABC transporter (the TM system). TrmB also represses the genes encoding the high affinity maltodextrin-specific ABC transporter (the MD system) with maltodextrin and sucrose as inducers. In addition, TrmB binds glucose leading to an increased repression of both, the TM and the MD system. Thus, TrmB recognizes different promoters and depending on the promoter it will be activated or inactivated for promoter binding by different sugar effectors. The TrmB-like protein TrmBL1 of P. furiosus is a global regulator and recognizes preferentially, but not exclusively, the TGM (for Thermococcales-glycolytic motif) sequence that is found upstream of the MD system as well as of genes encoding enzymes involved in the glycolytic and the gluconeogenic pathway. It responds to maltose and maltotriose as inducers and functions as repressor for the genes encoding the MD system and glycolytic enzymes, but as activator for genes encoding gluconeogenic enzymes. The TrmB-like protein TrmBL2 of P. furiosus lacks the sugar-binding domain that has been determined in TrmB. It recognizes the MD promoter, but not all TGM harboring promoters. It is evolutionary the most conserved among the Thermococcales. The regulatory range of TrmBL2 remains unclear.

  18. Enhancing Heat Tolerance of the Little Dogwood Cornus canadensis L. f. with Introduction of a Superoxide Reductase Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Geng, Xing-Min; Liu, Xiang; Ji, Mikyoung; Hoffmann, William A; Grunden, Amy; Xiang, Qiu-Yun J

    2016-01-01

    Production of reactive oxygen species (ROS) can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR) is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP) was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA), and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR.

  19. Enhancing heat tolerance of the little dogwood Cornus canadensis L. f. with introduction of a superoxide reductase gene from the hyperthermophilic archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Xinmin eGeng

    2016-01-01

    Full Text Available Production of reactive oxygen species (ROS can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA, and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR.

  20. Normal mode analysis of pyrococcus furiosus rubredoxin via nuclear resonance vibrational spectroscopy (NRVS) and resonance raman spectroscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Y.; Wang, H.; George, S.J.; Smith, M.C.; Adams, M.W.W.; Jenney, F.E., Jr.; Sturhahn, W.; Alp, E.E.; Zhao, J.; Yoda, Y.; Dey, A.; Solomon, E.I.; Cramer, S.P.; Experimental Facilities Division (APS); Univ. of California; LBNL; Stanford Univ.; Univ. of Georgia; SPring-8

    2005-10-26

    We have used {sup 57}Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(S{sub cys})4 site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also been investigated by resonance Raman spectroscopy. In the oxidized Rd NRVS, strong asymmetric Fe-S stretching modes are observed between 355 and 375 cm{sup -1}; upon reduction these modes shift to 300-320 cm{sup -1}. This is the first observation of Fe-S stretching modes in a reduced Rd. The peak in S-Fe-S bend mode intensity is at {approx}150 cm{sup -1} for the oxidized protein and only slightly lower in the reduced case. A third band occurs near 70 cm{sup -1} for both samples; this is assigned primarily as a collective motion of entire cysteine residues with respect to the central Fe. The {sup 57}Fe partial vibrational density of states (PVDOS) were interpreted by normal mode analysis with optimization of Urey-Bradley force fields. The three main bands were qualitatively reproduced using a D{sub 2d} Fe(SC){sub 4} model. A C{sub 1} Fe(SCC){sub 4} model based on crystallographic coordinates was then used to simulate the splitting of the asymmetric stretching band into at least 3 components. Finally, a model employing complete cysteines and 2 additional neighboring atoms was used to reproduce the detailed structure of the PVDOS in the Fe-S stretch region. These results confirm the delocalization of the dynamic properties of the redox-active Fe site. Depending on the molecular model employed, the force constant KFe-S for Fe-S stretching modes ranged from 1.24 to 1.32 mdyn/Angstrom. KFe-S is clearly diminished in reduced Rd; values from {approx}0.89 to 1.00 mdyn/Angstrom were derived from different models. In contrast, in the final models the force constants for S-Fe-S bending motion, HS-Fe-S, were 0.18 mdyn/Angstrom for oxidized Rd and 0.15 mdyn/Angstrom for reduced Rd. The NRVS technique demonstrates great promise for the observation and quantitative

  1. DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs▿

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex. PMID:17496095

  2. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  3. Hydrolysis of flavanone glycosides by β-glucosidase from Pyrococcus furiosus and its application to the production of flavanone aglycones from citrus extracts.

    Science.gov (United States)

    Shin, Kyung-Chul; Nam, Hyun-Koo; Oh, Deok-Kun

    2013-11-27

    The hydrolytic activity of the recombinant β-glucosidase from Pyrococcus furiosus for the flavanone glycoside hesperidin was optimal at pH 5.5 and 95 °C in the presence of 0.5% (v/v) dimethyl sulfoxide (DMSO) and 0.1% (w/v) Tween 40 with a half-life of 88 h, a Km of 1.6 mM, and a kcat of 68.4 1/s. The specific activity of the enzyme for flavonoid glycosides followed the order hesperidin > neohesperidin > naringin > narirutin > poncirin > diosmin > neoponcirin > rutin. The specific activity for flavanone was higher than that for flavone or flavonol. DMSO at 10% (v/v) was used to increase the solubility of flavanone glycosides as substrates. The enzyme completely converted flavanone glycosides (1 g/L) to flavanone aglycones and disaccharides via one-step reaction. The major flavanone in grapefruit peel, grapefruit pulp, or orange peel extract was naringin (47.5 mg/g), naringin (16.6 mg/g), or hesperidin (18.2 mg/g), respectively. β-Glucosidase from P. furiosus completely converted naringin and narirutin in 100% (w/v) grapefruit peel extract to 22.5 g/L naringenin after 12 h, with a productivity of 1.88 g L(-1) h(-1); naringin and narirutin in 100% (w/v) grapefruit pulp extract to 8.1 g/L naringenin after 9 h, with a productivity of 0.90 g L(-1) h(-1); and hesperidin in 100% (w/v) orange peel extract to 9.0 g/L hesperetin after 9 h, with a productivity of 1.00 g L(-1) h(-1). The conversion yields, concentrations, and productivities of flavanone aglycones in this study are the highest among those obtained from citrus extracts. Thus, this enzyme may be useful for the industrial hydrolysis of flavanone glycosides in citrus extracts.

  4. TrmB, a sugar sensing regulator of ABC transporter genes in Pyrococcus furiosus exhibits dual promoter specificity and is controlled by different inducers.

    Science.gov (United States)

    Lee, Sung-Jae; Moulakakis, Christina; Koning, Sonja M; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2005-09-01

    TrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus (malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus (mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar-binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half-maximal binding occurring at 20, 60, 250 and 500 microM substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross-linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

  5. Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters.

    Science.gov (United States)

    Bevers, Loes E; Hagedoorn, Peter-Leon; Krijger, Gerard C; Hagen, Wilfred R

    2006-09-01

    A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

  6. Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.

    Science.gov (United States)

    Ma, Xin; Lai, Lien B; Lai, Stella M; Tanimoto, Akiko; Foster, Mark P; Wysocki, Vicki H; Gopalan, Venkat

    2014-10-20

    We demonstrate that surface-induced dissociation (SID) coupled with ion mobility mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry of a multi-subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg(2+). We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg(2+)-dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

  7. Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.

    Science.gov (United States)

    Hayashi, I; Morikawa, K; Ishino, Y

    1999-12-15

    Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions.

  8. Characterization of the TrmB-like protein, PF0124, a TGM-recognizing global transcriptional regulator of the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-07-01

    The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.

  9. Tungsten biochemistry of Pyrococcus furiosus

    NARCIS (Netherlands)

    Bevers, L.E.

    2008-01-01

    Tungsten is the heaviest element that exhibits biological activity (atomic number 74), when it is present in an enzyme. It is taken up by cells in the form of tungstate, and it is subsequently processed into an organic cofactor referred to as tungstopterin, which is found as active center in several

  10. Crystal structures of the all-cysteinyl-coordinated D14C variant of Pyrococcus furiosus ferredoxin: [4Fe–4S] ↔ [3Fe–4S] cluster conversion

    DEFF Research Database (Denmark)

    Løvgreen, Monika Nøhr; Martic, Maja; Windahl, Michael S.

    2011-01-01

    molecules have different crystal packing and intramolecular disulfide bond conformation. The crystal packing reveals a β-sheet interaction between A molecules in adjacent asymmetric units, whereas B molecules are packed as monomers in a less rigid position next to the A–A extended β-sheet dimers...

  11. Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    van Haaster, D.J.

    2007-01-01

    Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris

  12. Molecular characterization of glycolysis in Pyrococcus furiosus

    NARCIS (Netherlands)

    Verhees, C.H.

    2002-01-01

    In the last few decades microorganisms have been isolated from rather unknown and hostile locations, such as those with high salt concentrations, an extreme pH, or low or high temperatures. Microorganisms isolated from these environments are referred to as extremophiles (1). The most

  13. Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    van Haaster, D.J.

    2007-01-01

    Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris (Hilde

  14. Heterometallic [AgFe3S4] ferredoxin variants: synthesis, characterization, and the first crystal structure of an engineered heterometallic iron–sulfur protein

    DEFF Research Database (Denmark)

    Martic, Maja; Simon, Ida Noemi; Haahr, Lærke Tvedebrink

    2013-01-01

    Heterometallic [AgFe3S4] iron–sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe3S4] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate...... group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe3S4]2+/+ couple. The oxidized form of the [AgFe3S4] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms...

  15. Identification of a glycolytic regulon in the archaea Pyrococcus and Thermococcus.

    Science.gov (United States)

    van de Werken, Harmen J G; Verhees, Corné H; Akerboom, Jasper; de Vos, Willem M; van der Oost, John

    2006-07-01

    The glycolytic pathway of the hyperthermophilic archaea that belong to the order Thermococcales (Pyrococcus, Thermococcus and Palaeococcus) differs significantly from the canonical Embden-Meyerhof pathway in bacteria and eukarya. This archaeal glycolysis variant consists of several novel enzymes, some of which catalyze unique conversions. Moreover, the enzymes appear not to be regulated allosterically, but rather at transcriptional level. To elucidate details of the gene expression control, the transcription initiation sites of the glycolytic genes in Pyrococcus furiosus have been mapped by primer extension analysis and the obtained promoter sequences have been compared with upstream regions of non-glycolytic genes. Apart from consensus sequences for the general transcription factors (TATA-box and BRE) this analysis revealed the presence of a potential transcription factor binding site (TATCAC-N(5)-GTGATA) in glycolytic and starch utilizing promoters of P. furiosus and several thermococcal species. The absence of this inverted repeat in Pyrococcus abyssi and Pyrococcus horikoshii probably reflects that their reduced catabolic capacity does not require this regulatory system. Moreover, this phyletic pattern revealed a TrmB-like regulator (PF0124 and TK1769) which may be involved in recognizing the repeat. This Thermococcales glycolytic regulon, with more than 20 genes, is the largest regulon that has yet been described for Archaea.

  16. Synthesis, Purification and Characterization of Ferredoxins with Re-Designed Active Sites

    DEFF Research Database (Denmark)

    Kristensen, Jytte

    on the redox signals of the P. furiosus ferredoxins. The P. furiosus ferredoxin with the heterometallic [CoFe3S4] cluster was synthesized and purified in the oxidized [CoFe3S4]2+ state. The chromatographic, mass spectro¬metric and EPR spec¬troscopic results indicated that the [CoFe3S4]2+ ferredoxin...... was purified to high purity and that the pro¬tein was stable under the used conditions. These results are in dis¬agree¬ment with pre¬vious reports of readily oxidative degrada¬tion of the [CoFe3S4]2+ ferredoxin to [Fe3S4]+ ferredoxin. Experiments with chemical reduc¬tion and oxi¬dation sugges¬ted a redox...... active protein and this was confirmed by cyclic voltammetry. One well-defined pair of redox peaks appear¬ed and the pair was assig¬ned to the [CoFe3S4]2+/+ redox couple and had a formal potential of -177 mV versus SHE. Unlike the naturally occurring iron-sulfur cluster the molybdenum-sulfur cluster...

  17. Cloning, expression, and purification of the His(6)-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Ahring, Birgitte Kiær

    2003-01-01

    The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E coli BL21(DE3)pLysS and E. coli Rosetta(DE3)p......LysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni2+-IDA-Sepharose, dialyzed...

  18. Structural and functional studies of the iron storage protein ferritin from Pyrococcus furiosus

    NARCIS (Netherlands)

    Tatur, J.

    2007-01-01

    This research focuses on the iron storage protein ferritin. Ferritin is a protein involved in iron homeostasis by storing Fe(II) excess in the form of an Fe(III) mineral core in the presence of oxygen and by releasing iron during iron deficiency. Ferritins are vital for human health. Their malfuncti

  19. Evaluation of sulfur-reducing microorganisms for organic desulfurization. [Pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Miller, K.W.

    1991-01-01

    Because of substantial portion of the sulfur in Illinois coal is organic, microbial desulfurization of sulfidic and thiophenic functionalities could hold great potential for completing pyritic sulfur removal. We are testing the hypothesis that organic sulfur can be reductively removed as H{sub 2}S through the activities of anaerobic microorganisms. Our objectives for this year include the following: (1) To obtain cultures that will reductively desulfurize thiophenic model compounds. In addition to crude oil enrichments begun last year, we sampled municipal sewage sludge. (2) To continue to work toward optimizing the activity of the DBDS-reducing cultures obtained during the previous year. (3) To expand coal desulfurization work to include other coals including Illinois Basin Coal 101 and a North Dakota lignite, which might be more susceptible to the dibenzyldisulfide reducing cultures due to its lower rank. (4) To address the problem of sulfide sorption, by investigating the sorption capacity of coals in addition to Illinois Basin Coal 108.

  20. The ABC of ABC-transport in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, S

    2003-01-01

    Living organisms of our earth can be divided into two groups, the prokaryotes and the eukaryotes. Eukaryotic cells have a nucleus, a special compartment in the cell, where the genetic material, the DNA is located. The DNA in the prokaryotic cell is floating freely in the cell. The eukaryotes, that i

  1. The ABC of ABC-transport in the hyperthermophilic archaeon Pyrococcus furiosus

    OpenAIRE

    Koning, S.

    2003-01-01

    Living organisms of our earth can be divided into two groups, the prokaryotes and the eukaryotes. Eukaryotic cells have a nucleus, a special compartment in the cell, where the genetic material, the DNA is located. The DNA in the prokaryotic cell is floating freely in the cell. The eukaryotes, that is where we belong to, together with animals, plants and fungi. Bacteria and archaea belong to the prokaryotes. Archaea resemble bacteria but in certain features they resemble more the eukaryotes. T...

  2. Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi

    Directory of Open Access Journals (Sweden)

    Melissa G. eCastillo-Lizardo

    2014-08-01

    Full Text Available Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+ and exonuclease deficient (exo- forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity Pyrococcus furiosus (Pfu DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.

  3. Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato

    Science.gov (United States)

    Green, L. S.; Yee, B. C.; Buchanan, B. B.; Kamide, K.; Sanada, Y.; Wada, K.

    1991-01-01

    Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.

  4. Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi.

    Science.gov (United States)

    Castillo-Lizardo, Melissa; Henneke, Ghislaine; Viguera, Enrique

    2014-01-01

    Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab) slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+) and exonuclease deficient (exo-) forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity P. furiosus (Pfu) DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.

  5. Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

    Directory of Open Access Journals (Sweden)

    Casey M. Theriot

    2011-01-01

    Full Text Available Prolidases hydrolyze Xaa-Pro dipeptides and can also cleave the P-F and P-O bonds found in organophosphorus (OP compounds, including the nerve agents soman and sarin. Ph1prol (PH0974 has previously been isolated and characterized from Pyrococcus horikoshii and was shown to have higher catalytic activity over a broader pH range, higher affinity for metal, and increased thermostability compared to P. furiosus prolidase, Pfprol (PF1343. To obtain a better enzyme for OP nerve agent decontamination and to investigate the structural factors that may influence protein thermostability and thermoactivity, randomly mutated Ph1prol enzymes were prepared. Four Ph1prol mutants (A195T/G306S-, Y301C/K342N-, E127G/E252D-, and E36V-Ph1prol were isolated which had greater thermostability and improved activity over a broader range of temperatures against Xaa-Pro dipeptides and OP nerve agents compared to wild type Pyrococcus prolidases.

  6. Energetic selection of topology in ferredoxins.

    Directory of Open Access Journals (Sweden)

    J Dongun Kim

    Full Text Available Models of early protein evolution posit the existence of short peptides that bound metals and ions and served as transporters, membranes or catalysts. The Cys-X-X-Cys-X-X-Cys heptapeptide located within bacterial ferredoxins, enclosing an Fe₄S₄ metal center, is an attractive candidate for such an early peptide. Ferredoxins are ancient proteins and the simple α+β fold is found alone or as a domain in larger proteins throughout all three kingdoms of life. Previous analyses of the heptapeptide conformation in experimentally determined ferredoxin structures revealed a pervasive right-handed topology, despite the fact that the Fe₄S₄ cluster is achiral. Conformational enumeration of a model CGGCGGC heptapeptide bound to a cubane iron-sulfur cluster indicates both left-handed and right-handed folds could exist and have comparable stabilities. However, only the natural ferredoxin topology provides a significant network of backbone-to-cluster hydrogen bonds that would stabilize the metal-peptide complex. The optimal peptide configuration (alternating α(L,α(R is that of an α-sheet, providing an additional mechanism where oligomerization could stabilize the peptide and facilitate iron-sulfur cluster binding.

  7. Ferredoxin Gene Mutation in Iranian Trichomonas Vaginalis Isolates

    Directory of Open Access Journals (Sweden)

    Soudabeh Heidari

    2013-09-01

    Full Text Available Background: Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO to its active form (CPR. Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied.Methods: Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations.Results: In four isolates (8.69% point mutation at nucleotide position -239 (the translation start codon of the ferredoxin gene were detected in which adenosine were converted to thymine.Conclusion: Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein’s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.

  8. Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803.

    Science.gov (United States)

    Bottin, H; Lagoutte, B

    1992-07-06

    The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.

  9. Energy conservation via electron bifurcating ferredoxin reduction and proton/Na(+) translocating ferredoxin oxidation.

    Science.gov (United States)

    Buckel, Wolfgang; Thauer, Rudolf K

    2013-02-01

    The review describes four flavin-containing cytoplasmatic multienzyme complexes from anaerobic bacteria and archaea that catalyze the reduction of the low potential ferredoxin by electron donors with higher potentials, such as NAD(P)H or H(2) at ≤ 100 kPa. These endergonic reactions are driven by concomitant oxidation of the same donor with higher potential acceptors such as crotonyl-CoA, NAD(+) or heterodisulfide (CoM-S-S-CoB). The process called flavin-based electron bifurcation (FBEB) can be regarded as a third mode of energy conservation in addition to substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP). FBEB has been detected in the clostridial butyryl-CoA dehydrogenase/electron transferring flavoprotein complex (BcdA-EtfBC), the multisubunit [FeFe]hydrogenase from Thermotoga maritima (HydABC) and from acetogenic bacteria, the [NiFe]hydrogenase/heterodisulfide reductase (MvhADG-HdrABC) from methanogenic archaea, and the transhydrogenase (NfnAB) from many Gram positive and Gram negative bacteria and from anaerobic archaea. The Bcd/EtfBC complex that catalyzes electron bifurcation from NADH to the low potential ferredoxin and to the high potential crotonyl-CoA has already been studied in some detail. The bifurcating protein most likely is EtfBC, which in each subunit (βγ) contains one FAD. In analogy to the bifurcating complex III of the mitochondrial respiratory chain and with the help of the structure of the human ETF, we propose a conformational change by which γ-FADH(-) in EtfBC approaches β-FAD to enable the bifurcating one-electron transfer. The ferredoxin reduced in one of the four electron bifurcating reactions can regenerate H(2) or NADPH, reduce CO(2) in acetogenic bacteria and methanogenic archaea, or is converted to ΔμH(+)/Na(+) by the membrane-associated enzyme complexes Rnf and Ech, whereby NADH and H(2) are recycled, respectively. The mainly bacterial Rnf complexes couple ferredoxin oxidation by NAD(+) with

  10. The ferredoxin-NADP+ reductase/ferredoxin electron transfer system of Plasmodium falciparum.

    Science.gov (United States)

    Balconi, Emanuela; Pennati, Andrea; Crobu, Danila; Pandini, Vittorio; Cerutti, Raffaele; Zanetti, Giuliana; Aliverti, Alessandro

    2009-07-01

    In the apicoplast of apicomplexan parasites, plastidic-type ferredoxin and ferredoxin-NADP(+) reductase (FNR) form a short electron transport chain that provides reducing power for the synthesis of isoprenoid precursors. These proteins are attractive targets for the development of novel drugs against diseases such as malaria, toxoplasmosis, and coccidiosis. We have obtained ferredoxin and FNR of both Toxoplasma gondii and Plasmodium falciparum in recombinant form, and recently we solved the crystal structure of the P. falciparum reductase. Here we report on the functional properties of the latter enzyme, which differ markedly from those of homologous FNRs. In the physiological reaction, P. falciparum FNR displays a k(cat) five-fold lower than those usually determined for plastidic-type FNRs. By rapid kinetics, we found that hydride transfer between NADPH and protein-bound FAD is slower in the P. falciparum enzyme. The redox properties of the enzyme were determined, and showed that the FAD semiquinone species is highly destabilized. We propose that these two features, i.e. slow hydride transfer and unstable FAD semiquinone, are responsible for the poor catalytic efficiency of the P. falciparum enzyme. Another unprecedented feature of the malarial parasite FNR is its ability to yield, under oxidizing conditions, an inactive dimeric form stabilized by an intermolecular disulfide bond. Here we show that the monomerdimer interconversion can be controlled by oxidizing and reducing agents that are possibly present within the apicoplast, such as H(2)O(2), glutathione, and lipoate. This finding suggests that modulation of the quaternary structure of P. falciparum FNR might represent a regulatory mechanism, although this needs to be verified in vivo.

  11. Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC.

    Science.gov (United States)

    Hutson, K G; Rogers, L J; Haslett, B G; Boulter, D; Cammack, R

    1978-06-15

    Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was --350mV and that of ferredoxin II was --445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11--12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.

  12. Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties*

    Science.gov (United States)

    Korn, Anja; Ajlani, Ghada; Lagoutte, Bernard; Gall, Andrew; Sétif, Pierre

    2009-01-01

    In photosynthetic organisms, ferredoxin:NADP+ oxidoreductase (FNR) is known to provide NADPH for CO2 assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNRS) similar to the one found in plant plastids and a large one (FNRL) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNRL exhibits a higher NADP+/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNRL, named FNRL-PC. The enzymatic activities of FNRL-PC were compared with those of FNRS. During NADPH oxidation, FNRL-PC exhibits a 30% decrease in the Michaelis constant Km(NADPH), and a 70% increase in Km(ferredoxin), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP+ reduction, the FNRL-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP+. The increase in Km(ferredoxin) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNRL-PC. Both isoforms are capable of catalyzing the NADP+ reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo. PMID:19759024

  13. Fusion of ferredoxin and cytochrome P450 enables direct light-driven biosynthesis

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Nielsen, Agnieszka Janina Zygadlo; Burow, Meike

    2016-01-01

    , electrons captured by the fused ferredoxin moiety are directed more effectively towards P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin...

  14. Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin.

    Science.gov (United States)

    Huang, Haiyan; Hu, Liejie; Yu, Wenjun; Li, Huili; Tao, Fei; Xie, Huijun; Wang, Shuning

    2016-05-01

    Ferredoxins are small, acidic proteins containing iron-sulfur clusters that are widespread in living organisms. They play key roles as electron carriers in various metabolic processes, including respiration, photosynthesis, fermentation, nitrogen fixation, carbon dioxide fixation, and hydrogen production. However, only several kinds of ferredoxins are commercially available now, greatly limiting the investigation of ferredoxin-related enzymes and metabolic processes. Here we describe the heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin. Adding extra iron and sulfur sources to the medium in combination with using Escherichia coli C41(DE3) harboring pCodonplus and pRKISC plasmids as host greatly enhanced iron-sulfur cluster synthesis in the three ferredoxins. After induction for 12 h in terrific broth and purification by affinity chromatography and anion exchange chromatography, approximately 3.4 mg of streptavidin (Strep)-tagged and 3.7 mg of polyhistidine (His)-tagged clostridial 2[4Fe4S] ferredoxins were obtained from 1 l of culture. Excitingly, after induction for 24 h in terrific broth, around 40 mg of His-tagged clostridial [2Fe2S] and 23 mg of His-tagged agrobacterial [2Fe2S] ferredoxins were purified from 1 l of culture. The recombinant ferredoxins apparently exhibited identical properties and physiological function to native ferredoxins. No negative impact of two different affinity tags on ferredoxin activity was found. In conclusion, we successfully developed a convenient method for heterologous overproduction of the three kinds of ferredoxins with satisfactory yields and activities, which would be very helpful for the ferredoxin-related researches.

  15. Evolution of the acceptor side of photosystem I: ferredoxin, flavodoxin, and ferredoxin-NADP(+) oxidoreductase.

    Science.gov (United States)

    Pierella Karlusich, Juan José; Carrillo, Néstor

    2017-02-01

    The development of oxygenic photosynthesis by primordial cyanobacteria ~2.7 billion years ago led to major changes in the components and organization of photosynthetic electron transport to cope with the challenges of an oxygen-enriched atmosphere. We review herein, following the seminal contributions as reported by Jaganathan et al. (Functional genomics and evolution of photosynthetic systems, vol 33, advances in photosynthesis and respiration, Springer, Dordrecht, 2012), how these changes affected carriers and enzymes at the acceptor side of photosystem I (PSI): the electron shuttle ferredoxin (Fd), its isofunctional counterpart flavodoxin (Fld), their redox partner ferredoxin-NADP(+) reductase (FNR), and the primary PSI acceptors F x and F A/F B. Protection of the [4Fe-4S] centers of these proteins from oxidative damage was achieved by strengthening binding between the F A/F B polypeptide and the reaction center core containing F x, therefore impairing O2 access to the clusters. Immobilization of F A/F B in the PSI complex led in turn to the recruitment of new soluble electron shuttles. This function was fulfilled by oxygen-insensitive [2Fe-2S] Fd, in which the reactive sulfide atoms of the cluster are shielded from solvent by the polypeptide backbone, and in some algae and cyanobacteria by Fld, which employs a flavin as prosthetic group and is tolerant to oxidants and iron limitation. Tight membrane binding of FNR allowed solid-state electron transfer from PSI bridged by Fd/Fld. Fine tuning of FNR catalytic mechanism led to formidable increases in turnover rates compared with FNRs acting in heterotrophic pathways, favoring Fd/Fld reduction instead of oxygen reduction.

  16. Multiple orientations in a physiological complex: the pyruvate-ferredoxin oxidoreductase-ferredoxin system.

    Science.gov (United States)

    Pieulle, Laetitia; Nouailler, Matthieu; Morelli, Xavier; Cavazza, Christine; Gallice, Philippe; Blanchet, Stéphane; Bianco, Pierre; Guerlesquin, Françoise; Hatchikian, E Claude

    2004-12-14

    Ferredoxin I from Desulfovibrio africanus (Da FdI) is a small acidic [4Fe-4S] cluster protein that exchanges electrons with pyruvate-ferredoxin oxidoreductase (PFOR), a key enzyme in the energy metabolism of anaerobes. The thermodynamic properties and the electron transfer between PFOR and either native or mutated FdI have been investigated by microcalorimetry and steady-state kinetics, respectively. The association constant of the PFOR-FdI complex is 3.85 x 10(5) M(-1), and the binding affinity has been found to be highly sensitive to ionic strength, suggesting the involvement of electrostatic forces in formation of the complex. Surprisingly, the punctual or combined neutralizations of carboxylate residues surrounding the [4Fe-4S] cluster slightly affect the PFOR-FdI interaction. Furthermore, hydrophobic residues around the cluster do not seem to be crucial for the PFOR-FdI system activity; however, some of them play an important role in the stability of the FeS cluster. NMR restrained docking associated with site-directed mutagenesis studies suggested the presence of various interacting sites on Da FdI. The modification of additional acidic residues at the interacting interface, generating a FdI pentamutant, evidenced at least two distinct FdI binding sites facing the distal [4Fe-4S] cluster of the PFOR. We also used a set of various small acidic partners to investigate the specificity of PFOR toward redox partners. The remarkable flexibility of the PFOR-FdI system supports the idea that the specificity of the physiological complex has probably been "sacrificed" to improve the turnover rate and thus the efficiency of bacterial electron transfer.

  17. Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

    NARCIS (Netherlands)

    Machielsen, M.P.; Leferink, N.G.H.; Hendriks, A.; Brouns, S.J.J.; Hennemann, H.; Daussmann, T.; Oost, van der J.

    2008-01-01

    There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with

  18. The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose.

    Science.gov (United States)

    Krug, Michael; Lee, Sung-Jae; Boos, Winfried; Diederichs, Kay; Welte, Wolfram

    2013-06-01

    TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α-glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar-degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three-dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N-terminal DNA binding domain containing a winged-helix-turn-helix (wHTH) domain followed by an amphipathic helix with a coiled-coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter.

  19. Spectrophotometric studies on alkaline isomerization of spinach ferredoxin.

    Science.gov (United States)

    Hasumi, H; Nagata, E; Nakamura, S

    1985-10-01

    The gross protein structure, the microenvironment of the iron-sulfur cluster, and the effect of neutral salts on the molecular structure of spinach ferredoxin were studied by CD and absorption spectroscopy in the alkaline pH range. In the pH range of 7-11, the existence of reversible isomerization which consisted of at least two proton dissociation processes was indicated by the statical CD and absorption spectra. The CD changes in the visible and far-UV regions were dramatic upon elevation of the pH from neutral to alkaline, indicating a significant alteration of the microenvironment of the cluster and a decrease in the ordered secondary structures. The absorption change in the visible region due to pH elevation was small but clearly observed with a high signal-to-noise ratio. The numbers of protons involved in the respective processes and the apparent pK values obtained from the pH-dependence of the CD changes were in good agreement with those obtained from the pH-dependence of the absorption changes in the visible region. In addition, the rate constants obtained from the time courses of the CD and absorption changes agreed with one another. By the addition of 1 M NaCl, the CD and absorption spectra at alkaline pH were reversed almost to those at neutral pH without significant pH change. On the other hand, above pH 11, ferredoxin was found to be irreversibly denatured. Based on analyses of the statical CD and absorption spectra and of the time courses of the CD changes, the probable mechanism of the isomerization was considered to be as follows: (Formula: see text) where H stands for a proton, N-form for native ferredoxin at neutral pH, N*-form for alkaline ferredoxin below pH 11 which still has the iron-sulfur cluster but with disordered secondary structures of the polypeptide chain, and D-form for completely denatured ferredoxin above pH 11. These results lead to the conclusions that (1) the interaction between the protein moiety and the iron-sulfur cluster is

  20. Identification of a glycolytic regulon in the Archaea Pyrococcus and Thermococcus

    NARCIS (Netherlands)

    Werken, van de H.J.G.; Verhees, C.H.; Akerboom, A.P.; Vos, de W.M.; Oost, van der J.

    2006-01-01

    The glycolytic pathway of the hyperthermophilic archaea that belong to the order Thermococcales (Pyrococcus, Thermococcus and Palaeococcus) differs significantly from the canonical Embden-Meyerhof pathway in bacteria and eukarya. This archaeal glycolysis variant consists of several novel enzymes, so

  1. Amino acid sequences of heterotrophic and photosynthetic ferredoxins from the tomato plant (Lycopersicon esculentum Mill.).

    Science.gov (United States)

    Kamide, K; Sakai, H; Aoki, K; Sanada, Y; Wada, K; Green, L S; Yee, B C; Buchanan, B B

    1995-11-01

    Several forms (isoproteins) of ferredoxin in roots, leaves, and green and red pericarps in tomato plants (Lycopersicon esculentum Mill.) were earlier identified on the basis of N-terminal amino acid sequence and chromatographic behavior (Green et al. 1991). In the present study, a large scale preparation made possible determination of the full length amino acid sequence of the two ferredoxins from leaves. The ferredoxins characteristic of fruit and root were sequenced from the amino terminus to the 30th residue or beyond. The leaf ferredoxins were confirmed to be expressed in pericarp of both green and red fruit. The ferredoxins characteristic of fruit and root appeared to be restricted to those tissue. The results extend earlier findings in demonstrating that ferredoxin occurs in the major organs of the tomato plant where it appears to function irrespective of photosynthetic competence.

  2. Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP⁺-oxidoreductase (FNR) enzymes in vitro

    National Research Council Canada - National Science Library

    Iftach Yacoby; Sergii Pochekailov; Hila Toporik; Maria L. Ghirardi; Paul W. King; Shuguang Zhang

    2011-01-01

    .... To elucidate the basis for competition, we bioengineered a ferredoxin-hydrogenase fusion and characterized hydrogen production kinetics in the presence of Fd, ferredoxin:NADP⁺-oxidoreductase (FNR), and NADP...

  3. Studies of ferredoxins and dCTP deaminase:dUTPase

    DEFF Research Database (Denmark)

    Helt, Signe Smedegaard

    2008-01-01

    Ferredoxiner tilhører klassen jern-svovl proteiner, og menes at være opstået under den tidlige evolution. Arbejdet omhandler primært rekonstruktion og karakterisering af to oldgamle ferredoxiner, der tidligere er blevet udledt baseret på fylogenetisk analyse. Proteinerne udgør modelsystemer til s...

  4. The key to the extraordinary thermal stability of P. furiosus holo-rubredoxin: iron binding-guided packing of a core aromatic cluster responsible for high kinetic stability of the native structure.

    Science.gov (United States)

    Prakash, Satya; Sundd, Monica; Guptasarma, Purnananda

    2014-01-01

    Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.

  5. Structural and functional diversity of ferredoxin-NADP(+) reductases.

    Science.gov (United States)

    Aliverti, Alessandro; Pandini, Vittorio; Pennati, Andrea; de Rosa, Matteo; Zanetti, Giuliana

    2008-06-15

    Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.

  6. Characterization of Ancient Ferredoxins & Chlamydia Trachomatis Ribonucleotide Reductase

    DEFF Research Database (Denmark)

    Nørgaard, Hanne

    Part I Two evolutionary old ferredoxins were characterized. LCA Fd – a 55 AA ferredoxin – is believed to have function in the last common ancestor of life on Earth. An internal repeat in the sequence of LCA Fd was phylogenetically traced even further back in evolution resulting in a 23 AA peptide...... and a significantly weaker signal probably from a small contribution of [3Fe-4S] at 47 mV vs. NHE. EPR-monitored redox titration showed one major transition at -345 mV vs. NHE. X-band EPR spectra from the titration revealed spectral changes depending on degree of reduction, and a complex spectrum was observed...... was relatively weaker at these conditions. Simulation of partly reduced LCA Fd1 X-band EPR spectrum resulted in g = [2.065; 1.926; 1.88]. Mass spectrometry confirmed that LCA Fd1 contains two [4Fe-4S] clusters. LCA Fd2 was also studied using EPR-monitored redox titration. The X-band EPR spectra showed a weakly...

  7. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  8. Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in Pectobacterium spp.

    Science.gov (United States)

    Grinter, Rhys; Milner, Joel; Walker, Daniel

    2012-01-01

    In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium Pectobacterium carotovorum carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of Pectobacterium carotovorum and Pectobacterium atrosepticum with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that Pectobacterium spp. carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of Pectobacterium carotovorum and atrosepticum that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells.

  9. Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in Pectobacterium spp.

    Directory of Open Access Journals (Sweden)

    Rhys Grinter

    Full Text Available In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium Pectobacterium carotovorum carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of Pectobacterium carotovorum and Pectobacterium atrosepticum with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that Pectobacterium spp. carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of Pectobacterium carotovorum and atrosepticum that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells.

  10. High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

    Science.gov (United States)

    Michoud, Grégoire; Jebbar, Mohamed

    2016-06-01

    Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

  11. High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

    KAUST Repository

    Michoud, Gregoire

    2016-06-02

    Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

  12. Ferredoxin-NADP reductase from the thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6.

    Science.gov (United States)

    Ikeda, Takeshi; Nakamura, Miyuki; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2009-08-01

    The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. Small iron-sulfur proteins, ferredoxins, play a central role as low-potential electron donors for this cycle. The fpr gene of this bacterium, encoding a putative ferredoxin-NADP(+) reductase (FNR, EC 1.18.1.2), was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Unexpectedly, the monomeric Fpr protein contained one molecule of FMN as a prosthetic group, although FNRs from other organisms are known to contain FAD. The FMN-containing Fpr was shown to be a bona fide FNR that catalyzes a reversible redox reaction between NADP(+)/NADPH and ferredoxins.

  13. Water-assisted proton transfer in ferredoxin I.

    Science.gov (United States)

    Lutz, Stephan; Tubert-Brohman, Ivan; Yang, Yonggang; Meuwly, Markus

    2011-07-08

    The role of water molecules in assisting proton transfer (PT) is investigated for the proton-pumping protein ferredoxin I (FdI) from Azotobacter vinelandii. It was shown previously that individual water molecules can stabilize between Asp(15) and the buried [3Fe-4S](0) cluster and thus can potentially act as a proton relay in transferring H(+) from the protein to the μ(2) sulfur atom. Here, we generalize molecular mechanics with proton transfer to studying proton transfer reactions in the condensed phase. Both umbrella sampling simulations and electronic structure calculations suggest that the PT Asp(15)-COOH + H(2)O + [3Fe-4S](0) → Asp(15)-COO(-) + H(2)O + [3Fe-4S](0) H(+) is concerted, and no stable intermediate hydronium ion (H(3)O(+)) is expected. The free energy difference of 11.7 kcal/mol for the forward reaction is in good agreement with the experimental value (13.3 kcal/mol). For the reverse reaction (Asp(15)-COO(-) + H(2)O + [3Fe-4S](0)H(+) → Asp(15)-COOH + H(2)O + [3Fe-4S](0)), a larger barrier than for the forward reaction is correctly predicted, but it is quantitatively overestimated (23.1 kcal/mol from simulations versus 14.1 from experiment). Possible reasons for this discrepancy are discussed. Compared with the water-assisted process (ΔE ≈ 10 kcal/mol), water-unassisted proton transfer yields a considerably higher barrier of ΔE ≈ 35 kcal/mol.

  14. Design and Synthesis of Chalcone Derivatives as Inhibitors of the FerredoxinFerredoxin-NADP+ Reductase Interaction of Plasmodium falciparum: Pursuing New Antimalarial Agents

    Directory of Open Access Journals (Sweden)

    Hery Suwito

    2014-12-01

    Full Text Available Some chalcones have been designed and synthesized using Claisen-Schmidt reactions as inhibitors of the ferredoxin and ferredoxin-NADP+ reductase interaction to pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition interactions between PfFd-PfFNR in the range of 10.94%–50%. The three strongest inhibition activities were shown by (E-1-(4-aminophenyl-3-(4-methoxyphenylprop-2-en-1-one (50%, (E-1-(4-aminophenyl-3-(2,4-dimethoxyphenylprop-2-en-1-one (38.16%, and (E-1-(4-aminophenyl-3-(2,3-dimethoxyphenylprop-2-en-1-one (31.58%. From the docking experiments we established that the amino group of the methoxyamino chlacone derivatives plays an important role in the inhibition activity by electrostatic interaction through salt bridges and that it forms more stable and better affinity complexes with FNR than with Fd.

  15. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

    OpenAIRE

    1997-01-01

    The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conse...

  16. Determination of nonligand amino acids critical to [4Fe-4S]2+/+ assembly in ferredoxin maquettes.

    Science.gov (United States)

    Mulholland, S E; Gibney, B R; Rabanal, F; Dutton, P L

    1999-08-10

    The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single [4Fe-4S]2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins. Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster. In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM. FdM-7 consists solely of the. CIACGAC. consensus ferredoxin core motif observed in natural protein sequences. Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences. Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little [4Fe-4S]2+/+ cluster, 6 and 7%, respectively. A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC. or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7. Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of [4Fe-4S] affinity. The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the [4Fe-4S] cluster by bulky hydrophobic amino acids. Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of [4Fe-4S] cluster binding relative to that of the glycine variant. The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence

  17. Ferredoxin:NAD + Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Liang; Lo, Jonathan; Shao, Xiongjun; Zheng, Tianyong; Olson, Daniel G.; Lynd, Lee R.; Atomi, H.

    2016-09-30

    ABSTRACT

    Ferredoxin:NAD+oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD+. This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway inThermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activityin vitrowith a ferredoxin-based FNOR assay. To determine its role in metabolism, thetsac_1705gene was deleted in different strains ofT. saccharolyticum. In wild-typeT. saccharolyticum, deletion oftsac_1705resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR inT.saccharolyticum. When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 inClostridium thermocellumand found improvements in both the titer and the yield of ethanol.

    IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD+, a key reaction in the

  18. The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

    Science.gov (United States)

    Huppe, H. C.; de Lamotte-Guery, F.; Buchanan, B. B.

    1990-01-01

    The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

  19. [Effects of the symbiosis of Trichomonas vaginalis with Mycoplasma hominis on ferredoxin gene].

    Science.gov (United States)

    Liu, Xiaodong; Wen, Wenjing; Xue, Changgui

    2011-08-01

    We isolated 30 Trichomonas vaginalis for the PCR detection from the gynecological outpatients in the Affiliated Hospital of Zhengzhou University using the specific 16s rDNA primers of Mycoplasma hominis. The results showed that there were 25 cases of Mycoplasma hominis infection, with the infection rate of 83.33%. This gave a clew that the symbiosis of Trichomonas vaginalis with Mycoplasma hominis may be of certain generality in China. We sequenced the ferredoxin gene of 10 Trichomonas vaginalis where 5 Mycoplasma hominis were positive and five negative, and found that the ferredoxin (Fd) gene of the 10 Trichomonas vaginalis were exactly the same. But compared to the genes in the GenBank, a comparative analysis of the gene revealed that there were 3 more ctg bases at the 200th position of encoding leucine, but this did not lead to changes in reading frame. The gene homology was 99%.

  20. Isolation and characterization of two ferredoxin-NADP+ reductases from Spirulina platensis.

    Science.gov (United States)

    Masaki, R; Wada, K; Matsubara, H

    1979-10-01

    Two ferredoxin-NADP+ reductases (FNRs I and II) [EC 1.6.7.1] were purified from a blue-green alga, Spirulina platensis, by (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 chromatography. FNRs I and II were both FAD-containing enzymes with molecular weights of 33,000, and could photochemically reduce NADP+ to the same extent in the presence of S. platensis ferredoxin, using FNR-depleted membrane fragments of S. platensis. They had similar physical and enzymatic properties, except for chemical properties such as the amino (N)-terminal sequences and the patterns of their peptide maps. The significance of the presence of two FNRs in S. platensis as as of the multiple forms found in other organisms is discussed.

  1. Schistosoma mansoni ferredoxin NADP(H) oxidoreductase and its role in detoxification.

    Science.gov (United States)

    Girardini, Javier E; Dissous, Colette; Serra, Esteban

    2002-01-01

    Ferredoxin NADP(H) oxidoreductases (FNR) are flavoenzymes that catalyze the electron transfer between NADP(H) and a wide range of compounds including ferredoxins and bacterial flavodoxins. FNRs are classified into two major groups: plant- and vertebrate-type. Plant-type FNRs are implicated in photosynthesis and nitrogen fixation in plastids and photosynthetic bacteria, and were recently implicated in cell protection against reactive oxygen species (ROS). Vertebrate-type FNRs are mitochondrial enzymes implicated in steroid hormone biosynthesis in mammals and in Fe(+) uptake and metabolism in yeasts. We have cloned and sequenced a cDNA coding for the vertebrate-type Schistosoma mansoni FNR. Gel diaphorase activity and western blot assays demonstrated that SmFNR represented the major diaphorase activity of adult worms. An active recombinant SmFNR was expressed in Escherichia coli that made the bacteria tolerant to oxygen peroxide, cumene hydroperoxide and the superoxide-generating herbicide, methyl viologen (MV).

  2. A new concept for ferredoxin-NADP(H) oxidoreductase binding to plant thylakoids.

    Science.gov (United States)

    Benz, J Philipp; Lintala, Minna; Soll, Jürgen; Mulo, Paula; Bölter, Bettina

    2010-11-01

    During the evolution of photosynthesis, regulatory circuits were established that allow the precise coupling of light-driven electron transfer chains with downstream processes such as carbon fixation. The ferredoxin (Fd):ferredoxin-NADP(+) oxidoreductase (FNR) couple is an important mediator for these processes because it provides the transition from exclusively membrane-bound light reactions to the mostly stromal metabolic pathways. Recent progress has allowed us to revisit how FNR is bound to thylakoids and to revaluate the current view that only membrane-bound FNR is active in photosynthetic reactions. We argue that the vast majority of thylakoid-bound FNR of higher plants is not necessary for photosynthesis. We furthermore propose that the correct distribution of FNR between stroma and thylakoids is used to efficiently regulate Fd-dependent electron partitioning in the chloroplast. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Identification of the specific electron transfer proteins, ferredoxin, and ferredoxin reductase, for CYP105D7 in Streptomyces avermitilis MA4680.

    Science.gov (United States)

    Pandey, Bishnu Prasad; Lee, Nahum; Choi, Kwon-Young; Kim, Ji-Nu; Kim, Eun-Jung; Kim, Byung-Gee

    2014-06-01

    It was previously proposed that regiospecific hydroxylation of daidzein at 3'-position is mediated by cytochrome P450 hydroxylase (CYP105D7) in the presence of putidaredoxin (CamB) and putidaredoxin reductase (CamA) as electron transfer proteins from Pseudomonas putida. The genome sequence of Streptomyces avermitilis MA4680 revealed 33 P450 (CYPs) with 6 ferredoxin reductases (Fprs) and 9 ferredoxins (Fdxs) as their putative electron transfer partner proteins. To identify right endogenous electron transfer proteins for CYP105D7 activity, in vitro reconstitution, gene disruption, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mRNA expression profile analysis were examined. The most effective electron transfer proteins for CYP105D7 appear to be FdxH (SAV7470), which is located downstream to CYP105D7 as a cluster, and FprD (SAV5675). Throughout our overall analysis, we proposed that the primary electron transfer pathway for CYP105D7 follows as such NAD(P)H→FdxH→FprD→CYP105D7.

  4. Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.

    Science.gov (United States)

    Seo, Daisuke; Soeta, Takahiro; Sakurai, Hidehiro; Sétif, Pierre; Sakurai, Takeshi

    2016-06-01

    Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (FNRs. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Determinació de l'estructura tridimensional de la glicogen sintasa de "Pyrococcus abyssi"

    OpenAIRE

    2005-01-01

    Les glicogen i midó sintases són glicosiltransferases que catalitzen la transferència de residus glucosil a l'extrem no reductor d'una cadena creixent d'un glucà α-1,4, retenint la configuració del carboni anomèric del sucre transferit. Aquest procès és central en el metabolisme energètic de la majoria d'èssers vius.En aquest treball presentem l'estructura cristal·logràfica de la glicogen sintasa de Pyrococcus abyssi (PaGS). Aquest enzim és termoestable i presenta una activitat màxima a ...

  6. Theoretical Study on the Allosteric Regulation of an Oligomeric Protease from Pyrococcus horikoshii by Cl− Ion

    Directory of Open Access Journals (Sweden)

    Dongling Zhan

    2014-02-01

    Full Text Available The thermophilic intracellular protease (PH1704 from Pyrococcus horikoshii that functions as an oligomer (hexamer or higher forms has proteolytic activity and remarkable stability. PH1704 is classified as a member of the C56 family of peptidases. This study is the first to observe that the use of Cl− as an allosteric inhibitor causes appreciable changes in the catalytic activity of the protease. Theoretical methods were used for further study. Quantum mechanical calculations indicated the binding mode of Cl− with Arg113. A molecular dynamics simulation explained how Cl− stabilized distinct contact species and how it controls the enzyme activity. The new structural insights obtained from this study are expected to stimulate further biochemical studies on the structures and mechanisms of allosteric proteases. It is clear that the discovery of new allosteric sites of the C56 family of peptidases may generate opportunities for pharmaceutical development and increases our understanding of the basic biological processes of this peptidase family.

  7. Superoxide generated from the glutathione-mediated reduction of selenite damages the iron-sulfur cluster of chloroplastic ferredoxin.

    Science.gov (United States)

    Fisher, Brian; Yarmolinsky, Dmitry; Abdel-Ghany, Salah; Pilon, Marinus; Pilon-Smits, Elizabeth A; Sagi, Moshe; Van Hoewyk, Doug

    2016-09-01

    Selenium assimilation in plants is facilitated by several enzymes that participate in the transport and assimilation of sulfate. Manipulation of genes that function in sulfur metabolism dramatically affects selenium toxicity and accumulation. However, it has been proposed that selenite is not reduced by sulfite reductase. Instead, selenite can be non-enzymatically reduced by glutathione, generating selenodiglutathione and superoxide. The damaging effects of superoxide on iron-sulfur clusters in cytosolic and mitochondrial proteins are well known. However, it is unknown if superoxide damages chloroplastic iron-sulfur proteins. The goals of this study were twofold: to determine whether decreased activity of sulfite reductase impacts selenium tolerance in Arabidopsis, and to determine if superoxide generated from the glutathione-mediated reduction of selenite damages the iron-sulfur cluster of ferredoxin. Our data demonstrate that knockdown of sulfite reductase in Arabidopsis does not affect selenite tolerance or selenium accumulation. Additionally, we provide in vitro evidence that the non-enzymatic reduction of selenite damages the iron-sulfur cluster of ferredoxin, a plastidial protein that is an essential component of the photosynthetic light reactions. Damage to ferredoxin's iron-sulfur cluster was associated with formation of apo-ferredoxin and impaired activity. We conclude that if superoxide damages iron-sulfur clusters of ferredoxin in planta, then it might contribute to photosynthetic impairment often associated with abiotic stress, including toxic levels of selenium.

  8. Roles of the species-specific subdomain and the N-terminal peptide of Toxoplasma gondii ferredoxin-NADP+ reductase in ferredoxin binding.

    Science.gov (United States)

    Pandini, Vittorio; Caprini, Gianluca; Tedeschi, Gabriella; Seeber, Frank; Zanetti, Giuliana; Aliverti, Alessandro

    2006-03-21

    The plant-type ferredoxin/ferredoxin-NADP(+) reductase (Fd/FNR) redox system found in parasites of the phylum Apicomplexa has been proposed as a target for novel drugs used against life-threatening diseases such as malaria and toxoplasmosis. Like many proteins from these protists, apicomplexan FNRs are characterized by the presence of unique peptide insertions of variable length and yet unknown function. Since three-dimensional data are not available for any of the parasite FNRs, we used limited proteolysis to carry out an extensive study of the conformation of Toxoplasma gondii FNR. This led to identification of 11 peptide bonds susceptible to the action of four different proteases. Cleavage sites are clustered in four regions of the enzyme, which include two of its three species-specific insertions. Such regions are thus predicted to form flexible surface loops. The protein substrate Fd protected FNR against cleavage both at its N-terminal peptide and at its largest sequence insertion (28 residues). Deletion by protein engineering of the species-specific subdomain containing the latter insertion resulted in an enzyme form that, although catalytically active, displayed a 10-fold decreased affinity for Fd. In contrast, removal of the first 15 residues of the enzyme unexpectedly enhanced its interaction with Fd. Thus, two flexible polypeptide regions of T. gondii FNR are involved in Fd interaction but have opposite roles in modulating the binding affinity for the protein ligand. In this respect, T. gondii FNR differs from plant FNRs, where the N-terminal peptide contributes to the stabilization of their complex with Fd.

  9. Arabidopsis FNRL protein is an NADPH-dependent chloroplast oxidoreductase resembling bacterial ferredoxin-NADP(+) reductases.

    Science.gov (United States)

    Koskela, Minna M; Dahlström, Käthe M; Goñi, Guillermina; Lehtimäki, Nina; Nurmi, Markus; Velazquez-Campoy, Adrian; Hanke, Guy; Bölter, Bettina; Salminen, Tiina A; Medina, Milagros; Mulo, Paula

    2017-08-18

    Plastidic ferredoxin-NADP(+) oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of FAD dependent oxidoreductases. In the present study, we show that the Arabidopsis thaliana At1g15140 (FERREDOXIN-NADP(+) OXIDOREDUCTASE -LIKE, FNRL) is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with kcat = 3.24 ± 0.17 s(-1) , Km(NADPH) = 1.6 ± 0.3 μM and kcat / Km(NADPH) = 2.0 ± 0.4 μM(-1) s(-1) . Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1 (AtFd1), which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. Additionally, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded β-sheet and one α-helix, and a C-terminal NADP(+) -binding α/β domain with a five-stranded β-sheet with a pair of α-helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs. This article is protected by copyright. All rights reserved.

  10. Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases.

    Science.gov (United States)

    Musumeci, Matías A; Botti, Horacio; Buschiazzo, Alejandro; Ceccarelli, Eduardo A

    2011-03-29

    Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.

  11. Cloning and sequencing of the ferredoxin gene of blue-green alga Anabaena siamensis

    Science.gov (United States)

    Li, Shou-Dong; Song, Li-Rong; Liu, Yong-Ding; Zhao, Jin-Dong

    1998-03-01

    The structure gene for ferredoxin, petFI, from Anabaena siamensis has been amplified by polymerase chain reaction(PCR) and cloned into cloning vector pGEM-3zf(+). The nucleotide sequence of petFI has been determined with silver staining sequencing method. There is 96.8% homology between coding region of petFI from A. siamensis and that of petFI from A. sp. 7120. Amino acid sequences of seven strains of blue-green algae are compared.

  12. A STD-NMR Study of the Interaction of the Anabaena Ferredoxin-NADP+ Reductase with the Coenzyme

    Directory of Open Access Journals (Sweden)

    Lara V. Antonini

    2014-01-01

    Full Text Available Ferredoxin-NADP+ reductase (FNR catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.

  13. Mechanostability of the Single-Electron-Transfer Complexes of Anabaena Ferredoxin-NADP(+) Reductase.

    Science.gov (United States)

    Marcuello, Carlos; de Miguel, Rocío; Martínez-Júlvez, Marta; Gómez-Moreno, Carlos; Lostao, Anabel

    2015-10-26

    The complexes formed between the flavoenzyme ferredoxin-NADP(+) reductase (FNR; NADP(+) =nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron-deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one-step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.

  14. Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins.

    Science.gov (United States)

    Ewen, Kerstin Maria; Kleser, Michael; Bernhardt, Rita

    2011-01-01

    Adrenodoxin is probably the best characterized member of the vertebrate-type [2Fe-2S]-cluster ferredoxins. It has been in the spotlight of scientific interest for many years due to its essential role in mammalian steroid hormone biosynthesis, where it acts as electron mediator between the NADPH-dependent adrenodoxin reductase and several mitochondrial cytochromes P450. In this review we will focus on the present knowledge about protein-protein recognition in the mitochondrial cytochrome P450 system and the modulation of the electron transfer between Adx and its redox partners, AdR and CYP(s). We also intend to point out the potential biotechnological applications of Adx as a versatile electron donor to different cytochromes P450, both in vitro and in vivo. Finally we will address the comparison between the mammalian cytochrome P450-associated adrenodoxin and ferredoxins involved in iron-sulfur-cluster biosynthesis. Despite their different functions, these proteins display an amazing similarity regarding their primary sequence, tertiary structure and biophysical features.

  15. Functional plasticity and catalytic efficiency in plant and bacterial ferredoxin-NADP(H) reductases.

    Science.gov (United States)

    Ceccarelli, Eduardo A; Arakaki, Adrián K; Cortez, Néstor; Carrillo, Néstor

    2004-05-06

    Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. Two great families of FAD-containing proteins displaying FNR activity have evolved from different and independent origins. The enzymes present in mitochondria and some bacterial genera are members of the structural superfamily of disulfide oxidoreductases whose prototype is glutathione reductase. A second group, comprising the FNRs from plastids and most eubacteria, constitutes a unique family, the plant-type FNRs, totally unrelated in sequence with the former. The two-domain structure of the plant family of FNR also provides the basic scaffold for an extended superfamily of electron transfer flavoproteins. In this article we compare FNR flavoenzymes from very different origins and describe how the natural history of these reductases shaped structure, flavin conformation and catalytic activity to face the very different metabolic demands they have to deal with in their hosts. We show that plant-type FNRs can be classified into a plastidic class, characterised by extended FAD conformation and high catalytic efficiency, and a bacterial class displaying a folded FAD molecule and low turnover rates. Sequence alignments supported this classification, providing a criterion to predict the structural and biochemical properties of newly identified members of the family.

  16. Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase.

    Science.gov (United States)

    Bortolotti, Ana; Pérez-Dorado, Inmaculada; Goñi, Guillermina; Medina, Milagros; Hermoso, Juan A; Carrillo, Néstor; Cortez, Néstor

    2009-02-01

    Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1-5 s(-1) while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2'-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2'-P-AMP and NADP(+) allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR.

  17. Crystallization and preliminary X-ray studies of ferredoxin-NAD(P)+ reductase from Chlorobium tepidum.

    Science.gov (United States)

    Muraki, Norifumi; Seo, Daisuke; Shiba, Tomoo; Sakurai, Takeshi; Kurisu, Genji

    2008-03-01

    Ferredoxin-NAD(P)(+) reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P)(+) to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222(1) and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4(1)22 or P4(3)22. Diffraction data were collected from a form I crystal to 2.4 A resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

  18. Complete Genome Sequence of the Hyperthermophilic Archaeon Pyrococcus sp. Strain ST04, Isolated from a Deep-Sea Hydrothermal Sulfide Chimney on the Juan de Fuca Ridge

    Science.gov (United States)

    Jung, Jong-Hyun; Lee, Ju-Hoon; Holden, James F.; Seo, Dong-Ho; Shin, Hakdong; Kim, Hae-Yeong; Kim, Wooki; Ryu, Sangryeol

    2012-01-01

    Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na+ gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential. PMID:22843576

  19. A highly stable plastidic-type ferredoxin-NADP(H reductase in the pathogenic bacterium Leptospira interrogans.

    Directory of Open Access Journals (Sweden)

    Daniela L Catalano-Dupuy

    Full Text Available Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H reductase (FNR with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107 encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP(+ domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature.

  20. A highly stable plastidic-type ferredoxin-NADP(H) reductase in the pathogenic bacterium Leptospira interrogans.

    Science.gov (United States)

    Catalano-Dupuy, Daniela L; Musumeci, Matías A; López-Rivero, Arleth; Ceccarelli, Eduardo A

    2011-01-01

    Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP(+) domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature.

  1. Substrate recognition of N,N'-diacetylchitobiose deacetylase from Pyrococcus horikoshii.

    Science.gov (United States)

    Nakamura, Tsutomu; Yonezawa, Yasushige; Tsuchiya, Yuko; Niiyama, Mayumi; Ida, Kurumi; Oshima, Maki; Morita, Junji; Uegaki, Koichi

    2016-09-01

    Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N'-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N'-diacetylchitobiose (GlcNAc2), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-d-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc2 complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc2. This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes.

  2. Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots.

    Science.gov (United States)

    Kim, Han-Woo; Ishikawa, Kazuhiko

    2011-07-15

    A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.

  3. Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin

    Energy Technology Data Exchange (ETDEWEB)

    Dey, A.; Francis, E.J.; Adams, M.W.W.; Babini, E.; Takahashi, Y.; Fukuyama, K.; Hodgson, K.O.; Hedman, B.; Solomon, E.I.; /Stanford U., Chem. Dept. /Georgia U. /Bologna U. /Osaka U. /SLAC, SSRL

    2009-04-29

    A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.

  4. Molecular Dynamics Simulations of Trichomonas vaginalis Ferredoxin Show a Loop-Cap Transition

    Energy Technology Data Exchange (ETDEWEB)

    Weksberg, Tiffany E; Lynch, Gillian C; Krause, Kurt; Pettitt, Bernard M

    2007-05-01

    The crystal structure of the oxidized Trichomonas vaginalis ferredoxin (Tvfd) showed a unique crevice that exposed the redox center. Here we have examined the dynamics and solvation of the active site of Tvfd using molecular dynamics simulations of both the reduced and oxidized states. The oxidized simulation stays true to the crystal form with a heavy atom root mean-squared deviation of 2Å . However, within the reduced simulation of Tvfd a profound loop-cap transition into the redox center occurred within 6-ns of the start of the simulation and remained open throughout the rest of the 20-ns simulation. This large opening seen in the simulations supports the hypothesis that the exceptionally fast electron transfer rate between Tvfd and the drug metronidazole is due to the increased access of the antibiotic to the redox center of the protein and not due to the reduction potential.

  5. Molecular Dynamics Simulations of Trichomonas vaginalis Ferredoxin Show a Loop-Cap Transition.

    Energy Technology Data Exchange (ETDEWEB)

    Weksberg, Tiffany E; Lynch, Gillian C; Krause, Kurt; Pettitt, Bernard M

    2007-05-01

    The crystal structure of the oxidized Trichomonas vaginalis ferredoxin (Tvfd) showed a unique crevice that exposed the redox center. Here we have examined the dynamics and solvation of the active site of Tvfd using molecular dynamics simulations of both the reduced and oxidized states. The oxidized simulation stays true to the crystal form with a heavy atom root mean-squared deviation of 2Å. However, within the reduced simulation of Tvfd a profound loop-cap transition into the redox center occurred within 6-ns of the start of the simulation and remained open throughout the rest of the 20-ns simulation. This large opening seen in the simulations supports the hypothesis that the exceptionally fast electron transfer rate between Tvfd and the drug metronidazole is due to the increased access of the antibiotic to the redox center of the protein and not due to the reduction potential.

  6. Characterization of cyanobacterial ferredoxin-NADP+ oxidoreductase molecular heterogeneity using chromatofocusing.

    Science.gov (United States)

    Serrano, A

    1986-05-01

    Chromatofocusing has been used as an analytical tool to check preparations of the enzyme ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) purified in either the presence or absence of the serine protease inhibitor phenylmethylsulfonyl fluoride from the cyanobacterium Anabaena sp. strain 7119. Only one isoelectric species was found when the crude extract was processed in the presence of the protease inhibitor. Nevertheless, when the inhibitor was omitted, four ionic forms of the enzyme--showing apparent pI's in the range 4.3-4.6--were separated after chromatofocusing of the purified preparation. These forms were found to differ in their specific activities, exhibiting, on the other hand, lower values than the single one obtained in the presence of the protease inhibitor. Analysis by acrylamide gel electrophoresis revealed virtually a single main protein band except for the ionic form of pI 4.39, which was clearly resolved into two active components. Except for the more basic form, which seems to be an homodimer of Mr 80,000, all the protein components were found to be monomeric species in the range Mr 33,000-38,000. These results indicate that the molecular heterogeneity of the ferredoxin-NADP+ oxidoreductase purified from the cyanobacterium Anabaena sp. strain 7119 may result from the activity of a protease present in the whole cell homogenates. On the other hand, these data also point out that chromatofocusing should be considered as an effective technique in the isolation and characterization of the different molecular forms of this enzyme.

  7. Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis.

    Science.gov (United States)

    Grazina, Raquel; de Sousa, Patrícia M Paes; Brondino, Carlos D; Carepo, Marta S P; Moura, Isabel; Moura, José J G

    2011-08-01

    The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S](+1/0) (E(0')=-158±5mV); [4Fe-4S](+2/+1) (E(0')=-474±5mV) and [3Fe-4S](0/-2) (E(0')=-660±5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=5.1±0.1, the [4Fe-4S](+2/+1) centre is pH independent, and the [3Fe-4S](0/-2) reduction is accompanied by the binding of two protons. The ability of the [3Fe-4S](0) cluster to be converted into a new [4Fe-4S] cluster was proven. The redox potential of the original [4Fe-4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70mV) of the redox potential of the original centre. Being most [Fe-S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.

  8. Induced fit and equilibrium dynamics for high catalytic efficiency in ferredoxin-NADP(H) reductases.

    Science.gov (United States)

    Paladini, Darío H; Musumeci, Matías A; Carrillo, Néstor; Ceccarelli, Eduardo A

    2009-06-23

    Ferredoxin-NADP(H) reductase (FNR) is a FAD-containing protein that catalyzes the reversible transfer of electrons between NADP(H) and ferredoxin or flavodoxin. This enzyme participates in the redox-based metabolism of plastids, mitochondria, and bacteria. Plastidic plant-type FNRs are very efficient reductases in supporting photosynthesis. They have a strong preference for NADP(H) over NAD(H), consistent with the main physiological role of NADP(+) photoreduction. In contrast, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover rates that are up to 100-fold lower than those of their plastidic and cyanobacterial counterparts. With the aim of elucidating the mechanisms by which plastidic enzymes achieve such high catalytic efficiencies and NADP(H) specificity, we investigated the manner in which the NADP(H) nicotinamide enters and properly binds to the catalytic site. Analyzing the interaction of different nucleotides, substrate analogues, and aromatic compounds with the wild type and the mutant Y308S-FNR from pea, we found that the interaction of the 2'-P-AMP moiety from NADP(+) induces a change that favors the interaction of the nicotinamide, thereby facilitating the catalytic process. Furthermore, the main role of the terminal tyrosine, Y308, is to destabilize the interaction of the nicotinamide with the enzyme, inducing product release and favoring discrimination of the nucleotide substrate. We determined that this function can be replaced by the addition of aromatic compounds that freely diffuse in solution and establish a dynamic equilibrium, reversing the effect of the mutation in the Y308S-FNR mutant.

  9. Two isoforms of ferredoxin:NADP(+) oxidoreductase from wheat leaves: purification and initial biochemical characterization.

    Science.gov (United States)

    Grzyb, Joanna; Malec, Przemysław; Rumak, Izabela; Garstka, Maciej; Strzałka, Kazimierz

    2008-04-01

    Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.

  10. Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi

    Directory of Open Access Journals (Sweden)

    N. Leulliot

    2013-01-01

    Full Text Available Pyrococcus abyssi virus 1 (PAV1 was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23 that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137 at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

  11. Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Terada, Atsushi; Honda, Takashi; Fukuhara, Hideo; Hada, Kazumasa; Kimura, Makoto

    2006-08-01

    Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.

  12. Regulation of cyclic photophosphorylation during ferredoxin-mediated electron transport. Effect of DCMU and the NADPH/NADP/sup +/ ratio

    Energy Technology Data Exchange (ETDEWEB)

    Hosler, J.P.; Yocum, C.F.

    1987-04-01

    Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O/sub 2/ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP/sup +/ ratio is varied, as is the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.

  13. Chlamydomonas reinhardtii Chloroplasts Contain a Homodimeric Pyruvate:Ferredoxin Oxidoreductase That Functions with FDX11[W][OA

    Science.gov (United States)

    van Lis, Robert; Baffert, Carole; Couté, Yohann; Nitschke, Wolfgang; Atteia, Ariane

    2013-01-01

    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 Km 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. PMID:23154536

  14. Crystallization and preliminary X-ray studies of ferredoxin-NAD(P){sup +} reductase from Chlorobium tepidum

    Energy Technology Data Exchange (ETDEWEB)

    Muraki, Norifumi [Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Seo, Daisuke [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Shiba, Tomoo [Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan); Sakurai, Takeshi [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Kurisu, Genji, E-mail: gkurisu@xtal.c.u-tokyo.ac.jp [Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902 (Japan)

    2008-03-01

    Ferredoxin-NAD(P){sup +} reductase from C. tepidum has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. Ferredoxin-NAD(P){sup +} reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P){sup +} to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222{sub 1} and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4{sub 1}22 or P4{sub 3}22. Diffraction data were collected from a form I crystal to 2.4 Å resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

  15. Control of reduction thermodynamics in [2Fe-2S] ferredoxins Entropy-enthalpy compensation and the influence of surface mutations.

    Science.gov (United States)

    Bellei, Marzia; Battistuzzi, Gianantonio; Wu, Shu-pao; Mansy, Sheref S; Cowan, James A; Sola, Marco

    2010-06-01

    The reaction thermodynamics for the one-electron reduction of the [2Fe-2S] cluster of both human ferredoxin and various surface point mutants, in which each of the negatively charged residues Asp72, Glu73, Asp76, and Asp79 were converted to Ala, have been determined by variable temperature spectroelectrochemical measurements. The above are conserved residues that have been implicated in interactions between the vertebrate-type ferredoxins and their redox partners. In all cases, and similar to other 2Fe-ferredoxins, the reduction potentials are negative as a result of both an enthalpic and entropic stabilization of the oxidized state. Although all Hs Fd mutants, with the exception of Asp72Ala, show slightly higher E degrees ' values than that of wild type Hs Fd, according to expectations for a purely electrostatic model, they exhibit changes in the H degrees '(rc) values that are electrostatically counter-intuitive. The observation of enthalpy-entropy compensation within the protein series indicates that the mutation-induced changes in H degrees '(rc) and S degrees '(rc) are dominated by reduction-induced solvent reorganization effects. Protein-based entropic effects are likely to be responsible for the low E degrees ' value of D72A.

  16. A larger transcript is required for the synthesis of the smaller isoform of ferredoxin:NADP oxidoreductase.

    Science.gov (United States)

    Omairi-Nasser, Amin; de Gracia, Adrienne Gomez; Ajlani, Ghada

    2011-09-01

    Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyse the exchange of electrons between ferredoxin and NADP(H). In cyanobacteria FNR provides NADPH for photoautotrophic metabolism, but the enzyme is also capable of oxidizing NADPH providing reduced ferredoxin. In the cyanobacterium Synechocystis sp. strain PCC6803, the unique petH gene has two translation products depending on growth conditions. As a consequence two isoforms of the FNR accumulate - FNR(L) and FNR(S) . In the present work, analysis of petH expression reveals that different transcriptional start points (tsp) are responsible for this differential translation initiation. Under standard conditions (where FNR(L) accumulates), two tsps were found at -52 and -34 relative to the first translation start site. Under nitrogen-starvation conditions (where FNR(S) accumulates) a tsp was mapped at -126 relative to the first translation start site. Therefore, the transcript responsible for FNR(S) translation is longer than that producing FNR(L) . In addition, expression of the short or long transcript in E. coli resulted in the accumulation of FNR(L) or FNR(S) respectively. This result demonstrates that translation can initiate at two different sites, 336-bases apart (ATG-1 to ATG-113), depending only on the 5'UTR structure. © 2011 Blackwell Publishing Ltd.

  17. Dynamics of Rhodobacter capsulatus [2Fe-2S] Ferredoxin VI and Aquifex aeolicus Ferredoxin 5 Via Nuclear Resonance Vibrational Spectroscopy (NRVS) and Resonance Raman Spectroscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Yuming; Tan, Ming-Liang; Ichiye, Toshiko; Wang, Hongxin; Guo, Yisong; Smith, Matt C.; Meyer, Jacques; Sturhahn, Wolfgang; Alp, E. E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2008-06-24

    We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(2)S(2)(Cys)(4) sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm(-1), and additional features observed as high as the B(2u) mode at approximately 421 cm(-1). Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B(3u) mode at approximately 288 cm(-1), attributed to an asymmetric combination of the two FeS(4) breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS(4) breathing. NRVS and RR analysis of isotope shifts with (36)S-substituted into bridging S(2-) ions in Rc FdVI allowed quantitation of S(2-) motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at approximately 190 cm(-1) in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields. Progressively more complex D(2h) Fe(2)S(2)S'(4), C(2h) Fe(2)S(2)(SCC)(4), and C(1) Fe(2)S(2)(Cys)(4) models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated

  18. Evidence for Lateral Transfer of Genes Encoding Ferredoxins, Nitroreductases, NADH Oxidase, and Alcohol Dehydrogenase 3 from Anaerobic Prokaryotes to Giardia lamblia and Entamoeba histolytica

    Science.gov (United States)

    Nixon, Julie E. J.; Wang, Amy; Field, Jessica; Morrison, Hilary G.; McArthur, Andrew G.; Sogin, Mitchell L.; Loftus, Brendan J.; Samuelson, John

    2002-01-01

    Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica. PMID

  19. Mapping cellular Fe-S cluster uptake and exchange reactions - divergent pathways for iron-sulfur cluster delivery to human ferredoxins.

    Science.gov (United States)

    Fidai, Insiya; Wachnowsky, Christine; Cowan, J A

    2016-12-07

    Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe-2S] or [4Fe-4S] clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe-2S] cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular cluster trafficking. This is the first step toward building a network map for all of the possible iron-sulfur cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

  20. Structural-functional characterization and physiological significance of ferredoxin-NADP reductase from Xanthomonas axonopodis pv. citri.

    Science.gov (United States)

    Tondo, María Laura; Musumeci, Matías A; Delprato, María Laura; Ceccarelli, Eduardo A; Orellano, Elena G

    2011-01-01

    Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP(+) reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of (•)O(2)(-). Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and

  1. Structural-functional characterization and physiological significance of ferredoxin-NADP reductase from Xanthomonas axonopodis pv. citri.

    Directory of Open Access Journals (Sweden)

    María Laura Tondo

    Full Text Available Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP(+ reductases (FNRs are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr that encodes for a FNR (Xac-FNR that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of (•O(2(-. Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762 as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv

  2. Structural and mutational studies of an electron transfer complex of maize sulfite reductase and ferredoxin.

    Science.gov (United States)

    Kim, Ju Yaen; Nakayama, Masato; Toyota, Hiroshi; Kurisu, Genji; Hase, Toshiharu

    2016-08-01

    The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å. Mutational analysis of a total of seven basic residues of SiR distributed widely at the interface of the complex showed their importance for supporting an efficient Fd-dependent activity and a strong physical binding to Fd. These combined results suggest that the productive electron transfer complex of SiR and Fd could be formed through multiple processes of the electrostatic intermolecular interaction and this implication is discussed in terms of the multi-functionality of Fd in various redox metabolisms.

  3. Overexpression of Ferredoxin, PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Li-Fen Huang

    2013-10-01

    Full Text Available Reactive oxygen species (ROS produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX. Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H2O2 levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H2O2 and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.

  4. Protein motifs involved in coenzyme interaction and enzymatic efficiency in anabaena ferredoxin-NADP+ reductase.

    Science.gov (United States)

    Peregrina, José R; Herguedas, Beatriz; Hermoso, Juan A; Martínez-Júlvez, Marta; Medina, Milagros

    2009-04-14

    Ferredoxin-NADP+ reductases (FNRs) must determine the coenzyme specificity and allow the transient encounter between N5 of its flavin cofactor and C4 of the coenzyme nicotinamide for efficient hydride transfer. Combined site-directed replacements in different putative determinants of the FNR coenzyme specificity were simultaneously produced. The resulting variants were structurally and functionally analyzed for their binding and hydride transfer abilities to the FNR physiological coenzyme NADP+/H, as well as to NAD+/H. The previously studied Y303S mutation is the only one that significantly enhances specificity for NAD+. Combination of mutations from the pyrophosphate or 2'-phosphate regions, even including Y303S, does not improve activity with NAD+, despite structures of these FNRs show how particular coenzyme-binding regions resembled motifs found in NAD+/H-dependent enzymes of the FNR family. Therefore, the "rational approach" did not succeed well, and coenzyme specificity redesign in the FNR family will be more complex than that anticipated in other NADP+/NAD+ families.

  5. Subcellular localization of ferredoxin-NADP(+) oxidoreductase in phycobilisome retaining oxygenic photosysnthetic organisms.

    Science.gov (United States)

    Morsy, Fatthy Mohamed; Nakajima, Masato; Yoshida, Takayuki; Fujiwara, Tatsuki; Sakamoto, Toshio; Wada, Keishiro

    2008-01-01

    Ferredoxin-NADP(+) oxidoreductase (FNR) catalyzing the terminal step of the linear photosynthetic electron transport was purified from the cyanobacterium Spirulina platensis and the red alga Cyanidium caldarium. FNR of Spirulina consisted of three domains (CpcD-like domain, FAD-binding domain, and NADP(+)-binding domain) with a molecular mass of 46 kDa and was localized in either phycobilisomes or thylakoid membranes. The membrane-bound FNR with 46 kDa was solublized by NaCl and the solublized FNR had an apparent molecular mass of 90 kDa. FNR of Cyanidium consisted of two domains (FAD-binding domain and NADP(+)-binding domain) with a molecular mass of 33 kDa. In Cyanidium, FNR was found on thylakoid membranes, but there was no FNR on phycobilisomes. The membrane-bound FNR of Cyanidium was not solublized by NaCl, suggesting the enzyme is tightly bound in the membrane. Although both cyanobacteria and red algae are photoautotrophic organisms bearing phycobilisomes as light harvesting complexes, FNR localization and membrane-binding characteristics were different. These results suggest that FNR binding to phycobilisomes is not characteristic for all phycobilisome retaining oxygenic photosynthetic organisms, and that the rhodoplast of red algae had possibly originated from a cyanobacterium ancestor, whose FNR lacked the CpcD-like domain.

  6. A New Type of YumC-Like Ferredoxin (Flavodoxin) Reductase Is Involved in Ribonucleotide Reduction

    DEFF Research Database (Denmark)

    Chen, Jun; Shen, Jing; Solem, Christian;

    2015-01-01

    The trxB2 gene, which is annotated as a thioredoxin reductase, was found to be essential for growth of Lactococcus lactis in the presence of oxygen. The corresponding protein (TrxB2) showed a high similarity with Bacillus subtilis YumC (E value = 4.0E-88), and YumC was able to fully complement....... Genome sequencing of two independent isolates, which were able to grow as well as the wild-type strain under aerated conditions, revealed the importance of mutations in nrdI, encoding a flavodoxin involved in aerobic ribonucleotide reduction. We suggest a role for TrxB2 in nucleotide metabolism, where....... subtilis but that the addition of deoxynucleosides cannot compensate for the lethal phenotype displayed by the B. subtilis yumC knockout mutant. Ferredoxin (flavodoxin) reductase (FdR) is involved in many important reactions in both eukaryotes and prokaryotes, such as photosynthesis, nitrate reduction, etc. The recently...

  7. Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk

    2016-02-01

    Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

  8. Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering

    NARCIS (Netherlands)

    Koutsopoulos, S; van der Oost, J; Norde, W

    2005-01-01

    The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational. and functional characteristics of the enzyme. The onset of

  9. Temperature-dependent structural and functional features of a hyperthermostable enzyme using elastic neutron scattering

    NARCIS (Netherlands)

    Koutsopoulos, S.; Oost, van der J.; Norde, W.

    2005-01-01

    The dynamic behavior of an endoglucanase from the hyperthermophilic microorganism Pyrococcus furiosus was investigated using elastic neutron scattering. The temperature dependence of the atomic motions was correlated with conformational and functional characteristics of the enzyme. The onset of

  10. Microbial alcohol dehydrogenases: identification, characterization and engineering

    NARCIS (Netherlands)

    Machielsen, M.P.

    2007-01-01

    Keywords: alcohol dehydrogenase, laboratory evolution, rational protein engineering, Pyrococcus furiosus, biocatalysis, characterization, computational design, thermostability.   Alcohol dehydrogeases (ADHs) catalyze the interconversion of alcohols, aldehydes and ketones. They display a wide variety

  11. Maillard reactions and increased enzyme inactivation during oligosaccharide synthesis by a hyperthermophilic glycosidase

    NARCIS (Netherlands)

    Bruins, M.E.; Hellemond, van E.W.; Janssen, A.E.M.; Boom, R.M.

    2003-01-01

    The thermostable Pyrococcus furiosus beta-glycosidase was used for oligosaccharide production from lactose in a kinetically controlled reaction. Our experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield. However, at reaction

  12. Complete Genome Sequence of the Hyperthermophilic Piezophilic Archaeon Pyrococcus kukulkanii NCB100 Isolated from the Rebecca's Roost Hydrothermal Vent in the Guaymas Basin.

    Science.gov (United States)

    Oger, Philippe M; Callac, Nolwenn; Oger-Desfeux, Christine; Hughes, Sandrine; Gillet, Benjamin; Jebbar, Mohamed; Godfroy, Anne

    2017-02-16

    Members of the order Thermococcales are common inhabitants of high-temperature hydrothermal vent systems (black smokers) that are represented in clone libraries mostly by isolates from the Thermococcus genus. We report the complete sequence of a novel species from the Pyrococcus genus, P. kukulkanii strain NCB100, which has been isolated from a flange fragment of the Rebecca's Roost hydrothermal vent system in the Guaymas Basin.

  13. cDNA cloning, expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Venegas-Calerón, M; Zambelli, A; Ruiz-López, N; Youssar, L; León, A; Garcés, R; Martínez-Force, Enrique

    2009-03-01

    Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5'-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.

  14. Association of Ferredoxin:NADP(+) oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Mosebach, Laura; Heilmann, Claudia; Mutoh, Risa; Gäbelein, Philipp; Steinbeck, Janina; Happe, Thomas; Ikegami, Takahisa; Hanke, Guy; Kurisu, Genji; Hippler, Michael

    2017-06-07

    Ferredoxins (FDX) and the FDX:NADP(+) oxidoreductase (FNR) represent a key junction of electron transport downstream of photosystem I (PSI). Dynamic recruitment of FNR to the thylakoid membrane has been considered as a potential mechanism to define the fate of photosynthetically derived electrons. In this study, we investigated the functional importance of the association of FNR with the photosynthetic apparatus in Chlamydomonas reinhardtii. In vitro assays based on NADP(+) photoreduction measurements as well as NMR chemical shift perturbation analyses showed that FNR preferentially interacts with FDX1 compared to FDX2. Notably, binding of FNR to a PSI supercomplex further enhanced this preference for FDX1 over FDX2, suggesting that FNR is potentially capable of channelling electrons towards distinct routes. NADP(+) photoreduction assays and immunoblotting revealed that the association of FNR with the thylakoid membrane including the PSI supercomplex is impaired in the absence of Proton Gradient Regulation 5 (PGR5) and/or Proton Gradient Regulation 5-Like photosynthetic phenotype 1 (PGRL1), implying that both proteins, directly or indirectly, contribute to the recruitment of FNR to the thylakoid membrane. As assessed via in vivo absorption spectroscopy and immunoblotting, PSI was the primary target of photodamage in response to high-light stress in the absence of PGR5 and/or PGRL1. Anoxia preserved the activity of PSI, pointing to enhanced electron donation to O2 as the source of the observed PSI inactivation and degradation. These findings establish another perspective on PGR5/PGRL1 knockout-related phenotypes and potentially interconnect FNR with the regulation of photosynthetic electron transport and PSI photoprotection in C. reinhardtii.

  15. Dynamics of the active site architecture in plant-type ferredoxin-NADP(+) reductases catalytic complexes.

    Science.gov (United States)

    Sánchez-Azqueta, Ana; Catalano-Dupuy, Daniela L; López-Rivero, Arleth; Tondo, María Laura; Orellano, Elena G; Ceccarelli, Eduardo A; Medina, Milagros

    2014-10-01

    Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP(+) reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP(+) coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor-acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. A [2Fe-2S] ferredoxin (FdVI) is essential for growth of the photosynthetic bacterium Rhodobacter capsulatus.

    OpenAIRE

    Armengaud, J.; Meyer, C.; Jouanneau, Y

    1997-01-01

    The physiological function of Rhodobacter capsulatus FdVI, a [2Fe-2S] ferredoxin, was investigated by the cloning, sequence analysis, and mutagenesis of its structural gene, called fdxE. The DNA region surrounding fdxE was mapped, and the nucleotide sequence of a 4.2-kb fragment was determined. fdxE is preceded by a sequence that is very similar to a sigma54 recognition site and is followed by a putative transcription stop signal, suggesting that fdxE forms a separate cistron. Two open readin...

  17. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    Energy Technology Data Exchange (ETDEWEB)

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  18. Studies of interaction of homo-dimeric ferredoxin-NAD(P)+ oxidoreductases of Bacillus subtilis and Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes.

    Science.gov (United States)

    Seo, Daisuke; Okabe, Seisuke; Yanase, Mitsuhiro; Kataoka, Kunishige; Sakurai, Takeshi

    2009-04-01

    Ferredoxin-NADP(+) oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris CGA009 (RPA3954) belong to a novel homo-dimeric type of FNR with high amino acid sequence homology to NADPH-thioredoxin reductases. These FNRs were purified from expression constructs in Escherichia coli cells, and their steady-state reactions with [2Fe-2S] type ferredoxins (Fds) from spinach and R. palustris, [4Fe-4S] type Fd from B. subtilis, NAD(P)(+)/NAD(P)H and ferricyanide were studied. From the K(m) and k(cat) values for the diaphorase activity with ferricyanide, it is demonstrated that both FNRs are far more specific for NADPH than for NADH. The UV-visible spectral changes induced by NADP(+) and B. subtilis Fd indicated that both FNRs form a ternary complex with NADP(+) and Fd, and that each of the two ligands decreases the affinities of the others. The steady-state kinetics of NADPH-cytochrome c reduction activity of YumC is consistent with formation of a ternary complex of NADPH and Fd during catalysis. These results indicate that despite their low sequence homology to other FNRs, these enzymes possess high FNR activity but with measurable differences in affinity for different types of Fds as compared to other more conventional FNRs.

  19. HC-Pro protein of sugar cane mosaic virus interacts specifically with maize ferredoxin-5 in vitro and in planta.

    Science.gov (United States)

    Cheng, Yu-Qin; Liu, Zhong-Mei; Xu, Jian; Zhou, Tao; Wang, Meng; Chen, Yu-Ting; Li, Huai-Fang; Fan, Zai-Feng

    2008-08-01

    Symptom development of a plant viral disease is a result of molecular interactions between the virus and its host plant; thus, the elucidation of specific interactions is a prerequisite to reveal the mechanism of viral pathogenesis. Here, we show that the chloroplast precursor of ferredoxin-5 (Fd V) from maize (Zea mays) interacts with the multifunctional HC-Pro protein of sugar cane mosaic virus (SCMV) in yeast, Nicotiana benthamiana cells and maize protoplasts. Our results demonstrate that the transit peptide rather than the mature protein of Fd V precursor could interact with both N-terminal (residues 1-100) and C-terminal (residues 301-460) fragments, but not the middle part (residues 101-300), of HC-Pro. In addition, SCMV HC-Pro interacted only with Fd V, and not with the other two photosynthetic ferredoxin isoproteins (Fd I and Fd II) from maize plants. SCMV infection significantly downregulated the level of Fd V mRNA in maize plants; however, no obvious changes were observed in levels of Fd I and Fd II mRNA. These results suggest that SCMV HC-Pro interacts specifically with maize Fd V and that this interaction may disturb the post-translational import of Fd V into maize bundle-sheath cell chloroplasts, which could lead to the perturbation of chloroplast structure and function.

  20. Insight Into the Radical Mechanism of Phycocyanobilin-Ferredoxin Oxidoreductase (Pcya) Revealed By X-Ray Crystallography And Biochemical Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Tu, S.-L.; Rockwell, N.; Lagarias, J.C.; Fisher, A.J.; /Inst. Plant Microb. Biol., Taipei /UC, Davis

    2007-07-13

    The X-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) from the cyanobacterium Nostoc sp. PCC7120 has been solved at 2.5 angstrom resolution. A comparative analysis of this structure with those recently reported for substrate-bound and substrate-free forms of PcyA from the cyanobacterium Synechocystis sp. PCC6803 (Hagiwara et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 27-32; Hagiwara et al. (2006) FEBS Lett. 580, 3823-3828) provides a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. On the basis of these structures and the biochemical analysis of site-directed mutants of Nostoc PcyA, including mutants reported in recent studies (Tu et al. (2006) J. Biol. Chem. 281, 3127-3136) as well as mutants described in this study, a revised mechanism for the PcyA-mediated four-electron reduction of biliverdin IX{alpha} to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates is proposed. The mechanistic insight of these studies, along with homology modeling, have provided new insight into the catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family that are widespread in oxygenic photosynthetic organisms.

  1. Modulation of the enzymatic efficiency of ferredoxin-NADP(H) reductase by the amino acid volume around the catalytic site.

    Science.gov (United States)

    Musumeci, Matías A; Arakaki, Adrián K; Rial, Daniela V; Catalano-Dupuy, Daniela L; Ceccarelli, Eduardo A

    2008-03-01

    Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases. In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20- to 100-fold lower than those of their plastidic and cyanobacterial counterparts. Several structural features of these enzymes have yet to be explained. The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the flavin and is highly conserved amongst members of the family. By computing the relative free energy for the lumiflavin-phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine. This effect is probably caused by amino acids C266 and L268, which face the other side of this tyrosine. Simple and double FNR mutants of these amino acids were obtained and characterized. It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure. Our results provide experimental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme.

  2. Mechanistic insights into ferredoxin-NADP(H) reductase catalysis involving the conserved glutamate in the active site.

    Science.gov (United States)

    Dumit, Verónica I; Essigke, Timm; Cortez, Néstor; Ullmann, G Matthias

    2010-04-02

    Plant-type ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes harboring one molecule of noncovalently bound flavin adenine dinucleotide that catalyze reversible reactions between obligatory one-electron carriers and obligatory two-electron carriers. A glutamate next to the C-terminus is strictly conserved in FNR and has been proposed to function as proton donor/acceptor during catalysis. However, experimental studies of this proposed function led to contradicting conclusions about the role of this glutamate in the catalytic mechanism. In the present work, we study the titration behavior of the glutamate in the active site of FNR using theoretical methods. Protonation probabilities for maize FNR were computed for the reaction intermediates of the catalytic cycle by Poisson-Boltzmann electrostatic calculations and Metropolis Monte Carlo titration. The titration behavior of the highly conserved glutamate was found to vary depending on the bound substrates NADP(H) and ferredoxin and also on the redox states of these substrates and the flavin adenine dinucleotide. Our results support the involvement of the glutamate in the FNR catalytic mechanism not only as a proton donor but also as a key residue for stabilizing and destabilizing reaction intermediates. On the basis of our findings, we propose a model rationalizing the function of the glutamate in the reaction cycle, which allows reinterpretation of previous experimental results. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.

    Science.gov (United States)

    Zhan, Dongling; Bai, Aixi; Yu, Lei; Han, Weiwei; Feng, Yan

    2014-01-01

    The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of ∼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters Kcat and Kcat/Km of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, Kcat and Kcat/Km is 10- and 21-fold higher than that of WT. Experimental data indicate the important functions of Tyr120: involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily.

  4. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 A resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 A. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V(M) = 2.24 A3 Da(-1)). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 A.

  5. Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-02-01

    Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.

  6. Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Bagautdinov, Bagautdin; Kunishima, Naoki

    2006-04-01

    Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 angstroms resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3(2)21, with unit-cell parameters a = b = 96.06, c = 298.90 angstroms. Assuming the presence of one hexamer in the asymmetric unit gives a V(M) value of 2.36 angstroms3 Da(-1) and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 angstroms resolution.

  7. Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2006-04-01

    RecA superfamily ATPase PH0284 from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with ATP. Both crystal forms belong to the trigonal space group P3{sub 2}21 and diffract X-rays to 2.0 and 2.3 Å resolution, respectively. Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 Å resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3{sub 2}21, with unit-cell parameters a = b = 96.06, c = 298.90 Å. Assuming the presence of one hexamer in the asymmetric unit gives a V{sub M} value of 2.36 Å{sup 3} Da{sup −1} and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 Å resolution.

  8. Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Sugahara, Mitsuaki [Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2007-01-01

    An archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue from P. horikoshii OT3 was overexpressed as native and selenomethionine-substituted protein, purified and crystallized. The native and selenomethionine-derivative crystals are isomorphous and diffract X-rays to 2.1 and 2.9 Å resolution, respectively. 6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 Å resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V{sub M} = 2.24 Å{sup 3} Da{sup −1}). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 Å.

  9. Purification, crystallization and preliminary crystallographic analysis of the biotin–protein ligase from Pyrococcus horikoshii OT3

    Science.gov (United States)

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki

    2005-01-01

    Biotin–protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin–protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 Å resolution from a native crystal and to 1.55 Å resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 38.601, b = 78.264, c  =  70.147 Å, β = 101.48°. Assuming a homodimer per asymmetric unit gives a V M value of 2.14 Å3 Da−1 and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5′-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 Å resolution, respectively. PMID:16510991

  10. Structural basis for the transglycosylase activity of a GH57-type glycogen branching enzyme from Pyrococcus horikoshii.

    Science.gov (United States)

    Na, Soohui; Park, Minjeong; Jo, Inseong; Cha, Jaeho; Ha, Nam-Chul

    2017-03-18

    Glycogen branching enzyme (GBE) catalyzes the formation of α-1,6-branching points during glycogenesis by cleaving α-1,4 bonds and making new α-1,6 bonds. Most GBEs belong to the glycoside hydrolase 13 family (GH13), but new GBEs in the GH57 family have been isolated from Archaea. Here, we determined the crystal structure of a GH57 GBE from the hyperthermophilic archaeon Pyrococcus horikoshii (PhGBE) at a resolution of 2.3 Å. PhGBE exhibits both α-1,6-branching activity and endo-α-1,4 hydrolytic activity. PhGBE has a central (β/α)7-barrel domain that contains an embedded helix domain and an α-helix-rich C-terminal domain. The active-site cleft is located at the interface of the central and C-terminal domains. Amino acid substitution at Trp22, which is separate from the catalytic nucleophilic residue, abolished both enzymatic activities, indicating that Trp22 might be responsible for substrate recognition. We also observed that shortening of the flexible loop near the catalytic residue changed branched chain lengths of the reaction products with increased hydrolytic activity. Taken together, our findings propose a molecular mechanism for how GH57 GBEs exhibit the two activities and where the substrate binds the enzyme.

  11. Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii.

    Science.gov (United States)

    Shen, Y; Tang, X-F; Matsui, E; Matsui, I

    2004-04-01

    Family D DNA polymerase (PolD) has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. We successfully cloned, expressed, and purified the family D DNA polymerase from Pyrococcus horikoshii (PolDPho). By site-directed mutagenesis, we identified amino acid residues Asp-1122 and Asp-1124 of a large subunit as the essential residues responsible for DNA-polymerizing activity. We analysed the domain structure using proteins truncated at the N- and C-termini of both small and large subunits (DP1Pho and DP2Pho), and identified putative regions responsible for subunit interaction, oligomerization and regulation of the 3'-5' exonuclease activity in PolDPho. It was also found that the internal region of the putative zinc finger motif (cysteine cluster II) at the C-terminal of DP2Pho is involved in the 3'-5' exonuclease activity. Using gel filtration analysis, we determined the molecular masses of the recombinant PolDPho and the N-terminal putative dimerization domain of the large subunit, and proposed that PolD from P. horikoshii probably forms a heterotetrameric structure in solution. Based on these results, a model regarding the subunit interaction and regulation of activity of PolDPho is proposed.

  12. Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

    Science.gov (United States)

    Matsunaga, Fujihiko; Glatigny, Annie; Mucchielli-Giorgi, Marie-Hélène; Agier, Nicolas; Delacroix, Hervé; Marisa, Laetitia; Durosay, Patrice; Ishino, Yoshizumi; Aggerbeck, Lawrence; Forterre, Patrick

    2007-01-01

    The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.

  13. Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction

    Science.gov (United States)

    Diakonova, A. N.; Khrushchev, S. S.; Kovalenko, I. B.; Riznichenko, G. Yu; Rubin, A. B.

    2016-10-01

    Ferredoxin (Fd) protein transfers electrons from photosystem I (PSI) to ferredoxin:NADP+-reductase (FNR) in the photosynthetic electron transport chain, as well as other metabolic pathways. In some photosynthetic organisms including cyanobacteria and green unicellular algae under anaerobic conditions Fd transfers electrons not only to FNR but also to hydrogenase—an enzyme which catalyzes reduction of atomic hydrogen to H2. One of the questions posed by this competitive relationship between proteins is which characteristics of thylakoid stroma media allow switching of the electron flow between the linear path PSI-Fd-FNR-NADP+ and the path PSI-Fd-hydrogenase-H2. The study was conducted using direct multiparticle simulation approach. In this method protein molecules are considered as individual objects that experience Brownian motion and electrostatic interaction with the surrounding media and each other. Using the model we studied the effects of pH and ionic strength (I) upon complex formation between ferredoxin and FNR and ferredoxin and hydrogenase. We showed that the rate constant of Fd-FNR complex formation is constant in a wide range of physiologically significant pH values. Therefore it can be argued that regulation of FNR activity doesn’t involve pH changes in stroma. On the other hand, in the model rate constant of Fd-hydrogenase interaction dramatically depends upon pH: in the range 7-9 it increases threefold. It may seem that because hydrogenase reduces protons it should be more active when pH is acidic. Apparently, regulation of hydrogenase’s affinity to both her reaction partners (H+ and Fd) is carried out by changes in its electrostatic properties. In the dark, the protein is inactive and in the light it is activated and starts to interact with both Fd and H+. Therefore, we can conclude that in chloroplasts the rate of hydrogen production is regulated by pH through the changes in the affinity between hydrogenase and ferredoxin.

  14. Spectroscopic and Redox Studies of Valence-Delocalized [Fe2S2]+ Centers in Thioredoxin-Like Ferredoxins

    Science.gov (United States)

    Subramanian, Sowmya; Duin, Evert C.; Fawcett, Sarah E. J.; Armstrong, Fraser A.; Meyer, Jacques; Johnson, Michael K.

    2015-01-01

    Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2]+ clusters, which constitute a fundamental building block of all higher nuclearity Fe-S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2]2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV-visible-NIR absorption, CD and variable-temperature MCD, and protein-film electrochemistry. The results indicate that the [Fe2S2]+ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0, due to protonation of the coordinated serinate residue. However, freezing high-pH samples results in partial or full conversion from valence-delocalized S = 9/2 to valence-localized S = 1/2 [Fe2S2]+ clusters. MCD saturation magnetization data for valence-delocalized S = 9/2 [Fe2S2]+ centers facilitated determination of transition polarizations and thereby assignments of low-energy MCD bands associated with the Fe−Fe interaction. The assignments provide experimental assessment of the double exchange parameter, B, for valence-delocalized [Fe2S2]+ centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized S = 9/2 [Fe2S2]+ fragments in higher nuclearity Fe-S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe-S clusters in general is discussed in light of these results. PMID:25790339

  15. Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

    Science.gov (United States)

    Kemper, M A; Stout, C D; Lloyd, S J; Prasad, G S; Fawcett, S E; Armstrong, F A; Shen, B; Burgess, B K; Lloyd, S E; Fawcett, S

    1997-06-20

    Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

  16. Crystal Structure of the FAD-Containing Ferredoxin-NADP+ Reductase from the Plant Pathogen Xanthomonas axonopodis pv. citri

    Directory of Open Access Journals (Sweden)

    María Laura Tondo

    2013-01-01

    Full Text Available We have solved the structure of ferredoxin-NADP(H reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5 Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen.

  17. Ab initio single and multideterminant methods used in the determination of reduction potentials and magnetic properties of Rieske ferredoxins

    Science.gov (United States)

    Powers, Nathan Lee

    2008-10-01

    The [Fe2S2]2+/[Fe2S 2]+ electronic structure of seven Rieske protein active sites (bovine mitochondrial cytochrome bc1 complex, spinach chloroplast cytochrome b6f complex, Rieske-type ferredoxin associated with biphenyl dioxygenase from Burkholderia cepacia, yeast cytochrome bcl complex from Saccharomyces cerevisiae, Rieske subunit of arsenite oxidase from Alcaligenes faecalis, respiratory-type Rieske protein from Thermus thermophilus, and Rieske protein II (soxF) from Sulfolobus acidocaldarius), which lie in a reduction potential range from -150 mV to 375 mV, have been studied by both single and multi-determinant quantum mechanical methods. Calculated reduction potentials and magnetic properties are found comparable to experimental values.

  18. Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Pampa, K.J., E-mail: sagarikakj@gmail.com [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India); Lokanath, N.K. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Girish, T.U. [Department of General Surgery, JSS Medical College and Hospital, JSS University, Mysore 570 015 (India); Kunishima, N. [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148 (Japan); Rai, V.R. [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India)

    2014-10-24

    Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme by X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.

  19. Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+.

    Science.gov (United States)

    Nascimento, Alessandro S; Catalano-Dupuy, Daniela L; Bernardes, Amanda; Neto, Mario de Oliveira; Santos, Maria Auxiliadora M; Ceccarelli, Eduardo A; Polikarpov, Igor

    2007-10-24

    Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

  20. Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+

    Directory of Open Access Journals (Sweden)

    Ceccarelli Eduardo A

    2007-10-01

    Full Text Available Abstract Background Ferredoxin-NADP(H reductases (FNRs are flavoenzymes that catalyze the electron transfer between NADP(H and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR displays low sequence identity with plant (34% with Zea mays and bacterial (31% with Escherichia coli FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. Results The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. Conclusion LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

  1. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) from Bacillus cereus.

    Science.gov (United States)

    Skråmo, Silje; Hersleth, Hans Petter; Hammerstad, Marta; Andersson, K Kristoffer; Røhr, Åsmund K

    2014-06-01

    Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. In Bacillus cereus there are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space group P2₁2₁2, with unit-cell parameters a=57.2, b=164.3, c=95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.

  2. Reduction of the pea ferredoxin-NADP(H) reductase catalytic efficiency by the structuring of a carboxyl-terminal artificial metal binding site.

    Science.gov (United States)

    Catalano-Dupuy, Daniela L; Orecchia, Martín; Rial, Daniela V; Ceccarelli, Eduardo A

    2006-11-21

    Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, and adrenodoxin) to redox-based metabolisms in plastids, mitochondria, and bacteria. The FNRs from plants and most eubacteria constitute a unique family, the plant-type ferredoxin-NADP(H) reductases. Plastidic FNRs are quite efficient at sustaining the demands of the photosynthetic process. At variance, FNRs from organisms with heterotrophic metabolisms or anoxygenic photosynthesis display turnover numbers that are 20-100-fold lower than those of their plastidic and cyanobacterial counterparts. To gain insight into the FNR structural features that modulate enzyme catalytic efficiency, we constructed a recombinant FNR in which the carboxyl-terminal amino acid (Tyr308) is followed by an artificial metal binding site of nine amino acids, including four histidine residues. This added structure binds Zn2+ or Co2+ and, as a consequence, significantly reduces the catalytic efficiency of the enzyme by decreasing its kcat. The Km for NADPH and the Kd for NADP+ were increased 2 and 3 times, respectively, by the addition of the amino acid extension in the absence of Zn2+. Nevertheless, the structuring of the metal binding site did not change the Km for NADPH or the Kd for NADP+ of the FNR-tail enzyme. Our results provide experimental evidence which indicates that mobility of the carboxyl-terminal backbone region of the FNR, mainly Tyr308, is essential for obtaining an FNR enzyme with high catalytic efficiency.

  3. The Rnf Complex of Clostridium ljungdahlii Is a Proton-Translocating Ferredoxin:NAD(+) Oxidoreductase Essential for Autotrophic Growth

    Energy Technology Data Exchange (ETDEWEB)

    Tremblay, PL; Zhang, T; Dar, SA; Leang, C; Lovley, DR

    2012-12-26

    It has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin: NAD(+) oxidoreductase which contributes to ATP synthesis by an H+-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H-2 as the electron source and CO2 as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation. IMPORTANCE Mechanisms for energy conservation in the acetogen Clostridium ljungdahlii are of interest because of its potential value as a chassis for the production of biocommodities with novel electron donors such as carbon monoxide, syngas, and electrons derived from electrodes. Characterizing the components implicated in the chemiosmotic ATP synthesis during acetogenesis by C. ljungdahlii is a prerequisite for the development of highly productive strains. The Rnf complex has been considered the prime candidate to be the pump responsible for the formation of an ion gradient coupled with ATP synthesis in multiple acetogens. However, experimental evidence for a proton-pumping Rnf complex has been lacking. This study establishes the C. ljungdahlii Rnf complex as

  4. Science Letters:Preparation, crystallization and preliminary X-ray diffraction analysis of PH1948, predicted RNA methyltransferase from Pyrococcus horikoshii

    Institute of Scientific and Technical Information of China (English)

    GAO Yong-gui; YAO Min; TANAKA Isao

    2005-01-01

    RNA methyltransferase is responsible for transferring methyl and resulting in methylation on the bases or ribose ring of RNA, which existed widely but mostly remains an open question. A recombinant protein PH1948 predicting RNA methyltransferase from Pyrococcus horikoshii OT3 has been crystallized. The crystals of selenomethionyl PH1948 belong to space group C2, with unit-cell parameters a=207.0 (A),b=43.1 (A), c= 118.2 (A), β=92.1°, and diffract X-rays to 2.2(A) resolution. The VM value was determined to be 2.8(A)3/Da, indicating the presence of four protein molecules in the asymmetric unit.

  5. Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

    Science.gov (United States)

    Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

    2016-12-27

    The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

  6. Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O 2-accumulating Brassica napus leaf protoplasts.

    Science.gov (United States)

    Tewari, Rajesh Kumar; Satoh, Mamoru; Kado, Sayaka; Mishina, Kohei; Anma, Misato; Enami, Kazuhiko; Hanaoka, Mitsumasa; Watanabe, Masami

    2014-12-01

    The isolation of Brassica napus leaf protoplasts induces reactive oxygen species generation and accumulation in the chloroplasts. An activated isoform of NADPH oxidase-like protein was detected in the protoplasts and the protoplast chloroplasts. The purpose of this study is to define the NADH oxidase-like activities in the H2O2-accumulating protoplast chloroplasts. Proteomic analysis of this protein revealed an isoform of ferredoxin:NADPH oxidoreductase (FNR1). While leaves highly expressed the LFNR1 transcript, protoplasts decreased the expression significantly. The protoplast chloroplasts predominantly expressed soluble FNR1 proteins. While the albino leaves of white kale (Brassica oleracea var. acephala f. tricolor cv. white pigeon) expressed FNR1 protein at the same level as B. napus leaves, the protoplasts of albino leaves displayed reduced FNR1 expression. The albino leaf protoplasts of white kale generated and accumulated H2O2 in the cytoplasm and on the plasma membrane. Intracellular pH showed that the chloroplasts were acidic, which suggest that excess H(+) was generated in chloroplast stroma. NADPH content of the protoplast chloroplasts increased by over sixfold during the isolation of protoplasts. This study reports a possibility of mediating electrons to oxygen by an overproduced soluble FNR, and suggests that the FNR has a function in utilizing any excess reducing power of NADPH.

  7. The chloroplast NifS-like protein of Arabidopsis thaliana is required for iron-sulfur cluster formation in ferredoxin.

    Science.gov (United States)

    Ye, Hong; Garifullina, Gulnara F; Abdel-Ghany, Salah E; Zhang, Lihong; Pilon-Smits, Elizabeth A H; Pilon, Marinus

    2005-02-01

    Plastids are known to be able to synthesize their own iron-sulfur clusters, but the biochemical machinery responsible for this process is not known. In this study it is investigated whether CpNifS, the chloroplastic NifS-like cysteine desulfurase of Arabidopsis thaliana (L.) Heynh. is responsible for the release of sulfur from cysteine for the biogenesis of iron-sulfur (Fe-S) clusters in chloroplasts. Using an in vitro reconstitution assay it was found that purified CpNifS was sufficient for Fe-S cluster formation in ferredoxin in the presence of cysteine and a ferrous iron salt. Antibody-depletion experiments using stromal extract showed that CpNifS is also essential for the Fe-S cluster formation activity of chloroplast stroma. The activity of CpNifS in the stroma was 50- to 80-fold higher than that of purified CpNifS on a per-protein basis, indicating that other stromal factors cooperate in Fe-S cluster formation. When stromal extract was separated on a gel-filtration column, most of the CpNifS eluted as a dimer of 86 kDa, but a minor fraction of the stromal CpNifS eluted at a molecular weight of approx. 600 kDa, suggesting the presence of a multi-protein complex. The possible nature of the interacting proteins is discussed.

  8. Atomic-resolution structure of the phycocyanobilin:ferredoxin oxidoreductase I86D mutant in complex with fully protonated biliverdin.

    Science.gov (United States)

    Hagiwara, Yoshinori; Wada, Kei; Irikawa, Teppei; Sato, Hideaki; Unno, Masaki; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu

    2016-10-01

    Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the reduction of biliverdin (BV) to produce phycocyanobilin, a linear tetrapyrrole pigment used for light harvesting and light sensing. Spectroscopic and HPLC analyses inidicate that BV bound to the I86D mutant of PcyA is fully protonated (BVH(+) ) and can accept an electron, but I86D is unable to donate protons for the reduction; therefore, compared to the wild-type PcyA, the I86D mutant stabilizes BVH(+) . To elucidate the structural basis of the I86D mutation, we determined the atomic-resolution structure of the I86D-BVH(+) complex and the protonation states of the essential residues Asp105 and Glu76 in PcyA. Our study revealed that Asp105 adopted a fixed conformation in the I86D mutant, although it had dual conformations in wild-type PcyA which reflected the protonation states of BV. Taken together with biochemical/spectroscopic results, our analysis of the I86D-BVH(+) structure supports the hypothesis that flexibility of Asp105 is essential for the catalytic activity of PcyA.

  9. N-terminal structure of maize ferredoxin:NADP+ reductase determines recruitment into different thylakoid membrane complexes.

    Science.gov (United States)

    Twachtmann, Manuel; Altmann, Bianca; Muraki, Norifumi; Voss, Ingo; Okutani, Satoshi; Kurisu, Genji; Hase, Toshiharu; Hanke, Guy T

    2012-07-01

    To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

  10. A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation.

    Science.gov (United States)

    Thomas, Jean-Claude; Ughy, Bettina; Lagoutte, Bernard; Ajlani, Ghada

    2006-11-28

    Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNR(S) approximately 34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNR(L) approximately 46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNR(S), is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNR(L) is an NADP(+) reductase, whereas FNR(S) is an NADPH oxidase.

  11. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

    Science.gov (United States)

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen

    2015-12-01

    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

  12. Inactivation of Escherichia coli glutamine synthetase by xanthine oxidase, nicotinate hydroxylase, horseradish peroxidase, or glucose oxidase: effects of ferredoxin, putidaredoxin, and menadione.

    Science.gov (United States)

    Stadtman, E R; Wittenberger, M E

    1985-06-01

    Previous studies have shown that several mixed-function oxidation (MFO) systems are capable of catalyzing the inactivation of glutamine synthetase (GS) [R.L. Levine, C. N. Oliver, R. M. Fulks, and E. R. Stadtman (1978) Proc. Natl. Acad. Sci. USA 78, 2120-2124] and a number of the other enzymes [L. Fucci, C. N. Oliver, M. J. Coon, and E. R. Stadtman (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525]. It has now been found that in the presence of Fe(III), O2, and an appropriate electron donor (hypoxanthine or NADPH, respectively) glutamine synthetase is also inactivated by either milk xanthine oxidase or Clostridial nicotinate hydroxylase. Inactivation of glutamine synthetase by either of these flavoproteins is greatly stimulated by the presence of electron carrier proteins possessing nonheme-iron-sulfur (NHIS) clusters (i.e., ferredoxin or putidaredoxin) or by the presence of menadione. The inactivation reactions are partially inhibited by free radical scavengers, superoxide dismutase, (SOD), histidine, mannitol, dimethyl sulfoxide, and dimethylthiourea, and are inhibited completely by either Mn(II), EDTA, or catalase. The sensitivity to SOD inhibition is greatly suppressed when the xanthine oxidase system is supplemented with either ferredoxin or redoxin. In the presence of the latter NHIS-proteins (and only when they are present), MFO systems, comprised of either horseradish peroxidase and H2O2 or glucose oxidase, O2, and glucose, can also catalyze the inactivation of GS. The ability of ferredoxin and putidaredoxin to promote oxidation modification of GS by any one of these MFO systems suggests that proteins with NHIS centers may mediate the generation (or stabilization) of highly reactive radical intermediates.

  13. Mutated forms of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to the iron-sulfur cluster.

    Science.gov (United States)

    Meyer, J; Fujinaga, J; Gaillard, J; Lutz, M

    1994-11-22

    The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, 60). Thus, no homologous sequences are available to infer, by comparison, the identity of the ligands of the iron-sulfur cluster. Therefore, in order to obtain information on the latter point, a combination of site-directed mutagenesis and UV-vis, EPR, and resonance Raman spectroscopy has been implemented. All of the cysteine residues have individually been replaced by serine and two of them by alanine. Cysteine 14 could be replaced by either serine or alanine without any modification of the spectroscopic properties of the protein and was therefore dismissed as a ligand of the [2Fe-2S] cluster. The C56S, and C60S-mutated proteins were both found to display UV-vis, EPR, and resonance Raman spectra consistent with serine-coordinated [2Fe-2S] clusters. The C11S-mutated protein was considerably less stable than the wild type ferredoxin. This observation, together with the hypsochromic shifts of UV-visible absorption features upon cysteine 11-->serine mutation, suggested cysteine 11 to be a ligand of the [2Fe-2S] cluster. Cysteine 24 could be replaced by either serine or alanine without decreasing the stability of the protein and without dramatically changing its spectroscopic properties. Thus, either cysteine 24 is not a ligand of the [2Fe-2S] cluster or it is replaced by another ligand in the C24A mutated protein. A [2Fe-2S] cluster was also assembled in the C14A/C24A doubly mutated protein, i.e., in a polypeptide chain containing only three cysteine residues.2+ off

  14. Mapping of protein-protein interaction sites in the plant-type [2Fe-2S] ferredoxin.

    Directory of Open Access Journals (Sweden)

    Haruka Kameda

    Full Text Available Knowing the manner of protein-protein interactions is vital for understanding biological events. The plant-type [2Fe-2S] ferredoxin (Fd, a well-known small iron-sulfur protein with low redox potential, partitions electrons to a variety of Fd-dependent enzymes via specific protein-protein interactions. Here we have refined the crystal structure of a recombinant plant-type Fd I from the blue green alga Aphanothece sacrum (AsFd-I at 1.46 Å resolution on the basis of the synchrotron radiation data. Incorporating the revised amino-acid sequence, our analysis corrects the 3D structure previously reported; we identified the short α-helix (67-71 near the active center, which is conserved in other plant-type [2Fe-2S] Fds. Although the 3D structures of the four molecules in the asymmetric unit are similar to each other, detailed comparison of the four structures revealed the segments whose conformations are variable. Structural comparison between the Fds from different sources showed that the distribution of the variable segments in AsFd-I is highly conserved in other Fds, suggesting the presence of intrinsically flexible regions in the plant-type [2Fe-2S] Fd. A few structures of the complexes with Fd-dependent enzymes clearly demonstrate that the protein-protein interactions are achieved through these variable regions in Fd. The results described here will provide a guide for interpreting the biochemical and mutational studies that aim at the manner of interactions with Fd-dependent enzymes.

  15. Crystal structure analysis of Bacillus subtilis ferredoxin-NADP(+) oxidoreductase and the structural basis for its substrate selectivity.

    Science.gov (United States)

    Komori, Hirofumi; Seo, Daisuke; Sakurai, Takeshi; Higuchi, Yoshiki

    2010-12-01

    Bacillus subtilis yumC encodes a novel type of ferredoxin-NADP+ oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP+ has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH-thioredoxin reductase (TdR) and TdR-like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP+ binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C-terminal extension, not found in E. coli TdR but in TdR-like protein from T. thermophilus HB8, covers the re-face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD-binding region and His324 in the C-terminal extension stack on the si- and re-faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid-type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid-type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2'-phosphate of NADP+, drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re-face of FAD is hardly relevant to the nucleotide selectivity. Copyright © 2010 The Protein Society.

  16. A single tyrosine hydroxyl group almost entirely controls the NADPH specificity of Plasmodium falciparum ferredoxin-NADP+ reductase.

    Science.gov (United States)

    Baroni, Sara; Pandini, Vittorio; Vanoni, Maria Antonietta; Aliverti, Alessandro

    2012-05-08

    Plasmodium falciparum ferredoxin-NADP(+) reductase (FNR) is a FAD-containing enzyme that, in addition to be a promising target of novel antimalarial drugs, represents an excellent model of plant-type FNRs. The cofactor specificity of FNRs depends on differences in both k(cat) and K(m) values for NADPH and NADH. Here, we report that deletion of the hydroxyl group of the conserved Y258 of P. falciparum FNR, which interacts with the 2'-phosphate group of NADPH, selectively decreased the k(cat) of the NADPH-dependent reaction by a factor of 2 to match that of the NADH-dependent one. Rapid-reaction kinetics, active-site titrations with NADP(+), and anaerobic photoreduction experiments indicated that this effect may be the consequence of destabilization of the catalytically competent conformation of bound NADPH. Moreover, because the Y258F replacement increased the K(m) for NADPH 4-fold and decreased that for NADH 3-fold, it led to a drop in the ability of the enzyme to discriminate between the coenzymes from 70- to just 1.5-fold. The impact of the Y258F change was not affected by the presence of the H286Q mutation, which is known to enhance the catalytic activity of the enzyme. Our data highlight the major role played by the Y258 hydroxyl group in determining the coenzyme specificity of P. falciparum FNR. From the general standpoint of engineering the kinetic properties of plant-type FNRs, although P. falciparum FNR is less strictly NADPH-dependent than its homologues, the almost complete abolishment of coenzyme selectivity reported here has never been accomplished before through a single mutation.

  17. External loops at the ferredoxin-NADP(+) reductase protein-partner binding cavity contribute to substrates allocation.

    Science.gov (United States)

    Sánchez-Azqueta, Ana; Martínez-Júlvez, Marta; Hervás, Manuel; Navarro, José A; Medina, Milagros

    2014-02-01

    Ferredoxin-NADP(+) reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)(+)/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet-loop-sheet motif, loop102-114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261-269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102-114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261-269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261-269 appears a determinant to ensure reversibility in photosynthetic FNRs. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system.

    Science.gov (United States)

    Boyer, Marcus E; Wang, Chia-Wei; Swartz, James R

    2006-05-05

    The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.

  19. Simultaneous involvement of a tungsten-containing aldehyde:ferredoxin oxidoreductase and a phenylacetaldehyde dehydrogenase in anaerobic phenylalanine metabolism.

    Science.gov (United States)

    Debnar-Daumler, Carlotta; Seubert, Andreas; Schmitt, Georg; Heider, Johann

    2014-01-01

    Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.

  20. Expression of the Minor Isoform Pea Ferredoxin in Tobacco Alters Photosynthetic Electron Partitioning and Enhances Cyclic Electron Flow1[W

    Science.gov (United States)

    Blanco, Nicolás E.; Ceccoli, Romina D.; Vía, María V. Dalla; Voss, Ingo; Segretin, María E.; Bravo-Almonacid, Fernando F.; Melzer, Michael; Hajirezaei, Mohammad-Reza; Scheibe, Renate; Hanke, Guy T.

    2013-01-01

    Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms. PMID:23370717

  1. An analysis of g strain in the EPR of two [2Fe2S] ferredoxins. Evidence for a protein rigidity model

    Science.gov (United States)

    Hearshen, D. O.; Hagen, W. R.; Sands, R. H.; Grande, H. J.; Crespi, H. L.; Gunsalus, I. C.; Dunham, W. R.

    Replacing current notions of a paramagnetic center in a metalloprotein as a single entity in vivo with the more realistic concept of an ensemble of spin systems, each uniquely disturbed by its own surrounding protein, leads to a rigorous description of the spectroscopic factor, g, as a random variable whose statistical properties contain information on the rigidity of the protein. Generation of a consistent set of accurate simulations of very low-noise, multifrvquency (3, 9, 15 GHz) EPR data from selected S = {1}/{2} proteins has now been achieved. This consistency lends support to the physical and biological inferences drawn from such simulations. The spectral contribution of magnetic hyperfine line-broadening is minimized by studying the 56Fe reconstituted [2Fe2S] cluster in fully deuterated ferredoxin from Synechococcus lividus and the 2H 2O exchanged [2Fe2S] ferredoxin from Pseudomonas putida. High-resolution Mössbauer data on oxidized and reduced 57Fe reconstituted S. lividus ferredoxin are also presented. The oxidized spectrum shows that the inequivalence of the two ferric ions in a [2Fe2S] cluster can be resolved as two Mössbauer lines. The complete absence of this splitting in the ferric fines of the reduced spectrum is definitive proof that the reducing electron always resides at the same 56Fe atom in frozen aqueous solutions. To explain the distributed nature of the paramagnetic site in the ferredoxins, three models are considered: (1) a multiplicity of EPR states; (2) external perturbations to the molecular Hamiltonian; (3) a distribution in the crystal field Hamiltonian parameters. The first model is discarded, the second is possible but difficult to verify, and the third model is shown to fit the data well. The latter comparison requires a correction to literature expressions for the g and A tensors in [2Fe2S] clusters. Statistical analysis strongly suggests that the EPR of metalloproteins in its details is a reflection of protein

  2. Antiferromagnetic exchange interaction in the two-iron-two-sulphur ferredoxin from the blue-green alga Spirulina maxima studied with a highly sensitive magnetic balance.

    Science.gov (United States)

    Petersson, L; Cammack, R; Rao, K K

    1980-03-26

    1. A highly sensitive magnetic balance of the Faraday type is described. 2. The magnetic susceptibility of the oxidized and reduced forms of the two-iron-two-sulphur ferredoxin from the blue-green alga Spirulina maxima has been measured over a wide temperature range. 3. The results can be interpreted within a simple model involving antiferromagnetically coupled iron atoms at the active site. The coupling, expressed as --J, is estimated to be 182 +/- 20/cm and 98 +5/-10 /cm for the oxidized and reduced forms, respectively.

  3. A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

    Science.gov (United States)

    Cho, Min Seok; Lee, Jang Ha; Her, Nam Han; Kim, Changkug; Seol, Young-Joo; Hahn, Jang Ho; Baeg, Ji Hyoun; Kim, Hong Gi; Park, Dong Suk

    2012-06-01

    The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

  4. Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

    Directory of Open Access Journals (Sweden)

    Li-Fen Huang

    2015-08-01

    Full Text Available Ferredoxins (FDX are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC; power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

  5. Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (Oryza sativa).

    Science.gov (United States)

    Chang, Hsiang; Huang, Hsiang-En; Cheng, Chin-Fu; Ho, Mei-Hsuan; Ger, Mang-Jye

    2017-04-01

    The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by

  6. Salt shock-inducible Photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin : NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain

    NARCIS (Netherlands)

    van Thor, JJ; Jeanjean, R; Havaux, M; Sjollema, KA; Joset, F; Hellingwerf, KJ; Matthijs, HCP

    2000-01-01

    Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terninus, The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The pheno

  7. Salt shock-inducible Photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin : NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain

    NARCIS (Netherlands)

    van Thor, JJ; Jeanjean, R; Havaux, M; Sjollema, KA; Joset, F; Hellingwerf, KJ; Matthijs, HCP

    2000-01-01

    Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terninus, The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The pheno

  8. Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04.

    Science.gov (United States)

    Park, Kwang-Hyun; Jung, Jong-Hyun; Park, Sung-Goo; Lee, Myeong-Eun; Holden, James F; Park, Cheon-Seok; Woo, Eui-Jeon

    2014-06-01

    A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

  9. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-04-01

    Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein-protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPDeltaN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 complexes as well as crystals of BPL*, BPL** and BCCPDeltaN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 crystals were collected at 100 K to 2.7 and 2.0 A resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2(1), with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 A, beta = 95.9 degrees . Assuming two subunits of the complex per asymmetric unit gives a V(M) value of 2.45 A(3) Da(-1) and a solvent content of 50%.

  10. Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

    Science.gov (United States)

    Kurdrid, Pavinee; Subudhi, Sanjukta; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Hongsthong, Apiradee

    2007-12-01

    When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

  11. Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles

    Directory of Open Access Journals (Sweden)

    Mohammad S. Eram

    2013-08-01

    Full Text Available Pyruvate decarboxylase (PDC encoded by pdc is a thiamine pyrophosphate (TPP-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE. Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL, and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter. However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles.

  12. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Carte, Jason; Wang, Ruiying; Li, Hong; Terns, Rebecca M.; Terns, Michael P. (FSU); (Georgia)

    2010-11-09

    An RNA-based gene silencing pathway that protects bacteria and archaea from viruses and other genome invaders is hypothesized to arise from guide RNAs encoded by CRISPR loci and proteins encoded by the cas genes. CRISPR loci contain multiple short invader-derived sequences separated by short repeats. The presence of virus-specific sequences within CRISPR loci of prokaryotic genomes confers resistance against corresponding viruses. The CRISPR loci are transcribed as long RNAs that must be processed to smaller guide RNAs. Here we identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targeting RNAs. Cas6 interacts with a specific sequence motif in the 5{prime} region of the CRISPR repeat element and cleaves at a defined site within the 3{prime} region of the repeat. The 1.8 angstrom crystal structure of the enzyme reveals two ferredoxin-like folds that are also found in other RNA-binding proteins. The predicted active site of the enzyme is similar to that of tRNA splicing endonucleases, and concordantly, Cas6 activity is metal-independent. cas6 is one of the most widely distributed CRISPR-associated genes. Our findings indicate that Cas6 functions in the generation of CRISPR-derived guide RNAs in numerous bacteria and archaea.

  13. The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa

    Science.gov (United States)

    Boonma, Siriwan; Romsang, Adisak; Duang-nkern, Jintana; Atichartpongkul, Sopapan; Trinachartvanit, Wachareeporn; Vattanaviboon, Paiboon

    2017-01-01

    Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes. PMID:28187184

  14. Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

    Science.gov (United States)

    Jouanneau, Y; Meyer, C; Asso, M; Guigliarelli, B; Willison, J C

    2000-02-01

    A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.

  15. The Flavoenzyme Ferredoxin (Flavodoxin)-NADP(H) Reductase Modulates NADP(H) Homeostasis during the soxRS Response of Escherichia coli

    Science.gov (United States)

    Krapp, Adriana R.; Rodriguez, Ramiro E.; Poli, Hugo O.; Paladini, Dar|$$|Aa|fio H.; Palatnik, Javier F.; Carrillo, Néstor

    2002-01-01

    Escherichia coli cells from strain fpr, deficient in the soxRS-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV). Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in fpr cells exposed to MV. FPR inactivation did not affect the MV-driven soxRS response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool. Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on soxRS induction and failed to protect fpr cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria. PMID:11844783

  16. Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin-NAD(P)+ reductase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

    Science.gov (United States)

    Liauw, Pasqual; Mashiba, Tomohiro; Kopczak, Marta; Wiegand, Katrin; Muraki, Norifumi; Kubota, Hisako; Kawano, Yusuke; Ikeuchi, Masahiko; Hase, Toshiharu; Rögner, Matthias; Kurisu, Genji

    2012-09-01

    Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, β = 98.21, γ = 90°.

  17. Functional analysis of two isoforms of leaf-type ferredoxin-NADP(+)-oxidoreductase in rice using the heterologous expression system of Arabidopsis.

    Science.gov (United States)

    Higuchi-Takeuchi, Mieko; Ichikawa, Takanari; Kondou, Youichi; Matsui, Keiko; Hasegawa, Yukako; Kawashima, Mika; Sonoike, Kintake; Mori, Masaki; Hirochika, Hirohiko; Matsui, Minami

    2011-09-01

    Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1- and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation.

  18. Ectopically expressed sweet pepper ferredoxin PFLP enhances disease resistance to Pectobacterium carotovorum subsp. carotovorum affected by harpin and protease-mediated hypersensitive response in Arabidopsis.

    Science.gov (United States)

    Ger, Mang-Jye; Louh, Guan-Yu; Lin, Yi-Hsien; Feng, Teng-Yung; Huang, Hsiang-En

    2014-12-01

    Plant ferredoxin-like protein (PFLP) is a photosynthesis-type ferredoxin (Fd) found in sweet pepper. It contains an iron-sulphur cluster that receives and delivers electrons between enzymes involved in many fundamental metabolic processes. It has been demonstrated that transgenic plants overexpressing PFLP show a high resistance to many bacterial pathogens, although the mechanism remains unclear. In this investigation, the PFLP gene was transferred into Arabidopsis and its defective derivatives, such as npr1 (nonexpresser of pathogenesis-related gene 1) and eds1 (enhanced disease susceptibility 1) mutants and NAHG-transgenic plants. These transgenic plants were then infected with the soft-rot bacterial pathogen Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora ssp. carotovora, ECC) to investigate the mechanism behind PFLP-mediated resistance. The results revealed that, instead of showing soft-rot symptoms, ECC activated hypersensitive response (HR)-associated events, such as the accumulation of hydrogen peroxide (H2 O2 ), electrical conductivity leakage and expression of the HR marker genes (ATHSR2 and ATHSR3) in PFLP-transgenic Arabidopsis. This PFLP-mediated resistance could be abolished by inhibitors, such as diphenylene iodonium (DPI), 1-l-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E64) and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), but not by myriocin and fumonisin. The PFLP-transgenic plants were resistant to ECC, but not to its harpin mutant strain ECCAC5082. In the npr1 mutant and NAHG-transgenic Arabidopsis, but not in the eds1 mutant, overexpression of the PFLP gene increased resistance to ECC. Based on these results, we suggest that transgenic Arabidopsis contains high levels of ectopic PFLP; this may lead to the recognition of the harpin and to the activation of the HR and other resistance mechanisms, and is dependent on the protease-mediated pathway.

  19. Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

    Science.gov (United States)

    Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joël; Dietrich, Jacques; Hübscher, Ulrich; Raffin, Jean-Paul

    2002-11-08

    The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

  20. The structures of the CutA1 proteins from Thermus thermophilus and Pyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly

    Science.gov (United States)

    Bagautdinov, Bagautdin

    2014-01-01

    CutA1 (copper tolerance A1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X-ray structures of CutA1 from the thermophilic bacterium Thermus thermophilus (TtCutA1) with and without bound Na+ at 1.7 and 1.9 Å resolution, respectively, and from the hyperthermophilic archaeon Pyrococcus horikoshii (PhCutA1) in complex with Na+ at 1.8 Å resolution have been determined. Both are short and rigid proteins of about 12 kDa that form intertwined compact trimers in the crystal and solution. The main difference in the structures is a wide-type β-bulge on top of the TtCutA1 trimer. It affords a mechanism for lodging a single-residue insertion in the middle of β2 while preserving the interprotomer main-chain hydrogen-bonding network. The liganded forms of the proteins provide new structural information about the metal-binding sites and CutA1 assembly. The Na+–TtCutA1 structure unveils a dodecameric assembly with metal ions in the trimer–trimer interfaces and the lateral clefts of the trimer. For Na+–PhCutA1, the metal ion associated with six waters in an octahedral geometry. The structures suggest that CutA1 may contribute to regulating intracellular metal homeostasis through various binding modes. PMID:24699729

  1. Crystal structure of Pyrococcus horikoshii tryptophanyl-tRNA synthetase and structure-based phylogenetic analysis suggest an archaeal origin of tryptophanyl-tRNA synthetase.

    Science.gov (United States)

    Dong, Xianchi; Zhou, Minyun; Zhong, Chen; Yang, Bei; Shen, Ning; Ding, Jianping

    2010-03-01

    The ancient and ubiquitous aminoacyl-tRNA synthetases constitute a valuable model system for studying early evolutionary events. So far, the evolutionary relationship of tryptophanyl- and tyrosyl-tRNA synthetase (TrpRS and TyrRS) remains controversial. As TrpRS and TyrRS share low sequence homology but high structural similarity, a structure-based method would be advantageous for phylogenetic analysis of the enzymes. Here, we present the first crystal structure of an archaeal TrpRS, the structure of Pyrococcus horikoshii TrpRS (pTrpRS) in complex with tryptophanyl-5' AMP (TrpAMP) at 3.0 A resolution which demonstrates more similarities to its eukaryotic counterparts. With the pTrpRS structure, we perform a more complete structure-based phylogenetic study of TrpRS and TyrRS, which for the first time includes representatives from all three domains of life. Individually, each enzyme shows a similar evolutionary profile as observed in the sequence-based phylogenetic studies. However, TyrRSs from Archaea/Eucarya cluster with TrpRSs rather than their bacterial counterparts, and the root of TrpRS locates in the archaeal branch of TyrRS, indicating the archaeal origin of TrpRS. Moreover, the short distance between TrpRS and archaeal TyrRS and that between bacterial and archaeal TrpRS, together with the wide distribution of TrpRS, suggest that the emergence of TrpRS and subsequent acquisition by Bacteria occurred at early stages of evolution.

  2. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

    Science.gov (United States)

    Kawakami, Ryushi; Ohmori, Taketo; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2015-08-01

    To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

  3. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2007-04-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2{sub 1} and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2{sub 1}, with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V{sub M} value of 2.45 Å{sup 3} Da{sup −1} and a solvent content of 50%.

  4. A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea

    Science.gov (United States)

    Yan, Zhen

    2017-01-01

    ABSTRACT Heterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO2-reducing methanogenic anaerobes (methanogens) from the domain Archaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F420 (F420H2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F420H2 and reduction of ferredoxin with the exergonic oxidation of F420H2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting. PMID:28174314

  5. Kinetically controlled refolding of a heat-denatured hyperthermostable protein

    NARCIS (Netherlands)

    Koutsopoulos, Sotirios; van der Oost, John; Norde, Willem

    2007-01-01

    The thermal denaturation of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 degrees C, corresponding to protein denaturation and complete unfolding, respectively, as

  6. Kinetically controlled refolding of a heat denatured hyperthermostable protein

    NARCIS (Netherlands)

    Koutsopoulos, S.; Oost, van der J.; Norde, W.

    2007-01-01

    The thermal denaturation of endo-ß-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144¿°C, corresponding to protein denaturation and complete unfolding, respectively, as shown by

  7. One- and two-electron reduction of molybdate reversibly bound to the archaeal tungstate/molybdate transporter WtpA

    NARCIS (Netherlands)

    Bevers, L.E.; Hagen, W.R.

    2009-01-01

    Reversible binding of the tetrahedral oxoanions MoO4 2- and WO4 2- to two carboxylato ligands of the soluble scavenger protein WtpA from the hyperthermophilic archaeon Pyrococcus furiosus enforces a quasi-octahedral MO6 coordination in which the +VI oxidation state is destabilized.

  8. A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes

    NARCIS (Netherlands)

    Kanai, T.; Akerboom, A.P.; Takedomi, S.; Werken, van de H.J.G.; Blombach, F.; Oost, van der J.; Murakami, T.; Atomi, H.; Imanaka, T.

    2007-01-01

    We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a tr

  9. The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.

    Science.gov (United States)

    Souza, André L F; Invitti, Adriana L; Rego, Fabiane G M; Monteiro, Rose A; Klassen, Giseli; Souza, Emanuel M; Chubatsu, Leda S; Pedrosa, Fábio O; Rigo, Liu U

    2010-02-01

    The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.

  10. Evaluation of the protection exerted by Pisum sativum Ferredoxin-NADP(H) Reductase against injury induced by hypothermia on Cos-7 cells.

    Science.gov (United States)

    Pucci Molineris, M; Di Venanzio, G; Mamprin, M E; Mediavilla, M G

    2013-08-01

    Hypothermia is employed as a method to diminish metabolism rates and preserve tissues and cells. However, low temperatures constitute a stress that produces biochemical changes whose extension depends on the duration and degree of cold exposure and is manifested when physiological temperature is restored. For many cellular types, cold induces an oxidative stress that is dependent on the elevation of intracellular iron, damages macromolecules, and is prevented by the addition of iron chelators. Pisum sativum Ferredoxin-NADP(H) Reductase (FNR) has been implicated in protection from injury mediated by intracellular iron increase and successfully used to reduce oxidative damage on bacterial, plant and mammalian systems. In this work, FNR was expressed in Cos-7 cells; then, they were submitted to cold incubation and iron overload to ascertain whether this enzyme was capable of diminishing the harm produced by these challenges. Contrary to expected, FNR was not protective and even exacerbated the damage under certain circumstances. It was also found that the injury induced by hypothermia in Cos-7 cells presented both iron-dependent and iron-independent components of damage when cells were actively dividing but only iron-independent component when cells were in an arrested state. This is in agreement with previous findings which showed that iron-dependent damage is also an energy-dependent process.

  11. FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin:NADP+ reductase and its Glu301Ala mutant.

    Science.gov (United States)

    Anusevicius, Zilvinas; Miseviciene, Lina; Medina, Milagros; Martinez-Julvez, Marta; Gomez-Moreno, Carlos; Cenas, Narimantas

    2005-05-15

    Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.

  12. Functional Inactivation of Putative Photosynthetic Electron Acceptor Ferredoxin C2 (FdC2) Induces Delayed Heading Date and Decreased Photosynthetic Rate in Rice.

    Science.gov (United States)

    Zhao, Juan; Qiu, Zhennan; Ruan, Banpu; Kang, Shujing; He, Lei; Zhang, Sen; Dong, Guojun; Hu, Jiang; Zeng, Dali; Zhang, Guangheng; Gao, Zhenyu; Ren, Deyong; Hu, Xingming; Chen, Guang; Guo, Longbiao; Qian, Qian; Zhu, Li

    2015-01-01

    Ferredoxin (Fd) protein as unique electron acceptor, involved in a variety of fundamental metabolic and signaling processes, which is indispensable for plant growth. The molecular mechanisms of Fd such as regulation of electron partitioning, impact of photosynthetic rate and involvement in the carbon fixing remain elusive in rice. Here we reported a heading date delay and yellowish leaf 1 (hdy1) mutant derived from Japonica rice cultivar "Nipponbare" subjected to EMS treatment. In the paddy field, the hdy1 mutant appeared at a significantly late heading date and had yellow-green leaves during the whole growth stage. Further investigation indicated that the abnormal phenotype of hdy1 was connected with depressed pigment content and photosynthetic rate. Genetic analysis results showed that the hdy1 mutant phenotype was caused by a single recessive nuclear gene mutation. Map-based cloning revealed that OsHDY1 is located on chromosome 3 and encodes an ortholog of the AtFdC2 gene. Complementation and overexpression, transgenic plants exhibited the mutant phenotype including head date, leaf color and the transcription levels of the FdC2 were completely rescued by transformation with OsHDY1. Real-time PCR revealed that the expression product of OsHDY1 was detected in almost all of the organs except root, whereas highest expression levels were observed in seeding new leaves. The lower expression levels of HDY1 and content of iron were detected in hdy1 than WT's. The FdC2::GFP was detected in the chloroplasts of rice. Real-time PCR results showed that the expression of many photosynthetic electron transfer related genes in hdy1 were higher than WT. Our results suggest that OsFdC2 plays an important role in photosynthetic rate and development of heading date by regulating electron transfer and chlorophyll content in rice.

  13. Pseudomonas aeruginosa IscR-Regulated Ferredoxin NADP(+ Reductase Gene (fprB Functions in Iron-Sulfur Cluster Biogenesis and Multiple Stress Response.

    Directory of Open Access Journals (Sweden)

    Adisak Romsang

    Full Text Available P. aeruginosa (PAO1 has two putative genes encoding ferredoxin NADP(+ reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein's physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection.

  14. Functional Inactivation of Putative Photosynthetic Electron Acceptor Ferredoxin C2 (FdC2 Induces Delayed Heading Date and Decreased Photosynthetic Rate in Rice.

    Directory of Open Access Journals (Sweden)

    Juan Zhao

    Full Text Available Ferredoxin (Fd protein as unique electron acceptor, involved in a variety of fundamental metabolic and signaling processes, which is indispensable for plant growth. The molecular mechanisms of Fd such as regulation of electron partitioning, impact of photosynthetic rate and involvement in the carbon fixing remain elusive in rice. Here we reported a heading date delay and yellowish leaf 1 (hdy1 mutant derived from Japonica rice cultivar "Nipponbare" subjected to EMS treatment. In the paddy field, the hdy1 mutant appeared at a significantly late heading date and had yellow-green leaves during the whole growth stage. Further investigation indicated that the abnormal phenotype of hdy1 was connected with depressed pigment content and photosynthetic rate. Genetic analysis results showed that the hdy1 mutant phenotype was caused by a single recessive nuclear gene mutation. Map-based cloning revealed that OsHDY1 is located on chromosome 3 and encodes an ortholog of the AtFdC2 gene. Complementation and overexpression, transgenic plants exhibited the mutant phenotype including head date, leaf color and the transcription levels of the FdC2 were completely rescued by transformation with OsHDY1. Real-time PCR revealed that the expression product of OsHDY1 was detected in almost all of the organs except root, whereas highest expression levels were observed in seeding new leaves. The lower expression levels of HDY1 and content of iron were detected in hdy1 than WT's. The FdC2::GFP was detected in the chloroplasts of rice. Real-time PCR results showed that the expression of many photosynthetic electron transfer related genes in hdy1 were higher than WT. Our results suggest that OsFdC2 plays an important role in photosynthetic rate and development of heading date by regulating electron transfer and chlorophyll content in rice.

  15. Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.).

    Science.gov (United States)

    Feraud, Magali; Masclaux-Daubresse, Céline; Ferrario-Méry, Sylvie; Pageau, Karine; Lelandais, Maud; Ziegler, Christine; Leboeuf, Edouard; Jouglet, Tiphaine; Viret, Lauriane; Spampinato, Axelle; Paganelli, Vanina; Hammouda, Mounir Ben; Suzuki, Akira

    2005-11-01

    GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5' flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, (15)NH4(+) was incorporated into [5-(15)N]glutamine and [2-(15)N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2-(15)N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2-(15)N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15-20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides

  16. A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

    Science.gov (United States)

    Shen, Yulong; Tang, Xiao-Feng; Yokoyama, Hideshi; Matsui, Eriko; Matsui, Ikuo

    2004-01-01

    Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.

  17. The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota.

    Science.gov (United States)

    Anderson, Iain J; Dharmarajan, Lakshmi; Rodriguez, Jason; Hooper, Sean; Porat, Iris; Ulrich, Luke E; Elkins, James G; Mavromatis, Kostas; Sun, Hui; Land, Miriam; Lapidus, Alla; Lucas, Susan; Barry, Kerrie; Huber, Harald; Zhulin, Igor B; Whitman, William B; Mukhopadhyay, Biswarup; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2009-04-02

    Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced -- Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

  18. The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Lakshmi, Lakshmi Dharmarajan [Virginia Polytechnic Institute and State University (Virginia Tech); Rodriquez, Jason [Virginia Polytechnic Institute and State University (Virginia Tech); Hooper, Sean [U.S. Department of Energy, Joint Genome Institute; Porat, I. [University of Georgia, Athens, GA; Ulrich, Luke [ORNL; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Sun, Hui [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Barry, Kerrie [U.S. Department of Energy, Joint Genome Institute; Huber, Harald [Universitat Regensburg, Regensburg, Germany; Zhulin, Igor B [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Whitman, W. B. [University of Georgia, Athens, GA; Mukhopadhyay, Biswarup [Virginia Polytechnic Institute and State University (Virginia Tech); Woese, Carl [University of Illinois, Urbana-Champaign; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute

    2009-01-01

    Background Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. Results The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. Conclusion The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

  19. The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota

    Directory of Open Access Journals (Sweden)

    Barry Kerrie

    2009-04-01

    Full Text Available Abstract Background Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. Results The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced – Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. Conclusion The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

  20. The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, iain J.; Dharmarajan, Lakshmi; Rodriguez, Jason; Hooper, Sean; Porat, Iris; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Sun, Hui; Land, Miriam; Lapidus, Alla; Lucas, Susan; Barry, Kerrie; Huber, Harald; Zhulin, Igor B.; Whitman, William B.; Mukhopadhyay, Biswarup; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2008-09-05

    Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced - Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

  1. Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

    Science.gov (United States)

    Seo, Daisuke; Naito, Hiroshi; Nishimura, Erika; Sakurai, Takeshi

    2015-08-01

    Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.

  2. 拟南芥铁氧还蛋白基因缺失促进花期提前的初步研究%Loss of Function of Ferredoxin2 Can Promote Flowering in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    刘兵; 魏海轩; 苏建斌; 张洋; 王金发; 王宏斌; 冯冬茹

    2013-01-01

    In Arabidopsis thaliana, the red and far-red light photoreceptors phytochromes (PHYs) act to involve in regulating flowering.Phytochromobilin synthase (HY2) synthesizes the open chain tetrapyrrole chromophore which is essential to the light-sensing function of phytochromes , and it is a member of the ferredoxin-dependent bilin reductases (FDBRs).Here, we found a Ds-T-DNA insertion line of Arabidop-sis thaliana for the gene encoding the most major ferredoxin (Fd2, At1g60950), which can promote flowering in the process of growth both under long-day and short-day conditions.In this report we show that loss of AtFd2c an promote flowering, AtFd2 interacts with AtHY2 in the chloroplast, and Fd2-KO mutants are impaired in the responses mediated by phytochromes .Together, these results implicate that loss of AtFd2 may promote flowering by impairing the physiology function of phytochromes .%  拟南芥的红光/远红光受体光敏色素(PHYs)参与花期调节过程,而铁氧还蛋白色素还原酶(FD-BRs)的一种---植物色素合成酶(HY2)对于光敏色素的合成是必不可少的。研究发现拟南芥铁氧还蛋白---AtFd2的基因缺失突变体(Fd2-KO突变体)在长日照与短日照培养条件下,较其野生型而言均表现出花期提前的表型,而且显示AtFd2与AtHY2在叶绿体中发生互作,并且Fd2突变体对光敏色素的反应受到抑制。推测At-Fd2基因的缺失可能通过影响光敏色素介导的相关生理功能进而对植株的花期进行调节。

  3. 阴道毛滴虫与人型支原体共生对铁氧还蛋白基因影响%Effects of the Symbiosis of Trichomonas Vaginalis with Mycoplasma Hominis on Ferredoxin Gene

    Institute of Scientific and Technical Information of China (English)

    刘晓东; 温雯静; 薛长贵

    2011-01-01

    We isolated 30 Trichomonas vaginalis for the PCR detection from the gynecological outpatients in the Affiliated Hospital of Zhengzhou University using the specific 16s rDNA primers of Mycoplasma hominis. The results showed that there were 25 cases of Mycoplasma hominis infection, with the infection rate of 83. 33%. This gave a clew that the symbiosis of Trichomonas vaginalis with Mycoplasma hominis may be of certain generality in China. We sequenced the ferredoxin gene of 10 Trichomonas vaginalis where 5 Mycoplasma hominis were positive and five negative, and found that the ferredoxin (Fd) gene of the 10 Trichomonas vaginalis were exactly the same. But compared to the genes in the GenBank. A comparative analysis of the gene revealed that there were 3 more ctg bases at the 200th position of encoding leucine, but this did not lead to changes in reading frame. The gene homology was 99%.%采用人型支原体16s rDNA的特异引物,对从郑州大学附属医院妇科门诊患者分离到的30株阴道毛滴虫进行PCR检测,结果有25株感染人型支原体,感染率为83.33%,这显示了阴道毛滴虫和人型支原体之间的共生关系在中国具有普遍性.并对10株阴道毛滴虫(5株人型支原体阳性和5株人型支原体阴性)的铁氧还蛋白(Fd)基因进行测序,探讨人型支原体对Fd基因的影响,结果发现10株阴道毛滴虫的Fd基因完全相同,但与GenBank中的基因进行比较分析,在第200位多出ctg三个碱基,编码亮氨酸,但并未导致读码框架改变.基因同源性为99%.

  4. Single-particle cryo-electron microscopy of macromolecular assemblies

    OpenAIRE

    Cheng, Kimberley

    2009-01-01

    In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli. Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric st...

  5. A T14C variant of Azotobacter vinelandii ferredoxin I undergoes facile [3Fe-4S]0 to [4Fe-4S]2+ conversion in vitro but not in vivo.

    Science.gov (United States)

    Gao-Sheridan, H S; Kemper, M A; Khayat, R; Tilley, G J; Armstrong, F A; Sridhar, V; Prasad, G S; Stout, C D; Burgess, B K

    1998-12-11

    [4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.

  6. Bioenergetic and physiological studies of hyperthermophilic archaea. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, R.M.

    1999-03-01

    This project focuses on physiological and bioenergetic characteristics of two representative hyperthermophilic archaea: Thermococcus litoralis (T{sub opt} 88 C) and Pyrococcus furiosus (T{sub opt} 98 C). Both are obligately anaerobic heterotrophs which grow in the presence or absence of reducible sulfur compounds. T. litoralis was studied in relation to information previously developed for P. furiosus: effect of sulfur reduction on bioenergetics, preferred fermentation patterns, tungsten requirement, etc. A defined medium was developed for T. litoralis consisting of amino acids, vitamins and nucleotides. This serves as the basis for continuous culture studies probing metabolic response to media changes. P. furiosus and T. litoralis have also been found to produce a polysaccharide in the presence of maltose and yeast extract. The composition and chemical structure of this polysaccharide was investigated as well as the metabolic motivation for its production. A novel and, perhaps, primitive intracellular proteolytic complex (previously designated as protease S66) in P. furiosus was isolated and the gene encoding the subunit of the complex was cloned, sequenced and the protease expressed in active form in Eschericia coli. Among other issues, the role of this complex in protein turnover and stress response was examined in the context of this organism in addition to comparing it to other complexes in eubacterial and eukaryotic cells. Biochemical characteristics of the protease have been measured in addition to examining other proteolytic species in P. furiosus.

  7. Response of Chloroplast NAD(P)H Dehydrogenase-Mediated Cyclic Electron Flow to a Shortage or Lack in Ferredoxin-Quinone Oxidoreductase-Dependent Pathway in Rice Following Short-Term Heat Stress.

    Science.gov (United States)

    Essemine, Jemaa; Qu, Mingnan; Mi, Hualing; Zhu, Xin-Guang

    2016-01-01

    Cyclic electron flow (CEF) around photosystem I (PSI) can protect photosynthetic electron carriers under conditions of stromal over-reduction. The goal of the research reported in this paper was to investigate the responses of both PSI and photosystem II (PSII) to a short-term heat stress in two rice lines with different capacities of cyclic electron transfer, i.e., Q4149 with a high capacity (hcef) and C4023 with a low capacity (lcef). The absorbance change at 820 nm (ΔA820) was used here to assess the charge separation in the PSI reaction center (P700). The results obtained show that short-term heat stress abolishes the ferredoxin-quinone oxidoreductase (FQR)-dependent CEF in rice and accelerates the initial rate of P700 (+) re-reduction. The P700 (+) amplitude was slightly increased at a moderate heat-stress (35°C) because of a partial restriction of FQR but it was decreased following high heat-stress (42°C). Assessment of PSI and PSII activities shows that PSI is more susceptible to heat stress than PSII. Under high temperature, FQR-dependent CEF was completely removed and NDH-dependent CEF was up-regulated and strengthened to a higher extent in C4023 than in Q4149. Specifically, under normal growth temperature, hcef (Q4149) was characterized by higher FQR- and chloroplast NAD(P)H dehydrogenase (NDH)-dependent CEF rates than lcef (C4023). Following thermal stress, the activation of NDH-pathway was 130 and 10% for C4023 and Q4149, respectively. Thus, the NDH-dependent CEF may constitute the second layer of plant protection and defense against heat stress after the main route, i.e., FQR-dependent CEF, reaches its capacity. We discuss the possibility that under high heat stress, the NDH pathway serves as a safety valve to dissipate excess energy by cyclic photophosphorylation and overcome the stroma over-reduction following inhibition of CO2 assimilation and any shortage or lack in the FQR pathway. The potential role of the NDH-dependent pathway during the

  8. Formate production through carbon dioxide hydrogenation with recombinant whole cell biocatalysts.

    Science.gov (United States)

    Alissandratos, Apostolos; Kim, Hye-Kyung; Easton, Christopher J

    2014-07-01

    The biological conversion of CO2 and H2 into formate offers a sustainable route to a valuable commodity chemical through CO2 fixation, and a chemical form of hydrogen fuel storage. Here we report the first example of CO2 hydrogenation utilising engineered whole-cell biocatalysts. Escherichia coli JM109(DE3) cells transformed for overexpression of either native formate dehydrogenase (FDH), the FDH from Clostridium carboxidivorans, or genes from Pyrococcus furiosus and Methanobacterium thermoformicicum predicted to express FDH based on their similarity to known FDH genes were all able to produce levels of formate well above the background, when presented with H2 and CO2, the latter in the form of bicarbonate. In the case of the FDH from P. furiosus the yield was highest, reaching more than 1 g L(-1)h(-1) when a hydrogen-sparging reactor design was used.

  9. Electroanalytical determination of tungsten and molybdenum in proteins.

    Science.gov (United States)

    Hagedoorn, P L; van't Slot, P; van Leeuwen, H P; Hagen, W R

    2001-10-01

    Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.

  10. NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

    Science.gov (United States)

    Abbas, Shahzada Nadeem; Mok, Kenneth Hun; Rashid, Naeem; Xie, Yongjing; Ruether, Manuel; O'Brien, John; Akhtar, Muhammad

    2016-06-01

    The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

  11. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J.; Mattingly, S.M. [Oak Ridge National Lab., TN (United States); Danson, M. [Univ. of Bath (United Kingdom)] [and others

    1996-07-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity. 23 refs., 5 figs.

  12. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  13. Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid [Emim][Ac].

    Science.gov (United States)

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Kim, Han-Woo; Ishikawa, Kazuhiko; Endo, Takashi; Lee, Seung-Hwan; Bon, Elba P S

    2013-12-01

    This study investigated the requirement of cellobiohydrolases (CBH) for saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus β-glucosidase (EGPh/BGPf) or Optimash™ BG while Acremonium Cellulase was used as control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free enzymes than were the BM-pretreated substrates. IL-treatment decreased the crystallinity and increased the specific surface area (SSA), whereas BM-treatment decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/Lh and a glucose yield of 81% within 9h. These results indicate the efficiency of CBH-free enzymes for the hydrolysis of IL-treated substrates. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Domain motions of Argonaute, the catalytic engine of RNA interference

    Directory of Open Access Journals (Sweden)

    Wall Michael E

    2007-11-01

    Full Text Available Abstract Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

  15. Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.

    Directory of Open Access Journals (Sweden)

    Junsong Sun

    Full Text Available Hydrogen gas is a major biofuel and is metabolized by a wide range of microorganisms. Microbial hydrogen production is catalyzed by hydrogenase, an extremely complex, air-sensitive enzyme that utilizes a binuclear nickel-iron [NiFe] catalytic site. Production and engineering of recombinant [NiFe]-hydrogenases in a genetically-tractable organism, as with metalloprotein complexes in general, has met with limited success due to the elaborate maturation process that is required, primarily in the absence of oxygen, to assemble the catalytic center and functional enzyme. We report here the successful production in Escherichia coli of the recombinant form of a cytoplasmic, NADP-dependent hydrogenase from Pyrococcus furiosus, an anaerobic hyperthermophile. This was achieved using novel expression vectors for the co-expression of thirteen P. furiosus genes (four structural genes encoding the hydrogenase and nine encoding maturation proteins. Remarkably, the native E. coli maturation machinery will also generate a functional hydrogenase when provided with only the genes encoding the hydrogenase subunits and a single protease from P. furiosus. Another novel feature is that their expression was induced by anaerobic conditions, whereby E. coli was grown aerobically and production of recombinant hydrogenase was achieved by simply changing the gas feed from air to an inert gas (N2. The recombinant enzyme was purified and shown to be functionally similar to the native enzyme purified from P. furiosus. The methodology to generate this key hydrogen-producing enzyme has dramatic implications for the production of hydrogen and NADPH as vehicles for energy storage and transport, for engineering hydrogenase to optimize production and catalysis, as well as for the general production of complex, oxygen-sensitive metalloproteins.

  16. Ni l-edge soft x-ray spectroscopy of ni-fe hydrogenases and modelcompounds--evidence for high-spin ni(ii) in the active enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hongxin; Ralston, C.Y.; Patil, D.S.; Jones, R.M.; Gu, M.; Verhagen, M.; Adams, M.; Ge, P.; Riordan, C.; Marganian, C.A.; Mascharak,P.; Kovacs, J.; Miller, C.G.; Collins, T.J.; Brooker, S.; Croucher, P.D.; Wang, Kun; Stiefel, E.I.; Cramer, S.P.

    2000-03-15

    L-edge X-ray absorption spectroscopy has been used to study, under a variety of conditions, the electronic structure of Ni in the Ni-Fe hydrogenases from Desulfovibrio gigas, Desulfovibrio baculatus, and Pyrococcus furiosus. The status of the enzyme films used for these measurements was monitored by FT-IR spectroscopy. The L-edge spectra were interpreted by ligand field multiplet simulations and by comparison with data for Ni model complexes. The spectrum for Ni in D. gigas enzyme ''form A'' is consistent with a covalent Ni(III) species. In contrast, all of the reduced enzyme samples exhibited high spin Ni(II) spectra. The significance of the Ni(II) spin state for the structure of the hydrogenase active site is discussed.

  17. DJ-1 family Maillard deglycases prevent acrylamide formation.

    Science.gov (United States)

    Richarme, Gilbert; Marguet, Evelyne; Forterre, Patrick; Ishino, Sonoko; Ishino, Yoshizumi

    2016-09-23

    The presence of acrylamide in food is a worldwide concern because it is carcinogenic, reprotoxic and neurotoxic. Acrylamide is generated in the Maillard reaction via condensation of reducing sugars and glyoxals arising from their decomposition, with asparagine, the amino acid forming the backbone of the acrylamide molecule. We reported recently the discovery of the Maillard deglycases (DJ-1/Park7 and its prokaryotic homologs) which degrade Maillard adducts formed between glyoxals and lysine or arginine amino groups, and prevent glycation damage in proteins. Here, we show that these deglycases prevent acrylamide formation, likely by degrading asparagine/glyoxal Maillard adducts. We also report the discovery of a deglycase from the hyperthermophilic archaea Pyrococcus furiosus, which prevents acrylamide formation at 100 °C. Thus, Maillard deglycases constitute a unique enzymatic method to prevent acrylamide formation in food without depleting the components (asparagine and sugars) responsible for its formation. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Enzymatic production of hydrogen gas from glucose and cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, S.M.; Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    An enzymatic process has been used to convert glucose to molecular hydrogen with the ultimate goal of converting cellulose to hydrogen. Two enzymes from the Archae, Thermoplasma acidophilium glucose dehydrogenase (GDH) and Pyrococcus furiosus hydrogenase, were used to oxidize glucose and NADPH respectively, resulting in the formation of molecular hydrogen. The stoichiometric yield of hydrogen from glucose was close to the theoretical maximum expected. Further, the molar amount of hydrogen produced was greater than the molar equivalent of NADP{sup +} present in the reaction mixture indicating that this GDH cofactor was regenerated throughout the course of the reaction. Hydrogen was also shown to be produced from cellulose if cellulase was included in the reaction mixture.

  19. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    Energy Technology Data Exchange (ETDEWEB)

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  20. Programmable DNA-Guided Artificial Restriction Enzymes.

    Science.gov (United States)

    Enghiad, Behnam; Zhao, Huimin

    2017-02-06

    Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.

  1. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

    Energy Technology Data Exchange (ETDEWEB)

    Hura, Greg L.; Menon, Angeli L.; Hammel, Michal; Rambo, Robert P.; Poole II, Farris L.; Tsutakawa, Susan E.; Jenney Jr, Francis E.; Classen, Scott; Frankel, Kenneth A.; Hopkins, Robert C.; Yang, Sungjae; Scott, Joseph W.; Dillard, Bret D.; Adams, Michael W. W.; Tainer, John A.

    2009-07-20

    We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

  2. Global transcriptional regulator TrmB family members in prokaryotes.

    Science.gov (United States)

    Kim, Minwook; Park, Soyoung; Lee, Sung-Jae

    2016-10-01

    Members of the TrmB family act as global transcriptional regulators for the activation or repression of sugar ABC transporters and central sugar metabolic pathways, including glycolytic, gluconeogenic, and other metabolic pathways, and also as chromosomal stabilizers in archaea. As a relatively newly classified transcriptional regulator family, there is limited experimental evidence for their role in Thermococcales, halophilic archaeon Halobacterium salinarum NRC1, and crenarchaea Sulfolobus strains, despite being one of the extending protein families in archaea. Recently, the protein structures of Pyrococcus furiosus TrmB and TrmBL2 were solved, and the transcriptomic data uncovered by microarray and ChIP-Seq were published. In the present review, recent evidence of the functional roles of TrmB family members in archaea is explained and extended to bacteria.

  3. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  4. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.

    Science.gov (United States)

    Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver; Williams, R Scott; Kuhnlein, Jeff; Lee, Ji-Hoon; Classen, Scott; Guenther, Grant; Russell, Paul; Tainer, John A; Paull, Tanya T

    2014-03-03

    The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

  5. Active-site models for complexes of quinolinate synthase with substrates and intermediates

    Energy Technology Data Exchange (ETDEWEB)

    Soriano, Erika V.; Zhang, Yang; Colabroy, Keri L.; Sanders, Jennie M.; Settembre, Ethan C.; Dorrestein, Pieter C.; Begley, Tadhg P.; Ealick, Steven E., E-mail: see3@cornell.edu [Cornell University, Ithaca, NY 14853-1301 (United States)

    2013-09-01

    Structural studies of quinolinate synthase suggest a model for the enzyme–substrate complex and an enzyme–intermediate complex with a [4Fe–4S] cluster. Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Å resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel β-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe–4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005 ▶), J. Biol. Chem.280, 26645–26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe–4S] cluster, prior to cluster assembly.

  6. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  7. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  8. A First Analysis of Metallome Biosignatures of Hyperthermophilic Archaea

    Directory of Open Access Journals (Sweden)

    Vyllinniskii Cameron

    2012-01-01

    Full Text Available To date, no experimental data has been reported for the metallome of hyperthermophilic microorganisms although their metal requirements for growth are known to be unique. Here, experiments were conducted to determine (i cellular trace metal concentrations of the hyperthermophilic Archaea Methanococcus jannaschii and Pyrococcus furiosus, and (ii a first estimate of the metallome for these hyperthermophilic species via ICP-MS. The metal contents of these cells were compared to parallel experiments using the mesophilic bacterium Escherichia coli grown under aerobic and anaerobic conditions. Fe and Zn were typically the most abundant metals in cells. Metal concentrations for E. coli grown aerobically decreased in the order Fe > Zn > Cu > Mo > Ni > W > Co. In contrast, M. jannaschii and P. furiosus show almost the reverse pattern with elevated Ni, Co, and W concentrations. Of the three organisms, a biosignature is potentially demonstrated for the methanogen M. jannaschii that may, in part, be related to the metallome requirements of methanogenesis. The bioavailability of trace metals more than likely has varied through time. If hyperthermophiles are very ancient, then the trace metal patterns observed here may begin to provide some insights regarding Earth's earliest cells and in turn, early Earth chemistry.

  9. A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases.

    Science.gov (United States)

    Sun, Fei; Huang, Li

    2016-07-01

    Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

  10. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  11. Combining biomass wet disk milling and endoglucanase/β-glucosidase hydrolysis for the production of cellulose nanocrystals.

    Science.gov (United States)

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Jang, Jae-Hyuk; Kim, Han-Woo; Ishikawa, Kazuhiko; Endo, Takashi; Lee, Seung-Hwan; Bon, Elba P S

    2015-09-05

    Cellulose nanocrystals (CNCs), a biomaterial with high added value, were obtained from pure cellulose, Eucalyptus holocellulose, unbleached Kraft pulp, and sugarcane bagasse, by fibrillating these biomass substrates using wet disk milling (WDM) followed by enzymatic hydrolysis using endoglucanase/β-glucosidase. The hydrolysis experiments were conducted using the commercial enzyme OptimashBG or a blend of Pyrococcus horikoshii endoglucanase and Pyrococcus furiosus β-glucosidase. The fibrillated materials and CNCs were analyzed by X-ray diffraction, atomic force microscopy, scanning electron microscopy, and the specific surface area (SSA) was measured. WDM resulted in the formation of long and twisted microfibers of 1000-5000 nm in length and 4-35 nm in diameter, which were hydrolyzed into shorter and straighter CNCs of 500-1500 nm in length and 4-12 nm in diameter, with high cellulose crystallinity. Therefore, the CNC's aspect ratio was successfully adjusted by endoglucanases under mild reaction conditions, relative to the reported acidic hydrolysis method. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. The intein of the Thermoplasma A-ATPase A subunit: Structure, evolution and expression in E. coli

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    Gogarten J Peter

    2001-11-01

    Full Text Available Abstract Background Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. Results The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain. T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. Conclusions Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

  13. Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales

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    Gogarten J Peter

    2008-01-01

    Full Text Available Abstract Background The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. Results We demonstrate that the two maltose ATP binding cassette (ABC transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to

  14. Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi

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    Carpousis Agamemnon J

    2011-06-01

    Full Text Available Abstract Background Noncoding RNA (ncRNA has been recognized as an important regulator of gene expression networks in Bacteria and Eucaryota. Little is known about ncRNA in thermococcal archaea except for the eukaryotic-like C/D and H/ACA modification guide RNAs. Results Using a combination of in silico and experimental approaches, we identified and characterized novel P. abyssi ncRNAs transcribed from 12 intergenic regions, ten of which are conserved throughout the Thermococcales. Several of them accumulate in the late-exponential phase of growth. Analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel P. abyssi ncRNA families. The CRISPR family is comprised of crRNAs expressed from two of the four P. abyssi CRISPR cassettes. The 5'UTR derived family includes four conserved ncRNAs, two of which have features similar to known bacterial riboswitches. Several of the novel ncRNAs have sequence similarities to orphan OrfB transposase elements. Based on RNA secondary structure predictions and experimental results, we show that three of the twelve ncRNAs include Kink-turn RNA motifs, arguing for a biological role of these ncRNAs in the cell. Furthermore, our results show that several of the ncRNAs are subjected to processing events by enzymes that remain to be identified and characterized. Conclusions This work proposes a revised annotation of CRISPR loci in P. abyssi and expands our knowledge of ncRNAs in the Thermococcales, thus providing a starting point for studies needed to elucidate their biological function.

  15. Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Tang, Xiao-Feng; Shen, Yulong; Matsui, Eriko; Matsui, Ikuo

    2004-09-21

    Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.

  16. A c subunit with four transmembrane helices and one ion (Na+)-binding site in an archaeal ATP synthase: implications for c ring function and structure.

    Science.gov (United States)

    Mayer, Florian; Leone, Vanessa; Langer, Julian D; Faraldo-Gómez, José D; Müller, Volker

    2012-11-16

    The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na(+)-DCCD competition experiments revealed only one binding site for DCCD and Na(+), indicating that the mature c subunit of this A(1)A(O) ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na(+)-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na(+)-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na(+)-specific under in vivo conditions, comparable with the Na(+)-dependent V(1)V(O) ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na(+)-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A(1)A(O) ATP synthases.

  17. A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes.

    Science.gov (United States)

    Kanai, Tamotsu; Akerboom, Jasper; Takedomi, Shogo; van de Werken, Harmen J G; Blombach, Fabian; van der Oost, John; Murakami, Taira; Atomi, Haruyuki; Imanaka, Tadayuki

    2007-11-16

    We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (Deltatgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Deltatgr mutant even under gluconeogenic conditions. The Deltatgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.

  18. A Computational Framework for Proteome-Wide Pursuit and Prediction of Metalloproteins using ICP-MS and MS/MS Data

    Directory of Open Access Journals (Sweden)

    Trauger Sunia A

    2011-02-01

    Full Text Available Abstract Background Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins. Results We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein

  19. Magnetic resonance studies of isotopically labeled paramagnetic proteins: (2FE-2S) ferredoxins

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, H.; Xia, B.; Chae, Y.K.; Westler, W.M.; Markley, J.L. [Univ. of Wisconsin, Madison, WI (United States)

    1994-12-01

    Recent developments in NMR spectroscopy, especially multidimensional, multinuclear NMR techniques, have made NMR the most versatile tool available for studying protein structure and function in solution. Unlike diamagnetic proteins, paramagnetic proteins contain centers with unpaired electrons. These unpaired electrons interact with magnetic nuclei either through chemical bonds by a contact mechanism or through space by a pseudocontact mechanism. Such interactions make the acquisition and analysis of NMR spectra of paramagnetic proteins more challenging than those of diamagnetic proteins. Some NMR signals from paramagnetic proteins are shifted outside the chemical shift region characteristic of diamagnetic proteins; these {open_quotes}hyperfine-shifted{close_quotes} resonances originate from nuclei that interact with unpaired electrons from the paramagnetic center. The large chemical shift dispersion in spectra of paramagnetic proteins makes it difficult to excite the entire spectral window and leads to distortions in the baseline. Interactions with paramagnetic centers shorten T{sub 1} and T{sub 2} relaxation times of nuclei; the consequences are line broadening and lower spectral sensitivity. Scalar (through bond) and dipolar (through space) interactions between pairs of nuclei are what give rise to crosspeak signals in multi-dimensional NMR spectra of small diamagnetic proteins. When such interactions involve a nucleus that is strongly relaxed by interaction with a paramagnetic center, specialized methods may be needed for its detection or it may be completely undetectable by present nD NMR methods.

  20. Structure of the bacterial plant-ferredoxin receptor FusA

    NARCIS (Netherlands)

    Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Bonvin, Alexandre M J J; Milner, Joel J.; Kelly, Sharon M.; Byron, Olwyn; Smith, Brian O.; Walker, Daniel

    2016-01-01

    Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble

  1. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    Science.gov (United States)

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.

  2. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  3. Hydrogen production and enzyme activities in the hyperthermophile Thermococcus paralvinellae grown on maltose, tryptone and agricultural waste

    Directory of Open Access Journals (Sweden)

    Sarah A. Hensley

    2016-02-01

    Full Text Available Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2 environments such as some hydrothermal vents and oil reservoirs. It may also be useful in industry for rapid agricultural waste treatment and concomitant H2 production. Thermococcus paralvinellae grown at 82°C without sulfur produced up to 5 mmol of H2 L-1 at rates of 5-36 fmol H2 cell-1 h-1 on 0.5% (wt vol-1 maltose, 0.5% (wt vol-1 tryptone, and 0.5% maltose + 0.05% tryptone media. Two potentially inhibiting conditions, the presence of 10 mM acetate and low pH (pH 5 in maltose-only medium, did not significantly affect growth or H2 production. Growth rates, H2 production rates, and cell yields based on H2 production were the same as those for Pyrococcus furiosus grown at 95°C on the same media for comparison. Acetate, butyrate, succinate, isovalerate and formate were also detected as end products. After 100 h, T. paralvinellae produced up to 5 mmol of H2 L-1 of medium when grown on up to 70% (vol vol-1 waste milk from cows undergoing treatment for mastitis with the bacterial antibiotic Ceftiofur and from untreated cows. The amount of H2 produced by T. paralvinellae increased with increasing waste concentrations, but decreased in P. furiosus cultures supplemented with waste milk above 1% concentration. All mesophilic bacteria from the waste milk that grew on Luria Bertani, Sheep’s Blood (selective for Staphylococcus, the typical cause of mastitis, and MacConkey (selective for Gram-negative enteric bacteria agar plates were killed by heat during incubation at 82°C. Ceftiofur, which is heat labile, was below the detection limit following incubation at 82°C. T. paralvinellae also produced up to 6 mmol of H2 L-1 of medium when grown on 0.1-10% (wt vol-1 spent brewery grain while P. furiosus produced < 1 mmol of H2 L-1. Twelve of 13 enzyme activities in T. paralvinellae showed significant (p<0.05 differences across six different growth conditions

  4. New oligosaccharyltransferase assay method.

    Science.gov (United States)

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  5. Selective control of oligosaccharide transfer efficiency for the N-glycosylation sequon by a point mutation in oligosaccharyltransferase.

    Science.gov (United States)

    Igura, Mayumi; Kohda, Daisuke

    2011-04-15

    Asn-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-X-Ser/Thr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon. One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(M/I) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. This mutational approach is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells.

  6. Role of Mn2+ and Compatible Solutes in the Radiation Resistance of Thermophilic Bacteria and Archaea

    Directory of Open Access Journals (Sweden)

    Kimberly M. Webb

    2012-01-01

    Full Text Available Radiation-resistant bacteria have garnered a great deal of attention from scientists seeking to expose the mechanisms underlying their incredible survival abilities. Recent analyses showed that the resistance to ionizing radiation (IR in the archaeon Halobacterium salinarum is dependent upon Mn-antioxidant complexes responsible for the scavenging of reactive oxygen species (ROS generated by radiation. Here we examined the role of the compatible solutes trehalose, mannosylglycerate, and di-myo-inositol phosphate in the radiation resistance of aerobic and anaerobic thermophiles. We found that the IR resistance of the thermophilic bacteria Rubrobacter xylanophilus and Rubrobacter radiotolerans was highly correlated to the accumulation of high intracellular concentration of trehalose in association with Mn, supporting the model of Mn2+-dependent ROS scavenging in the aerobes. In contrast, the hyperthermophilic archaea Thermococcus gammatolerans and Pyrococcus furiosus did not contain significant amounts of intracellular Mn, and we found no significant antioxidant activity from mannosylglycerate and di-myo-inositol phosphate in vitro. We therefore propose that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell.

  7. Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB

    Energy Technology Data Exchange (ETDEWEB)

    Hung, Kuo-Wei; Chang, Yi-Wei; Eng, Edward T.; Chen, Jai-Hui; Chen, Yi-Chung; Sun, Yuh-Ju; Hsiao, Chwan-Deng; Dong, Gang; Spasov, Krasimir A.; Unger, Vinzenz M.; Huang, Tai-huang (Yale-MED); (Perutz Lab); (AS); (NTHU-Taiwan)

    2010-09-17

    FeoB is a G-protein coupled membrane protein essential for Fe(II) uptake in prokaryotes. Here, we report the crystal structures of the intracellular domain of FeoB (NFeoB) from Klebsiella pneumoniae (KpNFeoB) and Pyrococcus furiosus (PfNFeoB) with and without bound ligands. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (S-domain), which despite its lack of sequence similarity between species is structurally conserved. In the nucleotide-free state, the G-domain's two switch regions point away from the binding site. This gives rise to an open binding pocket whose shallowness is likely to be responsible for the low nucleotide-binding affinity. Nucleotide binding induced significant conformational changes in the G5 motif which in the case of GMPPNP binding was accompanied by destabilization of the switch I region. In addition to the structural data, we demonstrate that Fe(II)-induced foot printing cleaves the protein close to a putative Fe(II)-binding site at the tip of switch I, and we identify functionally important regions within the S-domain. Moreover, we show that NFeoB exists as a monomer in solution, and that its two constituent domains can undergo large conformational changes. The data show that the S-domain plays important roles in FeoB function.

  8. An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA.

    Science.gov (United States)

    Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

    2012-09-28

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

  9. IMAGINE: first neutron protein structure and new capabilities for neutron macromolecular crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Munshi, Parthapratim [ORNL; Myles, Dean A A [ORNL; Robertson, Lee [ORNL; Stoica, Alexandru Dan [ORNL; Crow, Lowell [ORNL; Kovalevskyi, Andrii Y [ORNL; Koritsanszky, Tibor S [ORNL; Chakoumakos, Bryan C [ORNL; Blessing, Robert [Hauptman-Woodward Medical Research Institute; Meilleur, Flora [ORNL

    2013-01-01

    We report the first high resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory. Neutron diffraction data extending to 1.65 resolution were collected from a relatively small 0.7 mm3 PfRd crystal using 2.5 days (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the deuterium atoms of the protein, and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolutions (1.5 ) from crystals with volume < 1.0 mm3 and with unit cell edges < 100 . Beam line features include elliptical focusing mirrors that deliver 3x107 n s-1 cm-2 into a 3.5 x 2.0 mm2 focal spot at the sample position, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength configurations ( min=2.0 , 2.8 , 3.3 - max =3.0 , 4.0 , 4.5 , ~20 ). Notably, the crystal used to collect this PfRd data is 5-10 times smaller than has been previously reported.

  10. Molecular basis of transcription initiation in Archaea.

    Science.gov (United States)

    De Carlo, Sacha; Lin, Shih-Chieh; Taatjes, Dylan J; Hoenger, Andreas

    2010-01-01

    Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB, and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65ºC. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L "foot" domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture.

  11. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology

    Directory of Open Access Journals (Sweden)

    Cecilia R. Chambers

    2015-01-01

    Full Text Available With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments. We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

  12. Swimming behavior of selected species of Archaea.

    Science.gov (United States)

    Herzog, Bastian; Wirth, Reinhard

    2012-03-01

    The swimming behavior of Bacteria has been studied extensively, at least for some species like Escherichia coli. In contrast, almost no data have been published for Archaea on this topic. In a systematic study we asked how the archaeal model organisms Halobacterium salinarum, Methanococcus voltae, Methanococcus maripaludis, Methanocaldococcus jannaschii, Methanocaldococcus villosus, Pyrococcus furiosus, and Sulfolobus acidocaldarius swim and which swimming behavior they exhibit. The two Euryarchaeota M. jannaschii and M. villosus were found to be, by far, the fastest organisms reported up to now, if speed is measured in bodies per second (bps). Their swimming speeds, at close to 400 and 500 bps, are much higher than the speed of the bacterium E. coli or of a very fast animal, like the cheetah, each with a speed of ca. 20 bps. In addition, we observed that two different swimming modes are used by some Archaea. They either swim very rapidly, in a more or less straight line, or they exhibit a slower kind of zigzag swimming behavior if cells are in close proximity to the surface of the glass capillary used for observation. We argue that such a "relocate-and-seek" behavior enables the organisms to stay in their natural habitat.

  13. Comparative Genomic and Transcriptional Analyses of CRISPR Systems Across the Genus Pyrobaculum

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    David L Bernick

    2012-07-01

    Full Text Available Within the domain Archaea, the CRISPR immune system appears to be nearly ubiquitous based on computational genome analyses. Initial studies in bacteria demonstrated that the CRISPR system targets invading plasmid and viral DNA. Recent experiments in the model archaeon Pyrococcus furiosus uncovered a novel RNA-targeting variant of the CRISPR system potentially unique to archaea. Because our understanding of CRISPR system evolution in other archaea is limited, we have taken a comparative genomic and transcriptomic view of the CRISPR arrays across six diverse species within the crenarchaeal genus Pyrobaculum. We present transcriptional data from each of four species in the genus (P. aerophilum, P. islandicum, P. calidifontis, P. arsenaticum, analyzing mature CRISPR-associated small RNA abundance from over 20 arrays. Within the genus, there is remarkable conservation of CRISPR array structure, as well as unique features that are have not been studied in other archaeal systems. These unique features include: a nearly invariant CRISPR promoter, conservation of direct repeat families, the 5' polarity of CRISPR-associated small RNA abundance, and a novel CRISPR-specific association with homologues of nurA and herA. These analyses provide a genus-level evolutionary perspective on archaeal CRISPR systems, broadening our understanding beyond existing non-comparative model systems.

  14. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    Energy Technology Data Exchange (ETDEWEB)

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong (FSU); (Georgia)

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  15. A Macrocyclic Peptide that Serves as a Cocrystallization Ligand and Inhibits the Function of a MATE Family Transporter

    Directory of Open Access Journals (Sweden)

    Hiroaki Suga

    2013-08-01

    Full Text Available The random non-standard peptide integrated discovery (RaPID system has proven to be a powerful approach to discover de novo natural product-like macrocyclic peptides that inhibit protein functions. We have recently reported three macrocyclic peptides that bind to Pyrococcus furiosus multidrug and toxic compound extrusion (PfMATE transporter and inhibit the transport function. Moreover, these macrocyclic peptides were successfully employed as cocrystallization ligands of selenomethionine-labeled PfMATE. In this report, we disclose the details of the RaPID selection strategy that led to the identification of these three macrocyclic peptides as well as a fourth macrocyclic peptide, MaD8, which is exclusively discussed in this article. MaD8 was found to bind within the cleft of PfMATE’s extracellular side and blocked the path of organic small molecules being extruded. The results of an ethidium bromide efflux assay confirmed the efflux inhibitory activity of MaD8, whose behavior was similar to that of previously reported MaD5.

  16. Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

    Science.gov (United States)

    Sinapah, Sylvie; Wu, Shiying; Chen, Yu; Pettersson, B M Fredrik; Gopalan, Venkat; Kirsebom, Leif A

    2011-02-01

    RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

  17. Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics.

    Science.gov (United States)

    Surade, Sachin; Klein, Markus; Stolt-Bergner, Peggy C; Muenke, Cornelia; Roy, Ankita; Michel, Hartmut

    2006-09-01

    Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.

  18. Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure

    Energy Technology Data Exchange (ETDEWEB)

    Nakabayashi, Makoto; Kataoka, Misumi; Mishima, Yumiko; Maeno, Yuka; Ishikawa, Kazuhiko, E-mail: kazu-ishikawa@aist.go.jp [National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan)

    2014-03-01

    Substitutive mutations that convert a tetrameric β-glucosidase into a dimeric state lead to improvement of its crystal quality. β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed.

  19. Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

    Science.gov (United States)

    Roovers, Martine; Hale, Caryn; Tricot, Catherine; Terns, Michael P; Terns, Rebecca M; Grosjean, Henri; Droogmans, Louis

    2006-01-01

    Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

  20. Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain; Ulrich, Luke E.; Lupa, Boguslaw; Susanti, Dwi; Porat, Iris; Hooper, Sean D.; Lykidis, Athanasios; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla; Saunders, Elizabeth; Han, Cliff; Land, Miriam; Lucas, Susan; Mukhopadhyay, Biswarup; Whitman, William B.; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2009-05-01

    Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  1. Structure of the Class IV Adenylyl Cyclase Reveals a Novel Fold

    Energy Technology Data Exchange (ETDEWEB)

    Gallagher,D.; Smith, N.; Kim, S.; Heroux, A.; Robinson, H.; Reddy, P.

    2006-01-01

    The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 {angstrom} resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.

  2. PDI improves secretion of redox-inactive beta-glucosidase.

    Science.gov (United States)

    Powers, Sara Lawrence; Robinson, Anne Skaja

    2007-01-01

    Although manipulation of the endoplasmic reticulum (ER) folding environment in the yeast Saccharomyces cerevisiae has been shown to increase the secretory productivity of recombinant proteins, the cellular interactions and processes of native enzymes and chaperones such as protein disulfide isomerase (PDI) are still unclear. Previously, we reported that overexpression of the ER chaperone PDI enabled up to a 3-fold increase in secretion levels of the Pyrococcus furiosus beta-glucosidase in the yeast S. cerevisiae. This result was surprising since beta-glucosidase contains only one cysteine per monomer and no disulfide bonds. Two possible mechanisms were proposed: PDI either forms a transient disulfide bond with the lone cysteine residue of the nascent beta-glucosidase during the folding and assembly process or acts as a chaperone to aid in proper folding. To discern between the two mechanisms, the single cysteine residue was mutated to serine, and the secretion of the two protein variants was determined. The serine mutant still showed increased secretion in vivo when PDI levels were elevated. When the folding bottleneck is removed by increasing expression temperatures to 37 degrees C rather than 30 degrees C, PDI no longer has an improvement on secretion. These results suggest that, unexpectedly, PDI acts in a chaperone-like capacity or possibly cooperates with the cell's folding or degradation mechanisms regardless of whether the protein is redox-active.

  3. Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex

    Directory of Open Access Journals (Sweden)

    Nancy F. Ramia

    2014-12-01

    Full Text Available The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes.

  4. Proton Inventory and Dynamics in the Nia-S to Nia-C Transition of a [NiFe] Hydrogenase.

    Science.gov (United States)

    Greene, Brandon L; Wu, Chang-Hao; Vansuch, Gregory E; Adams, Michael W W; Dyer, R Brian

    2016-03-29

    Hydrogenases (H2ases) represent one of the most striking examples of biological proton-coupled electron transfer (PCET) chemistry, functioning in facile proton reduction and H2 oxidation involving long-range proton and electron transport. Spectroscopic and electrochemical studies of the [NiFe] H2ases have identified several catalytic intermediates, but the details of their interconversion are still a matter of debate. Here we use steady state and time-resolved infrared spectroscopy, sensitive to the CO ligand of the active site iron, as a probe of the proton inventory as well as electron and proton transfer dynamics in the soluble hydrogenase I from Pyrococcus furiosus. Subtle shifts in infrared signatures associated with the Nia-C and Nia-S states as a function of pH revealed an acid-base equilibrium associated with an ionizable amino acid near the active site. Protonation of this residue was found to correlate with the photoproduct distribution that results from hydride photolysis of the Nia-C state, in which one of the two photoproduct states becomes inaccessible at low pH. Additionally, the ability to generate Nia-S via PCET from Nia-C was weakened at low pH, suggesting prior protonation of the proton acceptor. Kinetic and thermodynamic analysis of electron and proton transfer with respect to the various proton inventories was utilized to develop a chemical model for reversible hydride oxidation involving two intermediates differing in their hydrogen bonding character.

  5. Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes.

    Science.gov (United States)

    Thomsen, Malene S; Nidetzky, Bernd

    2009-01-01

    Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB.

  6. Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA.

    Directory of Open Access Journals (Sweden)

    Samantha A Wynne

    Full Text Available Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10 of Pyrococcus furiosus (Pfu polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

  7. Transcription start site associated RNAs (TSSaRNAs are ubiquitous in all domains of life.

    Directory of Open Access Journals (Sweden)

    Livia S Zaramela

    Full Text Available A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq and a primary-transcript enriched (dRNA-seq strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome. Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  8. Genomic Characterization of Methanomicrobiales Reveals Three Classes of Methanogens

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Ulrich, Luke [ORNL; Lupa, Boguslaw [University of Georgia, Athens, GA; Susanti, Dwi [Virginia Polytechnic Institute and State University (Virginia Tech); Porat, I. [University of Georgia, Athens, GA; Hooper, Sean [U.S. Department of Energy, Joint Genome Institute; Lykidis, A [U.S. Department of Energy, Joint Genome Institute; Sieprawska-Lupa, Magdalena [University of Georgia, Athens, GA; Dharmarajan, Lakshmi [Virginia Polytechnic Institute and State University (Virginia Tech); Goltsman, Eugene [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Saunders, Elizabeth H [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Land, Miriam L [ORNL; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Mukhopadhyay, Biswarup [Virginia Polytechnic Institute and State University (Virginia Tech); Whitman, William [ORNL; Woese, Carl [University of Illinois, Urbana-Champaign; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute

    2009-01-01

    Background Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. Methodology/Principal Findings In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Conclusions/Significance Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  9. Genomic characterization of methanomicrobiales reveals three classes of methanogens.

    Science.gov (United States)

    Anderson, Iain; Ulrich, Luke E; Lupa, Boguslaw; Susanti, Dwi; Porat, Iris; Hooper, Sean D; Lykidis, Athanasios; Sieprawska-Lupa, Magdalena; Dharmarajan, Lakshmi; Goltsman, Eugene; Lapidus, Alla; Saunders, Elizabeth; Han, Cliff; Land, Miriam; Lucas, Susan; Mukhopadhyay, Biswarup; Whitman, William B; Woese, Carl; Bristow, James; Kyrpides, Nikos

    2009-06-04

    Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

  10. Anaerobic High-Throughput Cultivation Method for Isolation of Thermophiles Using Biomass-Derived Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton-Brehm, Scott [ORNL; Vishnivetskaya, Tatiana A [ORNL; Allman, Steve L [ORNL; Mielenz, Jonathan R [ORNL; Elkins, James G [ORNL

    2012-01-01

    Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium ther- mocellum (Topt = 55 C), Caldicellulosiruptor obsidiansis (Topt = 78 C) and the fermentative hyperthermo- philes, Pyrococcus furiosus (Topt = 100 C) and Thermotoga maritima (Topt = 80 C). Multi-well plates were incubated at various temperatures for approximately 72 120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD600. Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

  11. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    Science.gov (United States)

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

  12. A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria.

    Science.gov (United States)

    Scott, Israel M; Rubinstein, Gabe M; Lipscomb, Gina L; Basen, Mirko; Schut, Gerrit J; Rhaesa, Amanda M; Lancaster, W Andrew; Poole, Farris L; Kelly, Robert M; Adams, Michael W W

    2015-10-01

    Caldicellulosiruptor bescii grows optimally at 78°C and is able to decompose high concentrations of lignocellulosic plant biomass without the need for thermochemical pretreatment. C. bescii ferments both C5 and C6 sugars primarily to hydrogen gas, lactate, acetate, and CO2 and is of particular interest for metabolic engineering applications given the recent availability of a genetic system. Developing optimal strains for technological use requires a detailed understanding of primary metabolism, particularly when the goal is to divert all available reductant (electrons) toward highly reduced products such as biofuels. During an analysis of the C. bescii genome sequence for oxidoreductase-type enzymes, evidence was uncovered to suggest that the primary redox metabolism of C. bescii has a completely uncharacterized aspect involving tungsten, a rarely used element in biology. An active tungsten utilization pathway in C. bescii was demonstrated by the heterologous production of a tungsten-requiring, aldehyde-oxidizing enzyme (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus. Furthermore, C. bescii also contains a tungsten-based AOR-type enzyme, here termed XOR, which is phylogenetically unique, representing a completely new member of the AOR tungstoenzyme family. Moreover, in C. bescii, XOR represents ca. 2% of the cytoplasmic protein. XOR is proposed to play a key, but as yet undetermined, role in the primary redox metabolism of this cellulolytic microorganism.

  13. Production of chitooligosaccharides from Rhizopus oligosporus NRRL2710 cells by chitosanase digestion.

    Science.gov (United States)

    Mahata, Maria; Shinya, Shoko; Masaki, Eiko; Yamamoto, Takashi; Ohnuma, Takayuki; Brzezinski, Ryszard; Mazumder, Tapan K; Yamashita, Kazuhiko; Narihiro, Kazue; Fukamizo, Tamo

    2014-01-13

    The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS (13)C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN-GlcNAc, (GlcN)2-GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosanase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact.

  14. Redox regulation of SurR by protein disulfide oxidoreductase in Thermococcus onnurineus NA1.

    Science.gov (United States)

    Lim, Jae Kyu; Jung, Hae-Chang; Kang, Sung Gyun; Lee, Hyun Sook

    2017-03-01

    Protein disulfide oxidoreductases are redox enzymes that catalyze thiol-disulfide exchange reactions. These enzymes include thioredoxins, glutaredoxins, protein disulfide isomerases, disulfide bond formation A (DsbA) proteins, and Pyrococcus furiosus protein disulfide oxidoreductase (PfPDO) homologues. In the genome of a hyperthermophilic archaeon, Thermococcus onnurineus NA1, the genes encoding one PfPDO homologue (TON_0319, Pdo) and three more thioredoxin- or glutaredoxin-like proteins (TON_0470, TON_0472, TON_0834) were identified. All except TON_0470 were recombinantly expressed and purified. Three purified proteins were reduced by a thioredoxin reductase (TrxR), indicating that each protein can form redox complex with TrxR. SurR, a transcription factor involved in the sulfur response, was tested for a protein target of a TrxR-redoxin system and only Pdo was identified to be capable of catalyzing the reduction of SurR. Electromobility shift assay demonstrated that SurR reduced by the TrxR-Pdo system could bind to the DNA probe with the SurR-binding motif, GTTttgAAC. In this study, we present the TrxR-Pdo couple as a redox-regulator for SurR in T. onnurineus NA1.

  15. Crystal structure of the sugar binding domain of the archaeal transcriptional regulator TrmB.

    Science.gov (United States)

    Krug, Michael; Lee, Sung-Jae; Diederichs, Kay; Boos, Winfried; Welte, Wolfram

    2006-04-21

    TrmB is an alpha-glucoside-sensing transcriptional regulator controlling two operons encoding maltose/trehalose and maltodextrin ABC transporters of Pyrococcus furiosus. The crystal structure of an N-terminal truncated derivative of TrmB (amino acids 2-109 deleted; TrmB(delta2-109)) was solved at 1.5 A resolution. This protein has lost its DNA binding domain but has retained its sugar recognition site. The structure represents a novel sugar-binding fold. TrmB(delta2-109) bound maltose, glucose, sucrose, and maltotriose, exhibiting Kd values of 6.8, 25, 34, and 160 microM, respectively. TrmB(delta2-109) behaved as a monomer in dilute buffer solution in contrast to the full-length protein, which is a dimer. Co-crystallization with bound maltose identified a binding site involving seven amino acid residues: Ser229, Asn305, Gly320, Met321, Val324, Ile325, and Glu326. Six of these residues interact with the nonreducing glucosyl residue of maltose. The nonreducing glucosyl residue is shared by all substrates bound to TrmB, suggesting it as a common recognition motif.

  16. TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon Thermococcus litoralis.

    Science.gov (United States)

    Lee, Sung-Jae; Engelmann, Afra; Horlacher, Reinhold; Qu, Qiuhao; Vierke, Gudrun; Hebbeln, Carina; Thomm, Michael; Boos, Winfried

    2003-01-10

    We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  17. Assembly of the Complex between Archaeal RNase P Proteins RPP30 and Pop5

    Directory of Open Access Journals (Sweden)

    Brandon L. Crowe

    2011-01-01

    Full Text Available RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae. Four of these proteins function in pairs (Pop5-RPP30 and RPP21–RPP29. We have used nuclear magnetic resonance (NMR spectroscopy and isothermal titration calorimetry (ITC to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu. NMR backbone resonance assignments of free RPP30 (25 kDa indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.

  18. Genomic characterization of methanomicrobiales reveals three classes of methanogens.

    Directory of Open Access Journals (Sweden)

    Iain Anderson

    Full Text Available BACKGROUND: Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. METHODOLOGY/PRINCIPAL FINDINGS: In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II, and the Methanosarcinales (Class III.

  19. Purification, crystallization and preliminary crystallographic analysis of the biotin–protein ligase from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Bagautdinov, Bagautdin; Kuroishi, Chizu; Sugahara, Mitsuaki; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp

    2005-02-01

    The biotin–protein ligase from P. horikoshii OT3 was overexpressed, purified, crystallized and cocrystallized with biotin, ADP and biotinyl-5′-AMP. The crystals belong to space group P2{sub 1} and diffract to beyond 1.6 Å resolution.

  20. Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase

    DEFF Research Database (Denmark)

    Auxilien, Sylvie; El Khadali, Fatima; Rasmussen, Anette;

    2007-01-01

    Members of the archease superfamily of proteins are represented in all three domains of life. Archease genes are generally located adjacent to genes encoding proteins involved in DNA or RNA processing. Archease have therefore been predicted to play a modulator or chaperone role in selected steps...

  1. Functional Genomic Analysis of the HY2 Family of Ferredoxin-Dependent Bilin Reductases from Oxygenic Photosynthetic Organisms

    National Research Council Canada - National Science Library

    Nicole Frankenberg; Keiko Mukougawa; Takayuki Kohchi; J. Clark Lagarias

    2001-01-01

    Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria...

  2. Characterization of two ferredoxin-dependent sulfite reductases having different substrate specificity in the red alga Cyanidioschyzon merolae.

    Science.gov (United States)

    Sekine, Kohsuke; Moriyama, Takashi; Kim, JuYaen; Hase, Toshiharu; Sato, Naoki

    2017-07-01

    Assimilatory sulfite reductase (SiR) and nitrite reductase (NiR), which are important determinants in biomass productivity, are homologous enzymes that catalyze the reduction of sulfite to sulfide and nitrite to ammonium, respectively. They have a siroheme and a [4Fe-4S] cluster as prosthetic groups in common. The red alga Cyanidioschyzon merolae encodes two SiR-like enzymes, CmSiRA and CmSiRB, which are likely products of recent gene duplication, but no homologues of NiR. The growth in a medium containing nitrate, however, must be supported by a nitrite reducing activity. CmSiRB was not detected in the ammonium medium, but, in the nitrate medium, it was present at a level 1/6 of that of constitutively expressed CmSiRA. Kinetic analysis of the two enzymes showed that CmSiRA has high kcat values with both sulfite and nitrite, but CmSiRB has virtually only the activity of nitrite reduction, although the Km value against nitrite was fairly high in both enzymes. The six amino acid residues that are specific to CmSiRB among various SiR-like enzymes in the active site were mutagenized to mimic partially CmSiRA. Among them, the mutation S217C in CmSiRB partially recovered sulfite reduction activity, suggesting that this residue is a major determinant of substrate specificity. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  3. Fiscal 2000 achievement report on project for research and development of technologies for intelligent infrastructure creation and utilization. Development of high-efficiency protein expression system (Development of high-efficiency protein expression system utilizing protein folding mechanism of hyperthermophile); 2000 nendo chiteki kiban sose riyo gijutsu kenkyu kaihatsu jigyo seika hokokusho. Kokoritsu tanpakushitsu hatsugen system no kaihatsu (Cho konetsukin no tanpakushitsu oritatami kiko wo riyo shita kokoritsu tanpaku hatsugen system no kaihatsu)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Studies were conducted aiming at a heterogenic expression system, with E. coli acting as the host, capable of high-efficiency expression of the protein of hyperthermophiles, hyperthermophilic archaebacteria in particular. In this fiscal year, 9 kinds of enzymes were caused by E. coli to express from the genomes of P. Horikoshi, A. pernix, etc., and examined for characteristics. Concerning the L-proline dehydrogenase obtained by the screening of hyperthermophiles of the Thermococcus profundus genus, it was noted that the ferredoxin was possibly a natural acceptor of electrons and that it was possibly an amino acid dehydrogenase of an utterly new category. Furthermore, it was found that the NADkinase of Pyrococcus horikoshii was a unique heat-resistant enzyme which used the inexpensive polyphosphoric acid instead of expensive ATP (adenosine triphosphate), and this justified a hope that it would be quite useful in the industrial production of NADP (H) (nicotinamide adenine dinucleotide phosphate). A success was attained in uniformly refining the glutamate dehydrogenase of hyperthermophilic and inactive Pyrobaculum islandicum produced by E. coli. (NEDO)

  4. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.

    Science.gov (United States)

    Matsumi, Rie; Manabe, Kenji; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

    2007-04-01

    We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.

  5. Temperature, pressure, and electrochemical constraints on protein speciation: Group additivity calculation of the standard molal thermodynamic properties of ionized unfolded proteins

    Directory of Open Access Journals (Sweden)

    J. M. Dick

    2006-01-01

    Full Text Available Thermodynamic calculations can be used to quantify environmental constraints on the speciation of proteins, such as the pH and temperature dependence of ionization state, and the relative chemical stabilities of proteins in different biogeochemical settings. These calculations depend in part on values of the standard molal Gibbs energies of proteins and their ionization reactions as a function of temperature and pressure. Because these values are not generally available, we calculated values of the standard molal thermodynamic properties at 25°C and 1 bar as well as the revised Helgeson-Kirkham-Flowers equations of state parameters of neutral and charged zwitterionic reference model compounds including aqueous amino acids, polypeptides, and unfolded proteins. The experimental calorimetric and volumetric data for these species taken from the literature were combined with group additivity algorithms to calculate the properties and parameters of neutral and ionized sidechain and backbone groups in unfolded proteins. The resulting set of group contributions enables the calculation of the standard molal Gibbs energy, enthalpy, entropy, isobaric heat capacity, volume, and isothermal compressibility of unfolded proteins in a range of proton ionization states to temperatures and pressures exceeding 100°C and 1000 bar. This approach provides a useful frame of reference for thermodynamic studies of protein folding and complexation reactions. It can also be used to assign provisional values of the net charge and Gibbs energy of ionized proteins as a function of temperature and pH. Using these values, an Eh-pH diagram for a reaction representing the speciation of extracellular proteins from Pyrococcus furiosus and Bacillus subtilis was generated. The predicted predominance limits of these proteins correspond with the different electrochemical conditions of hydrothermal vents and soils. More comprehensive calculations of this kind may reveal pervasive

  6. Enzymatic synthesis of beta-glucosylglycerol using a continuous-flow microreactor containing thermostable beta-glycoside hydrolase CelB immobilized on coated microchannel walls.

    Science.gov (United States)

    Schwarz, Alexandra; Thomsen, Malene S; Nidetzky, Bernd

    2009-08-01

    beta-Glucosylglycerol (betaGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable beta-glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of betaGG under conditions of continuous flow at 70 degrees C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 microL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2-nitrophenyl-beta-D-glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25-1.0 M), and the average residence time (0.2-90 s). Glycerol caused a concentration-dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of betaGG were > or =80% and approximately 60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (> or =80%), giving about 120 mM (30 g/L) of betaGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1-O-beta-D-glucopyranosyl-rac-glycerol (79%) and 2-O-beta-D-glucopyranosyl-sn-glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme.

  7. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    Science.gov (United States)

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.

  8. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    Science.gov (United States)

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.

  9. Temperature, pressure, and electrochemical constraints on protein speciation: Group additivity calculation of the standard molal thermodynamic properties of ionized unfolded proteins

    Science.gov (United States)

    Dick, J. M.; Larowe, D. E.; Helgeson, H. C.

    2006-07-01

    Thermodynamic calculations can be used to quantify environmental constraints on the speciation of proteins, such as the pH and temperature dependence of ionization state, and the relative chemical stabilities of proteins in different biogeochemical settings. These calculations depend in part on values of the standard molal Gibbs energies of proteins and their ionization reactions as a function of temperature and pressure. Because these values are not generally available, we calculated values of the standard molal thermodynamic properties at 25°C and 1 bar as well as the revised Helgeson-Kirkham-Flowers equations of state parameters of neutral and charged zwitterionic reference model compounds including aqueous amino acids, polypeptides, and unfolded proteins. The experimental calorimetric and volumetric data for these species taken from the literature were combined with group additivity algorithms to calculate the properties and parameters of neutral and ionized sidechain and backbone groups in unfolded proteins. The resulting set of group contributions enables the calculation of the standard molal Gibbs energy, enthalpy, entropy, isobaric heat capacity, volume, and isothermal compressibility of unfolded proteins in a range of proton ionization states to temperatures and pressures exceeding 100°C and 1000 bar. This approach provides a useful frame of reference for thermodynamic studies of protein folding and complexation reactions. It can also be used to assign provisional values of the net charge and Gibbs energy of ionized proteins as a function of temperature and pH. Using these values, an Eh-pH diagram for a reaction representing the speciation of extracellular proteins from Pyrococcus furiosus and Bacillus subtilis was generated. The predicted predominance limits of these proteins correspond with the different electrochemical conditions of hydrothermal vents and soils. More comprehensive calculations of this kind may reveal pervasive chemical potential

  10. The structures of the CutA1 proteins from Thermus thermophilus and Pyrococcus horikoshii: characterization of metal‐binding sites and metal‐induced assembly

    National Research Council Canada - National Science Library

    Bagautdinov, Bagautdin

    2014-01-01

    .... In Escherichia coli it is implicated in divalent metal tolerance, while the mammalian CutA1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. The X...

  11. Purification, crystallization and preliminary crystallographic analysis of archaeal 6‐pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3

    National Research Council Canada - National Science Library

    Bagautdinov, Bagautdin; Sugahara, Mitsuaki; Kunishima, Naoki

    2007-01-01

    .... The crystal belongs to the orthorhombic space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 35.83, b = 95.71, c = 105.65 Å. Threefold noncrystallographic symmetry was identified from self...

  12. Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures

    Science.gov (United States)

    2011-01-01

    Reactions contained Mutazyme II reaction buffer, 125 ng/μL of each primer, 40mM dNTP mix, and 2.5U of Mutazyme II DNA polymerase. Initial DNA template...there was interest in determining the relative activity of recombi - nant Ph1prol compared to Pf prol and Phprol against G- type nerve agent simulants DFP

  13. Cloning, expression, and purification of the His(6)-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Ahring, Birgitte Kiær

    2003-01-01

    LysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni2+-IDA-Sepharose, dialyzed...

  14. NCBI nr-aa BLAST: CBRC-PCAP-01-1724 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PCAP-01-1724 ref|ZP_05708570.1| ferredoxin/ferredoxin-NADP reductase [Corynebacterium genital...ium ATCC 33030] gb|EEV90099.1| ferredoxin/ferredoxin-NADP reductase [Corynebacterium genitalium ATCC 33030] ZP_05708570.1 1.1 33% ...

  15. Main: 1GAW [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GAW トウモロコシ Corn Zea mays L. Ferredoxin. Name=L-Fnri; Zea Mays Molecule: Ferredoxin...TSSSLLYKEEFGKMKERAPENFRVDYAVSREQTNAAGERMYIQTRMAEYKEELWELLKKDNTYVYMCGLKGMEKGIDDIMVSLAEKDGIDWFDYKKQLKRGDQWNVEVY corn_1GAW.jpg ...

  16. AcEST: DK948622 [AcEST

    Lifescience Database Archive (English)

    Full Text Available _BUMFI Ferredoxin OS=Bumilleriopsis filiformis PE=1... 77 9e-14 sp|P00222|FER_COLES Ferredoxin OS=Colocasia ...=petF... 78 4e-14 sp|P81373|FERB_ALOMA Ferredoxin-B OS=Alocasia macrorrhiza PE=1 SV=1 78 5e-14 sp|P13106|FER

  17. 4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate*

    Science.gov (United States)

    Perche-Letuvée, Phanélie; Kathirvelu, Velavan; Berggren, Gustav; Clemancey, Martin; Latour, Jean-Marc; Maurel, Vincent; Douki, Thierry; Armengaud, Jean; Mulliez, Etienne; Fontecave, Marc; Garcia-Serres, Ricardo; Gambarelli, Serge; Atta, Mohamed

    2012-01-01

    Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed. PMID:23043105

  18. Response of Chloroplast NAD(PH Dehydrogenase-Mediated Cyclic Electron Flow to a Shortage or Lack in Ferredoxin-Quinone Oxidoreductase-Dependent Pathway in Rice Following Short-Term Heat Stress

    Directory of Open Access Journals (Sweden)

    Jemaa eEssemine

    2016-03-01

    Full Text Available Cyclic electron flow around PSI can protect photosynthetic electron carriers under conditions of stromal over-reduction. The goal of the research reported in this paper was to investigate the responses of both PSI and PSII to a short-term heat stress in two rice lines with different capacities of cyclic electron transfer, i.e. Q4149 with a high capacity (hcef and C4023 with a low capacity (lcef. The absorbance change at 820 nm (ΔA820 was used here to assess the charge separation in the photosystem I (PSI reaction center (P700. The results obtained show that short-term heat stress abolishes the FQR-dependent CEF in rice and accelerates the initial rate of P700+ re-reduction. The P700+ amplitude was slightly increased at a moderate heat-stress (35°C because of a partial restriction of FQR but it was decreased following high heat-stress (42°C. Assessment of PSI and PSII activities shows that PSI is more susceptible to heat stress than photosystem II (PSII. Under high temperature, FQR-dependent CEF was completely removed and NDH-dependent CEF was up-regulated and strengthened to a higher extent in C4023 than in Q4149. Specifically, under normal growth temperature, hcef (Q4149 was characterized by higher FQR- and NDH-dependent CEF rates than lcef (C4023. Following thermal stress, the activation of NDH-pathway was 130% and 10% for C4023 and Q4149, respectively. Thus, the NDH-dependent CEF may constitute the second layer of plant protection and defence against heat stress after the main route, i.e. FQR-dependent CEF, reaches its capacity. We discuss the possibility that under high heat stress, the NDH pathway serves as a safety valve to dissipate excess energy by cyclic photophosphorylation and overcome the stroma over-reduction following inhibition of CO2 assimilation and any shortage or lack in the FQR pathway. The potential role of the NDH-dependent pathway during the evolution of C4 photosynthesis is briefly discussed.

  19. inducing medium

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... 2National Institute of Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, ... inserted in pET-22b(+) vector to produce the recombinant plasmid pET-SB. ... furiosus, which function at temperatures greater than.

  20. Mössbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of superoxide reductase from Desulfoarculus Baarsii. Density functional calculations on related models.

    Science.gov (United States)

    Horner, Olivier; Mouesca, Jean-Marie; Oddou, Jean-Louis; Jeandey, Claudine; Nivière, Vincent; Mattioli, Tony A; Mathé, Christelle; Fontecave, Marc; Maldivi, Pascale; Bonville, Pierre; Halfen, Jason A; Latour, Jean-Marc

    2004-07-13

    Superoxide reductase (SOR) is an Fe protein that catalyzes the reduction of superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Mathé et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a Mössbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the Mössbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The Mössbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction ( approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are

  1. Protein (Cyanobacteria): 40514 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available YP_007120166.1 1117:753 1150:5622 44471:543 1173027:1122 ferredoxin Microcoleus sp. PCC 7113 MSDSSPIPERS...GLEPELGGIFRDAPERSGFEPELGGALRQKGVYVDEITCIGCKHCAHVARNTFYIEEDYGRSRVIRQDGDSEEVIQEAIETCPVDCIHWVDYTELKQLEQERQYQVIPLVGYPIDHSLVAIQSRQKRLKQQHKKARKRH ...

  2. AcEST: DK956918 [AcEST

    Lifescience Database Archive (English)

    Full Text Available FLDDDQIEAGYVLTCVAYPTSDCTIQTHQEEGL 98 >sp|P0A3D1|FER_THEVL Ferredoxin-1 OS=Thermosynechococcus vulcanus GN=pe...-17 sp|P00245|FER_SPIMA Ferredoxin OS=Spirulina maxima PE=1 SV=2 88 4e-17 sp|P0A3D1|FER_THEVL Ferredoxin-1 OS=Thermosynechococcus vul...canus... 87 6e-17 sp|P0A3C9|FER_THEEB Ferredoxin-1 OS=Thermosynechococcus elongatu.

  3. GenBank blastx search result: AK062096 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062096 001-044-H12 AB075242.1 Terrabacter sp. YK3 plasmid pYK3 genes for alpha subunit of dibenzofuran... dioxygenase, beta subunit of dibenzofuran dioxygenase, ferredoxin component of dibenzofuran... dioxygenase, ferredoxin reductase component of dibenzofuran dioxygenase, complete cds.|BCT BCT 3e-17 +3 ...

  4. NCBI nr-aa BLAST: CBRC-XTRO-01-0090 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0090 ref|YP_732839.1| ferredoxin-type protein, NapH/MauN family [Shewanella... sp. MR-4] gb|ABI37782.1| ferredoxin-type protein, NapH/MauN family [Shewanella sp. MR-4] YP_732839.1 0.096 27% ...

  5. NCBI nr-aa BLAST: CBRC-TBEL-01-2325 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TBEL-01-2325 ref|YP_987657.1| 4Fe-4S ferredoxin, iron-sulfur binding domain protein [Acidovorax... sp. JS42] gb|ABM43581.1| 4Fe-4S ferredoxin, iron-sulfur binding domain protein [Acidovorax sp. JS42] YP_987657.1 0.87 41% ...

  6. AcEST: BP912632 [AcEST

    Lifescience Database Archive (English)

    Full Text Available maxima PE=1 SV=2 88 2e-17 sp|P0A3D1|FER_THEVL Ferredoxin-1 OS=Thermosynechococcus vulcanus... 87 4e-17 sp|P...GYVLTCVAYPTSDCTIQTHQEEGL 98 >sp|P0A3D1|FER_THEVL Ferredoxin-1 OS=Thermosynechococcus vulcan

  7. Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake.

    Science.gov (United States)

    Grinter, Rhys; Josts, Inokentijs; Zeth, Kornelius; Roszak, Aleksander W; McCaughey, Laura C; Cogdell, Richard J; Milner, Joel J; Kelly, Sharon M; Byron, Olwyn; Walker, Daniel

    2014-07-01

    The colicin-like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram-negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M-like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α-helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin-containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.

  8. Sodium ion pumps and hydrogen production in glutamate fermenting anaerobic bacteria.

    Science.gov (United States)

    Boiangiu, Clara D; Jayamani, Elamparithi; Brügel, Daniela; Herrmann, Gloria; Kim, Jihoe; Forzi, Lucia; Hedderich, Reiner; Vgenopoulou, Irini; Pierik, Antonio J; Steuber, Julia; Buckel, Wolfgang

    2005-01-01

    Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B12-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na+ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na+ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na+ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP 'richer' than that of NADH.

  9. Reference: 76 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-fe... accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of fe...rredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vi...tro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells,...rredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an import

  10. Main: 1JB9 [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1JB9 トウモロコシ Corn Zea mays L. Ferredoxin-Nadp Reductase Precursor Zea Mays Molecule:...SLLYDEEFTSYLKQYPDNFRYDKALSREQKNRSGGKMYVQDKIEEYSDEIFKLLDGGAHIYFCGLKGMMPGIQDTLKKVAERRGESWDQKLAQLKKNKQWHVEVY corn_1JB9.jpg ...

  11. Main: 1GAQ [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GAQ トウモロコシ Corn Zea mays L. Ferredoxin I, Chloroplast Precursor Name=Fdx1; Synonym...DDVYILDQAEEDGIDLPYSCRAGSCSSCAGKVVSGSVDQSDQSYLDDGQIADGWVLTCHAYPTSDVVIETHKEEELTGA corn_1GAQ.jpg ...

  12. Method of controlled reduction of nitroaromatics by enzymatic reaction with oxygen sensitive nitroreductase enzymes

    Science.gov (United States)

    Shah, Manish M.; Campbell, James A.

    1998-01-01

    A method for the controlled reduction of nitroaromatic compounds such as nitrobenzene and 2,4,6-trinitrotoluene by enzymatic reaction with oxygen sensitive nitroreductase enzymes, such as ferredoxin NADP oxidoreductase.

  13. Domain Modeling: NP_115604.1 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_115604.1 chr11 Crystal structure of hypothetical protein PH0414 from Pyrococcus ...horikoshii OT3 p2hunb_ chr11/NP_115604.1/NP_115604.1_holo_12-402.pdb psi-blast 16T,17G,19T,20G,21F,22L,23G,5

  14. Conformational heterogeneity of the aspartate transporter Glt(Ph)

    NARCIS (Netherlands)

    Hänelt, Inga; Wunnicke, Dorith; Bordignon, Enrica; Steinhoff, Heinz-Juergen; Slotboom, Dirk Jan

    2013-01-01

    Glt(Ph) is a Pyrococcus horikoshii homotrimeric Na+-coupled aspartate transporter that belongs to the glutamate transporter family. Each protomer consists of a trimerization domain involved in subunit interaction and a transporting domain with the substrate-binding site. Here, we have studied the co

  15. Na+ : Aspartate Coupling Stoichiometry in the Glutamate Transporter Homologue Glt(Ph)

    NARCIS (Netherlands)

    Groeneveld, Maarten; Slotboom, Dirk-Jan

    2010-01-01

    The Na+ aspartate symporter Glt(Ph) from Pyrococcus horikoshil is the only member of the glutamate transporter family for which crystal structures have been determined. The cation:aspartate coupling stoichiometry is unknown, thus hampering the elucidation of the ion coupling mechanism. Here we measu

  16. AcEST: DK946632 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ta OS=Methanobrevibacte... 42 0.001 sp|Q8C2K1|DEFI6_MOUSE Differentially expressed in FDCP 6 OS=Mus ... 42 0... subunit beta OS=Pyrococcus abyssi... 41 0.003 sp|Q9H4E7|DEFI6_HUMAN Differentially expressed in FDCP 6 homo

  17. Domain Modeling: NP_054887.2 [SAHG[Archive

    Lifescience Database Archive (English)

    Full Text Available NP_054887.2 chr2 crystal structure of a putative RNA methyltransferase PH1948 from ...Pyrococcus horikoshii c1wy7d_ chr2/NP_054887.2/NP_054887.2_holo_5-208.pdb blast 19F,26L,27E,28Q,29Y,30P,31T,

  18. Iron-Sulfur Proteins Investigated by EPR-, Moessbauer- and EXAFS-Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wegner, P.; Bever, M.; Schuenemann, V.; Trautwein, A. X. [University of Luebeck, Institute of Physics (Germany); Schmidt, C. [University of Luebeck, Institute of Biochemistry (Germany); Boenisch, H. [Center for Structural Biochemistry, Karolinska Institutet, Dept. of Biosciences at NOVUM (Sweden); Gnida, M.; Meyer-Klaucke, W. [DESY, EMBL Outstation Hamburg (Germany)

    2004-12-15

    The structural and spectroscopic properties of the biologically active [Fe-4S] site of three different mutants of the wild-type rubredoxin from the archaeon Pyrococcus abyssi were investigated and compared with each other and additionally with those of the rubredoxin from the bacterium Clostridium pasteurianum.

  19. AcEST: BP921295 [AcEST

    Lifescience Database Archive (English)

    Full Text Available B Rubredoxin OS=Pyrococcus abyssi GN=rub PE=1... 32 0.76 sp|P00267|RUBR_PEPAS Rubredoxin OS=Peptostreptoco...ccus asaccharol... 32 0.76 sp|P15412|RUBR_DESVM Rubredoxin OS=Desulfovibrio vulgari

  20. Low affinity and slow Na+-binding precedes high affinity aspartate binding in GltPh

    NARCIS (Netherlands)

    Hänelt, Inga; Jensen, Sonja; Wunnicke, Dorith; Slotboom, Dirk Jan

    2015-01-01

    GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters (EAATs) that take up the neurotransmitter glutamate. Each protomer in GltPh consis

  1. Na+ : Aspartate Coupling Stoichiometry in the Glutamate Transporter Homologue Glt(Ph)

    NARCIS (Netherlands)

    Groeneveld, Maarten; Slotboom, Dirk-Jan

    2010-01-01

    The Na+ aspartate symporter Glt(Ph) from Pyrococcus horikoshil is the only member of the glutamate transporter family for which crystal structures have been determined. The cation:aspartate coupling stoichiometry is unknown, thus hampering the elucidation of the ion coupling mechanism. Here we measu

  2. Novel Three-Component Rieske Non-Heme Iron Oxygenase System Catalyzing the N-Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6 and DC-2

    Science.gov (United States)

    Chen, Qing; Wang, Cheng-Hong; Deng, Shi-Kai; Wu, Ya-Dong; Li, Yi; Yao, Li; Jiang, Jian-Dong; Yan, Xin; Li, Shun-Peng

    2014-01-01

    Sphingomonads DC-6 and DC-2 degrade the chloroacetanilide herbicides alachlor, acetochlor, and butachlor via N-dealkylation. In this study, we report a three-component Rieske non-heme iron oxygenase (RHO) system catalyzing the N-dealkylation of these herbicides. The oxygenase component gene cndA is located in a transposable element that is highly conserved in the two strains. CndA shares 24 to 42% amino acid sequence identities with the oxygenase components of some RHOs that catalyze N- or O-demethylation. Two putative [2Fe-2S] ferredoxin genes and one glutathione reductase (GR)-type reductase gene were retrieved from the genome of each strain. These genes were not located in the immediate vicinity of cndA. The four ferredoxins share 64 to 72% amino acid sequence identities to the ferredoxin component of dicamba O-demethylase (DMO), and the two reductases share 62 to 65% amino acid sequence identities to the reductase component of DMO. cndA, the four ferredoxin genes, and the two reductases genes were expressed in Escherichia coli, and the recombinant proteins were purified using Ni-affinity chromatography. The individual components or the components in pairs displayed no activity; the enzyme mixture showed N-dealkylase activities toward alachlor, acetochlor, and butachlor only when CndA-His6 was combined with one of the four ferredoxins and one of the two reductases, suggesting that the enzyme consists of three components, a homo-oligomer oxygenase, a [2Fe-2S] ferredoxin, and a GR-type reductase, and CndA has a low specificity for the electron transport component (ETC). The N-dealkylase utilizes NADH, but not NADPH, as the electron donor. PMID:24928877

  3. Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82.

    OpenAIRE

    Takizawa, N; Kaida, N; Torigoe, S; Moritani,T.; Sawada, T.; Satoh, S.; Kiyohara, H

    1994-01-01

    Naphthalene and phenanthrene are transformed by enzymes encoded by the pah gene cluster of Pseudomonas putida OUS82. The pahA and pahB genes, which encode the first and second enzymes, dioxygenase and cis-dihydrodiol dehydrogenase, respectively, were identified and sequenced. The DNA sequences showed that pahA and pahB were clustered and that pahA consisted of four cistrons, pahAa, pahAb, pahAc, and pahAd, which encode ferredoxin reductase, ferredoxin, and two subunits of the iron-sulfur prot...

  4. Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

    OpenAIRE

    Moreno-Vivian, C; Hennecke, S; Pühler, A.; Klipp, W

    1989-01-01

    DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of...

  5. AcEST: BP918943 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ition tr|Q0W0W8|Q0W0W8_UNCMA Putative tungsten-containing aldehyde:ferredoxin oxidoreductase OS=Uncultured m...e Score E Sequences producing significant alignments: (bits) Value tr|Q0W0W8|Q0W0W8_UNCMA Putative tungsten-...containing aldehyde:fer... 33 5.5 >tr|Q0W0W8|Q0W0W8_UNCMA Putative tungsten-containing aldehyde:ferredoxin o

  6. Rôles respectifs des isoformes de ferrédoxine-NADP-oxydoréductase dans la cyanobactérie Synechocystis sp. PCC 6803

    OpenAIRE

    Korn, Anja

    2010-01-01

    In photosynthetic organisms, ferredoxin:NADP oxidoreductase (FNR) provides NADPH for CO2 assimilation, but it also utilizes NADPH to provide reduced ferredoxin (Fd). The cyanobacterium Synechocystis sp. strain PCC6803 contains two FNR isoforms, a small (FNRS, 34 kDa) and a large one (FNRL, 46 kDa) that is associated with the phycobilisome (PBS), a light-harvesting complex. We purified a PBS subcomplex comprising FNRL (FNRL-PC) and compared the enzymatic properties of FNRL-PC to FNRS. FNRL-PC ...

  7. New Therapeutic Strategies for Antibiotic-Resistant Select Agents

    Science.gov (United States)

    2007-12-31

    Soriano, A., Zhao, W., Gullo, V. P., and Chan, T.-M. (2004) Two new bacterial DNA primase inhibitors from the plant Polygonum cuspidatum, Bioorg...hyperthermophilic archaeon Pyrococcus horikoshii, J. Biochem. 130, 727-730. 26. Sheaff, R. J., and Kuchta, R. D. (1993) Mechanism of calf thymus DNA primase...Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA-polymerase-alpha, J

  8. A Self-Assembling Protein Hydrogel Technology for Enzyme Incorporation onto Electrodes in Biofuel Cells

    Science.gov (United States)

    2015-10-26

    is in preparation. Overall, we developed novel highly stable and flexible protein hydrogels, and applied these protein hydrogels in the synthesis of...punctiform (NpuN, NpuC) (Fig. 1). CutA is a small trimeric protein (12 kDa) from thermophilic bacteria pyrococcus horihoshhi4, 5 that exhibits ultrahigh...on graphene and application to glucose detection. Electrochim Acta 55, 8606-8614 (2010). 16. Zhang, J., Feng, M. & Tachikawa, H. Layer-by-layer

  9. Biogeography and evolution of Thermococcus isolates from hydrothermal vent systems of the Pacific

    Directory of Open Access Journals (Sweden)

    Mark Thomas Price

    2015-09-01

    Full Text Available Thermococcus is a genus of hyperthermophilic archaea that is ubiquitous in marine hydrothermal environments growing in anaerobic subsurface habitats but able to survive in cold oxygenated seawater. DNA analyses of Thermococcus isolates were applied to determine the relationship between geographic distribution and relatedness focusing primarily on isolates from the Juan de Fuca Ridge and South East Pacific Rise. Amplified fragment length polymorphism (AFLP analysis and multilocus sequence typing (MLST were used to resolve genomic differences in 90 isolates of Thermococcus, making biogeographic patterns and evolutionary relationships apparent. Isolates were differentiated into regionally endemic populations however there was also evidence in some lineages of cosmopolitan distribution. The biodiversity identified in Thermococcus isolates and presence of distinct lineages within the same vent site suggests the utilization of varying ecological niches in this genus. In addition to resolving biogeographic patterns in Thermococcus, this study has raised new questions about the closely related Pyrococcus genus. The phylogenetic placement of Pyrococcus type strains shows the close relationship between Thermococcus and Pyrococcus and the unresolved divergence of these two genera.

  10. The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20

    Directory of Open Access Journals (Sweden)

    Morgan N Price

    2014-10-01

    Full Text Available Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13 is sometimes important alongside Rnf and may perform an electron bifurcation to generate more reduced ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1 is important and may generate reduced ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions, although we cannot rule out genetic redundancy.

  11. Sequence Classification: 53127 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|50084183|ref|YP_045693.1| vanil...late O-demethylase oxidoreductase (Vanillate degradation ferredoxin-like protein) || http://www.ncbi.nlm.nih.gov/protein/50084183 ...

  12. AcEST: BP914233 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 5.7 sp|Q7S6P8|BFR2_NEUCR Protein bfr-2 OS=Neurospora crassa GN=bfr-2... 30 7.5 sp|Q56303|FOR_THELI Tungst...en-containing formaldehyde ferredoxin ... 30 9.7 sp|Q8WN22|PRKDC_CANFA DNA-dependen

  13. Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9 alpha-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

    NARCIS (Netherlands)

    Petrusma, M.; Dijkhuizen, L.; van der Geize, R.

    2009-01-01

    This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9 alpha-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in

  14. Gclust Server: 19843 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 19843 Bja_blr6743 Cluster Sequences Related Sequences(57) 640 putative ferredoxin o...xidoreductase alpha subunit 5 1.00e-35 0.0 0.0 0.0 6.67 9.68 0.0 Show 19843 Cluster ID 19843 Sequence ID Bja

  15. Probing the redox metabolism in the strictly anaerobic, extremely thermophilic, hydrogen-producing Caldicellulosiruptor saccharolyticus using amperometry

    DEFF Research Database (Denmark)

    Kostesha, Natalie; Willquist, Karin; Emnéus, Jenny;

    2011-01-01

    flow between cells harvested in the exponential and stationary growth phases. The electron flow of C. saccharolyticus was dependent on the NADH- and reduced ferredoxin generation flux and the competitive behavior of cytosolic and membrane-associated oxidoreductases. Sodium oxamate was used to inhibit...

  16. Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase

    Science.gov (United States)

    Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterised with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion ha...

  17. Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9 alpha-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

    NARCIS (Netherlands)

    Petrusma, M.; Dijkhuizen, L.; van der Geize, R.

    2009-01-01

    This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9 alpha-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichi

  18. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor

    DEFF Research Database (Denmark)

    Jensen, Kenneth; Johnston, Jonathan B.; Montellano, Paul R. Ortiz de;

    2012-01-01

    ) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP+ to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron...

  19. New enzymatic pathways for the reduction of reactive oxygen species in Entamoeba histolytica.

    Science.gov (United States)

    Cabeza, Matías S; Guerrero, Sergio A; Iglesias, Alberto A; Arias, Diego G

    2015-06-01

    Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified. The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica. We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin. The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species. To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20.

    Science.gov (United States)

    Price, Morgan N; Ray, Jayashree; Wetmore, Kelly M; Kuehl, Jennifer V; Bauer, Stefan; Deutschbauer, Adam M; Arkin, Adam P

    2014-01-01

    Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13) is sometimes important alongside Rnf and may perform an electron bifurcation to generate more reduced ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1) is important and may generate reduced ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions, although we cannot rule out genetic redundancy.

  1. Open reading frame ssr2016 is required for antimycin A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803

    NARCIS (Netherlands)

    Yeremenko, N.; Jeanjean, R.; Prommeenate, P.; Krasikov, V.; Nixon, P.J.; Vermaas, W.F.J.; Havaux, M.; Matthijs, H.C.P.

    2005-01-01

    Open reading frame ssr2016 encodes a protein with substantial sequence similarities to PGR5 identified as a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in PSI cyclic photophosphorylation in Arabidopsis thaliana. We studied cyclic electron flow in Synechocystis sp.

  2. CHARACTERIZATION OF HYDROGENOSOMES AND THEIR ROLE IN GLUCOSE-METABOLISM OF NEOCALLIMASTIX SP L2

    NARCIS (Netherlands)

    MARVINSIKKEMA, FD; GOMES, TMP; GRIVET, JP; GOTTSCHAL, JC; PRINS, RA

    1993-01-01

    In the anaerobic fungus Neocallimastix. sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme', NAD(P)H:ferredoxin

  3. Chemotherapy of Leishmaniasis.

    Science.gov (United States)

    1979-09-01

    action have indicated that Trichomonas probably possesses a ferredoxin or flavodoxin which is characteristic of anaerobic organisms (Muller et al...1976) but whether such compounds exist in Leishmania has not yet been explored. Nitroimidazoles exert their toxic action on T. vaginalis through the

  4. GenBank blastx search result: AK062096 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062096 001-044-H12 AF060489.1 Sphingomonas sp. CB3 initial dioxygenase large subu...nit (carAa) gene, partial cds; and initial dioxygenase small subunit (carAb), initial dioxygenase ferredoxin subunit (carAc), initial

  5. The Effect of Iron Fertilization on Nodulation, Yield and Yield Traits ...

    African Journals Online (AJOL)

    bacteria, which is an essential component of nitrogenase, leghaemoglobin and ferredoxins. (Evans and ... The site has been used for maize (Zea mays L.) and tomato production in the ... Nitrogen (i.e. 20 kg ha-1 based) in the form of urea was.

  6. Thioredoxins in evolutionarily primitive organisms

    Science.gov (United States)

    Buchanan, B. B.

    1986-01-01

    Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

  7. Le recours à l’expertise psychiatrique dans les juridictions ecclésiastiques (1850-1930) The Appeal to the Psychiatric Expertise in the Ecclesiastical Jurisdictions (1850-1930)

    OpenAIRE

    Laurent Kondratuk

    2011-01-01

    Les juridictions ecclésiastiques, à l'instar des juridictions civiles, usent de l'expertise psychiatrique dans les procès pénaux mais surtout matrimoniaux. Les juges demandent des avis pour déterminer si l'un des conjoints, présumé déficient mental (furiosus) était en mesure de manifester un consentement et d'assumer les obligations du mariage. Il est rappelé tout d'abord les cas où les juridictions ecclésiastiques recourent à l'expertise, psychiatrique ou même gynécologique. Ensuite, il est ...

  8. An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis.

    Directory of Open Access Journals (Sweden)

    Anja Poehlein

    Full Text Available Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf. The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.

  9. Fat Metabolism in Higher Plants: LXII. Stearl-acyl Carrier Protein Desaturase from Spinach Chloroplasts.

    Science.gov (United States)

    Jacobson, B S; Jaworski, J G; Stumpf, P K

    1974-10-01

    Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds.

  10. The antimycin A-sensitive pathway of cyclic electron flow

    DEFF Research Database (Denmark)

    Labs, Mathias; Rühle, Thilo; Leister, Dario Michael

    2016-01-01

    severe effects on plant growth. One of the two pathways mediating cyclic electron flow can be inhibited by antimycin A, a chemical that has also widely been used to characterize the mitochondrial respiratory chain. For the characterization of cyclic electron flow, antimycin A has been used since 1963......Cyclic electron flow has puzzled and divided the field of photosynthesis researchers for decades. This mainly concerns the proportion of its overall contribution to photosynthesis, as well as its components and molecular mechanism. Yet, it is irrefutable that the absence of cyclic electron flow has......, when ferredoxin was found to be the electron donor of the pathway. In 2013, antimycin A was used to identify the PGRL1/PGR5 complex as the ferredoxin:plastoquinone reductase completing the last puzzle piece of this pathway. The controversy has not ended, and here, we review the history of research...

  11. An atypical segment swap in the DN and DC domains of the Acr_tran family resistance-nodulation-cell division pump.

    Science.gov (United States)

    Acharya, Giriraj; Kaur, Gurmeet; Subramanian, Srikrishna

    2016-12-01

    Domain/segment swapping is an exchange of equivalent secondary structure element(s) among two or more protein domains resulting in the reconstitution of the original fold while simultaneously causing oligomerization. Here we report an example of the outer membrane factor docking region of the Acr_tran family (PF00873) resistance-nodulation-cell division pump, in which a swapped, misfolded state, of the ferredoxin-like fold of the DN and DC domains, effectuates oligomerization. The atypical segment swap and the associated displacement of a region of the ferredoxin-like fold leads to a topology that is distinct from the original fold. To our knowledge, such segment swaps and associated fold change are rare. This exemplifies the role of functional constraints including oligomerization that determine the interplay between sequence and the three-dimensional structure of proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Sequence Classification: 509333 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|73539581|ref|YP_299948.1| Ferredoxin:Oxido...reductase FAD/NAD(P)-binding:Molybdopterin oxidoreductase:Molydopterin dinucleotide-binding region:Molybdopterin oxido...reductase Fe4S4 region:Oxidoreductase FAD-binding region || http://www.ncbi.nlm.nih.gov/protein/73539581 ...

  13. Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean)--coordination structure of the heme complex and catabolism of heme.

    Science.gov (United States)

    Gohya, Tomohiko; Zhang, Xuhong; Yoshida, Tadashi; Migita, Catharina T

    2006-12-01

    Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IX(alpha) as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO-1 and heme oxygenases from other biological species is low, and GmHO-1 binds heme with 1 : 1 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid-base transition and is converted to biliverdin IX(alpha) in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme-heme oxygenase or CO-verdoheme-heme oxygenase complexes was observed and was extracted as a bis-imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system.

  14. GenBank blastx search result: AK103964 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103964 001-015-F12 U04052.1 Pseudomonas pickettii PKO1 toluene-3-monooxygenase ox...ygenase subunit (tbuA1) and (tbuA2) genes, (tbuU) gene, (tbuB) gene, toluene-3-monooxygenase ferredoxin protein (tbuV) gene, and tolu...ene-3-monooxygenase oxidoreductase (tbuC) gene, complete cds.|BCT BCT 2e-12 +3 ...

  15. GenBank blastx search result: AK058594 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058594 001-017-H01 U04052.1 Pseudomonas pickettii PKO1 toluene-3-monooxygenase ox...ygenase subunit (tbuA1) and (tbuA2) genes, (tbuU) gene, (tbuB) gene, toluene-3-monooxygenase ferredoxin protein (tbuV) gene, and tolu...ene-3-monooxygenase oxidoreductase (tbuC) gene, complete cds.|BCT BCT 2e-12 +3 ...

  16. Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity.

    Science.gov (United States)

    Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

    2017-03-21

    A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

  17. Structural Analysis of Proteins in Extreme Saline Environments

    Science.gov (United States)

    1989-03-16

    ferredoxinz with the same 2Fe-2S prosthetic group trom the blue-green algae Spirulina platensis (SplFd) (K. Fukuyama et al., 1980; Tsukihara et al...homologous 2Fe-2S ferredoxin from a nonhalophilic source, the algae Spirulinaplatensis. We wanted to understand what sort of adaptation, on the...thermophilic organisms are known today, among them bacteria, algae and fungi. The group of true thermophiles, i.e. organisms that thrive in high temperatures and

  18. Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite.

    Science.gov (United States)

    Payne, M J; Woods, L F; Gibbs, P; Cammack, R

    1990-10-01

    The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the iron-sulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sporogenes in liquid cultures at pH 7.4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0.5 mM-nitrite with 10 mM-ascorbate stopped growth completely. In partially-purified preparations 4.1 mM-NaNO2 and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78%) inactivation of pyruvate-ferredoxin oxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO)2(SR)2] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxin were shown by EPR spectroscopy to be resistant to treatment with 3.6 mM-NaNO2 and 3.6 mM-ascorbate. It is concluded that the effects of nitrite on pre-formed iron-sulphur proteins are not convincing as a basis for the lethal effects on bacterial cells.

  19. GenBank blastx search result: AK059171 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059171 001-023-F01 AF119361.1 Frankia sp. EuIK1 NifK (nifK), NifE (nifE), NifN (nifN), NifX (nifX), Nif...W (nifW), NifZ (nifZ), NifB (nifB), NifV (nifV), NifS (nifS), ferredoxin oxidoreductas

  20. GenBank blastx search result: AK058791 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058791 001-002-F06 AF119361.1 Frankia sp. EuIK1 NifK (nifK), NifE (nifE), NifN (nifN), NifX (nifX), Nif...W (nifW), NifZ (nifZ), NifB (nifB), NifV (nifV), NifS (nifS), ferredoxin oxidoreductas

  1. Studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) of Acidaminococcus fermentans.

    Science.gov (United States)

    Chowdhury, Nilanjan Pal; Mowafy, Amr M; Demmer, Julius K; Upadhyay, Vikrant; Koelzer, Sebastian; Jayamani, Elamparithi; Kahnt, Joerg; Hornung, Marco; Demmer, Ulrike; Ermler, Ulrich; Buckel, Wolfgang

    2014-02-21

    Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

  2. Crystal Structures of the Iron–Sulfur Cluster-Dependent Quinolinate Synthase in Complex with Dihydroxyacetone Phosphate, Iminoaspartate Analogues, and Quinolinate

    Energy Technology Data Exchange (ETDEWEB)

    Fenwick, Michael K. [Cornell Univ., Ithaca, NY (United States); Ealick, Steven E. [Cornell Univ., Ithaca, NY (United States)

    2016-07-12

    The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.

  3. Bioenergetics of Monoterpenoid Essential Oil Biosynthesis in Non-Photosynthetic Glandular Trichomes.

    Science.gov (United States)

    Johnson, Sean R; Lange, Iris; Srividya, Narayanan; Lange, Bernd Markus

    2017-08-24

    The commercially important essential oils of peppermint (Mentha x piperita L.) and its relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical structures called glandular trichomes (GTs). A genome-scale stoichiometric model of secretory phase metabolism in peppermint GTs was constructed based on current biochemical and physiological knowledge. Fluxes through the network were predicted based on metabolomic and transcriptomic data. Using simulated reaction deletions, this model predicted that two processes, the regeneration of ATP and ferredoxin (in its reduced form), exert substantial control over flux toward monoterpenes. Follow-up biochemical assays with isolated GTs indicated that oxidative phosphorylation and ethanolic fermentation were active, and that cooperation to provide ATP depended on the concentration of the carbon source. We also report that GTs with high flux toward monoterpenes express, at very high levels, genes coding for a unique pair of ferredoxin and ferredoxin-NADP+ reductase isoforms. This study provides the first evidence how bioenergetic processes determine flux through monoterpene biosynthesis in GTs. {copyright, serif} 2017 American Society of Plant Biologists. All rights reserved.

  4. Iron effect on the fermentative metabolism of Clostridium acetobutylicum ATCC 824 using cheese whey as substrate

    Directory of Open Access Journals (Sweden)

    Victoria Rosalía Durán-Padilla

    2014-12-01

    Full Text Available Butanol is considered a superior liquid fuel that can replace gasoline in internal combustion engines. It is produced by acetone-butanol-ethanol (ABE fermentation using various species of solventogenic clostridia. Performance of ABE fermentation process is severely limited mostly by high cost of substrate, substrate inhibition and low solvent tolerance; leading to low product concentrations, low productivity, low yield, and difficulty in controlling culture metabolism. In order to decrease the cost per substrate and exploit a waste generated by dairy industry, this study proposes using cheese whey as substrate for ABE fermentation. It was observed that the addition of an iron source was strictly necessary for the cheese whey to be a viable substrate because this metal is needed to produce ferredoxin, a key protein in the fermentative metabolism of Clostridium acetobutylicum serving as a temporary electron acceptor. Lack of iron in the cheese whey impedes ferredoxin synthesis and therefore, restricts pyruvate-ferredoxin oxidoreductase activity leading to the production of lactic acid instead of acetone, butanol and ethanol. Moreover, the addition of FeSO4 notably improved ABE production performance by increasing butanol content (7.13 ± 1.53 g/L by 65% compared to that of FeCl3 (4.32 ± 0.94 g/L under the same fermentation conditions.

  5. Detection of the gene encoding the small subunit of the CO dehydrogenase enzyme in the H{sub 2}-evolving bacterium Rubrivivax gelatinosus CBS

    Energy Technology Data Exchange (ETDEWEB)

    Kish, A.; Levin, D. [Victoria Univ., BC (Canada)]|[Victoria Univ., BC (Canada)

    2001-06-01

    A purple non-sulfur bacterium, Rubrivivax gelatinosus CBS presents great opportunities, on a commercial scale, for the biological hydrogen production. A water-gas shift reaction is catalyzed when the bacterium is cultured in the presence of carbon oxide in the dark. The result is carbon monoxide (and water) being shifted into hydrogen (H{sub 2}) and carbon dioxide in near stoichiometric quantities. The production of hydrogen as a clean alternative fuel could be accomplished by using carbon monoxide generated from gasified waste biomass, using the bacterial water-gas shift reaction for that purpose. The characterization of three key enzymes and the genes encoding them was performed in a closely related purple non-sulfur bacterium called Rhodospirillum rubrum. They were: (1) a carbon monoxide dehydrogenase (CODH), (2) the ferredoxin-like electron-carrier small subunit of the CODH enzyme, and (3) an hydrogen-evolving hydrogenase. A transcriptional unit separate from the genes encoding the CODH and its ferredoxin-like small subunit encode the genes for the hydrogenase. A fragment of the Rhodospirillum rubrum ferredoxin-like subunit gene was amplified through the use of a polymerase chain reaction. Southern blots of restriction endonuclease digested genomic deoxyribonucleic acid (DNA) extracted from Rubrivivax gelatinosus CBS was probed with the fragment of the Rhodospirillum rubrum previously amplified using the polymerase chain reaction. Confirmation of the identification is being confirmed, while the gene is sequenced. 25 refs., 2 figs.

  6. Inorganic Fe2+ formation upon Fe-S protein thermodestruction in the membranes of thermophilic cyanobacteria: Mössbauer spectroscopy study.

    Science.gov (United States)

    Kaurov YuN; Novakova, A A; Davletshina, L N; Aleksandrov AYu; Khval'kovskaya, E A; Semin, B K; Belevich, N P; Ivanov, I I; Rubin, A B

    1999-04-30

    A model description of the Mössbauer spectrum (80 K) of native membranes of the thermophilic cyanobacterium Synechococcus elongatus is suggested on the basis of the known values of quadrupole splitting (deltaE(Q)) and isomer shift (deltaFe) for the iron-containing components of the photosynthetic apparatus. Using this approach, we found that heating the membranes at 70-80 K results in a decrease of doublet amplitudes belonging to F(X), F(A), F(B) and ferredoxin and simultaneous formation of a new doublet with deltaE(Q) = 3.10 mm/s and delta-Fe = 1.28 mm/s, typical of inorganic hydrated forms of Fe2+. The inhibition of electron transfer via photosystem I to oxygen, catalyzed by ferredoxin, occurs within the same range of temperatures. The data demonstrate that the processes of thermoinduced Fe2+ formation and distortions in the photosystem I electron transport in the membranes are interrelated and caused mainly by the degradation of ferredoxin. The possible role of Fe2+ formation in the damage of the photosynthetic apparatus resulting from heating and the action of other extreme factors is discussed.

  7. Relationships between H{sub 2} photoproduction and different electron transport pathways in sulfur-deprived Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Antal, Taras K.; Volgusheva, Alena A.; Kukarskih, Galina P.; Krendeleva, Tatyana E.; Rubin, Andrej B. [Faculty of Biology, Moscow State University, Vorobyevi Gory 119992, Moscow (Russian Federation)

    2009-11-15

    In this study the relationships between photosystem (PS) II dependent and independent pathways of H{sub 2} photoproduction, cyclic electron transport around PS I, chloro- and mitorespiration, and transmembrane {delta}pH were examined by inhibitor analysis in S deprived Chlamydomonas reinhardtii. The rate of non-photochemical reduction of plastoquinones in photosynthetic membranes was significantly diminished under starvation which may explain the minor contribution of the PS II independent pathway of H{sub 2} photoproduction in starved cells. The suppressive effect of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea on the long-term H{sub 2} photoproduction was shown to be entirely attributed to the inhibition of electron transport in PS II, whereas non-specific interactions did not take place. Ferredoxin-quinone reductase - dependent cyclic electron transport around PS I slowed down H{sub 2} photoproduction more than two fold. This result was related to the competition between ferredoxin-quinone reductase and hydrogenase for the reduced ferredoxin and to the decrease in transmembrane {delta}pH induced by the cyclic electron flow. The {delta}pH gradient was shown to down regulate the PS II independent pathway of H{sub 2} photoproduction in starved cells. (author)

  8. Chloroplast thioredoxin systems: prospects for improving photosynthesis.

    Science.gov (United States)

    Nikkanen, Lauri; Toivola, Jouni; Diaz, Manuel Guinea; Rintamäki, Eevi

    2017-09-26

    Thioredoxins (TRXs) are protein oxidoreductases that control the structure and function of cellular proteins by cleavage of a disulphide bond between the side chains of two cysteine residues. Oxidized thioredoxins are reactivated by thioredoxin reductases (TR) and a TR-dependent reduction of TRXs is called a thioredoxin system. Thiol-based redox regulation is an especially important mechanism to control chloroplast proteins involved in biogenesis, in regulation of light harvesting and distribution of light energy between photosystems, in photosynthetic carbon fixation and other biosynthetic pathways, and in stress responses of plants. Of the two plant plastid thioredoxin systems, the ferredoxin-dependent system relays reducing equivalents from photosystem I via ferredoxin and ferredoxin-thioredoxin reductase (FTR) to chloroplast proteins, while NADPH-dependent thioredoxin reductase (NTRC) forms a complete thioredoxin system including both reductase and thioredoxin domains in a single polypeptide. Chloroplast thioredoxins transmit environmental light signals to biochemical reactions, which allows fine tuning of photosynthetic processes in response to changing environmental conditions. In this paper we focus on the recent reports on specificity and networking of chloroplast thioredoxin systems and evaluate the prospect of improving photosynthetic performance by modifying the activity of thiol regulators in plants.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Authors.

  9. Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis.

    Science.gov (United States)

    Nikkanen, Lauri; Toivola, Jouni; Rintamäki, Eevi

    2016-08-01

    Thioredoxins (TRXs) mediate light-dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox-regulated enzymes. Of the two plastid TRX systems, the ferredoxin-TRX system consists of ferredoxin-thioredoxin reductase (FTR) and multiple TRXs, while the NADPH-dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd-TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO2 fixation rate and lowered non-photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX-regulated enzymes in Calvin-Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose-1,6-bisphosphatase, phosphoribulokinase and CF1 γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC-mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin-TRX system and is crucial when availability of light is limiting photosynthesis. © 2016 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

  10. Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7.

    Science.gov (United States)

    Lantsman, Yelena; Tan, Kevin S W; Morada, Mary; Yarlett, Nigel

    2008-09-01

    A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

  11. Le recours à l’expertise psychiatrique dans les juridictions ecclésiastiques (1850-1930 The Appeal to the Psychiatric Expertise in the Ecclesiastical Jurisdictions (1850-1930

    Directory of Open Access Journals (Sweden)

    Laurent Kondratuk

    2011-03-01

    Full Text Available Les juridictions ecclésiastiques, à l'instar des juridictions civiles, usent de l'expertise psychiatrique dans les procès pénaux mais surtout matrimoniaux. Les juges demandent des avis pour déterminer si l'un des conjoints, présumé déficient mental (furiosus était en mesure de manifester un consentement et d'assumer les obligations du mariage. Il est rappelé tout d'abord les cas où les juridictions ecclésiastiques recourent à l'expertise, psychiatrique ou même gynécologique. Ensuite, il est fait état tant de la doctrine que de la jurisprudence regardant la question de la folie et des psychopathies sexuelles (homosexualité dans les procès en nullité de mariage, principalement dans la première moitié du XXe siècle.Ecclesiastical courts, like civil courts, call on psychiatric expertise in criminal trials, especially where spouses are involved.  Judges seek expert advice in determining whether one of the parties, presumably mentally deficient (furiosus, was competent to consent to, and take on, the obligations of marriage.  We review cases in which ecclesiastical courts resorted to psychiatric and even gynecological expertise.  Next, we consider both doctrine and jurisprudence concerning the question of madness and sexual psychopathology (as was deemed homosexuality in the nullification procedure, mainly during the first half of the twentieth century.

  12. Enzymological studies of one-carbon reactions in the pathway of acetate utilization by methanogenic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Ferry, J.G.

    1991-12-31

    Several enzymes in the pathway of acetate conversion to methane and carbon dioxide have been purified from Methanosarcina thermophila. The mechanisms of these enzymes are under investigation utilizing biochemical, biophysical and molecular genetic approaches. Acetate kinase and phosphotransacetylase catalyzes the activation of acetate to acetyl-CoA. The primary structure of these enzymes will be determined through cloning and sequencing of the genes. Two protein components of the CO dehydrogenase complex are under investigations. The metal centers of each component have been characterized using EPR. Cloning and sequencing of the genes for the two subunits of each component is in progress. Results indicate that the Ni/Fe-S component cleaves the C-C and C-S bonds of acetyl-CoA followed by oxidation of the carbonyl group to carbon dioxide and transfer of the methyl group to the Co/Fe-S component. The enzymes and cofactors involved in transfer of the methyl group from the Co/Fe-S component to coenzyme M will be purified and characterized. Ferredoxin is an electron acceptor for the Ni/Fe-S component and also serves to reductively reactivate methylreductase which catalyzes the demethylation of methyl coenzyme M to methane. This ferredoxin is being characterized utilizing EPR and RR spectroscopic methods to determine the properties of the Fe-S centers. Genes encoding this and other ferredoxins have been cloned and sequenced to determine the primary structures. Carbonic anhydrase is being purified and characterized to determine the function of this enzyme in the pathway.

  13. Enzymological studies of one-carbon reactions in the pathway of acetate utilization by methanogenic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Ferry, J.G.

    1991-01-01

    Several enzymes in the pathway of acetate conversion to methane and carbon dioxide have been purified from Methanosarcina thermophila. The mechanisms of these enzymes are under investigation utilizing biochemical, biophysical and molecular genetic approaches. Acetate kinase and phosphotransacetylase catalyzes the activation of acetate to acetyl-CoA. The primary structure of these enzymes will be determined through cloning and sequencing of the genes. Two protein components of the CO dehydrogenase complex are under investigations. The metal centers of each component have been characterized using EPR. Cloning and sequencing of the genes for the two subunits of each component is in progress. Results indicate that the Ni/Fe-S component cleaves the C-C and C-S bonds of acetyl-CoA followed by oxidation of the carbonyl group to carbon dioxide and transfer of the methyl group to the Co/Fe-S component. The enzymes and cofactors involved in transfer of the methyl group from the Co/Fe-S component to coenzyme M will be purified and characterized. Ferredoxin is an electron acceptor for the Ni/Fe-S component and also serves to reductively reactivate methylreductase which catalyzes the demethylation of methyl coenzyme M to methane. This ferredoxin is being characterized utilizing EPR and RR spectroscopic methods to determine the properties of the Fe-S centers. Genes encoding this and other ferredoxins have been cloned and sequenced to determine the primary structures. Carbonic anhydrase is being purified and characterized to determine the function of this enzyme in the pathway.

  14. Unique flexibility in energy metabolism allows mycobacteria to combat starvation and hypoxia.

    Directory of Open Access Journals (Sweden)

    Michael Berney

    Full Text Available Mycobacteria are a group of obligate aerobes that require oxygen for growth, but paradoxically have the ability to survive and metabolize under hypoxia. The mechanisms responsible for this metabolic plasticity are unknown. Here, we report on the adaptation of Mycobacterium smegmatis to slow growth rate and hypoxia using carbon-limited continuous culture. When M. smegmatis is switched from a 4.6 h to a 69 h doubling time at a constant oxygen saturation of 50%, the cells respond through the down regulation of respiratory chain components and the F1Fo-ATP synthase, consistent with the cells lower demand for energy at a reduced growth rate. This was paralleled by an up regulation of molecular machinery that allowed more efficient energy generation (i.e. Complex I and the use of alternative electron donors (e.g. hydrogenases and primary dehydrogenases to maintain the flow of reducing equivalents to the electron transport chain during conditions of severe energy limitation. A hydrogenase mutant showed a 40% reduction in growth yield highlighting the importance of this enzyme in adaptation to low energy supply. Slow growing cells at 50% oxygen saturation subjected to hypoxia (0.6% oxygen saturation responded by switching on oxygen scavenging cytochrome bd, proton-translocating cytochrome bc1-aa3 supercomplex, another putative hydrogenase, and by substituting NAD+-dependent enzymes with ferredoxin-dependent enzymes thus highlighting a new pattern of mycobacterial adaptation to hypoxia. The expression of ferredoxins and a hydrogenase provides a potential conduit for disposing of and transferring electrons in the absence of exogenous electron acceptors. The use of ferredoxin-dependent enzymes would allow the cell to maintain a high carbon flux through its central carbon metabolism independent of the NAD+/NADH ratio. These data demonstrate the remarkable metabolic plasticity of the mycobacterial cell and provide a new framework for understanding their

  15. Antiprotozoal Constituents from Annona cherimola Miller, a Plant Used in Mexican Traditional Medicine for the Treatment of Diarrhea and Dysentery

    Science.gov (United States)

    Calzada, Fernando; Correa-Basurto, Jose; Barbosa, Elizabeth; Mendez-Luna, David; Yepez-Mulia, Lilian

    2017-01-01

    Background: Annona cherimola Miller (Annonaceae) is a medicinal plant frequently recommended in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. Objective: This work was undertaken to obtain information that support the traditional use of A. cherimola, on pharmacological basis using in vitro and computational experiments. Material and Methods: Bioassay-guided fractionation of the ethanol extract of the leaves of A. cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin, and rutin. Results: The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different than metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase (PFOR). Conclusion: Considering that PFOR is a target of metronidazole; kaempferol may be a lead compound for the development of novel antiprotozoal agent. Also, these findings give support to the use of A. cherimola in the traditional medicine from México for the treatment of diarrhea and dysentery. SUMMARY Bioassay-guided fractionation of the ethanol extract of the leaves of Annona cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin and rutin. The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different that metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase. Abbreviations used: PFOR:Pyruvate:ferredoxin oxidoreductase, G: lamblia: Giardia lamblia, E: histolytica: Entamoeba histolytica PMID:28216899

  16. The drug ornidazole inhibits photosynthesis in a different mechanism described for protozoa and anaerobic bacteria.

    Science.gov (United States)

    Marcus, Yehouda; Tal, Noam; Ronen, Mordechai; Carmieli, Raanan; Gurevitz, Michael

    2016-12-01

    Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP(+) reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  17. Effect of tungstate on acetate and ethanol production by the electrosynthetic bacterium Sporomusa ovata

    DEFF Research Database (Denmark)

    Ammam, Fariza; Tremblay, Pier-Luc; Lizak, Dawid Mariusz

    2016-01-01

    successfully converted to their corresponding alcohols 1-propanol and 1-butanol by S. ovata during gas fermentation. Increasing tungstate concentration enhanced conversion efficiency for both propionate and butyrate. Gene expression analysis suggested that tungsten-containing aldehyde ferredoxin...... oxidoreductases (AORs) and a tungsten-containing formate dehydrogenase (FDH) were involved in the improved biosynthesis of acetate, ethanol, 1-propanol, and 1-butanol. AORs and FDH contribute to the fatty acids re-assimilation pathway and the Wood-Ljungdahl pathway, respectively. This study presented here shows...

  18. NMR of proteins (4Fe-4S): structural properties and intramolecular electron transfer; RMN de proteines (4Fe-4S): proprietes structurales et transfert electronique intramoleculaire

    Energy Technology Data Exchange (ETDEWEB)

    Huber, J.G.

    1996-10-17

    NMR started to be applied to Fe-S proteins in the seventies. Its use has recently been enlarged as the problems arising from the paramagnetic polymetallic clusters ware overcome. Applications to [4Fe-4S] are presented herein. The information derived thereof deepens the understanding of the redox properties of these proteins which play a central role in the metabolism of bacterial cells. The secondary structure elements and the overall folding of Chromatium vinosum ferredoxin (Cv Fd) in solution have been established by NMR. The unique features of this sequence have been shown to fold as an {alpha} helix at the C-terminus and as a loop between two cysteines ligand of one cluster: these two parts localize in close proximity from one another. The interaction between nuclear and electronic spins is a source of additional structural information for (4Fe-AS] proteins. The conformation of the cysteine-ligands, as revealed by the Fe-(S{sub {gamma}}-C{sub {beta}}-H{sub {beta}})Cys dihedral angles, is related to the chemical shifts of the signals associated with the protons of these residues. The longitudinal relaxation times of the protons depend on their distance to the cluster. A quantitative relationship has been established and used to show that the solution structure of the high-potential ferredoxin from Cv differs significantly from the crystal structure around Phe-48. Both parameters (chemical shifts and longitudinal relaxation times) give also insight into the electronic and magnetic properties of the [4Fe-4S] clusters. The rate of intramolecular electron transfer between the two [4FE-4S] clusters of ferredoxins has been measured by NMR. It is far slower in the case of Cv Fd than for shorter ferredoxins. The difference may be associated with changes in the magnetic and/or electronic properties of one cluster. The strong paramagnetism of the [4Fe-4S] clusters, which originally limited the applicability of NMR to proteins containing these cofactors, has been proven

  19. AcEST: BP912260 [AcEST

    Lifescience Database Archive (English)

    Full Text Available efinition sp|P17847|NIR_MAIZE Ferredoxin--nitrite reductase, chloroplastic (Fragment) OS=Zea mays Align length 31 Score............................................done Score E Sequences producing signifi...tic (Fragment) OS=Zea mays GN=NIR PE=2 SV=1 Length = 569 Score = 46.2 bits (108), Expect = 9e-05 Identities ...Y+ ++ +GN +ITNL Sbjct: 196 PHEIVDTRPYTNLLSSYVTNNSQGNPTITNL 226 Score = 43.9 bits ...ctase, chloroplastic OS=Arabidopsis thaliana GN=NIR1 PE=1 SV=1 Length = 586 Score = 45.8 bits (107), Expect

  20. Influence of charged microenvironment on redox potential and diffusion coefficient of [Fe4S4(SPh)4](NBu4)2 in DMF and inside CTAB film on electrode surface

    Indian Academy of Sciences (India)

    Raben Ch Roy; Diganta Kumar Das

    2005-11-01

    Redox potential of [Fe4S4(SPh)4]2-/3-, a model of the active centre of ferredoxin, in DMF solution shows a 90-mV positive shift, when the charged microenvironment provided by the surfactant is changed from negative to positive. Inside the positive surfactant film on GC electrode there is a 235-mV positive shift in redox potential compared to that in neutral DMF solution. Diffusion coefficient of the reduced cluster onto the electrode surface is also found to be 102 times greater in the presence of negative surfactant compared to that in positive surfactant.

  1. A proteome investigation of roxarsone degradation by Alkaliphilus oremlandii strain OhILAs.

    Science.gov (United States)

    Chovanec, Peter; Stolz, John F; Basu, Partha

    2010-02-01

    Clostridial species predominate in both chicken gastrointestinal tract as well as litter where the organoarsenical roxarsone (3-nitro 4-hydroxybenzenearsonic acid) is anaerobically transformed releasing the more recognized toxic inorganic arsenic. 2D-gel electrophoresis and mass spectrometry were used to evaluate the changes in protein expression of Alkaliphilus oremlandii in response to different growth conditions (e.g., terminal electron acceptors) in order to explore the mechanism of microbial biotransformation of roxarsone. Aldehyde ferredoxin oxidoreductase, the enzyme that belongs to the xanthine oxidase family of molybdoenzymes was significantly overexpressed in the presence of roxarsone suggesting a role in the anaerobic metabolism of this substituted nitrophenol.

  2. Protein-based multi-bit biomemory device consisting of various metalloproteins on self-assembled 11-MUA layer.

    Science.gov (United States)

    Lee, Taek; Min, Junhong; Lee, Jin-Ho; Choi, Jeong-Woo

    2011-01-01

    A multi-bit biomemory device was devised by introducing 4 different metalloproteins (azurin, cytochrome c, ferredoxin, myoglobin) to an electronic device using 11-MUA (11-mercapto-undecanoic acid) as the chemical linker. The immobilization of the 4 different self-assembled protein layers on a Au substrate via 11-MUA were confirmed by Raman spectroscopy and atomic force microscopy (AFM). The redox properties of these 4 different protein layers immobilized onto Au surface were assessed by cyclic voltammetry (CV). In addition, their memory functions were verified by chronoamperometry (CA). Based on these results, we demonstrated that a multi-bit biomemory concept could be realized using various metalloproteins as active materials.

  3. Assignment of Individual Metal Redox States in a Metalloprotein By Crystallographic Refinement at Multiple X-Ray Wavelengths

    Energy Technology Data Exchange (ETDEWEB)

    Einsle, O.; Andrade, S.L.A.; Dobbek, H.; Meyer, J.; Rees, D.C.; /Gottingen U. /Bayreuth U. /DRDC, Grenoble /Caltech

    2007-07-09

    A method is presented to derive anomalous scattering contributions for individual atoms within a protein crystal by collecting several sets of diffraction data at energies spread along an X-ray absorption edge of the element in question. The method has been applied to a [2Fe:2S] ferredoxin model system with localized charges in the reduced state of the iron-sulfur cluster. The analysis shows that upon reduction the electron resides at the iron atom closer to the protein surface. The technique should be sufficiently sensitive for more complex clusters with noninteger redox states and is generally applicable given that crystals are available.

  4. Cofactor engineering for advancing chemical biotechnology.

    Science.gov (United States)

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.

  5. Recycling of carbon dioxide and acetate as lactic acid by the hydrogen-producing bacterium Thermotoga neapolitana.

    Science.gov (United States)

    d'Ippolito, Giuliana; Dipasquale, Laura; Fontana, Angelo

    2014-09-01

    The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of sugars. Under capnophilic (carbon dioxide requiring) conditions, the process is preferentially associated with the production of lactic acid, which, as shown herein, is synthesized by reductive carboxylation of acetyl coenzyme A. The enzymatic coupling is dependent on the carbon dioxide stimulated activity of heterotetrameric pyruvate:ferredoxin oxidoreductase. Under the same culture conditions, T. neapolitana also operates the unfavorable synthesis of lactic acid from an exogenous acetate supply. This process, which requires carbon dioxide (or carbonate) and an unknown electron donor, allows for the conversion of carbon dioxide into added-value chemicals without biomass deconstruction.

  6. Improving Delivery of Photosynthetic Reducing Power to Cytochrome P450s

    DEFF Research Database (Denmark)

    Mellor, Silas Busck

    Oxygenic photosynthesis allows plants, algae and cyanobacteria to depend primarily on readily available light, carbon dioxide and water, in turn generating the chemical energy required for complex metabolism. This makes photosynthetic organisms ideal hosts for metabolic engineering aimed...... at sustainable production of high-value and commodity products. Cytochrome P450 enzymes play key roles in the biosynthesis of important natural products. The electron carrier ferredoxin can couple P450s non-natively to photosynthetic electron supply, providing ample reducing power for catalysis. However...

  7. Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

    OpenAIRE

    Madden, M S; Kindon, N D; Ludden, P W; Shah, V K

    1990-01-01

    In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analogs allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (1R,2S)-threo- and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrat...

  8. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima

    DEFF Research Database (Denmark)

    Worning, Peder; Jensen, Lars Juhl; Nelson, K. E.

    2000-01-01

    The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters......, which brings independent evidence for the lateral gene transfer in the genome of T.maritima, The structural analysis relates the Archaea-like DNA sequences to the genome of Pyrococcus horikoshii. Analysis of 24 complete genomic DNA sequences shows different periodicity patterns for organisms...... of different origin, The typical genomic periodicity for Bacteria is 11 bp whilst it is 10 bp for Archaea, Eukaryotes have more complex spectra but the dominant period in the yeast Saccharomyces cerevisiae is 10.2 bp. These periodicities are most likely reflective of differences in chromatin structure....

  9. Orientation of growing crystals of Co- or Gd-containing L-threonine dehydrogenase by magnetic fields

    Science.gov (United States)

    Maki, Syou; Ishikawa, Kazuhiko; Ataka, Mitsuo

    2009-12-01

    L-Threonine dehydrogenase from Pyrococcus horikoshii (TDH) is a water-soluble metalloenzyme, the molecular structure of which has been unknown until recently. The Zn 2+ ion in the native TDH, prepared as a recombinant protein, is replaced artificially with Co 2+, Ni 2+ or Gd 3+. These samples are crystallized in homogeneous magnetic fields of 2-10 T. Half of the Co- or Gd-substituted crystals show magnetic orientation in a field of 2 T at 278 K whereas the crystals of the native TDH require a 4 T magnetic field for half orientation. The sensitivity to magnetic orientation can thus be increased by metal substitution. On the other hand, we cannot assign clear changes in the size, number, and quality of the native and metal-substituted crystals with and without the presence of the magnetic field.

  10. Investigating the mechanism of ADP-forming acetyl-CoA synthetase from the protozoan parasite Entamoeba histolytica.

    Science.gov (United States)

    Jones, Cheryl P; Khan, Kirin; Ingram-Smith, Cheryl

    2017-02-01

    ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. The related succinyl-CoA synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II. © 2017 Federation of European Biochemical Societies.

  11. AcEST: DK959559 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TST39A01NGRL0005_A12 632 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0005_A12. 5' end sequence. DK95...9559 CL61Contig1 Show DK959559 Clone id TST39A01NGRL0005_A12 Library TST39 Length 63...2 Definition Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0005_A12. 5' end sequence. Accession DK95955...pped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402. Query= DK9595...caldoc... 91 3e-18 sp|Q5JDH1|RL23_PYRKO 50S ribosomal protein L23P OS=Pyrococcus ko... 86 2e-16 sp|O74095

  12. Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450

    Directory of Open Access Journals (Sweden)

    Choi Kwon-Young

    2012-06-01

    Full Text Available Abstract A cytochrome P450 (CYP enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH, responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8 μM-1 min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6% was 5.2-fold higher than that of the wild-type.

  13. Biotransformation of Isoflavone Using Enzymatic Reactions

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    Changhyun Roh

    2013-03-01

    Full Text Available The roles of cytochrome P450 monooxygenases (CYPs from Streptomyces spp. which are called the “treasure islands” for natural products for medicine and antibiotics are not well understood. Substrate specificity studies on CYPs may give a solution for elucidation of their roles. Based on homology sequence information, the CYP105D7 of a soluble cytochrome P450 known as heme protein from Streptomyces avermitilis MA4680 was expressed using the T7 promoter of the bacterial expression vector pET24ma, over-expressed in Escherichia coli system and characterized. An engineered whole cell system for daidzein hydroxylation was constructed using an exogenous electron transport system from ferredoxin reductase (PdR and ferredoxin (Pdx. Also, an in vitro reaction study showed the purified CYP105D7 enzyme, using NADH-dependent-reducing equivalents of a redox partner from Pseudomonas putida, hydroxylated daidzein at the 3' position of the B ring to produce 7,3,'4' trihydroxyisoflavone. The hydroxylated position was confirmed by GC-MS analysis. The turnover number of the enzyme was 0.69 μmol 7,3,'4'-trihydroxyisoflavone produced per μmol P450 per min. This enzyme CYP105D7 represents a novel type of 3'-hydroxylase for daidzein hydroxylation. A P450 inhibitor such as coumarin significantly (ca.98% inhibited the daidzein hydroxylation activity.

  14. Metabolism of lactose by Clostridium thermolacticum growing in continuous culture.

    Science.gov (United States)

    Collet, Christophe; Girbal, Laurence; Péringer, Paul; Schwitzguébel, Jean-Paul; Soucaille, Philippe

    2006-06-01

    The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l(-1)) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h(-1). Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h(-1). The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganisms.

  15. Anaerobic respiration: In vitro efficacy of Nitazoxanide against mitochondriate Acanthamoeba castellanii of the T4 genotype.

    Science.gov (United States)

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Farooq, Maria; Khan, Naveed Ahmed

    2015-10-01

    Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba.

  16. Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser.

    Science.gov (United States)

    Coe, Jesse; Kupitz, Christopher; Basu, Shibom; Conrad, Chelsie E; Roy-Chowdhury, Shatabdi; Fromme, Raimund; Fromme, Petra

    2015-01-01

    Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question. © 2015 Elsevier Inc. All rights reserved.

  17. Sunlight mediated synthesis of silver nanoparticles using redox phytoprotein and their application in catalysis and colorimetric mercury sensing.

    Science.gov (United States)

    Ahmed, Khan Behlol Ayaz; Senthilnathan, Rajendran; Megarajan, Sengan; Anbazhagan, Veerappan

    2015-10-01

    Owing to the benign nature, plant extracts mediated green synthesis of metal nanoparticles (NPs) is rapidly expanding. In this study, we demonstrated the successful green synthesis of silver nanoparticles (AgNPs) by utilizing natural sunlight and redox protein complex composed of ferredoxin-NADP(+) reductase (FNR) and ferredoxin (FD). The capping and stabilization of the AgNPs by the redox protein was confirmed by Fourier transform infrared spectroscopy. Light and redox protein is the prerequisite factor for the formation of AgNPs. The obtained result shows that the photo generated free radicals by the redox protein is responsible for the reduction of Ag(+) to Ag(0). Transmission electron microscopy revealed the formation of spherical AgNPs with size ranging from 10 to 15 nm. As-prepared AgNPs exhibit excellent catalytic activity toward the degradation of hazardous organic dyes, such as methylene blue, methyl orange and methyl red. These bio-inspired AgNPs is highly sensitive and selective in sensing hazardous mercury ions in the water at micromolar concentration. In addition, FNR/FD extract stabilized AgNPs showed good antimicrobial activity against gram positive and gram negative bacteria.

  18. Genes involved in the degradation of ether fuels by bacteria of the Mycobacterium / Rhodococcus group; Genes impliques dans la degradation des ethers carburants par des bacteries du groupe Mycobacterium/Rhodococcus

    Energy Technology Data Exchange (ETDEWEB)

    Beguin, P.; Chauvaux, S.; Miras, I. [Institut Pasteur, Unite Microbiologie et Environnement, URA 2172 CNRS, 75 - Paris (France); Francois, A.; Fayolle, F.; Monot, F. [Institut Francais du Petrole (IFP), Dept. Biotechnologie et Chimie de la Biomasse, 92 - Rueil-Malmaison (France)

    2003-08-01

    A cluster of genes encoding a cytochrome P-450 monooxygenase system involved in the utilisation of ethyl tert-butyl ether (ETBE) was cloned in Rhodococcus ruber IFP 2001. This cluster includes ethR, a putative regulator gene of the araC/xylS family; ethA, encoding a ferredoxin reductase; ethB, encoding a cytochrome P-450, ethC, encoding a ferredoxin; and ethD, which is required for the function of the monooxygenase system, but whose exact role is unknown. The ethRABCD cluster is flanked on either side by two identical copies of a class II transposon, which explains that it is lost at high frequency by homologous recombination when the strain is grown under non selective conditions. Two other, highly conserved clusters of eth genes were detected in the ETBE-utilizing strains Rhodococcus zopfii IFP 2005 and Mycobacterium sp. IFP 2009. In all cases, the eth locus is inserted in a different genomic context, suggesting that it may be transferred horizontally between different species and inserted at different sites in the genome. In addition, in R. zopfii IFP 2005, the downstream copy of the transposon carries a 117-bp (base pairs) deletion; in Mycobacterium sp. IFP 2009, the upstream copy is absent and the downstream copy is inserted 2771 bp closer to the ethRABCD cluster. (authors)

  19. Examination of the ligand-binding and enzymatic properties of a bilin-binding protein from the poisonous caterpillar Lonomia obliqua.

    Directory of Open Access Journals (Sweden)

    Ana B G Veiga

    Full Text Available The bilin-binding proteins (BBP from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1:1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH(+-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.

  20. Examination of the ligand-binding and enzymatic properties of a bilin-binding protein from the poisonous caterpillar Lonomia obliqua.

    Science.gov (United States)

    Veiga, Ana B G; Ribeiro, José M C; Francischetti, Ivo M B; Xu, Xueqing; Guimarães, Jorge A; Andersen, John F

    2014-01-01

    The bilin-binding proteins (BBP) from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1:1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH(+)-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II) state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.

  1. A novel nematode effector suppresses plant immunity by activating host reactive oxygen species-scavenging system.

    Science.gov (United States)

    Lin, Borong; Zhuo, Kan; Chen, Shiyan; Hu, Lili; Sun, Longhua; Wang, Xiaohong; Zhang, Lian-Hui; Liao, Jinling

    2016-02-01

    Evidence is emerging that plant-parasitic nematodes can secrete effectors to interfere with the host immune response, but it remains unknown how these effectors can conquer host immune responses. Here, we depict a novel effector, MjTTL5, that could suppress plant immune response. Immunolocalization and transcriptional analyses showed that MjTTL5 is expressed specifically within the subventral gland of Meloidogyne javanica and up-regulated in the early parasitic stage of the nematode. Transgenic Arabidopsis lines expressing MjTTL5 were significantly more susceptible to M. javanica infection than wild-type plants, and vice versa, in planta silencing of MjTTL5 substantially increased plant resistance to M. javanica. Yeast two-hybrid, coimmunoprecipitation and bimolecular fluorescent complementation assays showed that MjTTL5 interacts specifically with Arabidopsis ferredoxin : thioredoxin reductase catalytic subunit (AtFTRc), a key component of host antioxidant system. The expression of AtFTRc is induced by the infection of M. javanica. Interaction between AtFTRc and MjTTL could drastically increase host reactive oxygen species-scavenging activity, and result in suppression of plant basal defenses and attenuation of host resistance to the nematode infection. Our results demonstrate that the host ferredoxin : thioredoxin system can be exploited cunningly by M. javanica, revealing a novel mechanism utilized by plant-parasitic nematodes to subjugate plant innate immunity and thereby promoting parasitism. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  2. Patchwork assembly of nag-like nitroarene dioxygenase genes and the 3-chlorocatechol degradation cluster for evolution of the 2-chloronitrobenzene catabolism pathway in Pseudomonas stutzeri ZWLR2-1.

    Science.gov (United States)

    Liu, Hong; Wang, Shu-Jun; Zhang, Jun-Jie; Dai, Hui; Tang, Huiru; Zhou, Ning-Yi

    2011-07-01

    Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.

  3. Engineering the pentose phosphate pathway to improve hydrogen yield in recombinant Escherichia coli.

    Science.gov (United States)

    Kim, Young Mi; Cho, Han-Saem; Jung, Gyoo Yeol; Park, Jong Moon

    2011-12-01

    Among various routes for the biological hydrogen production, the NAD(P)H-dependent pentose phosphate (PP) pathway is the most efficient for the dark fermentation. Few studies, however, have focused on the glucose-6-phosphate 1-dehydrogenase, encoded by zwf, as a key enzyme activating the PP pathway. Although the gluconeogenic activity is essential for activating the PP pathway, it is difficult to enhance the NADPH production by regulating only this activity because the gluconeogenesis is robust and highly sensitive to concentrations of glucose and AMP inside the cell. In this study, the FBPase II (encoded by glpX), a regulation-insensitive enzyme in the gluconeogenic pathway, was activated. Physiological studies of several recombinant, ferredoxin-dependent hydrogenase system-containing Escherichia coli BL21(DE3) strains showed that overexpression of glpX alone could increase the hydrogen yield by 1.48-fold compared to a strain with the ferredoxin-dependent hydrogenase system only; the co-overexpression of glpX with zwf increased the hydrogen yield further to 2.32-fold. These results indicate that activation of the PP pathway by glpX overexpression-enhanced gluconeogenic flux is crucial for the increase of NAD(P)H-dependent hydrogen production in E. coli BL21(DE3).

  4. Analysis of multi-domain hypothetical proteins containing iron-sulphur clusters and fad ligands reveal rieske dioxygenase activity suggesting their plausible roles in bioremediation.

    Science.gov (United States)

    Sathyanarayanan, Nitish; Nagendra, Holenarasipur Gundurao

    2012-01-01

    'Conserved hypothetical' proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a 'complete' understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 40 proteins with N-terminus 2Fe-2S domain and C-terminus FNR domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicating their plausible roles in degradation of aromatic moieties.

  5. Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

    Directory of Open Access Journals (Sweden)

    Masaki Ihara

    Full Text Available The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I. Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH. We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd, and ferredoxin-NADP(+-reductase (FNR. Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

  6. Properties of immobilized flavodoxin from Peptostreptococcus elsdenii. An affinity ligand for the purification of riboflavin 5'-phosphate (FMN) and its analogues.

    Science.gov (United States)

    Mayhew, S G; Strating, M J

    1975-11-15

    The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr-activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin-free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively. Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (+/- 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N-10 side-chain of 5 carbon atoms and a C-5' phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12-15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months.

  7. Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB

    Directory of Open Access Journals (Sweden)

    Pratick Khara

    2014-01-01

    Full Text Available Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

  8. Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions.

    Science.gov (United States)

    Dani, Diksha Narhar; Sainis, Jayashree Krishna

    2005-05-15

    A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000 x g, followed by 90,000 x g and finally at 150,000 x g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000 x g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000 x g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO2 fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.

  9. Exploring the electron transfer pathway in the oxidation of avermectin by CYP107Z13 in Streptomyces ahygroscopicus ZB01.

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    Mei Li

    Full Text Available Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin. CYP107Z13 is responsible for this site-specific oxidation in ZB01. In the present study, we explored the electron transfer pathway in oxidation of avermectin by CYP107Z13 in ZB01. A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin. To clarify whether FdR18 and FdR28 participate in the electron transfer during avermectin oxidation by CYP107Z13, two whole-cell biocatalytic systems were designed in E. coli BL21 (DE3, with one co-expressing CYP107Z13, Fd68 and FdR18 and the other co-expressing CYP107Z13, Fd68 and FdR28. Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin. Thus, we propose an electron transfer pathway NADH→FdR18/FdR28→Fd68→CYP107Z13 for oxidation of avermectin to form 4″-oxo-avermectin in ZB01.

  10. Mitochondria as a Possible Place for Initial Stages of Steroid Biosynthesis in Plants

    Directory of Open Access Journals (Sweden)

    Elena K. Shematorova

    2014-12-01

    Full Text Available With the aim of thorough comparison of steroidogenic systems of plants and animals, transgenic plants of Solanaceae family expressing CYP11A1 cDNA encoding cytochrome P450SCC of mammalian mitochondria were further analysed. Positive effect of CYP11A1 on resistance of the transgenic tobacco plants to the infection by fungal phytopathogene Botrytis cinerea was for the first time detected. Subtle changes in mitochondria of the transgenic Nicotiana tabacum plants expressing mammalian CYP11A1 cDNA were demonstrated by transmissive electron microscopy. The main components of the electron transfer chain of plant mitochondria were for the first time cloned and characterized. It was established that plants from the Solanacea family (tomato, tobacco and potato contain two different genes with similar exon-intron structures (all contain 8 exons encoding mitochondrial type ferredoxins (MFDX, and one gene for mitochondrial ferredoxin reductase (MFDXR. The results obtained point out on profound relatedness of electron transfer chains of P450-dependent monooxygenases in mammalian and plant mitochondria and support our previous findings about functional compatability of steroidogenic systems of Plantae and Animalia.

  11. Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

    Science.gov (United States)

    Kelly, Ciarán L.; Pinske, Constanze; Murphy, Bonnie J.; Parkin, Alison; Armstrong, Fraser; Palmer, Tracy; Sargent, Frank

    2015-01-01

    Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme. PMID:26839796

  12. Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ciarán L. Kelly

    2015-12-01

    Full Text Available Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.

  13. Peculiar citric acid cycle of hydrothermal vent chemolithoautotroph Hydrogenovibrio crunogenus, and insights into carbon metabolism by obligate autotrophs.

    Science.gov (United States)

    Quasem, Ishtiaque; Achille, Alexandra N; Caddick, Brittany A; Carter, Travis A; Daniels, Camille; Delaney, Jennifer A; Delic, Vedad; Denton, Kimberly A; Duran, Martina C; Fatica, Marianne K; Ference, Christopher M; Galkiewicz, Julie P; Garcia, Ana M; Hendrick, Jacqueline D; Horton, Steven A; Kun, Mey S; Koch, Phoebe W; Lee, Tien Min; McCabe, Christie R; McHale, Sean; McDaniel, Lauren D; Menning, Damian M; Menning, Kristy J; Mirzaei-Souderjani, Hamed; Mostajabian, Salina; Nicholson, David A; Nugent, Courtney K; Osman, Nicholas P; Pappas, Desiree I; Rocha, Andrea M; Rosario, Karyna; Rubelmann, Haydn; Schwartz, Julie A; Seeley, Kent W; Staley, Christopher M; Wallace, Elizabeth M; Wong, Terianne M; Zielinski, Brian L; Hanson, Thomas E; Scott, Kathleen M

    2017-08-01

    The genome sequence of the obligate chemolithoautotroph Hydrogenovibrio crunogenus paradoxically predicts a complete oxidative citric acid cycle (CAC). This prediction was tested by multiple approaches including whole cell carbon assimilation to verify obligate autotrophy, phylogenetic analysis of CAC enzyme sequences and enzyme assays. Hydrogenovibrio crunogenus did not assimilate any of the organic compounds provided (acetate, succinate, glucose, yeast extract, tryptone). Enzyme activities confirmed that its CAC is mostly uncoupled from the NADH pool. 2-Oxoglutarate:ferredoxin oxidoreductase activity is absent, though pyruvate:ferredoxin oxidoreductase is present, indicating that sequence-based predictions of substrate for this oxidoreductase were incorrect, and that H. crunogenus may have an incomplete CAC. Though the H. crunogenus CAC genes encode uncommon enzymes, the taxonomic distribution of their top matches suggests that they were not horizontally acquired. Comparison of H. crunogenus CAC genes to those present in other 'Proteobacteria' reveals that H. crunogenus and other obligate autotrophs lack the functional redundancy for the steps of the CAC typical for facultative autotrophs and heterotrophs, providing another possible mechanism for obligate autotrophy. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Photoacclimation in the Red Alga Porphyridium cruentum1

    Science.gov (United States)

    Cunningham, Francis X.; Vonshak, Avigad; Gantt, Elisabeth

    1992-01-01

    Acclimation of the photosynthetic apparatus to changes in the light environment was studied in the unicellular red alga Porphyridium cruentum (American Type Culture Collection No. 50161). Absolute or relative amounts of four photosynthetic enzymes and electron carriers were measured, and the data were compared with earlier observations on light-harvesting components (F.X. Cunningham, Jr., R.J. Dennenberg, L. Mustárdy, P.A. Jursinic, E. Gantt [1989] Plant Physiol 91: 1179-1187; F.X. Cunningham, Jr., R.J. Dennenberg, P.A. Jursinic, E. Gantt [1990] Plant Physiol 93: 888-895) and with measurements of photosynthetic capacity. Pmax, the light-saturated rate of photosynthesis on a chlorophyll (Chl) basis, increased more than 4-fold with increase in growth irradiance from 6 to 280 μeinsteins·m−2·s−1. Amounts of ferredoxin-NADP+ reductase, ribulose-1,5-bisphosphate carboxylase, and cytochrome f increased in parallel with Pmax, whereas numbers of the light-harvesting complexes (photosystem [PS] I, PSII, and phycobilisomes) changed little, and ATP synthase increased 7-fold relative to Chl. The calculated minimal turnover time for PSII under the highest irradiance, 5 ms, was thus about 4-fold faster than that calculated for cultures grown under the lowest irradiance (19 ms). A change in the spectral composition of the growth light (irradiance kept constant at 15 μeinsteins·m−2·s−1) from green (absorbed predominantly by the phycobilisome antenna of PSII) to red (absorbed primarily by the Chl antenna of PSI) had little effect on the amounts of ribulose-1,5-bisphosphate carboxylase, ATP synthase, and phycobilisomes on a Chl, protein, or thylakoid area basis. However, the number of PSI centers declined by 40%, cytochrome f increased by 40%, and both PSII and ferredoxin-NADP+ reductase increased approximately 3-fold on a thylakoid area basis. The substantial increase in ferredoxin-NADP+ reductase under PSI light is inconsistent with a PSI-mediated reduction of NADP

  15. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    Directory of Open Access Journals (Sweden)

    Rydzak Thomas

    2012-09-01

    Full Text Available Abstract Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative

  16. The bifunctional aldehyde-alcohol dehydrogenase controls ethanol and acetate production in Entamoeba histolytica under aerobic conditions.

    Science.gov (United States)

    Pineda, Erika; Encalada, Rusely; Olivos-García, Alfonso; Néquiz, Mario; Moreno-Sánchez, Rafael; Saavedra, Emma

    2013-01-16

    By applying metabolic control analysis and inhibitor titration we determined the degree of control (flux control coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde-alcohol dehydrogenase (ADHE) over the fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The flux-control coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and -0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant flux-control on the carbon end-product formation of amoebas subjected to aerobic conditions. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Valeria R Turowski

    Full Text Available Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR and ferredoxin (Fd, two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

  18. Origins of prokaryotes, eukaryotes, mitochondria, and chloroplasts

    Science.gov (United States)

    Schwartz, R. M.; Dayhoff, M. O.

    1978-01-01

    A computer branching model is used to analyze cellular evolution. Attention is given to certain key amino acids and nucleotide residues (ferredoxin, 5s ribosomal RNA, and c-type cytochromes) because of their commonality over a wide variety of cell types. Each amino acid or nucleotide residue is a sequence in an inherited biological trait; and the branching method is employed to align sequences so that changes reflect substitution of one residue for another. Based on the computer analysis, the symbiotic theory of cellular evolution is considered the most probable. This theory holds that organelles, e.g., mitochondria and chloroplasts invaded larger bodies, e.g., bacteria, and combined functions to form eucaryotic cells.

  19. Carbon dioxide fixation and photoevolution of hydrogen and oxygen in a mutant of Chlamydomonas lacking Photosystem I

    Energy Technology Data Exchange (ETDEWEB)

    Greenbaum, E.; Lee, J.W.; Tevault, C.V. [Oak Ridge National Lab., TN (United States)] [and others

    1995-09-01

    Sustained photoassimilation of atmospheric CO{sub 2} and simultaneous photoevolution of molecular hydrogen and oxygen has been observed in a Photosystem I deficient mutant B4 of Chlamydomonas reinhardtii that contains only Photosystem II. The data indicate that Photosystem II alone is capable of spanning the potential difference between water oxidation/oxygen evolution and ferredoxin reduction. The rates of both CO{sub 2} fixation and hydrogen and oxygen evolution are similar in the mutant to that of the wild-type C. reinhardtii 137c containing both photosystems. The wild-type had stable photosynthetic activity, measured as CO{sub 2} fixation, under both air and anaerobic conditions, while the mutant was stable only under anaerobic conditions. The results are discussed in terms of the fundamental mechanisms and energetics of photosynthesis and possible implications for the evolution of oxygenic photosynthesis.

  20. AIFM1 mutation presenting with fatal encephalomyopathy and mitochondrial disease in an infant

    Science.gov (United States)

    Morton, Sarah U.; Prabhu, Sanjay P.; Lidov, Hart G.W.; Shi, Jiahai; Anselm, Irina; Brownstein, Catherine A.; Bainbridge, Matthew N.; Beggs, Alan H.; Vargas, Sara O.; Agrawal, Pankaj B.

    2017-01-01

    Apoptosis-inducing factor mitochondrion-associated 1 (AIFM1), encoded by the gene AIFM1, has roles in electron transport, apoptosis, ferredoxin metabolism, reactive oxygen species generation, and immune system regulation. Here we describe a patient with a novel AIFM1 variant presenting unusually early in life with mitochondrial disease, rapid deterioration, and death. Autopsy, at the age of 4 mo, revealed features of mitochondrial encephalopathy, myopathy, and involvement of peripheral nerves with axonal degeneration. In addition, there was microvesicular steatosis in the liver, thymic noninvolution, follicular bronchiolitis, and pulmonary arterial medial hypertrophy. This report adds to the clinical and pathological spectrum of disease related to AIFM1 mutations and provides insights into the role of AIFM1 in cellular function. PMID:28299359

  1. Identification and Characterization of Two Functionally Unknown Genes Involved in Butanol Tolerance of Clostridium acetobutylicum

    Science.gov (United States)

    Jia, Kaizhi; Zhang, Yanping; Li, Yin

    2012-01-01

    Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance. PMID:22768047

  2. Identification and characterization of two functionally unknown genes involved in butanol tolerance of Clostridium acetobutylicum.

    Directory of Open Access Journals (Sweden)

    Kaizhi Jia

    Full Text Available Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518 with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance.

  3. Metabolic flux network analysis of fermentative hydrogen production: using Clostridium tyrobutyricum as an example.

    Science.gov (United States)

    Cheng, Hai-Hsuan; Whang, Liang-Ming; Lin, Che-An; Liu, I-Chun; Wu, Chao-Wei

    2013-08-01

    This study applies metabolic flux network analysis (MFA) to evaluate the metabolic flux of fermentative hydrogen production (FHP) with the use of Clostridium tyrobutyricum fed with either glucose or lactate/acetate as substrates. The MFA results suggest that hydraulic retention time (HRT) presents significant impact on hydrogen production from glucose. At HRT between 4 and 18 h, increase of HRT increased hydrogen production but decreased lactate production, while at HRT below 4 h decrease of HRT increased hydrogen production but decreased lactate production. The flux for lactate, butyrate and acetate seemed to affect H₂ production, due presumably to their impacts on the balance of NADH, ferredoxin and ATP. It is suggested that the MFA can be a useful tool to provide valuable information for optimization and design of the fermentative hydrogen production process.

  4. The genetic basis for bacterial mercury methylation.

    Science.gov (United States)

    Parks, Jerry M; Johs, Alexander; Podar, Mircea; Bridou, Romain; Hurt, Richard A; Smith, Steven D; Tomanicek, Stephen J; Qian, Yun; Brown, Steven D; Brandt, Craig C; Palumbo, Anthony V; Smith, Jeremy C; Wall, Judy D; Elias, Dwayne A; Liang, Liyuan

    2013-03-15

    Methylmercury is a potent neurotoxin produced in natural environments from inorganic mercury by anaerobic bacteria. However, until now the genes and proteins involved have remained unidentified. Here, we report a two-gene cluster, hgcA and hgcB, required for mercury methylation by Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. In either bacterium, deletion of hgcA, hgcB, or both genes abolishes mercury methylation. The genes encode a putative corrinoid protein, HgcA, and a 2[4Fe-4S] ferredoxin, HgcB, consistent with roles as a methyl carrier and an electron donor required for corrinoid cofactor reduction, respectively. Among bacteria and archaea with sequenced genomes, gene orthologs are present in confirmed methylators but absent in nonmethylators, suggesting a common mercury methylation pathway in all methylating bacteria and archaea sequenced to date.

  5. Crystal structure and biochemical features of dye-decolorizing peroxidase YfeX from Escherichia coli O157 Asp(143) and Arg(232) play divergent roles toward different substrates.

    Science.gov (United States)

    Liu, Xiuhua; Yuan, Zenglin; Wang, Jiaxu; Cui, Yaqi; Liu, Shuang; Ma, Yinliang; Gu, Lichuan; Xu, Sujuan

    2017-02-26

    YfeX from Escherichia coli O157 is a bacterial dye-decolorizing peroxidase that represents both dye-decoloring activity and typical peroxidase activity. We reported the crystal structure of YfeX bound to heme at 2.09 Å resolution. The YfeX monomer resembles a ferredoxin-like fold and contains two domains. The three conserved residues surrounding the heme group are His(215), Asp(143) and Arg(232). His(215) functions as the proximal axial ligand of the heme iron atom. Biochemical data show that the catalytic significance of the conserved Asp(143) and Arg(232) depends on the substrate types and that YfeX may adopt various catalytic mechanisms toward divergent substrates. In addition, it is observed that an access tunnel spans from the protein molecular surface to the heme distal region, it serves as the passageway for the entrance and binding of the H2O2.

  6. Identification of a gene cluster associated with triclosan catabolism.

    Science.gov (United States)

    Kagle, Jeanne M; Paxson, Clayton; Johnstone, Precious; Hay, Anthony G

    2015-06-01

    Aerobic degradation of bis-aryl ethers like the antimicrobial triclosan typically proceeds through oxygenase-dependent catabolic pathways. Although several studies have reported on bacteria capable of degrading triclosan aerobically, there are no reports describing the genes responsible for this process. In this study, a gene encoding the large subunit of a putative triclosan oxygenase, designated tcsA was identified in a triclosan-degrading fosmid clone from a DNA library of Sphingomonas sp. RD1. Consistent with tcsA's similarity to two-part dioxygenases, a putative FMN-dependent ferredoxin reductase, designated tcsB was found immediately downstream of tcsA. Both tcsAB were found in the midst of a putative chlorocatechol degradation operon. We show that RD1 produces hydroxytriclosan and chlorocatechols during triclosan degradation and that tcsA is induced by triclosan. This is the first study to report on the genetics of triclosan degradation.

  7. Novel Insight into the Genetic Context of the cadAB Genes from a 4-chloro-2-methylphenoxyacetic Acid-Degrading Sphingomonas

    DEFF Research Database (Denmark)

    Nielsen, Tue Kjærgaard; Xu, Zhuofei; Gözdereliler, Erkin

    2013-01-01

    of IS3 elements. The canonical tfdA alpha-gene of group III 2,4-D degraders, encoding the first step in degradation of 2,4-D and related compounds, was not present in the chromosomal contigs. However, the alternative cadAB genes, also providing the initial degradation step, were found in Tn6228, along...... with the 2,4-D-degradation-associated genes tfdBCDEFKR and cadR. Putative reductase and ferredoxin genes cadCD of Rieske non-heme iron oxygenases were also present in close proximity to cadAB, suggesting that these might have an unknown role in the initial degradation reaction. Parts of the composite...

  8. The composition of upstream open reading frames (uORF) in four genes from Trypanosoma cruzi typical strains.

    Science.gov (United States)

    Jaeger, Lauren Hubert; Brandão, Adeilton

    2011-10-01

    Upstream open reading frames (uORF) are small open reading frames located in the 5' untranslated region (5' utr) of a mature mRNA. We analysed in four strains representing the Trypanosoma cruzi groups Tc I, Tc II, Tc IV and Tc VI the uORF present in 5' utr sequences of four genes: P-type H+-ATPase 1, DEAD/H RNA helicase, casein kinase 1.1 and ferredoxin-NADP+ reductase. A segment in the 5' utr at each of these genes encompassing one or more uORF was PCR amplified and sequenced. An analysis of these sequences reveals that the uORF in T. cruzi show minor variations; however, these nucleotide substitutions mirror the divergence of T. cruzi strains into major groups.

  9. Evolutionary connections of biological kingdoms based on protein and nucleic acid sequence evidence

    Science.gov (United States)

    Dayhoff, M. O.

    1983-01-01

    Prokaryotic and eukaryotic evolutionary trees are developed from protein and nucleic-acid sequences by the methods of numerical taxonomy. Trees are presented for bacterial ferredoxins, 5S ribosomal RNA, c-type cytochromes , cytochromes c2 and c', and 5.8S ribosomal RNA; the implications for early evolution are discussed; and a composite tree showing the branching of the anaerobes, aerobes, archaebacteria, and eukaryotes is shown. Single lines are found for all oxygen-evolving photosynthetic forms and for the salt-loving and high-temperature forms of archaebacteria. It is argued that the eukaryote mitochondria, chloroplasts, and cytoplasmic host material are descended from free-living prokaryotes that formed symbiotic associations, with more than one symbiotic event involved in the evolution of each organelle.

  10. Novel insight into the genetic context of the cadAB genes from a 4-chloro-2-methylphenoxyacetic acid-degrading Sphingomonas

    DEFF Research Database (Denmark)

    Nielsen, Tue Kjærgaard; Xu, Zhuofei; Gozdereliler, Erkin;

    2013-01-01

    of IS3 elements. The canonical tfdA alpha-gene of group III 2,4-D degraders, encoding the first step in degradation of 2,4-D and related compounds, was not present in the chromosomal contigs. However, the alternative cadAB genes, also providing the initial degradation step, were found in Tn6228, along...... with the 2,4-D-degradation-associated genes tfdBCDEFKR and cadR. Putative reductase and ferredoxin genes cadCD of Rieske non-heme iron oxygenases were also present in close proximity to cadAB, suggesting that these might have an unknown role in the initial degradation reaction. Parts of the composite...

  11. HHrep: de novo protein repeat detection and the origin of TIM barrels.

    Science.gov (United States)

    Söding, Johannes; Remmert, Michael; Biegert, Andreas

    2006-07-01

    HHrep is a web server for the de novo identification of repeats in protein sequences, which is based on the pairwise comparison of profile hidden Markov models (HMMs). Its main strength is its sensitivity, allowing it to detect highly divergent repeat units in protein sequences whose repeats could as yet only be detected from their structures. Examples include sequences with beta-propellor fold, ferredoxin-like fold, double psi barrels or (betaalpha)8 (TIM) barrels. We illustrate this with proteins from four superfamilies of TIM barrels by revealing a clear 4- and 8-fold symmetry, which we detect solely from their sequences. This symmetry might be the trace of an ancient origin through duplication of a betaalphabetaalpha or betaalpha unit. HHrep can be accessed at http://hhrep.tuebingen.mpg.de.

  12. Understanding drug resistance in human intestinal protozoa.

    Science.gov (United States)

    El-Taweel, Hend Aly

    2015-05-01

    Infections with intestinal protozoa continue to be a major health problem in many areas of the world. The widespread use of a limited number of therapeutic agents for their management and control raises concerns about development of drug resistance. Generally, the use of any antimicrobial agent should be accompanied by meticulous monitoring of its efficacy and measures to minimize resistance formation. Evidence for the occurrence of drug resistance in different intestinal protozoa comes from case studies and clinical trials, sometimes with a limited number of patients. Large-scale field-based assessment of drug resistance and drug sensitivity testing of clinical isolates are needed. Furthermore, the association of drug resistance with certain geographic isolates or genotypes deserves consideration. Drug resistance has been triggered in vitro and has been linked to modification of pyruvate:ferredoxin oxidoreductase, nitroreductases, antioxidant defense, or cytoskeletal system. Further mechanistic studies will have important implications in the development of second generation therapeutic agents.

  13. A trans-1,2 End-On Disulfide-Bridged Iron-Tetracarbene Dimer and Its Electronic Structure.

    Science.gov (United States)

    Meyer, Steffen; Krahe, Oliver; Kupper, Claudia; Klawitter, Iris; Demeshko, Serhiy; Bill, Eckhard; Neese, Frank; Meyer, Franc

    2015-10-19

    A disulfide-bridged diiron complex with [Fe-S-S-Fe] core, which represents an isomer of the common biological [2Fe-2S] ferredoxin-type clusters, was synthesized using strongly σ-donating macrocyclic tetracarbene capping ligands. Though the complex is quite labile in solution, single crystals were obtained, and the structure was elucidated by X-ray diffraction. The electron-rich iron-sulfur core is found to show rather unusual magnetic and electronic properties. Experimental data and density functional theory studies indicate extremely strong antiferromagnetic coupling (-J > 800 cm(-1)) between two low-spin iron(III) ions via the S2(2-) bridge, and the intense near-IR absorption characteristic for the [Fe-S-S-Fe] core was assigned to a S → Fe ligand-to-metal charge transfer transition.

  14. The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

    Science.gov (United States)

    Crespi, M; Vereecke, D; Temmerman, W; Van Montagu, M; Desomer, J

    1994-01-01

    Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants. PMID:8169198

  15. Photobiological hydrogen production.

    Science.gov (United States)

    Asada, Y; Miyake, J

    1999-01-01

    The principles and recent progress in the research and development of photobiological hydrogen production are reviewed. Cyanobacteria produce hydrogen gas using nitrogenase and/or hydrogenase. Hydrogen production mediated by native hydrogenases in cyanobacteria occurs under in the dark under anaerobic conditions by degradation of intracellular glycogen. In vitro and in vivo coupling of the cyanobacterial photosynthetic system with a clostridial hydrogenase via cyanobacterial ferredoxin was demonstrated in the presence of light. Genetic transformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum was successful; the active enzyme was expressed in PCC7942. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Coculture of Rhodobacter and Clostriudium was applied for hydrogen production from glucose. A mutant strain of Rhodobacter sphaeroides RV whose light-harvesting proteins were altered was obtained by UV irradiation. Hydrogen productivity by the mutant was improved when irradiated with monochromatic light of some wavelengths. The development of photobioreactors for hydrogen production is also reviewed.

  16. Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes.

    Science.gov (United States)

    Liu, Xian-De; Shen, Yun-Gang

    2005-02-17

    This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.

  17. The problem of resistant Trichomonas vaginalis to antiprotozoal drugs

    Directory of Open Access Journals (Sweden)

    A. L. Poznyak

    2011-01-01

    Full Text Available This review presents recent data on the energy metabolism of Trichomonas vaginalis and ways the activation of metronidazole. The sensitivity of microorganisms to the 5-nitroimidazole by the presence of their enzyme systems, generating and transporting electrons, which can then transfer them to the nitro group of the drug. In T.vaginalis these are pyruvate ferredoxin-oxydoreductase, thioredoxin reductase and flavin reductase. The development of resistance T.vaginalis to metronidazole preparations of this multistep process, based on the gradual reduction (up to a loss activity hydrogenosomal enzymes and / or violation of the flavindependent metabolic pathways.

  18. Formate Formation and Formate Conversion in Biological Fuels Production

    Directory of Open Access Journals (Sweden)

    Bryan R. Crable

    2011-01-01

    Full Text Available Biomethanation is a mature technology for fuel production. Fourth generation biofuels research will focus on sequestering CO2 and providing carbon-neutral or carbon-negative strategies to cope with dwindling fossil fuel supplies and environmental impact. Formate is an important intermediate in the methanogenic breakdown of complex organic material and serves as an important precursor for biological fuels production in the form of methane, hydrogen, and potentially methanol. Formate is produced by either CoA-dependent cleavage of pyruvate or enzymatic reduction of CO2 in an NADH- or ferredoxin-dependent manner. Formate is consumed through oxidation to CO2 and H2 or can be further reduced via the Wood-Ljungdahl pathway for carbon fixation or industrially for the production of methanol. Here, we review the enzymes involved in the interconversion of formate and discuss potential applications for biofuels production.

  19. Phycoerythrobilin synthase (PebS) of a marine virus. Crystal structures of the biliverdin complex and the substrate-free form.

    Science.gov (United States)

    Dammeyer, Thorben; Hofmann, Eckhard; Frankenberg-Dinkel, Nicole

    2008-10-10

    The reddish purple open chain tetrapyrrole pigment phycoerythrobilin (PEB; A(lambdamax) approximately 550 nm) is an essential chromophore of the light-harvesting phycobiliproteins of most cyanobacteria, red algae, and cryptomonads. The enzyme phycoerythrobilin synthase (PebS), recently discovered in a marine virus infecting oceanic cyanobacteria of the genus Prochlorococcus (cyanophage PSSM-2), is a new member of the ferredoxin-dependent bilin reductase (FDBR) family. In a formal four-electron reduction, the substrate biliverdin IXalpha is reduced to yield 3Z-PEB, a reaction that commonly requires the action of two individual FDBRs. The first reaction catalyzed by PebS is the reduction of the 15,16-methine bridge of the biliverdin IXalpha tetrapyrrole system. This reaction is exclusive to PEB biosynthetic enzymes. The second reduction site is the A-ring 2,3,3(1),3(2)-diene system, the most common target of FDBRs. Here, we present the first crystal structures of a PEB biosynthetic enzyme. Structures of the substrate complex were solved at 1.8- and 2.1-A resolution and of the substrate-free form at 1.55-A resolution. The overall folding revealed an alpha/beta/alpha-sandwich with similarity to the structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA). The substrate-binding site is located between the central beta-sheet and C-terminal alpha-helices. Eight refined molecules with bound substrate, from two different crystal forms, revealed a high flexibility of the substrate-binding pocket. The substrate was found to be either in a planar porphyrin-like conformation or in a helical conformation and is coordinated by a conserved aspartate/asparagine pair from the beta-sheet side. From the alpha-helix side, a conserved highly flexible aspartate/proline pair is involved in substrate binding and presumably catalysis.

  20. Remodeling of Membrane Lipids in Iron-starved Chlamydomonas*

    Science.gov (United States)

    Urzica, Eugen I.; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M. Dudley; Casero, David; Gallaher, Sean D.; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S.

    2013-01-01

    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24–48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii. PMID:23983122