7 results on '"Jenney, Francis E."'
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2. Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.
- Author
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Chandrayan SK, McTernan PM, Hopkins RC, Sun J, Jenney FE Jr, and Adams MW
- Subjects
- Archaeal Proteins genetics, Biofuels, Catalytic Domain physiology, Cytoplasm genetics, Gene Expression, Hydrogenase genetics, Maltose metabolism, Maltose pharmacology, Operon physiology, Pyrococcus furiosus genetics, Pyrococcus furiosus growth & development, Sweetening Agents metabolism, Sweetening Agents pharmacology, Archaeal Proteins biosynthesis, Cytoplasm enzymology, Hydrogen metabolism, Hydrogenase biosynthesis, Metabolic Engineering, Pyrococcus furiosus enzymology
- Abstract
The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.
- Published
- 2012
- Full Text
- View/download PDF
3. Homologous expression of a subcomplex of Pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase.
- Author
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Hopkins RC, Sun J, Jenney FE Jr, Chandrayan SK, McTernan PM, and Adams MW
- Subjects
- Catalysis, Catalytic Domain, Protein Binding, Recombination, Genetic, Hydrogenase metabolism, Pyrococcus furiosus enzymology, Pyruvate Synthase metabolism
- Abstract
Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O(2)-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining 'minimal' hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg(-1) in H(2) production using the artificial electron donor methyl viologen) and thermostable (t(1/2) ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H(2) in vitro without the need for an intermediate electron carrier.
- Published
- 2011
- Full Text
- View/download PDF
4. Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production.
- Author
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Sun J, Hopkins RC, Jenney FE, McTernan PM, and Adams MW
- Subjects
- Amino Acid Sequence, Base Sequence, Escherichia coli genetics, Molecular Sequence Data, Plasmids chemistry, Plasmids genetics, Promoter Regions, Genetic genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, beta-Galactosidase genetics, Biofuels microbiology, Escherichia coli metabolism, Hydrogenase metabolism, NADP metabolism, Pyrococcus furiosus enzymology
- Abstract
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.
- Published
- 2010
- Full Text
- View/download PDF
5. Hydrogenases of the model hyperthermophiles.
- Author
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Jenney FE Jr and Adams MW
- Subjects
- Archaeal Proteins metabolism, Bacterial Proteins metabolism, Energy Metabolism, Fuel Oils, Hydrogen metabolism, Hydrogenase metabolism, Thermofilaceae enzymology, Thermotoga maritima enzymology
- Abstract
Hydrogenases are enzymes found in all domains of life that catalyze a remarkably simple chemistry, the reversible oxidation of molecular hydrogen to protons and electrons. In order to perform this chemistry, cells have evolved, several different times, intricate organometal complexes built around a binuclear Ni-Fe or Fe-Fe center, with bound CO and CN(-) groups, as well as multiple FeS centers. These complicated enzymes have been an area of intense study for many decades, with interest peaking on the occasions of major increases in national energy costs. Interest in biologically generated hydrogen as a potential substitute for fossil fuels is again at the forefront, and the new tools of the postgenomic world available for manipulating these enzymes make it a truly viable possibility. Hydrogenases from hyperthermophilic microorganisms such as Pyrococcus furiosus and Thermotoga maritima, with optimal growth temperatures near 100 degrees C, are of particular interest and promise for elucidating and manipulating these enzymatic mechanisms.
- Published
- 2008
- Full Text
- View/download PDF
6. Observation of Fe-H/D modes by nuclear resonant vibrational spectroscopy.
- Author
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Bergmann U, Sturhahn W, Linn DE Jr, Jenney FE Jr, Adams MW, Rupnik K, Hales BJ, Alp EE, Mayse A, and Cramer SP
- Subjects
- Pyrococcus furiosus chemistry, Rubredoxins chemistry, Scattering, Radiation, Spectrum Analysis methods, Sulfides chemistry, Vibration, X-Rays, Hydrogen chemistry, Hydrogenase chemistry, Iron Compounds chemistry, Nitrogenase chemistry
- Abstract
Metal-hydrogen bonding is important in chemistry and catalysis, but H atoms are often difficult to observe, especially in metalloproteins. In this work we show that Fe-H interactions can be probed by nuclear resonance vibrational spectroscopy at the 14.4 keV 57Fe nuclear resonance. An important advantage of this method, compared to Raman and IR spectroscopy, is the selectivity for modes that involve 57Fe motion. We present data on the FeS4 site in rubredoxin and the [FeH(D)6]2- ion. Prospects for studying more complex systems are discussed.
- Published
- 2003
- Full Text
- View/download PDF
7. Hydrogenesis in hyperthermophilic microorganisms: Implications for biofuels
- Author
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Chou, Chung-Jung, Jenney, Francis E., Adams, Michael W.W., and Kelly, Robert M.
- Subjects
- *
THERMOPHILIC microorganisms , *BIOMASS energy , *FERREDOXIN-NADP reductase , *ETHANOL as fuel , *HYDROGEN production , *BIOMASS production , *HYDROGENASE , *PHYSIOLOGY - Abstract
Abstract: Hydrothermal microbiotopes are characterized by the consumption and production of molecular hydrogen. Heterotrophic hyperthermophilic microorganisms (growth T opt⩾80°C) actively participate in the production of H2 in these environments through the fermentation of peptides and carbohydrates. Hyperthermophiles have been shown to approach the theoretical (Thauer) limit of 4mol of H2 produced per mole of glucose equivalent consumed, albeit at lower volumetric productivities than observed for mesophilic bacteria, especially enterics and clostridia. Potential advantages for biohydrogen production at elevated temperatures include fewer metabolic byproducts formed, absence of catabolic repression for growth on heterogeneous biomass substrates, and reduced loss of H2 through conversion to H2S and CH4 by mesophilic consortia containing sulfate reducers and methanogens. To fully exploit the use of these novel microorganisms and their constituent hydrogenases for biohydrogen production, development of versatile genetic systems and improvements in current understanding of electron flux from fermentable substrates to H2 in hyperthermophiles are needed. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
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