scholarly journals Purification of the membrane-bound hydrogenase of Escherichia coli

1979 ◽  
Vol 183 (1) ◽  
pp. 11-22 ◽  
Author(s):  
M W W Adams ◽  
D O Hall
Keyword(s):  
Escherichia Coli ◽  
Methyl Viologen ◽  
Hydrophobic Interaction Chromatography ◽  
H2 Evolution ◽  
Ph Optimum ◽  
Benzyl Viologen ◽  
Electron Carrier ◽  
Membrane Bound ◽  
High Concentrations ◽  
Electron Carriers

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.

scholarly journals The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli

1980 ◽  
Vol 188 (2) ◽  
pp. 345-350 ◽  
Author(s):  
R W Jones
Keyword(s):  
Escherichia Coli ◽  
Electron Acceptor ◽  
Cytoplasmic Membrane ◽  
Proton Translocation ◽  
Terminal Electron Acceptor ◽  
Benzyl Viologen ◽  
Carbonyl Cyanide ◽  
Energy Conserving ◽  
Membrane Bound ◽  
Hydroxy Quinoline

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.

scholarly journals Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans

1983 ◽  
Vol 209 (2) ◽  
pp. 445-454 ◽  
Author(s):  
W V Lalla-Maharajh ◽  
D O Hall ◽  
R Cammack ◽  
K K Rao ◽  
J Le Gall
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Desulfovibrio Desulfuricans ◽  
Relative Molecular Mass ◽  
H2 Evolution ◽  
Cytochrome C3 ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Heavy Metal Salts ◽  
Membrane Bound

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.

scholarly journals Characterization of Two β-1,3-Glucosyltransferases from Escherichia coli Serotypes O56 and O152

2008 ◽  
Vol 190 (14) ◽  
pp. 4922-4932 ◽  
Author(s):  
Inka Brockhausen ◽  
Bo Hu ◽  
Bin Liu ◽  
Kenneth Lau ◽  
Walter A. Szarek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Clusters ◽  
Aliphatic Chain ◽  
Ph Optimum ◽  
Acceptor Substrate ◽  
C Terminus ◽  
Membrane Bound ◽  
O Antigens ◽  
Phosphate Groups

ABSTRACT The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-β1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-β1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

scholarly journals Localization of hydrogenase in Thiocapsa roseopersicina photosynthetic membrane

1982 ◽  
Vol 202 (1) ◽  
pp. 255-258 ◽  
Author(s):  
Csaba Bagyinka ◽  
Kornel L. Kovács ◽  
Erika Rak
Keyword(s):  
Cell Membrane ◽  
Methyl Viologen ◽  
Photosynthetic Membrane ◽  
Benzyl Viologen ◽  
Thiocapsa Roseopersicina ◽  
Membrane Bound ◽  
Reduced Forms

The photosynthetic cell membrane is impermeable to the oxidized redox dyes Methyl Viologen and Benzyl Viologen, whereas the reduced forms easily penetrate into the cells. By exploiting this permeability difference, the orientation of the membrane-bound hydrogenase has been determined.

A ferredoxin-Iinked sulfite reductase from Clostridium pasteurianum

1971 ◽  
Vol 17 (7) ◽  
pp. 889-895 ◽  
Author(s):  
E. J. Laishley ◽  
Po-Min Lin ◽  
H. D. Peck Jr.
Keyword(s):  
Phosphate Buffer ◽  
Molecular Hydrogen ◽  
Methyl Viologen ◽  
Hydrogen Uptake ◽  
Electron Donors ◽  
Sulfite Reductase ◽  
Clostridium Pasteurianum ◽  
Ph Optimum ◽  
Benzyl Viologen ◽  
Dissimilatory Sulfite Reductase

A soluble sulfite reductase (EC. 1.8.1.2) system is present in cell-free extracts of Clostridium pasteurianum that reduces sulfite to sulfide in the presence of molecular hydrogen. The natural electron donor for this reductase has been found to be ferredoxin, which can be completely replaced by methyl viologen or partially by benzyl viologen. The physiological electron donors NAD, NADP, FMN, and FAD were not active in the transfer of electrons from hydrogen for sulfite reduction. The pH optimum of the sulfite reductase was found to be 7.0 using phosphate buffer, and the extracts reduced nitrite and hydroxyl-amine in addition to sulfite. The stoichiometry of the reaction in terms of hydrogen uptake to sulfide formation was 2.7, which is very close to the theoretical ratio of 3. The above properties of the system indicate that it is an assimilatory rather than a dissimilatory sulfite reductase, as outlined in the Discussion.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

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