Maturation of hydrogenase enzymes in thiocapsa roseopersicina gergely maroti supervisors. The membranebound hydrogenase from thiocapsa roseopersicina is composed of two subunits and contains two fes centres and one ni per molecule. Towards understanding hydrogenase maturation in thiocapsa roseopersicina ph. Strong inhibition was shown for polyllysine bearing positive charge. The effect of polypeptides having different charge on the activity of thiocapsa roseopersicina hydsl hydrogenase was studied. Heterologous expression of alteromonas macleodii and. Electrontransfer subunits of the nife hydrogenases in thiocapsa roseopersicina bbs l. A second soluble hoxtype nife enzyme completes the hydrogenase set in thiocapsa roseopersicina bbs. The purple sulfur bacterium thiocapsa roseopersicina is known to synthesize several nife hydrogenases 2.
Thiocapsa hydrogenase and examined its cellular location in the recombinant cyanobacterial strain. Threedimensional structure of the nickelcontaining. Pdf the hydrogenases of thiocapsa roseopersicina gabor. Thiocapsa roseopersicina was discovered by sergei winogradsky in the 1880s when he was studying sulfur bacteria. In addition to the small and large structural subunits, additional proteins are usually associated with the nife hydrogenases, connecting their activity to other redox processes in. The soluble and chromatophorebound hydrogenases from the purple sulphur bacterium thiocapsa roseopersicina strain bbs were purified up to homogeneity and the properties studied. These enzymes consist of a large subunit containing the catalytic center with nife atoms, coordinated by. Structural genes coding for two membraneassociated nife hydrogenases in the phototrophic purple sulfur bacterium thiocapsa roseopersicina hupsl and hynsl have recently been isolated and characterized. Thiocapsa roseopersicina bbs belongs to the family of purple sulfur photosynthetic bacteria, the chromatiaceae 5. The thermostable hydsl hydrogenase from the purple sulfur bacterium thiocapsa roseopersicina bbs belongs to the family of nife hydrogenases. In thiocapsa roseopersicina bbs, a remarkably stable hydrogenase activity has been found and characterized 2022, 24. In this paper, we report the kinetic mechanism of the unidirectional h,oxidizing hydrogenase from the department of biochemistry, university of wisconsin madison, madison, wisconsin 53706. Monoclonal antibodies to the hydrogenase of thiocapsa roseopersicina article pdf available in current microbiology 116.
Thiocapsa roseopersicina bbs is a photosynthetic purple sulfur bacterium belonging to the chromatiaceae. An environmental alteromonas hydrogenase showing 99% identity to the altde hydrogenase was heterologously expressed in thiocapsa roseopersicina and was confirmed to be active maroti et al. A moving front has been observed as a special pattern during the hydrogenase catalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thinlayer reaction chamber. Pdf a second soluble hoxtype nife enzyme completes the. Protein immunoelectroblotting was used to identify the hydrogenase specific antibodies. Gabor rakhely institute of biophysics, biological research center, hungarian academy of sciences, and department of biotechnology, university of szeged szeged, hungary 2003. Threedimensional structure of nickelcontaining hydrogenase. It cannot use formate or glucose as substrates though. Cyanobacterial hydrogenases and hydrogen metabolism. In these organisms, each hydrogenase enzyme is assumed to have specific physiological functions. Threedimensional structure of the nickelcontaining hydrogenase from thiocapsa roseopersicina article pdf available in journal of bacteriology 1738. Thiocapsa hydrogenase carries a signal peptide in its small subunit and it is a membranebound hydrogenase in its original host thiocapsa roseopersicina.
The alteromonas hydrogenase largesubunit hyab was detected in t. Pdf improvement of biohydrogen production and intensification of. Hydrogenases, accessory genes and the regulation of 6nife9 hydrogenase biosynthesis in thiocapsa roseopersicina. Connection between the membrane electron transport system and. The first report that demonstrated expression of nonnative nife hydrogenase in cyanobacteria.
Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. Pdf the photosynthetic cell membrane is impermeable to the oxidized redox dyes methyl viologen and benzyl viologen, whereas the. Thiocapsa roseopersicina bbs contains at least three different active nife hydrogenases. The hoxefuhy in thiocapsa roseopersicina is a nife hydrogenase. Kovacs1,2 1 department of biotechnology, university of szeged, hungary. It has high catalytic activity and resistance to a number of denaturing agents 1. Kovacsa a institute of biophysics, biological research center, hungarian academy of sciences, department of biotechnology, university of szeged, szeged, hungary. Its two structural genes hynsl, encoding small and large hydrogenase subunits, are surrounded by eight genes hynd, huph and hypcabdfe predicted to encode accessory proteins involved in maturation of the hydrogenase. Pdf localization of hydrogenase in thiocapsa roseopersicina. Thiocapsa roseopersicina has spherical cells that can form tetrads.
Hynsl from alteromonas macleodii deep ecotype altde is an oxygentolerant and thermostable nife hydrogenase. We characterized the physiological h 2 consumptionevolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. As shown earlier 9, a number of metal cations have high affinity to t. However, thiocapsa rosea grows at low intensities, usually between 50300 lux. Hydrogenmetabolizing organisms use an nifehydrogenase to catalyze hydrogen oxidation. Structural properties, functional states and physiological. A hoxtype soluble hydrogenase was also identified in t. For example, thiocapsa roseopersicina prefers high amounts of light, between 1,000 and 2,000 lux.
In this report, we present molecular biology evidence that in the photosynthetic bacterium t. Pdf discovery of nife hydrogenase genes in metagenomic. Hydrogenase catalysis can also serve as an experimental example or model of a number of autocatalytic biological reactions prion, autophosphorylation, etc. This made possible the separation of the functionally distinct. Thiocapsa roseopersicina bbs contains at least three different active nife. The enzyme resists heat and proteolytic degradation, its activity is retained under sdspage conditions. This study represents the first conversion of an environmental dna into a hydrogenase vi. Concerted regulation of hupsl and hox1 hydrogenases in. Discovery of nife hydrogenase genes in metagenomic dna. European journal of biochemistry 1996, 241 2, 675681.
Towards understanding hydrogenase maturation in thiocapsa. Inhibition by iodoacetamide and acetylene of the hdexchange reaction catalyzed by thiocapsa roseopersicina hydrogenase. The inhibition was reversible and competitive to methyl viologen, an electron acceptor, in the reaction of hydrogen oxidation catalyzed by the hydrogenase. A moving front has been observed as a special pattern during the hydrogenasecatalyzed reaction of hydrogen uptake with benzyl viologen as electron acceptor in a thinlayer reaction chamber. During anoxygenic photosynthesis, this bacterium requires reduced sulfur compounds e. We used this genetic system to compare maturation mechanisms between altde hynsl and its thiocapsa roseopersicina homologue. The purple sulphur phototrophic bacterium, thiocapsa roseopersicina bbs contains several nife hydrogenases. Ptclusters deposited on the interior of a heat shock protein cage architecture 268 h 2 ptmin, methyl viologen varpness.
Thus, electrostatic interactions play a significant role in the interaction between the enzyme and its substrate acceptor. Protein immunoelectroblotting was used to identify the hydrogenasespecific antibodies. This novel hydrogenase was successfully heteroexpressed in. Pdf nife hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells.
An expression vector made for this organism is available and was tested with the gene for a green fluorescent protein. The role of hox hydrogenase in the h metabolism of thiocapsa. The characterization of a hyd gene cluster encoding the stable, bidirectional nifehydrogenase 1 enzyme in thiocapsa roseopersicina bbs, a purple sulfur photosynthetic bacterium belonging to the family chromatiaceae, is presented. The purple sulfur phototrophic bacterium thiocapsa roseopersicina bbs synthesizes at least three nife hydrogenases hox, hup, hyn. Pdf threedimensional structure of the nickelcontaining. Thiocapsa roseopersicina bbs is a purple sulfur photosynthetic bacterium 4. The enzyme forms a large, open, ringshaped complex, containing 6 molecules each of the large 62 kda and small 26 kda subunits. Electrontransfer subunits of the nife hydrogenases in. Monoclonal antibodies to the hydrogenase of thiocapsa. Sullivan for the degree of honors baccalaureate of. Kinetic mechanism the hydrogenoxidizing hydrogenase from.
Study of the mechanism of inactivation of hydrogenase at elevated temperatures 11. Using a metagenomics approach, we have cloned a piece of environmental dna from the sargasso sea that encodes an nife hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a thiocapsa roseopersicina o 2tolerant nife hydrogenase. Later, the altde hydrogenase was characterized and was found to. The stable nife hydrogenase from thiocapsa roseopersicina hynsl 18. The properties of hydrogenase from thiocapsa roseopersicina. Genetic analysis of the alteromonas macleodii nife. Concentrationdependent front velocity of the autocatalytic. In addition to the small and large structural subunits, additional proteins are usually associated with the nife hydrogenases, connecting their activity to other redox processes in the cells. Hydogenase enzymes may be sensitive to a number of denaturing factors, including heat 6, 18 and ph 18.
Various species require different amounts of light in order to undergo this process, which determines their location in the water column. Hydrogenases, accessory genes and the regulation of nife hydrogenase biosynthesis in thiocapsa roseopersicina. Thiocapsa roseopersicina, isolated from the north sea 10. We heterologously expressed the two structural genes hyaa and hyab and two accessory genes huph and hyad of this novel environmental hydrogenase in t.
Localization of hydrogenase in thiocapsa roseopersicina photosynthetic membrane article pdf available in biochemical journal 2021. Exafs investigations of the nickel site in thiocapsa. Investigation of the nature of this catalysis via the development of reduced benzyl viologen showed clearly the typical characteristics of an autocatalytic reaction. Hydrogenase in a twocompartment protonexchangemembrane pem sodium dithionite sd oh et al.
The purple sulfur phototrophic bacteria thiocapsa roseopersicina contains several hydrogenases. The role of hox hydrogenase in the h metabolism of. Xray absorption spectroscopic study of the reductive. Such fronts start spontaneously and at random times at different points of the reaction chamber. The threedimensional structure of the nickelcontaining hydrogenase from thiocapsa roseopersicina has beendeterminedat aresolution of2nmin theplaneand4nmin the vertical direction byelectronmicroscopy andcomputerized image processing on microcrystals of the enzyme. The dna sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to. Hynsl hydrogenase from thiocapsa roseopersicina was applied to catalyze the oxidation of molecular hydrogen in a new, improved, thinlayer reaction chamber. The hydrogenases of thiocapsa roseopersicina article pdf available in biochemical society transactions 33pt 1. The amino acid composition of hydrogenase preparations from different fractions of t. An autocatalytic step in the reaction cycle of hydrogenase. Hydrogenase immobilization, on porous pyrolytic carbon paper pcp and packed graphite columns pgc johnston et al. Pdf hydrogenases, accessory genes and the regulation of.
Obtaining crystals and study the spatial structure of hydrogenase of t. Nagy, gergely maroti, eva kondorosi, gabor rakhely and kornel l. Maturation of hydrogenase enzymes in thiocapsa roseopersicina. Hupsl is a membrane bound h 2 uptake hydrogenase responsible for h 2 consumption produced by nitrogenase enzyme complex. Pdf hupo, a novel regulator involved in thiosulfate. Among the 18 monoclonal antibodies selected by enzyme immunoassay, three were found to react with highly immunogenic trace contaminating proteins. Cloning and heterologous expression in thiocapsa roseopersicina. The role of hox hydrogenase in the h2 metabolism of thiocapsa. A second soluble hoxtype nife enzyme completes the. Structure of hydrogenase from thiocapsa roseopersicina. The role of hox hydrogenase in the h 2 metabolism of thiocapsa roseopersicina gabor rakhelya, tatyana v. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized k. To investigate whether heteroexpressed thiocapsa hydrogenase is a membranebound hydrogenase. This presentation does not contain any proprietary, confidential informati on, or otherwise restricted information.
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