The Human Pathogen Clostridium Dificle: A Look at a Putative Involved in Protection from Oxidative Stress

Background: Hospital acquired infections due to Clostridium difficile (C. diff) is associated with nosocomial diarrhea and pseudomembranous colitis. Using a published microarray analysis of C. diff strain 630, several open reading frames (ORFs) were noted for their upregulation under different environmental stresses. One of these genes (CD1134) is a putative glyoxalase I. The glyoxalase enzymes detoxify a side product of glycolysis, methylglyoxal, and use metals as a cofactor

Characterization of a Small Iron Protein, Pyrococcus Furiosus Rubredoxin, as a Potential Cancer Drug Delivery System

Background: Cancer is an elusive neoplastic disease that claims the lives of many people around the world every year. Though treatments have become more specific to the different types of cancer, the need for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity remains, and rubredoxin might be such a tool. Rubredoxin is a small (53 amino acids), water soluble, non-heme iron electron transfer protein that contains an iron atom cofactor, which can be substituted with various cytotoxic transition metals such as nickel and cobalt with little or no effect on the protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project is to incorporate tumor-specific binding sequences at several modifiable sites on the protein as well as substitute the iron-center with cytotoxic metals. Once a stable rubredoxin containing these characteristics is created, its effects and efficacy will be studied on specific cancer cells in vitro

Homologous Expression of a Subcomplex of Pyrococcus furiosus Hydrogenase that Interacts with Pyruvate Ferredoxin Oxidoreductase

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 O2-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 H2 production using the artificial electron donor methyl viologen) and thermostable (t1/2 ∼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 H2 in vitro without the need for an intermediate electron carrier

Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

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

Roles of the soluble cytochrome c2 and membrane-associated cytochrome cy of rhodobacter capsulatus in photosynthetic electron transfer

Genetic evidence indicates that Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center (RC) [Jenney, F. E., & Daldal, F. (1993) EMBOJ. 12, 1283-1292]. One pathway is via the water soluble cytochrome (cyt) c2, and the other is via a novel, membrane-associated c-type cytochrome, cyt cy, now believed to be identical to the cyt cx of Jones et al. [Jones, M. R., et al. (1990) Biochim. Biophys. Acta 975, 59-66] and c354 of Zannoni et al. [Zannoni, D., et al. (1992) Arch. Microbiol. 157, 367-374]. Mutants lacking either cyt c2, cyt cy, or the bc1 complex, as well as various combinations, were utilized to probe the functional role of these cytochromes in electron transfer. Data obtained by monitoring flash induced electron transfer kinetics in the RC, cyt c pool, cyt b, and the carotenoid band shift indicate that there are two pathways for electron transfer from the bc1 complex to the RC in R. capsulatus, one via cyt c2 and the other through cyt cy. The two pathways show strikingly different kinetics for RC reduction and cyt c oxidation, and both are present in the wild-type strain MT-1131. After genetic inactivation of both cyt c2 and cyt cy there remains no flash oxidizible c-type cytochrome, and inactivation of cyt cy rather than cyt c2 has a more pronounced effect on the extent of the light-induced membrane potential under the conditions tested. Finally, heme-stained SDS-PAGE and flash spectroscopy experiments indicate that cyt cy is detectable in strains lacking the bc1 complex when grown on minimal growth medium but not on rich medium. These findings complement the earlier genetic data and further establish that cyt cy is the electron carrier permitting soluble cyt c2-independent photosynthetic growth in R. capsulatus. © 1994 American Chemical Society

Evaluating Methods to Lessen Ischemic Reperfusion Injury by Testing the Effects in Vitro of Pyrococcus Furiosus Superoxide Reductase in Reducing Oxidative Damage in Superoxide Induced Cells

Background: Ischemic-Reperfusion (I/R) injuries are commonly associated with conditions such as heart attacks, liver disease, diabetes, and hypertension, due to the restoration of blood flow to oxygen-starved tissues. This restoration of oxygen has been known to cause oxidative damage within cells, producing an increased concentration of reactive oxygen species (ROS), such as superoxide (O2-). Cells use both chemical and enzymatic defenses to reduce the accumulation of ROS, although enzymes are faster at lowering this oxidative damage. Aerobic organisms use the enzyme superoxide dismutase (SOD) to eliminate superoxide by converting it to hydrogen peroxide and oxygen. Superoxide reductase (SOR) is an enzyme found in anaerobic microbes that has the ability to not only decrease the concentration of superoxide, but also decrease the concentration of reducing compounds that could create more superoxide by blocking the transfer of electrons to oxygen. The highly thermostable SOR from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) will be tested, in vitro, for its ability to protect human cells against superoxide toxicity. Method and Results: The gene encoding Pf SOR (Pf 1281) was expressed in E. coli, and the protein was purified using heat treatment, (due to its hyperthermophilic nature), followed by ion exchange, and hydrophobic interaction chromatography. Pf SOR was analyzed by SDS-PAGE, a protein assay, absorption spectroscopy, and activity tested using a SOR activity assay. Human Jurkat cancer cells are currently being incubated and are awaiting viability tests, such as the Trypan Blue Exclusion Test and the Lactate Dehydrogenase (LDH) Assay, to determine SOR effectiveness against induced superoxide damage in vitro

Use of Pyrococcus Furiosus Rubredoxin as a Potential targeted cancer therapeutic

Background: Cancer is a disease that affects approximately one million people around the world each year. Though there are both traditional and targeted therapies currently available, there is still a need for therapeutics that will specifically target cancer cells and not cause toxicity by killing normal cells in the body. Pyrococcus furiosus rubredoxin could potentially be the agent for such therapies as it is a small (5.9 kDa), water-soluble protein that is a hyperthermostable, non-heme iron protein with an iron cofactor bound by cysteine residues. The focus of this project is to use mutated forms of this protein and replace the iron cofactor with a cytotoxic metal, for example ruthenium, giving it the potential to be a successful cancer delivery system. Methods and Results: A mutant of P. furiosus rubredoxin that contains the integrin-binding tag, RGD, and another mutant that contains an E-tag that can be easily visualized are being used. RGD is a sequence that will bind to tumor cells overexpressing integrins. Both of these tags were previously incorporated after the D20 residue of the protein. These wild type and mutant proteins were purified using anion-exchange DEAE and size-exclusion G-75 Sephadex chromatography. The proteins were then analyzed via absorption spectroscopy, SDS-PAGE, and protein assays. Mutants were then denatured and refolded in the presence of ruthenium. The metal content of the proteins was analyzed via inductively coupled plasma mass spectrometry (ICP-MS). Analyses via ICP-MS also show that both the wild type and mutant rubredoxins were successfully able to unfold and refold with ruthenium as the new cofactor with few contaminants. Jurkat T-lymphocyte cancer cells will be stimulated with phorbol ester in order to overexpress integrins on the membrane. The cells will then be incubated with the ruthenium-containing wild type rubredoxin, the D20-RGD mutant, and the D20 E-tag mutant in varying concentrations and then assayed for cell viability using Trypan blue and for apoptosis using gel electrophoresis. We expect that the mutant RGD rubredoxin will have a higher affinity for the phorbol ester stimulated cells than the wild type rubredoxin and that it will kill the Jurkat cells. If the mutant rubredoxin that contains ruthenium is able to cause a reduction in the viability of the cancer cells in vitro, experiments will be further extended in vivo

The impact of extremophiles on structural genomics (and vice versa)

The advent of the complete genome sequences of various organisms in the mid-1990s raised the issue of how one could determine the function of hypothetical proteins. While insight might be obtained from a 3D structure, the chances of being able to predict such a structure is limited for the deduced amino acid sequence of any uncharacterized gene. A template for modeling is required, but there was only a low probability of finding a protein closely-related in sequence with an available structure. Thus, in the late 1990s, an international effort known as structural genomics (SG) was initiated, its primary goal to fill sequence-structure space by determining the 3D structures of representatives of all known protein families. This was to be achieved mainly by X-ray crystallography and it was estimated that at least 5,000 new structures would be required. While the proteins (genes) for SG have subsequently been derived from hundreds of different organisms, extremophiles and particularly thermophiles have been specifically targeted due to the increased stability and ease of handling of their proteins, relative to those from mesophiles. This review summarizes the significant impact that extremophiles and proteins derived from them have had on SG projects worldwide. To what extent SG has influenced the field of extremophile research is also discussed. © 2007 Springer