Search

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the [k.sub.cat]/[K.sub.m] value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108[degrees]C that increases to 112[degrees]C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-[angstrom] resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P.furiosus showed that spermidine, cadaverine, and sym.nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.

Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these [alpha]-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize altodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-[alpha]-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of 1-glucan substrates by P. furiosus.

Cyanide is added to oxidized 1Fe and 2Fe superoxide reductase (SOR) for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide by using vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Low temperature ENDOR data shows that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR.

The original genome annotation of the hyperthermophilic archaeon Pyrococcus furiosus contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two databases, many very significantly. For example, more than 170 of the predicted proteins differ at their N termini by more than 25 amino acids. Similar discrepancies were observed in the TIGR and NCBI databases with the other archaeal and bacterial genomes examined. In addition, the two databases contain 60 (NCBI) and 221 (TIGR) ORFs not present in the original annotation of P. furiosus. In the present study we have experimentally assessed the validity of 88 previously unannotated ORFs. Transcriptional analyses showed that 11 of 61 ORFs examined were expressed in P. furiosus when grown at either 95 or 72[degrees]C. In addition, 7 of 54 ORFs examined yielded heat-stable recombinant proteins when they were expressed in Escherichia coli, although only one of the seven ORFs was expressed in P. furiosus under the growth conditions tested. It is concluded that the P. furiosus genome contains at least 17 ORFs not previously recognized in the original annotation. This study serves to highlight the discrepancies in the public databases and the problems of accurately defining the number and sizes of ORFs within any microbial genome.

The main aim of the study is to obtain the recombinant form of Pyrococcus furiosus NAD(P)H:rubredoxin oxidoreductase (NROR), to reconstitute an in vitro recombinant superoxide reduction pathway and to show that NADPH can serve as an electron source for superoxide reduction to peroxide. It is demonstrated that the complete pathway from NAD(P)H to reduction of superoxide reductase (SOR) via NROR and rubredoxin can be reconstituted in an in vitro recombinant system.

Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 [+ or -] 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of [Co.sup.2+] ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100[degrees]C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100[degrees]C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 [+ or -] 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85[degrees]C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 [micro]mol of rubredoxin oxidized/min/mg with apparent [K.sub.m] values of 35 and 70 [micro]M for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 [micro]mol of [H.sub.2][O.sub.2] reduced/min/mg of rubrerythrin at 85[degrees]C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide: rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

The spectroscopic studies of pyrococcus furiosus superoxide reductase are detailed. The ability cyanide to bind exogenous ligands to both the ferrous and ferric sites of superoxide reductase is consistent with an inner-sphere catalytic mechanism involving superoxide binding at the ferrous site to yield a ferric-hydroperoxo intermediate.

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 subunits, 22 for nickel including seven from known nickel-proteins, and 20 for molybdenum including two from known molybdo-proteins. The uncharacterized proteins are prime candidates for metal-based purification or recombinant approaches to validate these predictions. Conclusions We conclude that the largely uncharacterized extent of native metalloproteomes can be revealed through analysis of the co-occurrence of metals and proteins across a fractionation space. This can significantly impact our understanding of metallobiochemistry, disease mechanisms, and metal toxicity, with implications for bioremediation, medicine and other fields.

[super 57]Fe Nuclear resonance vibrational spectroscopy (NRVS) is applied for identifying protein-bound dinitrosyl iron complexes. NRVS spectra for four iron dinitrosyl model compounds are used as benchmarks for identifying species formed in the reaction of Pyrrococcus furiosus ferredoxin D14C with nitric oxide.

A combination of sulfur K-edge X-ray absorption spectroscopy (XAS) and density functional theory is used to study the structure of superoxide reductase (SOR). The sites with high positive charge in these compounds are found to have higher affinity for binding another anion.

The solution structure of the PF0610, which is a novel winged helix-turn-helix (wHTH) protein with a rubredoxin-like Zn ribbon as its W1 segment, is determined by using nuclear magnetic resonance (NMR). The novel structure might help in understanding the transcription regulatory processes in Pyrococcus furiosus.

The properties of the iron ligands of CN-SOR and CN-P450cam are compared through a combination of [(super).1.H], [(super.13.C], and [(super).14.N] ENDOR spectroscopy. Results show that the difference between the in-plane heme and out-of-plane dihedral [His.sub.4] ligation of P450cam and SOR, respectively, causes real differences in the distribution of spin density over the on-axis atoms.

The Fe(S(sub cys))4 site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf) is studied using (super 57)Fe nuclear resonance vibrational spectroscopy (NRVS). A normal mode analysis using a Urey-Bradley force field is undertaken to quantitatively interpret the NRVS spectra and to set the stage for analysis of seemingly more complex problems such as hydrogenase and nitrogenase.

Nuclear resonant inelastic X-ray scatting (NRIXS) data on FeS4 and FeH(D)(sub 6) model systems is presented. Fe-H stretches have high frequencies and little Fe motion and both factors will reduce NIRXS intensity.

The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe–4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe–4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe–S clusters. [ABSTRACT FROM AUTHOR]

Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign FeCO and FeCN bending and stretching vibrations of the active site outside the spectral range of the FeS cluster normal modes. [ABSTRACT FROM AUTHOR]

Blick ins Zentrum: Die Methode der inelastischen kernresonanten Streuung (NRVS) wurde genutzt, um 57Fe ‐ markierte [NiFe] ‐ Hydrogenase in unterschiedlichen Zuständen des Enzyms zu untersuchen. Damit gelang es, die Fe ‐ CO ‐ und Fe ‐ CN ‐ Beuge ‐ und ‐ Streckschwingung außerhalb des Spektralbereichs der Normalmoden der Fe ‐ S ‐ Cluster zu detektieren und zuzuordnen (siehe Spektrum). [ABSTRACT FROM AUTHOR]

Structural and functional characterization of the entire protein complement (the proteome) of an organism can provide an infrastructure upon which questions about biological pathways and systems biology can be framed. The technology necessary to perform this proteome-level structural and functional characterization is under development in numerous structural genomics and functional genomics initiatives. Given the ubiquity of metal active sites in a proteome, it seems appropriate to ask whether comprehensive local structural characterization of metal sites within a proteome (metalloproteomics) is either a valid or obtainable goal. With a proteome-wide knowledge of the active-site structures of all metalloproteins, one could start to ask how metal insertion, cluster assembly and metalloprotein expression are affected by growth conditions or developmental status etc. High-throughput X-ray absorption spectroscopy (HTXAS) is being developed as a technology for investigating the metalloproteome. In creating a pipeline from genome to metalloproteome, several bottlenecks to high-throughput determination of metal-site structures must be overcome. For example, automation of arraying small samples for XAS examination must be invented, automation of rapid data collection of multiple low-volume low-concentration samples must be developed, automation of data reduction and analysis must be perfected. Discussed here are the promises and the pitfalls of HTXAS development, including the results of initial feasibility experiments. [ABSTRACT FROM AUTHOR]

Presents a study which concluded that cytochrome cy of Rhodobacter capsulatus is attached to the cytoplasmis membrane by an uncleaved signal sequence-like anchor. Research methodology; Statistical information gathered from the study; Analysis of results obtained.

Rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus maintains its native structure at high temperatures (373 K). In order to investigate the role of hydrogen bonding, hydration and chain dynamics in this thermostability, wavelength-resolved Laue neutron diffraction data have been collected from the W3Y single mutant (Trp3→Tyr3) on the spallation neutron protein crystallography station (PCS) at Los Alamos Neutron Science Center. Data were measured at room temperature from nine crystal settings, each of approximately 12 h duration. The total data-measurement period was less than 5 d from a single crystal that had undergone H[sub2]/D[sub2]O exchange. The nominal resolution of the data is 2.1 Å. [ABSTRACT FROM AUTHOR]

The active site of [NiFe] hydrogenase is an excellent catalyst for hydrogen conversion with an intriguing ability to recover from exposure to dioxygen. In their Communication on page 724 ff., W. Lubitz, S. P. Cramer, and co‐workers use the synchrotron technique of nuclear resonance vibrational spectroscopy to identify normal modes associated with active‐site FeCN and FeCO motion. [ABSTRACT FROM AUTHOR]

Das aktive Zentrum der [NiFe] ‐ Hydrogenase ist ein exzellenter Katalysator der Wasserstoffumsetzung mit der faszinierenden Fähigkeit, sich nach Sauerstoffexposition zurückzubilden. W. Lubitz, S. P. Cramer et al. nutzen in ihrer Zuschrift auf S. 752 ff. die Synchrotrontechnik der kernresonanten inelastischen Streuung, um Normalmoden der Fe ‐ CN ‐ und Fe ‐ CO ‐ Bewegung im aktiven Zentrum zu identifizieren. [ABSTRACT FROM AUTHOR]

Extremophile organisms are known that can metabolize at temperatures down to - 25 °C (psychrophiles) and up to 122 °C (hyperthermophiles). Understanding viability under extreme conditions is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Information about the stability and dynamics of proteins under environmental extremes is an important factor in this regard. Here we compare the dynamics of small Fe-S proteins - rubredoxins - from psychrophilic and hyperthermophilic microorganisms, using three different nuclear techniques as well as molecular dynamics calculations to quantify motion at the Fe site. The theory of 'corresponding states' posits that homologous proteins from different extremophiles have comparable flexibilities at the optimum growth temperatures of their respective organisms. Although 'corresponding states' would predict greater flexibility for rubredoxins that operate at low temperatures, we find that from 4 to 300 K, the dynamics of the Fe sites in these homologous proteins are essentially equivalent., (© 2024. The Author(s).)

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.

We used a novel experimental setup to conduct the first synchrotron-based (61)Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. (61)NiCr2O4 was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional (61)Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, (61)Ni{N(SiMe3)Dipp}2, where Dipp = C6H3-2,6-(i)Pr2, was chosen as a sample with an "extreme" geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based (61)Ni Mössbauer spectroscopy, we examined the spectra of (61)Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et4N]2[(61)Ni(SPh)4] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based (61)Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources.

The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe-4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe-4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe-S clusters.

Nuclear inelastic scattering of (57)Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.