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PubMed1996

2D 1H and 3D 1H-15N NMR of zinc-rubredoxins: contributions of the beta-sheet to thermostability.

Based on 2D 1H-1H and 2D and 3D 1H-15N NMR spectroscopies, complete 1H NMR assignments are reported for zinc-containing Clostridium pasteurianum rubredoxin (Cp ZnRd). Complete 1H NMR assignments are also reported for a mutated Cp ZnRd, in which residues near the N-terminus, namely, Met 1, Lys 2, and Pro 15, have been changed to their counterparts, (-), Ala and Glu, respectively, in rubredoxin from the hyperthermophilic archaeon, Pyrococcus furiosus (Pf Rd). The secondary structure of both wild-type and mutated Cp ZnRds, as determined by NMR methods, is essentially the same. However, the NMR data indicate an extension of the three-stranded beta-sheet in the mutated Cp ZnRd to include the N-terminal Ala residue and Glu 15, as occurs in Pf Rd. The mutated Cp Rd also shows more intense NOE cross peaks, indicating stronger interactions between the strands of the beta-sheet and, in fact, throughout the mutated Rd. However, these stronger interactions do not lead to any significant increase in thermostability, and both the mutated and wild-type Cp Rds are much less thermostable than Pf Rd. These correlations strongly suggest that, contrary to a previous proposal [Blake PR et al., 1992, Protein Sci 1:1508-1521], the thermostabilization mechanism of Pf Rd is not dominated by a unique set of hydrogen bonds or electrostatic interactions involving the N-terminal strand of the beta-sheet. The NMR results also suggest that an overall tighter protein structure does not necessarily lead to increased thermostability.

Amino Acid Sequence↗
PubMed2006

The role of backbone stability near Ala44 in the high reduction potential class of rubredoxins.

Rubredoxins may be separated into high and low reduction potential classes, with reduction potentials differing by approximately 50 mV. Our previous work showed that a local shift in the polar backbone due to an A(44) versus V(44) side-chain size causes this reduction potential difference. However, this work also indicated that in the low potential Clostridium pasteurianum (Cp) rubredoxin, a V(44) --> A(44) mutation causes larger local backbone flexibility, because the V(44) side-chain present in the wild-type (wt) is no longer present to interlock with neighboring residues to stabilize the subsequent G(45). Since Pyrococcus furiosus (Pf) and other high potential rubredoxins generally have a P(45), it was presumed that a G(45) --> P(45) mutation might stabilize a V(44) --> A(44) mutation in Cp rubredoxin. Here crystal structure analysis, energy minimization, and molecular dynamics (MD) were performed for wt V(44)G(45), single mutant A(44)G(45) and double mutant A(44)P(45) Cp, and for wt A(44)P(45) Pf rubredoxins. The local structural, dynamical, and electrostatic properties of Cp gradually approach wt Pf in the order wt Cp to single to double mutant because of greater sequence similarity, as expected. The double mutant A(44)P(45) Cp exhibits increased backbone stability near residue 44 and thus enhances the probability that the backbone dipoles point toward the redox site, which favors an increase in the electrostatic contribution to the reduction potential. It appears that the electrostatic potential of residue 44 and the solvent accessibility to the redox are both determinants for the reduction potentials of homologous rubredoxins. Overall, these results indicate that an A(44) in a rubredoxin may require a P(45) for backbone stability whereas a V(44) can accommodate a G(45), since the valine side-chain can interlock with its neighbors.

Alanine↗
PubMed1995

Molecular dynamics simulations of rubredoxin from Clostridium pasteurianum: changes in structure and electrostatic potential during redox reactions.

Molecular dynamics simulations of Clostridium pasteurianum rubredoxin in the oxidized and reduced forms have been performed. Good agreement between both forms and crystal data has been obtained (rms deviation of backbone atoms of 1.06 and 1.42 A, respectively), which was due in part to the use of explicit solvent and counterions. The reduced form exhibits an unexpected structural change: the redox site becomes much more solvent-accessible, so that water enters a channel between the surface and the site, but with little actual structural rearrangement (the rms deviation of backbone atoms between the oxidized and reduced is 0.77 A). The increase in solvent accessibility is also seen, although to a much lesser extent, between the oxidized and reduced crystal structures of Pyrococcus furiosus rubredoxin, but no high resolution crystal or nuclear magnetic resonance solution data exist for reduced C. pasteurianum rubredoxin. The electrostatic potential at the iron site and fluctuations in the potential, which contribute to both the redox and electron transfer properties, have also been evaluated for both the oxidized and the reduced simulations. These results show that the backbone plays a significant role (62-70 kcal/mol/e) and the polar side chains contribute relatively little (0-4 kcal/mol/e) to the absolute electrostatic potential at the iron of rubredoxin for both forms. However, both groups contribute significantly to the change in redox state by becoming more polarized and more densely packed around the redox site upon reduction. Furthermore, these results show that the solvent becomes much more polarized in the reduced form than in the oxidized form, even excluding the penetrating water. Finally, the simulation indicates that the contribution of the charged side chains to the electrostatic potential is largely canceled by that of the counterions.

Algorithms↗
PubMed2004

Unique sugar metabolism and novel enzymes of hyperthermophilic archaea.

Hyperthermophiles are a group of microorganisms that have their optimum growth temperature above 80 degrees C. More than 60 species of the hyperthermophiles have been isolated from marine and continental volcanic environments. Most hyperthermophiles belong to Archaea, the third domain of life, and are considered to be the most ancient of all extant life forms. Recent studies have revealed the presence of unusual sugar metabolic processes in hyperthermophilic archaea, for example, a modified Embden-Meyerhof pathway, that has so far not been observed in bacteria and eucarya. Several novel enzymes, such as ADP-dependent glucokinase, ADP-dependent phosphofructokinase, glyceraldehyde-3-phosphate ferredoxin oxidoreductase, phosphoenolpyruvate synthase, pyruvate : ferredoxin oxidoreductase, and ADP-forming acetyl-CoA synthetase, have been found to be involved in a modified Embden-Meyerhof pathway of the hyperthermophilic archaeon Pyrococcus furiosus. In addition, a unique mode of ATP regeneration has been postulated to exist in the pathway of P. furiosus. The metabolic design observed in this microorganism might reflect the situation at an early stage of evolution.

Amino Acid Sequence↗
PubMed1996

Identification of the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans as bis(molybdopterin guanine dinucleotide)molybdenum.

Chemical analysis of dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans has shown that its molybdenum center contains two molybdopterin guanine dinucleotide molecules and a single atom of molybdenum. The enzyme, which exists as a monomer of 86 kDa, was shown to contain 1 mol of molybdenum, 4 mol of organic phosphate, and 2 mol of guanine per mole of protein. In addition, the relative yield of Form A, a fluorescent derivative of molybdopterin, was twice that obtained from sulfite oxidase, a protein which contains a single molybdopterin per molybdenum. These findings correlate with the recent report of the presence of two molybdopterin ligands in the tungsten cofactor of aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus, providing the first example of a bis(pterin)molybdenum cofactor and extending this structural motif to the molybdopterin dinucleotide enzymes.

Guanine↗
PubMed1993

Low incorporation of dUMP by some thermostable DNA polymerases may limit their use in PCR amplifications.

Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.

Bacteria↗
PubMed1997

Crystal structure of glutamate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima at 3.0 A resolution.

The extremely thermostable glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been crystallized and the three-dimensional structure has been determined by X-ray diffraction methods. Crystals up to a maximum size of 1.2 mm have been grown in 3% polyethylene glycol, 120 mM ammonium acetate and 50 mM bis-tris propane (pH 6.5). The enzyme crystallized in the trigonal space group P3(1)21 with the cell dimensions a = b = 147.3 A, c = 273.6 A. The diffraction limit of these crystals is 3.0 A. Measured diffraction data have a completeness of 94% up to a resolution of 3.0 A and contain 75% of all possible data in the last resolution shell between 3.1 and 3.0 A. The crystal structure of T. maritima glutamate dehydrogenase has been solved by Patterson search methods using the hexameric Pyrococcus furiosus glutamate dehydrogenase as a search model. The crystallographic refinement has been carried out to a maximum resolution of 3.1 A and an crystallographic R-value of 22.5% (Rfree = 29.5%). The three-dimensional structure of the T. maritima enzyme shows typical features of hexameric glutamate dehydrogenases: six subunits are arranged in 32 symmetry. Each subunit consists of two domains connected by a flexible hinge region. Secondary structure elements as well as residues important for the catalytic activity of the enzyme are highly conserved. A structural comparison of the two glutamate dehydrogenases from the hyperthermophiles T. maritima and P. furiosus with the enzyme from the mesophilic bacterium Clostridium symbiosum has revealed that common as well as distinct mechanisms contribute to the thermal stability of these enzymes. The number of intrasubunit ion pairs is increased and the volume of intrasubunit cavities decreased in both thermostable enzymes, whereas striking differences have been observed in the subunit interfaces. In P. furiosus glutamate dehydrogenase the subunit interactions are dominated by ionic interactions realized by large saltbridge networks. However, in T. maritima glutamate dehydrogenase the number of intersubunit ion pairs is reduced and the hydrophobic interactions are increased.

Amino Acid Sequence↗
PubMed1998

Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. I. Introduction of a six-residue ion-pair network in the hinge region.

Comparison of the recently determined three-dimensional structures of several glutamate dehydrogenases allowed for the identification of a five-residue ion-pair network in the hinge region of Pyrococcus furiosus glutamate dehydrogenase (melting temperature 113 degrees C), that is not present in the homologous glutamate dehydrogenase from Thermotoga maritima (melting temperature 93 degrees C). In order to study the role of this ion-pair network, we introduced it into the T. maritima enzyme using a site-directed mutagenesis approach. The resulting T. maritima glutamate dehydrogenases N97D, G376 K and N97D/G376 K as well as the wild-type enzyme were overproduced in Escherichia coli and subsequently purified. Elucidation of the three-dimensional structure of the double mutant N97D/G376 K at 3.0 A, showed that the designed ion-pair interactions were indeed formed. Moreover, because of interactions with an additional charged residue, a six-residue network is present in this double mutant. Melting temperatures of the mutant enzymes N97D, G376 K and N97D/G376 K, as determined by differential scanning calorimetry, did not differ significantly from that of the wild-type enzyme. Identical transition midpoints in guanidinium chloride-induced denaturation experiments were found for the wild-type and all mutant enzymes. Thermal inactivation at 85 degrees C occured more than twofold faster for all mutant enzymes than for the wild-type glutamate dehydrogenase. At temperatures of 65 degrees C and higher, the wild-type and the three mutant enzymes showed identical specific activities. However, at 58 degrees C the specific activity of N97D/G376 K and G376 K was found to be significantly higher than that of the wild-type and N97D enzymes. These results suggest that the engineered ion-pair interactions in the hinge region do not affect the stability towards temperature or guanidinium chloride-induced denaturation but rather affect the specific activity of the enzyme and the temperature at which it functions optimally.

Enzyme Stability↗
PubMed1999

Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.

Amino Acid Substitution↗
PubMed2001

Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2.

In the final stages of genetic recombination, Holliday junction resolving enzymes transform the four-way DNA intermediate into two duplex DNA molecules by introducing pairs of staggered nicks flanking the junction. This fundamental process is apparently common to cells from all three domains of life. Two cellular resolving enzymes from extremely thermophilic representatives of both kingdoms of the domain Archaea, the euryarchaeon Pyrococcus furiosus and the crenarchaeon Sulfolobus solfataricus, have been described recently. Here we report for the first time the isolation, purification and characterization of Holliday junction cleaving enzymes (Hjc) from two archaeal viruses. Both viruses, SIRV1 and SIRV2, infect Sulfolobus islandicus. Their Hjcs both consist of 121 amino acid residues (aa) differing only by 18 aa. Both proteins bind selectively to synthetic Holliday-structure analogues with an apparent dissociation constant of 25 nM. In the presence of Mg(2+) the enzymes produce identical cleavage patterns near the junction. While S. islandicus shows optimal growth at about 80 degrees C, the nucleolytic activities of recombinant SIRV2 Hjc was highest between 45 degrees C and 70 degrees C. Based on their specificity for four-way DNA structures the enzymes may play a general role in genetic recombination, DNA repair and the resolution of replicative intermediates.

Amino Acid Sequence↗
PubMed1994

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes.

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-alpha and GSI-beta. GSI-alpha-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI-beta-type genes occur in all other bacteria. GSI-alpha- and GSI-beta-type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision.

Amino Acid Sequence↗
PubMed1995

Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments.

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon. In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism. The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating an alpha beta gamma delta structure. Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent Km for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent Vmax of 64 U/mg (at 65 degrees C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70 degrees C). The temperature optimum was above 90 degrees C. At 90 degrees C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the alpha-subunit had similarities to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate:ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the alpha-subunits of the enzyme from A. fulgidus and the heterodimeric pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

Amino Acid Sequence↗
PubMed1999

DNA repair systems in archaea: mementos from the last universal common ancestor?

DNA repair in the Archaea is relevant to the consideration of genome maintenance and replication fidelity in the last universal common ancestor (LUCA) from two perspectives. First, these prokaryotes embody a mix of bacterial and eukaryal molecular features. Second, DNA repair proteins would have been essential in LUCA to maintain genome integrity, regardless of the environmental temperature. Yet we know very little of the basic molecular mechanisms of DNA damage and repair in the Archaea in general. Many studies on DNA repair in archaea have been conducted with hyperthermophiles because of the additional stress imposed on their macromolecules by high temperatures. In addition, of the six complete archaeal genome sequences published so far, five are thermophilic archaea. We have recently shown that the hyperthermophile Pyrococcus furiosus has an extraordinarily high capacity for repair of radiation-induced double-strand breaks and we have identified and sequenced several genes involved in DNA repair in P. furiosus. At the sequence level, only a few genes share homology with known bacterial repair genes. For instance, our phylogenetic analysis indicates that archaeal recombinases occur in two paralogous gene families, one of which is very deeply branched, and both recombinases are more closely related to the eukaryotic RAD51 and Dmc1 gene families than to the Escherichia coli recA gene. We have also identified a gene encoding a repair endo/exonuclease in the genomes of several Archaea. The archaeal sequences are highly homologous to those of the eukaryotic Rad2 family and they cluster with genes of the FEN-1 subfamily, which are known to be involved in DNA replication and repair in eukaryotes. We argue that there is a commonality of mechanisms and protein sequences, shared between prokaryotes and eukaryotes for several modes of DNA repair, reflecting diversification from a minimal set of genes thought to represent the genome of the LUCA.

Amino Acid Sequence↗
PubMed2000

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation.

We have studied four proteins containing oxidized 3Fe clusters ([Fe3S4]+, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T1, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2-4.2 K. The T1, values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g3 values and in 57 Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T1 is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at approximately 20 cm(-1) above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported 57Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J13=J, J12=J(1+epsilon'), J23=J(1+epsilon), where Jij is the isotropic exchange coupling between ferric ions i and j, and epsilon' and epsilon' are measures of coupling inequivalence. We have extended their theory to include the effects of epsilon' not equal to 0 and thus derived an exact expression for the energy of the doublet excited state for any epsilon, epsilon'. This excited state energy corresponds roughly to epsilonJ and is in the range 5-10 cm(-1) for each of these four 3Fe proteins. This magnitude of the product epsilonJ, determined by our time-domain relaxation studies in the temperature range 2-4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5-50 K, NMR studies in the range 293-303 K, and static susceptibility measurements in the range 1.8-200 K. We suggest that an apparent disagreement as to the individual values of J and epsilon be resolved in favor of the values obtained by susceptibility and NMR (J > or approximately 200 cm(-1) and epsilon> or =0.02 cm(-1)). as opposed to a smaller J and larger r as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters.

Aconitate Hydratase↗
PubMed2002

Updating carbamoylphosphate synthase (CPS) phylogenies: occurrence and phylogenetic identity of archaeal CPS genes.

Among Bacteria the carA and carB genes encoding the small (CarA) and large (CarB) subunits of carbamoylphosphate synthase (CPS) have been lost in certain symbionts (Haemophylus influenzae) and in most obligate intracellular parasites (Chlamydiae, Spirochaetes, Mycoplasmatales, Rickettsiae) having genome sizes in the 0.7- to 1.1-Mb range. Compared to Bacteria, Archaea exhibit a more varied pattern of CPS gene losses and an unusual propensity to incorporate CPS genes derived from both Bacteria and other Archaea. Schematically they fall into three groups. Group 1 taxa (the crenarchaeon Aeropyrum pernix and the euryarchaea Pyrococcus horikoshi and Pyrococcus abyssii) lack CPS genes altogether. Group 2 taxa (comprising Halobacteriales, Thermoplasmales, Methanococcales, Methanomicrobiales, Archaeoglobales) harbor CPS genes whose encoded CarB and CarA subunit proteins are ostensibly bacterial in origin; that is, they are intermixed with bacterial homologues on a phylogeny of concatenated CarA and CarB sequences and are not distinguishable from bacterial sequences after searching for domain-specific amino acid residue positions. Group 3 taxa (the crenarchaea Pyrobaculum aerophilum, Sulfolobus solfataricus, and Sulfolobus tokodaii and the euryarchaeon Pyrococcus furiosus) harbor CPS genes whose encoded proteins appear to be archaeal: consistent with an archaeal origin, the CarA and CarB sequences in this group possess both unique signatures and signatures affiliating them to Eukarya. Based on the topology of the clade comprising the four Group 3 taxa, we argue that CPS genes of P. furiosus (a euryarchaeon) and those of the crenarchaea P. aerophilum, S. solfataricus, and S. tokodaii are of a single type, resulting from the two genes being laterally transferred from a crenarchaeon to P. furiosus.

Amino Acid Motifs↗
PubMed2001

Both pH and carbon flux influence the level of rubredoxin in Clostridium butyricum.

The rubredoxin expression level in Clostridium butyricum DSM 5431 grown in continuous culture was monitored using primer extension analyses of the rub gene and a specific enzymatic assay of the iron-sulfur protein. In this way, we showed that variations in rubredoxin content and in rub mRNA level were influenced by the pH of the culture and were directly dependent on the carbon flux. The maximum rubredoxin level reached 1227.3 pmol (mg of proteins)(-1) (i.e. 0.7% of the total protein content) under strictly anaerobic conditions when cells grew at pH 6.5 with an excess of glucose. In addition, primer extension analyses established that the control for all the variations observed operates at the level of gene transcription. Altogether, these results suggested a main function of rubredoxin in Clostridium butyricum independent of the protection against oxygen as has already been reported for Desulfovibrio gigas and Pyrococcus furiosus.

Bacterial Proteins↗
PubMed2004

Contribution of the [FeII(SCys)4] site to the thermostability of rubredoxins.

The thermostabilities of Fe(2+) ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH.S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)(4)] site, and substitutions at this location affect the redox properties of the [Fe(SCys)(4)] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V "exchange" mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)(4)] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn(2+), Cd(2+)) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and (1)H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [Fe(II)(SCys)(4)] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [Fe(II)(SCys)(4)] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)(4)] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H(+), Cd(2+)) on the Cys42 sulfur ligand at the [Fe(SCys)(4)] site occurs through the V8 side and not the V44 side of the iron site.

Amino Acid Sequence↗
PubMed2005

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum.

A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S](2+/1+) cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g(zyx) values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S](1+) cluster with S=3/2 and zero field splitting parameters D=7.5 cm(-1) and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at -306 and -445 mV. In the presence of 55 microM ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.

Aldehyde Oxidoreductases↗