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Background: Pyrrolidone carboxyl peptidases (pcps) are a group of exopeptidases responsible for the hydrolysis of N-terminal pyroglutamate residues from peptides and proteins. The bacterial and archaeal pcps are members of a conserved family of cysteine proteases. The pcp from the hyperthermophilic archaeon Thermococcus litoralis is more thermostable than the bacterial enzymes with which it has up to 40% sequence identity. The pcp activity in archaea and eubacteria is proposed to be involved in detoxification processes and in nutrient metabolism; eukaryotic counterparts of the enzyme are involved in the processing of biologically active peptides. Results: The crystal structure of pcp has been determined by multiple isomorphous replacement techniques at 1.73 A resolution and refined to an R factor of 18.7% (R free = 21.4%). The enzyme is a homotetramer of single open α / β domain subunits, with a prominent hydrophobic core formed from loops coming together from each monomer. The active-site residues have been identified as a Cys143–His167–Glu80 catalytic triad. Structural homology to enzymes of different specificity and mechanism has been identified. Conclusions: The Thermococcus pcp has no sequence or structural homology with other members of the cysteine protease family. It does, however, show considerable similarities to other hydrolytic enzymes of widely varying substrate specificity and mechanism, suggesting that they are the products of divergent evolution from a common ancestor. The enhanced thermostability of the T. litoralis pcp may arise from hydrophobic interactions between the subunits and the presence of intersubunit disulphide bridges.

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.

A molecular model for the three-dimensional solution structure of the paramagnetic, four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been constructed on the basis of the reported 1H NMR spectral parameters [Donaire, A. (1996) J. Biomol. NMR 7, 35-47]. The conventional use of long mixing time NOESY cross-peak intensity, backbone angles, and hydrogenbonding constraints for building the structure was augmented by short mixing time NOESY, steady-state NOE, paramagnetic relaxation constraints, and the angular dependence of the ligated Cys H beta contact shifts. Distance geometry was used to generate various initial structures, and these structures were refined with the simulated annealing protocol. The family of structures with inconsequential violations exhibited low RMS deviations for the backbone except for a few residues in the immediate cluster vicinity and traces out a secondary structure very similar to those of the structurally characterized single cubane cluster Fds. The ability to describe the cluster environment depended on the use of numerous paramagnetic relaxation constraints which resulted in even the cluster loop residues exhibiting well-defined orientations, with the exception of one residue (Ilel1) whose 1H signals have not been located. Comparison of the structure of Tl Fd to those of mesophilic ferredoxins reveals that Tl Fd possesses the same secondary structural elements, two beta-sheets, two helices, and four turns, with the exception that the beta-sheet involving the termini incorporates a third strand in Tl Fd. Several minor structural adjustments in Tl Fd relative to other Fds, in addition to the third strand for beta-sheet, include the incorporation of the termini into the beta-sheet, a likely salt bridge from the side chain of the third beta-strand to the N-terminus, and a more hydrophobic and compact interaction between the large beta-sheet and the long helix. It is likely that each of these modifications, among others not yet well-defined (i.e., surface salt bridges), contributes to the extraordinary thermostability of Tl Fd.

The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98 degrees C using peptides and proteins as the sole sources of carbon and nitrogen. Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity. Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa. Using 2-oxoglutarate as the amino acceptor, each catalyzed the pyridoxal-5'-phosphate-dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine. From the determination of Km and kcat values using 2-oxoglutarate, phenylalanine, tyrosine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcat/Km approximately 400 s-1 mM-1); the 47-kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45-kDa one. Kinetic analyses indicated a two-step mechanism with a pyridoxamine intermediate. Both enzymes were virtually inactive at 30 degrees C and exhibited maximal activity between 95-100 degrees C. They showed no N-terminal sequence similarity with each other (approximately 30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escherichia coli and rat liver. The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon. In contrast, it is proposed that one or both play a catabolic role in proteolytic T. litoralis in which they generate glutamate and an arylpyruvate. These serve as substrates for glutamate dehydrogenase and indolepyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids.

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)

The hyperthermophile Archaeon Thermococcus litoralis transports both Disaccharide Trehalose and maltose by the same transportation complex from the medium in the cell inside. This first in the group the Archaea discovered ABC transporters could be characterized in this work nearer. The besides won knowledge was compared to the information already known bacterial bandage protein of dependent ABC transporters. One of the settings of tasks of this work was in addition to the already known information common characteristics and differences of the archaeellen Trehalose/Maltose-bandage protein TMBP with that of its bacterial homolog to lay the table to the maltose / Maltodextrin-Bindeprotein MBP. That's way became on the one hand, that in the membrane anchored bandage protein cleaned. By a staining method specific for Glykoproteine turned out that the TMBP is present as a Glykoprotein. Around a three-dimensional structure of the TMBP became to determine for the X-raying's structural analysis one, after cloning and heterologer expression in E. coli, protein cleaning method establishes itself. The structure of TMBP installs, the already described bandage proteins, homologous folds sample on. In at beginning of this work to confessed malEFG-sequence section nobody could be identified to the Energetisierung of the transportation required ABC domain. Therefore, the central project was the 'search' for the ABC domain. The corresponding gene could take place kloniert and the first characteristic going out from the DNA sequence. From 1119 bp were determined for malK 372 amino acids big protein arise. The comparison of the T. litoralis of MalK protein with ABC domains from other organisms showed that all sequences typical for these proteins exist. After successful heterologer expression in E. Coli was cleaned the protein and a detailed biochemic characteristic was executed.

A novel trehalose glycosyl transferring synthase (TreT) from the hyperthermophilic archaeon T. litoralis was identified. This enzyme produces trehalose by the transfer of glucose from ADP-glucose (or, less effectively, UDP-glucose and GDP-glucose) onto the 1-position of glucose to form trehalose and ADP. Such an enzymatic activity in the synthesis of trehalose has not yet been described for any other organisms. It catalyzes a reversible reaction for trehalose synthesis and degradation by converting glucose and ADP-glucose to trehalose and ADP. The enzyme properties were characterized biochemically. The physiological function of TreT in vivo is still not completely understood.An ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) was also identified and characterized in T. litoralis, which gene locates divergently to the trehalose/maltose transporter. The biochemical properties of this enzyme were characterized in this study.TrmB is a novel archaeal sugar-specific transcriptional repressor regulating the expression of trehalose/maltose ABC transporter dependent on the presence of maltose or trehalose in medium. TrmB binds maltose much stronger than trehalose. TrmB binds to the DNA fragments of the TATA box promoter region and upstream motif of malEFG-operon. TrmB does not bind to MalK.Two enzymes (TreT and FrK) have been identified in this research work and their expressional regulation by TrmB also has been investigated. The further interesting work can focus on the sucrose or trehalose metabolism pathway and the net work of sugar metabolism regulation in T. litoralis.

The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by 1H NMR spectroscopy. TOCSY and NOESY experiments in H2O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H2O and D2O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified in Tl Fd are a three-stranded antiparallel beta-strand involving the termini of the protein, a double beta-strand (also antiparallel), two alpha-helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the 'diamagnetic' protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas [Kissinger et al. (1991) J. Mol. Biol., 219, 693-723]. The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.

4-Alpha-Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optimal temperature for its activity was 90 degrees C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive saccharides were produced. On incubation of amylose with the T. litoralis enzyme, glucoamylase-resistant but alpha-amylase-sensitive molecules were produced, but the amount of reducing sugar showed only slight increases. These results indicate that the T. litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear alpha-1,4-glucan, but also intramolecular transglycosylation to produce cyclic alpha-1,4-glucan (cycloamylose), similarly to potato 4-alpha-glucanotransferase (called disproportionating enzyme). The gene encoding the T. litoralis 4-alpha-glucanotransferase was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77,883 Da. The amino acid sequence of the T. litoralis enzyme showed high similarity with those of alpha-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-alpha-glucanotransferases.

The NADP(+)-dependent glutamate dehydrogenase from Thermococcus litoralis has been crystallized by the hanging-drop method of vapour diffusion using an ammonium sulfate and PEG mixture as the precipitant. The crystals belong to the monoclinic system and are in space group C2 with unit-cell dimensions a = 142.7, b = 202.0, c = 125.8 A with beta = 113.1 degrees with a hexamer in the asymmetric unit. T. Litoralis, a hyperthermophilic organism, belongs to the family of Archaea and has a maximum growth temperature of about 370 K. The glutamate dehydrogenase isolated from this organism has a half-life of 2 h at 373 K and a comparison of this structure with that of other GluDH's from hyperthermophilic organisms and from mesophiles will contribute to an understanding of the molecular mechanisms which underlie thermostability.

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. Its growth is stimulated by tungsten, and a tungsten-containing iron-sulfur protein that has formaldehyde ferredoxin oxidoreductase (FOR) activity has been purified. FOR is a homotetramer with a subunit M(r) of 70,000. It contains approximately four irons, four acid-labile sulfides, and one tungsten atom per subunit. The tungsten appears to be present as a pterin cofactor, and the Fe/S seems to comprise an unusual [4Fe-4S] cluster that in the reduced state exists in a pH-independent S = 3/2 form and a pH-dependent S = 1/2 form. FOR catalyzed the oxidation of C1-C3 aldehydes with a temperature optimumor = 90 degrees C and used T. litoralis ferredoxin as an electron acceptor. It did not oxidize aldehyde phosphates, utilize CoASH, or reduce NAD(P). The N-terminal sequence of FOR shows homology with the tungsto-iron-sulfur aldehyde ferredoxin oxidoreductase previously purified from the saccharolytic, hyperthermophilic archaeon Pyrococcus furiosus, in which it is proposed to function in a novel pyroglycolytic pathway (Mukund, S., and Adams, M. W. W. (1991) J. Biol. Chem. 266, 14208-14216). We show here that P. furiosus, which will also grow on peptides, albeit poorly, contains a second aldehyde-oxidizing enzyme analogous to FOR. Similarly, T. litoralis, which utilizes saccharides if limited for peptides, contains low concentrations of an enzyme analogous to AOR. It is proposed that formaldehyde (apparent Km, 62 mM) is not the true substrate for FOR; rather, the enzyme has an as yet unknown role in peptide fermentation in hyperthermophilic archaea.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli , and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase.

Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli. Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms. The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.

The crystal structure of a novel alanine:glyoxylate aminotransferase from the hyperthermophilic archaeon Thermococcus litoralis was determined at 2.3 A resolution. The asymmetric unit contains four homologous subunits and the functional tetramer is generated by noncrystallographic 222 symmetry. Although the main-chain coordinates of the monomer of the Thermococcus litoralis enzyme showed a high degree of similarity to those of aspartate aminotransferase from Thermus thermophilus HB8, the amino-acid residues involved in substrate binding in the aspartate aminotransferase are only partially conserved in the Thermococcus litoralis enzyme. This may account for the difference in the substrate specificities of the two enzymes.

A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis . The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.

The sequence of genes encoding extracellular amylopullulanase and putative maltodextrin ATP binding cassette (ABC) transporter of a hyperthermophilic archaeon, Thermococcus litoralis, was determined. The mdxK gene, which encodes an ATPase subunit of the putative maltodextrin ABC transporter, has extraordinarily high similarity with the malK gene, which encodes an ATPase subunit of trehalose/maltose ABC transporter of the same organism. DNA sequence comparison revealed that the malK gene was generated through the duplication of the mdxK gene before lateral gene transfer of the mal gene cluster from T. litoralis to Pyrococcus furiosus.

Publisher Summary Pyrrolidone carboxylpeptidases (pcps) are a group of enzymes that remove pyroglutamate (pGlu) from the amino terminus of peptides and proteins. The enzymatic activity was initially identified in bacteria of the genus Pseudornonas I and has since been found in a wide variety of prokaryotes and eukaryotes. The pcp enzymes form a new class of cysteine peptidase enzymes. During the screening of crude extracts of the hyperthermophilic archaea for esterase/amidase activity of commercial interest, an activity was isolated from the hyperthermophilic archaeon Thermococcus litoralis that was later identified as a “pcp.” The archaeal pcp appears to be related to the bacterial pcp enzymes and to the class I mammalian enzymes. The exact function of the enzyme in bacterial and archaeal systems is less well studied. It has been proposed that the pcp activity may serve to reduce toxicity of N-terminally blocked peptides 14 or to play a role in nutrient assimilation.