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The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5ʹ-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.

Highlights • Ornithine cyclodeaminase homolog from an archeon was characterized biochemically. • This protein functions as a novel Δ1-pyrroline-2-carboxylate reductase. • This enzyme is probably involved in trans-3-hydroxy-l-proline metabolism as in bacteria and mammals., l-Ornithine cyclodeaminase (OCD) is involved in l-proline biosynthesis and catalyzes the unique deaminating cyclization of l-ornithine to l-proline via a Δ1-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD+-dependent l-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-l-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to l-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240–250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-l-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.

Thermococcus litoralis, a hyperthermophilic Archaeon, is able to reduce elemental sulfur during fermentative growth. An unusual gene cluster (nsoABCD) was identified in this organism. In silico analysis suggested that three of the genes (nsoABC) probably originated from Eubacteria and one gene (nsoD) from Archaea. The putative NsoA and NsoB are similar to NuoE- and NuoF-type electron transfer proteins, respectively. NsoC has a unique domain structure and contains a GltD domain, characteristic of glutamate synthase small subunits, which seems to be integrated into a NuoG-type sequence. Flavin and NAD(P)H binding sites and conserved cysteines forming iron-sulfur clusters binding motifs were identified in the protein sequences deduced. The purified recombinant NsoC contains one FAD cofactor per protein molecule and catalyzes the reduction of polysulfide with NADPH as an electron donor and it also reduces oxygen. It was concluded that the Nso complex is a new type of NADPH-oxidizing enzyme using sulfur and/or oxygen as an electron acceptor.

We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5′-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on l-cysteic acid and l-cysteine sulfinic acid in addition to d- and l-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. d-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells.

초호열성 고세균 Thermococcus litoralis 유래의 4-α-glucanotransferase는 클로닝되어 염기배열이 밝혀졌으며, 대장균에서 발현되었다. 발현된 이 효소는 기능적인 면에서는 D-enzyme과 유사하지만 아미노산 배열에서는 큰 차이점을 나타내었다. 이 효소는 cycloamylose를 생산하는 새로운 기능적인 특성을 가지고 있어 당대사 관련 효소 단백질에 관한 연구의 중요성 때문에 산업적으로 많은 각광을 받고 있다. 본 연구는 초호열성 고세균 T. litoralis 유래 4-α-glucanotransferase 유전자를 부위 특이적 변이 방법으로 재조합하여 lac와 T7 프로모터를 이용해서 대장균 발현 벡터 시스템에서 대량발현 시켰다. 대장균에서 대량 발현된 재조합 효소 단백질은 열처리, Butyl-Toyopearl, Mono Q 크로마티그래피 방법에 의하여 간단히 정제되었다. 정제된 재조합 효소 단백질은 본래의 효소 단백질과 같은 기능을 가지고 있는 것으로 확인되었다.

The gene encoding pyrrolidone carboxyl peptidase (Pcp) has been cloned from the hyperthermophilic archaeon Thermococcus litoralis. The recombinant enzyme has been expressed in Escherichia coli, purified, and char-acterized. The T. litoralis Pcp demonstrates strong sequence homology to previously characterized bacterial Pcps. Some investigations have been carried out on enzyme substrate specificity and stability.

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.

Thermococcus litoralis 4-α-glucanotransferase (GTase) belongs to family 57 of glycosyl hydro-lases and catalyzes the disproportionation and cycloamylose synthesis reactions. Using 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (3 KBG 5 CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of 4-α-glucanotransferase involves a Ping-Pong Bi Bi mechanism. Based on this reaction mechanism, we trapped the glycosyl-enzyme intermediate by treating the enzyme with 3 KBG 5 CNP in the absence of an acceptor. MALDITOFMS and PSD analysis of a peptic digest of the intermediate and great decrease in activity of the E 123 Q mutant enzyme indicated that completely conserved Glu 123 was the catalytic amino acid. Next, we have determined the structure of GTase, the first structure of the family 57 of glycosyl hydrolases, at 2.8 A resolution. The structure of GTase is composed of three domains. N-terminal domain (domain A) contains two catalytic residues and has a novel pseudo-TIM barrel fold. C-terminal domain (domain C) is mainly made of β-strands arranged in two layered β-sheet sandwich. Domain B is located between domains A and C, and consists of α- and 310-helices. Active site cleft lies between domains A and B. The cleft is partially covered with aromatic residues to form a tunnel-like shape, which probably plays an important role in the formation of large cyclic glucans.

DNA polymerase of the archaeon Thermococcus litoralis can synthesize a long stretch of linear double-stranded DNA in the complete absence of added primer and template DNAs. This finding suggests that genetic information can potentially be created by protein. We report here the effects of temperature, ionic strength and pH on this ab initio DNA synthesis by the protein in vitro . When the temperature of the reaction was changed, the sequence of the product DNA changed markedly. For instance, the reaction products were (TAAT) n at 69 degrees C, (TATCCGGA) n at 84 degrees C and (TATCGCGATAGCGATCGC) n at 89 degrees C. The ionic strength of the reaction condition also affected the sequence: it was (TATCTAGA) n with 0 mM KCl, (TATATACG) n with 50 mM KCl and (TATAGTTATAAC) n with 100 mM KCl at 74 degrees C. When the pH of the reaction condition was changed from 6.8 to 10.8, the size of the product DNA decreased, but its sequence did not. These results demonstrate that DNA synthesized ab initio by DNA polymerase of T.litoralis is markedly influenced by the reaction conditions. The results also suggest that genetic information that might have been created by protein on the early earth is strongly influenced by environmental factors.

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.

Thermococcus litoralis is a hyperthermophilic archaeon that grows at temperatures up to 98 °C by fermentative metabolism and reduces elemental sulfur (S0) to H2S. A [NiFe] hydrogenase, responsible for H2S or H2 production, has been purified and characterized. The enzyme is composed of four subunits with molecular mass 46, 42, 34 and 32 kDa. Elemental analyses gave approximate values of 22 Fe, 22 S and 1 Ni per hydrogenase. EPR spectra at 70 and 5 K indicated the presence of four or five [4Fe-4S] and one [2Fe-2S] type clusters. The optimal temperature for both H2 evolution and oxidation, using artificial electron carriers, was around 80 °C. The operon encoding the T. litoralis enzyme is composed of four genes forming one transcriptional unit, and transcription is not regulated by S0. An unusual transcription-initiation site is located 139 bp upstream from the translational start point. Sequence analyses indicated the presence of new putative nucleotide-binding domains. Upstream from the hydrogenase operon, ORFs probably encoding a molybdopterin oxidoreductase enzyme have been identified. Based on sequence, biochemical and biophysical analyses, a model of the enzyme and the pathway of electron flow during catalysis is proposed.

Nutritional characteristics of the hyperthermophilic archaeon Thermococcus litoralis have been investigated with emphasis on the development of a sulfur-free, defined growth medium, analysis of an exocellular polysaccharide, and formation of a biofilm. An artificial-seawater-based medium, containing 16 amino acids, adenine, uracil, vitamins, and trace elements, allowed T. litoralis to attain growth rates and cell densities similar to those found with complex media. Four amino acids (alanine, asparagine, glutamine, and glutamate) were not included due to their lack of effect on growth rates and cell yields. In this medium, cultures reached densities of 10(sup8) cells per ml, with doubling times of 55 min (without maltose) or 43 min (with maltose). Neither the addition of elemental sulfur nor the presence of H(inf2) significantly affected cell growth. A sparingly soluble exopolysaccharide was produced by T. litoralis grown in either defined or complex media. Analysis of the acid-hydrolyzed exopolysaccharide yielded mannose as the only monosaccharidic constituent. This exopolysaccharide is apparently involved in the formation of a biofilm on polycarbonate filters and glass slides, which is inhabited by high levels of T. litoralis. Biofilm formation by hyperthermophilic microorganisms in geothermal environments has not been examined to any extent, but further work in this area may provide information related to the interactions among high-temperature organisms.

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 Thermococcus litoralis strain NS-C, first isolated in 1985, has been a foundational organism for archaeal research in biocatalysis, DNA replication, metabolism, and the discovery of inteins. Here, we present the genome sequence of T. litoralis with a focus on the replication machinery and inteins.

Glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from a hyperthermophilic archaeon, Thermococcus litoralis DSM 5473 was purified to homogeneity from the crude extract. The enzyme had a molecular mass of about 300 kDa and consisted of six subunits with identical molecular masses of 37 kDa. The enzyme was extremely themostable; the activity was not lost after incubation at 95°C for 30 min and at pH 7–11 at 80°C for 20 min. The maximum enzyme activity in L-glutamate deamination was obtained around 95°C. Both optimum pHs for L-glutamate deamination and a-ketoglutarate amination were around 7.8. The enzyme exclusively catalyzed the oxidative deamination of L-glutamate in the presence of NADP but showed low amino acceptor specificity; several α-keto acids such as α-ketocaporoate, α-ketovalerate and pyruvate were also aminated as well as α-ketoglutarate in the presence of NADPH and ammonia. Michaelis constants for the substrates were as follows: NADP, 0.045 mM; L-glutamate, 2.1...

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)

We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C. Our results show that at 25 degrees C the binding of Glc to TMBP is well represented by a bimodal model with apparent K(d) of 20 muM and approximately 3-8 mM for the first and the second binding step, respectively. At 60 degrees C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K(d) value of about 40 muM. Finally, at 85 degrees C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermococcus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Furthermore, we have tested the optical response of the protein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection.

Background Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis. Results An about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a f ormate d eh ydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, m embrane-bound hy drogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase. Conclusion The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY) complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose uptake system. In the hyperthermophilic archaeon T. litoralis this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity. In this work we used differential scanning calorimetry studies together with a detailed analysis, at the molecular level, of the three-dimensional protein structure to shed light on the basis of the high thermostability exhibited by the recombinant TMBP from the archaeon T. litoralis. The obtained data suggest that the presence of trehalose does not change the overall mechanism of the denaturation of this protein but it selectively modifies the stability of the TMBP structural domains. Proteins 2007. © 2007 Wiley-Liss, Inc.

The enzyme L-aminoacylase catalyses the hydrolysis of N-acyl-L-amino acids from peptides or proteins. The recombinant enzyme from the hyperthermophilic archaeon Thermococcus litoralis has been purified to homogeneity. This zinc-containing enzyme has been crystallized from ammonium sulfate using the sitting-drop vapour-diffusion method. The crystals diffract to 2.8 A resolution and belong to the rhombohedral space group R32, with unit-cell parameters a = b = 102.4, c = 178.5 A, gamma = 120 degrees in a hexagonal lattice setting. The asymmetric unit contains one enzyme monomer, containing a single zinc ion. Two synchrotron data sets have been collected at a remote wavelength and at the maximum f'wavelength for zinc. This has allowed the position of the metal to be identified in anomalous Patterson maps.

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium.

Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases. We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli. We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties. At its optimal temperature of 80 degrees C, the apparent Km and Vmax values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration. The enzyme did not lose activity at 80 degrees C for 4 h. Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa. Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe2+, but inhibited by Zn2+ and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in kcat. The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.

We report the cloning, sequencing, and expression of malK encoding the ATP-hydrolyzing subunit of the maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis. According to the deduced amino acid sequence, MalK consists of 372 amino acids with a calculated molecular weight of 41,787. It shows 47% identity with the MalK protein of Escherichia coli and high sequence conservation in important regions. C-terminal His-tagged MalK was purified. The soluble protein appeared monomeric by molecular sieve chromatography and showed ATPase activity. Enzymatic activity was highest at 80 degrees C with a Km of 150 microM and a Vmax of 0.55 micromol of ATP hydrolyzed/min/mg of protein. ADP was not a substrate but a competitive inhibitor (Ki 230 microM). GTP and CTP were also hydrolyzed. ATPase activity was inhibited by N-ethylmaleimide but not by vanadate. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the ABC transporters in these two phylogenetic branches.

We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 Å resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

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.

Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis . Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p -nitrophenyl-α- d -glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis , but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A K m of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a K d of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli . Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

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.

L-Hydroxyproline (L-Hyp) is a nonstandard amino acid that is present in certain proteins, in some antibiotics and in the cell-wall components of plants. L-Hyp is the product of the post-translational modification of protein prolines by prolyl hydroxylase enzymes, and the isomers trans-3-hydroxy-L-proline (T3LHyp) and trans-4-hydroxy-L-proline (T4LHyp) are major components of mammalian collagen. T4LHyp follows two distinct degradation pathways in bacteria and mammals, while T3LHyp is metabolized by a two-step metabolic pathway that is conserved in bacteria and mammals, which involves a T3LHyp dehydratase and a Δ1-pyrroline-2-carboxylate (Pyr2C) reductase. In order to shed light on the structure and catalysis of the enzyme involved in the second step of the T3LHyp degradation pathway, the crystal structure of Pyr2C reductase from the archaeon Thermococcus litoralis DSM 5473 complexed with NADH and L-proline is presented. The model allows the mapping of the residues involved in cofactor and product binding and represents a valid model for rationalizing the catalysis of Pyr2C reductases.

Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay. Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis the protocol outlined here allows real time coupling of ATP consumption to downstream signal change enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose-6-phosphate dehydrogenase to reduce NAD(+) to NADH which can be measured at 340 nm. We have shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable background from ATP.