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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. [ABSTRACT FROM AUTHOR]

Hydroxyprolines (Hyp) are non-standard amino acids derived from the post-translational modification of proteins by prolyl hydroxylase enzymes. Some plants and bacteria produce Hyp, and the isomers trans -3-Hydroxy- l -proline (T3LHyp) and trans -4-Hydroxy- l -proline (T4LHyp) are major components of mammalian collagen. While T4LHyp is metabolised following distinct degradative pathways in mammals and bacteria, T3LHyp metabolic pathway is conserved in bacteria, plants and mammals, and involves a T3LHyp dehydratase (T3LHypD) in the first degradation step. We report here the crystal structure of T3LHypD from the archaea Thermococcus litoralis in the free and substrate-complexed form. The model shows an "open" and a "closed" conformation depending on the presence (or absence) of the substrate in the catalytic site and allows the mapping of the residues involved in ligand recognition. Moreover, the structure highlights the presence of a water molecule interacting with the hydroxy group of the substrate and potentially involved in catalysis. The structure here reported is the first of its family to be elucidated, and represents a valid model for rationalising the substrate specificity and catalysis of T3LHyp dehydratases. • The metabolism of the collagen-derived trans -3-Hydroxy- l -proline involves a dehydratase reaction. • The paper reports the first crystal structure of a trans -3-Hydroxy- l -proline dehydratase. • The model shows an "open" and a "closed" conformation depending on the binding of the substrate. • The structure allows the mapping of the residues involved in substrate binding. • A water molecule interacts with the substrate –OH group, suggesting its involvement in catalysis. [ABSTRACT FROM AUTHOR]

We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product ( Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest k / K value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo- Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, K , of the FAD- Tl-LASPO complex was determined to be 5.9 × 10 M. [ABSTRACT FROM AUTHOR]

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A thermostable L-aminoacylase from Thermococcus litoralis was cloned, sequenced, and overexpressed in Escherichia coli. The enzyme is a homotetramer of 43 kDa monomers and has an 82% sequence identity to an aminoacylase from Pyrococcus horikoshii and 45% sequence identity to a carboxypeptidase from Sulfolobus solfataricus. It contains one cysteine residue that is highly conserved among aminoacylases. Cell-free extracts of the recombinant enzyme were characterized and were found to have optimal activity at 85°C in Tris-HCl at pH 8.0. The recombinant enzyme is thermostable, with a half-life of 25 h at 70°C. Aminoacylase inhibitors, such as mono-tert-butyl malonate, had only a slight effect on activity. The enzyme was partially inhibited by EDTA and p- hydroxymercuribenzoate, suggesting that the cysteine residue and a metal ion are important, but not essential, for activity. Addition of Zn2+ and Co2+ to the apoenzyme increased the enzyme activity, whereas Sn4+ and Cu2+ almost completely abolished enzyme activity. The enzyme was most specific for substrates containing N-benzoyl- or N-chloroacetyl-amino acids, preferring substrates containing hydrophobic, uncharged, or weakly charged amino acids such as phenylalanine, methionine, and cysteine. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effect of pressure on protein structure and stability at high temperature. Thermoinactivation experiments at 5 and 500 atm were performed using the wild-type (WT) enzyme and two single mutants (D167T and T138E) of the glutamate dehydrogenase (GDH) from the hyperthermophile Thermococcus litoralis. All three GDHs were stabilized, although to different degrees, by the application of 500 atm. Interestingly, the degree of pressure stabilization correlated with GDH stability as well as the magnitude of electrostatic repulsion created by residues at positions 138 and 167. Thermoinactivation experiments also were performed in the presence of trehalose. Addition of the sugar stabilized all three GDHs; the degree of sugar-induced thermostabilization followed the same order as pressure stabilization. Previous studies suggested a mechanism whereby the enzyme adopts a more compact and rigid structure and volume fluctuations away from the native state are diminished under pressure. The present results on the three GDHs allowed us to further confirm and refine the proposed mechanism for pressure-induced thermostabilization. In particular, we propose that pressure stabilizes against thermoinactivation by shifting the equilibrium between conformational substates of the GDH hexamer, thus inhibiting irreversible aggregation. [ABSTRACT FROM AUTHOR]

A novel NADH-dependent glyoxylate reductase has been found in a hyperthermophilic archaeon Thermococcus litoralis DSM 5473. This is the first evidence for glyoxylate metabolism and its corresponding enzyme in hyperthermophilic archaea. NADH-dependent glyoxylate reductase was purified ≈ 560-fold from a crude extract of the hyperthermophile by five successive column chromatographies and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 76 kDa, and the enzyme consisted of a homodimer with a subunit molecular mass of ≈ 37 kDa. The optimum pH and temperature for enzyme activity were ≈ 6.5 and 90 °C, respectively. The enzyme was extremely thermostable; the activity was stable up to 90 °C. The glyoxylate reductase catalyzed the reduction of glyoxylate and hydroxypyruvate, and the relative activity for hydroxypyruvate was ≈ one-quarter that of glyoxylate in the presence of NADH as an electron donor. NADPH exhibited rather low activity as an electron donor compared with NADH. The Km values for glyoxylate, hydroxypyruvate, and NADH were determined to be 0.73, 1.3 and 0.067 mm, respectively. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the glyoxylate reductase gene was determined and found to encode a peptide of 331 amino acids with a calculated relative molecular mass of 36 807. The amino-acid sequence of the T. litoralis enzyme showed high similarity with those of probable dehydrogenases in Pyrococcus horikoshii and P. abyssi. The purification of the enzyme from recombinant E. coli was much simpler compared with that from T. litoralis; only two steps of heat treatment and dye-affinity chromatography were needed. [ABSTRACT FROM AUTHOR]

The ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51,265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53,621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed. [ABSTRACT FROM AUTHOR]

4-α-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°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 α-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 α-l,4-glucan, but also intramolecular transglycosylation to produce cyclic α-l,4-glucan (cycloamylose), similarly to potato 4-α-glucanotransferase (called disproportionating enzyme). The gene encoding the Z litoralis 4-α-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. titoralis enzyme showed high similarity with those of α-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-α-glucanotransferases. [ABSTRACT FROM AUTHOR]