The Lreu_1276 protein from represents a third family of dihydroneopterin triphosphate pyrophosphohydrolases in bacteria.

Unlabelled: Tetrahydrofolate is a cofactor involved in C metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon , we identified an enzyme which we designate "arginine synthetase".

Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon , an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway.

Development of a rapid and highly accurate method for C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 μl of 100 nmol/l amino acids comparable to a mere 1 [Formula: see text] 10-10 prokaryotic cells.

Crystal structure of GTP-dependent dephospho-coenzyme A kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases.

Biochemical and genetic examination of two aminotransferases from the hyperthermophilic archaeon .

The hyperthermophilic archaeon utilizes amino acids as a carbon and energy source. Multiple aminotransferases, along with glutamate dehydrogenase, are presumed to be involved in the catabolic conversion of amino acids. harbors seven Class I aminotransferase homologs on its genome.

Structural Determination and Chemical Synthesis of the N-Glycan from the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Asparagine-linked protein glycosylations (N-glycosylations) are one of the most abundant post-translational modifications and are essential for various biological phenomena. Herein, we describe the isolation, structural determination, and chemical synthesis of the N-glycan from the hyperthermophilic archaeon Thermococcus kodakarensis. The N-glycan from the organism possesses a unique structure including myo-inositol, which has not been found in previously characterized N-glycans.

A non-carboxylating pentose bisphosphate pathway in halophilic archaea.

Bacteria and Eucarya utilize the non-oxidative pentose phosphate pathway to direct the ribose moieties of nucleosides to central carbon metabolism. Many archaea do not possess this pathway, and instead, Thermococcales utilize a pentose bisphosphate pathway involving ribose-1,5-bisphosphate (R15P) isomerase and ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Intriguingly, multiple genomes from halophilic archaea seem only to harbor R15P isomerase, and do not harbor Rubisco.

A Lipoate-Protein Ligase Is Required for Lipoyl-Protein Biosynthesis in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner.

Regulation of thermoregulatory behavior by commensal bacteria in Drosophila.

Commensal bacteria affect many aspects of host physiology. In this study, we focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. We demonstrated that the elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons.

Identification and Enzymatic Analysis of an Archaeal ATP-Dependent Serine Kinase from the Hyperthermophilic Archaeon .

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce -phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis.

Altering the Phosphorylation Position of Pyrophosphate-Dependent -Inositol-1-Kinase Based on Its Crystal Structure.

Most kinases utilize ATP as a phosphate donor and phosphorylate a wide range of phosphate acceptors. An alternative phosphate donor is inorganic pyrophosphate (PPi), which costs only 1/1000 of ATP. To develop a method to engineer PPi-dependent kinases, we herein aimed to alter the product of PPi-dependent -inositol kinase from d-inositol 1-phosphate to d-inositol 3-phosphate.

Degradation of complex arabinoxylans by human colonic Bacteroidetes.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood.

TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Members of harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon The TK1211 gene was expressed in under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P ). The purified protein did not display aminotransferase activity but exhibited racemase activity.

A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit.

Total Syntheses of C60- and C100-Dolichols.

C60- and C100-dolichols were synthesized. A -selective Wittig reaction was achieved with high selectivity in a microflow system to realize the scalable supply of the -isoprene unit. An isoprene chain was efficiently elongated by an S2-type coupling between allyl sulfone and allyl chloride using -BuOK.

Different Proteins Mediate Step-Wise Chromosome Architectures in and .

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in , histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown.

Effects of high-level expression of A-ATPase on H production in Thermococcus kodakarensis.

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H fermentation. H production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H production is primary metabolism, here we aimed to partially uncouple cell growth and H production of T.

Structural Insight into [NiFe] Hydrogenase Maturation by Transient Complexes between Hyp Proteins.

[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The core unit of [NiFe] hydrogenase consists of a large and a small subunit. The active site of the large subunit of [NiFe] hydrogenases contains a NiFe(CN)CO cluster.

Integration of large heterologous DNA fragments into the genome of Thermococcus kodakarensis.

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii.

Thermophilic Degradation of Hemicellulose, a Critical Feedstock in the Production of Bioenergy and Other Value-Added Products.

Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks.

Crystal structure of pantoate kinase from Thermococcus kodakarensis.

The coenzyme A biosynthesis pathways in most archaea involve two unique enzymes, pantoate kinase and phosphopantothenate synthetase, to convert pantoate to 4'-phosphopantothenate. Here, we report the first crystal structure of pantoate kinase from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complex with ATP and a magnesium ion. The electron density for the adenosine moiety of ATP was very weak, which most likely relates to its broad nucleotide specificity.

The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

TrpY from is a regulator that inhibits transcription of the Trp biosynthesis () operon. Here, we show that the TrpY homolog in is not involved in such regulation. There are 87 genes on the genome predicted to encode transcriptional regulators (TRs).

Microbe Profile: : the model hyperthermophilic archaeon.

is a hyperthermophilic that grows well under laboratory conditions and, being naturally competent for genetic transformation, it has become a widely studied experimental model species. With the genome sequence available since 2004, combining genetic, enzymological and structural biochemical approaches has revealed previously unknown and unanticipated features of archaeal molecular biology and metabolism. DNA polymerase is already commercialized and with the details of metabolism and hydrogenase available, generating H from biopolymers solubilized at high temperatures, most notably chitin, now seems a very attractive possibility as a renewable energy bioprocess.

Distinct Modified Nucleosides in tRNA from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA mG10/mG10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures.

tRNA mG10/mG10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms ,-dimethylguanosine (mG10) via -methylguanosine (mG10). We determined the complete sequence of tRNA, one of the substrate tRNAs for archaeal Trm11 from , a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNA identified 15 types of modified nucleoside at 21 positions.