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".

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.

Structure and function of an ancestral-type β-decarboxylating dehydrogenase from Thermococcus kodakarensis.

β-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity. In a molecular phylogenetic analysis of 183 β-decarboxylating dehydrogenase homologs from 84 species, TK0280 from Thermococcus kodakarensis was selected as a candidate for an ancestral-type β-decarboxylating dehydrogenase. The biochemical characterization of recombinant TK0280 revealed that the enzyme exhibited dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively.

Characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a hyperthermophilic archaeon Thermoproteus sp. GDH-1.

A hyperthermophilic archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity.

Crystal structure of phosphopantothenate synthetase from Thermococcus kodakarensis.

Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes to convert pantoate to 4'-phosphopantothenate. These two enzymes are absent in almost all archaea. Recently, it was reported that two novel enzymes, pantoate kinase, and phosphopantothenate synthetase (PPS), are responsible for this conversion in archaea.

An archaeal glutamate decarboxylase homolog functions as an aspartate decarboxylase and is involved in β-alanine and coenzyme A biosynthesis.

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear.

Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis.

Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis.

CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes.

Genetic examination of initial amino acid oxidation and glutamate catabolism in the hyperthermophilic archaeon Thermococcus kodakarensis.

Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T.

CoA biosynthesis in archaea.

CoA is a ubiquitous molecule in all three domains of life and is involved in various metabolic pathways. The enzymes and reactions involved in CoA biosynthesis in eukaryotes and bacteria have been identified. By contrast, the proteins/genes involved in CoA biosynthesis in archaea have not been fully clarified, and much has to be learned before we obtain a general understanding of how this molecule is synthesized.

A detailed biochemical characterization of phosphopantothenate synthetase, a novel enzyme involved in coenzyme A biosynthesis in the Archaea.

We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4'-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion.

Biochemical characterization of pantoate kinase, a novel enzyme necessary for coenzyme A biosynthesis in the Archaea.

Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4'-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T.

Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate.

In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T.

Thermococcus kodakarensis as a host for gene expression and protein secretion.

Taking advantage of the gene manipulation system developed in Thermococcus kodakarensis, here, we developed a system for gene expression and efficient protein secretion using this hyperthermophilic archaeon as a host cell. DNA fragments encoding the C-terminal domain of chitinase (ChiAΔ4), which exhibits endochitinase activity, and the putative signal sequence of a subtilisin-like protease (TK1675) were fused and positioned under the control of the strong constitutive promoter of the cell surface glycoprotein gene. This gene cassette was introduced into T.

Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis in the Archaea.

Bacteria/eukaryotes share a common pathway for coenzyme A (CoA) biosynthesis. Although archaeal genomes harbor homologs for most of these enzymes, homologs of bacterial/eukaryotic pantothenate synthetase (PS) and pantothenate kinase (PanK) are missing. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to produce pantothenate, whereas PanK catalyzes the ATP-dependent phosphorylation of pantothenate to produce 4'-phosphopantothenate.