Tamotsu Kanai

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.

A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes.

We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (Δtgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Δtgr mutant even under gluconeogenic conditions. The Δtgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.

The Unique Pentagonal Structure of an Archaeal Rubisco Is Essential for Its High Thermostability

We have previously determined the crystal structure of a novel pentagonal ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. Here we have carried out biochemical studies to identify the necessities and/or advantages of this intriguing pentagonal structure. The structure indicated the presence of three neighboring residues (Glu-63, Arg-66, and Asp-69), participating in ionic interactions within unique dimer-dimer interfaces. We constructed three single mutant proteins (E63S, R66S, and D69S) and one triple mutant protein (E63S/R66S/D69S) by replacing the charged residues with serine. The wild type (WT) and all mutant proteins were purified and subjected to gel permeation chromatography at various temperatures. WT and D69S proteins were decameric at all temperatures examined between 30 and 90 °C. The majority of E63S and R66S were decamers at 30 °C but were found to gradually disassemble with the elevation in temperature. E63S/R66S/D69S was found in a dimeric form even at 30 °C. An interesting correlation was found between the subunit assembly and thermostability of the proteins. Circular dichroism and differential scanning calorimetry analyses indicated that the denaturation temperatures of dimeric enzymes (E63S, R66S, and E63S/R66S/D69S) were ∼95 °C, whereas those of the enzymes retaining a decameric structure (WT and D69S) were ∼110 °C. Disassembly into tetramer or dimer units did not alter the slopes of the Arrhenius plots, indicating that the decameric structure had no effect on catalytic performance per se. The results indicate that the decameric assembly of Tk-Rubisco contributes to enhance the thermostability of the enzyme. Taking into account the growth temperature of strain KOD1 (65–100 °C), the decameric structure of Tk-Rubisco can be considered essential for the stable presence of the enzyme in the host cells. This study provides an interesting example in which the thermostability of a protein can be enhanced by formation of a unique quaternary structure not found in mesophilic enzymes.

A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis

We have found that the upstream region of the isocitrate lyase gene (UPR-ICL) from then-alkane-utilizing yeastCandida tropicalis was functional inSaccharomyces cerevisiae as a novel promoter with non-fermentable carbon sources, such as oleic acid, acetate, ethanol, and glycerol/lactate. The expression of two foreign genes coding for β-galactosidase fromEscherichia coli (LacZ) and glutamate decarboxylase from rat brain was carried out under the control of UPR-ICL. Expression ofLacZ was repressed by glucose and enhanced over 300-fold by acetate. When an expression vector pW13 containing multicloning sites between UPR-ICL and the transcriptional terminator of the isocitrate lyase gene (TERM-ICL) was used, the smaller isoform of glutamate decarboxylase (GAD65) was highly produced in a soluble and active form. These results demonstrate that the novel expression system using UPR-ICL and TERM-ICL fromC. tropicalis is useful for the production of heterologous proteins inS. cerevisiae.

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the alpha-1,4 linkage and its subsequent transfer to the alpha-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain alpha-glucans, indicating that the reaction product contained many alpha-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70 degrees C, and thermostability up to 90 degrees C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436DeltaH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436DeltaH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms.

Application of hyperthermophiles and their enzymes

Enzymes from hyperthermophiles display extreme (thermo)stability and a wide range of enzymes have been examined to explore their potential for various biotechnological processes. In addition, recent years have witnessed the development of genetic systems in a number of hyperthermophilic archaea. This has provided the means to initiate cell engineering studies in these organisms. Biofuel production is now an important topic in microbial biotechnology, and the hydrogen producing capabilities of (hyper)thermophiles, as well as their thermostable hydrogenases, are now attracting much attention.

Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H₂) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S⁰) or H₂ generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H₂S- and H₂-evolving conditions. H₂S generation in PHY1 was equivalent to that of the host strain, and H₂ generation was higher in PHY1, suggesting that Hyh functions in the direction of H₂ uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H₂-evolving conditions. This fact, as well as the impaired H₂ generation activity in MHD1, indicated that Mbh is mainly responsible for H₂ evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H₂ transfer (i.e., H₂ evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H₂S-evolving conditions and exhibited a lower H₂S generation activity, indicating the involvement of Mbx in the S⁰ reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.

Characterization of a cytosolic NiFe-hydrogenase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (T. kodakaraensis hydrogenase). A gene cluster encoding T. kodakaraensis hydrogenase was composed of four open reading frames (hyhBGSL(Tk)), where the hyhS(Tk) and hyhL(Tk) gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (hybD(Tk)) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL(Tk) were used for immunoaffinity purification of T. kodakaraensis hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified T. kodakaraensis hydrogenase was composed of four subunits (beta, gamma, delta, and alpha), corresponding to the products of hyhBGSL(Tk), respectively. Each alphabetagammadelta unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the T. kodakaraensis hydrogenase was 95 degrees C for H(2) uptake and 90 degrees C for H(2) production with methyl viologen as the electron carrier. We found that NADP(+) and NADPH promoted high levels of uptake and evolution of H(2), respectively, suggesting that the molecule is the electron carrier for the T. kodakaraensis hydrogenase.

Structural basis for branching‐enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1

Branching enzymes (BEs) catalyze the formation of branch points in glycogen and amylopectin by cleavage of α‐1,4 glycosidic bonds and subsequent transfer to a new α‐1,6 position. BEs generally belong to glycoside hydrolase family 13 (GH13); however TK1436, isolated from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, is the first GH57 member, which possesses BE activity. To date, the only BE structure that had been determined is a GH13‐type from Escherichia coli. Herein, we have determined the crystal structure of TK1436 in the native state and in complex with glucose and substrate mimetics that permitted mapping of the substrate‐binding channel and identification of key residues for glucanotransferase activity. Its structure encompasses a distorted (β/α)7‐barrel juxtaposed to a C‐terminal α‐helical domain, which also participates in the formation of the active‐site cleft. The active site comprises two acidic catalytic residues (Glu183 and Asp354), the polarizer His10, aromatic gate‐keepers (Trp28, Trp270, Trp407, and Trp416) and the residue Tyr233, which is fully conserved among GH13‐ and GH57‐type BEs. Despite TK1436 displaying a completely different fold and domain organization when compared to E. coli BE, they share the same structural determinants for BE activity. Structural comparison with AmyC, a GH57 α‐amylase devoid of BE activity, revealed that the catalytic loop involved in substrate recognition and binding, is shortened in AmyC structure and it has been addressed as a key feature for its inability for glucanotransferase activity. The oligomerization has also been pointed out as a possible determinant for functional differentiation among GH57 members. Proteins 2011.

Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature‐sensitive

All archaeal genomes encode RNA polymerase (RNAP) subunits E and F that share a common ancestry with the eukaryotic RNAP subunits A43 and A14 (Pol I), Rpb7 and Rpb4 (Pol II), and C25 and C17 (Pol III). By gene replacement, we have isolated archaeal mutants of Thermococcus kodakarensis with the subunit F‐encoding gene (rpoF) deleted, but we were unable to isolate mutants lacking the subunit E‐encoding gene (rpoE). Wild‐type T. kodakarensis grows at temperatures ranging from 60°C to 100°C, optimally at 85°C, and the ΔrpoF cells grew at the same rate as wild type at 70°C, but much slower and to lower cell densities at 85°C. The abundance of a chaperonin subunit, CpkB, was much reduced in the ΔrpoF strain growing at 85°C and increased expression of cpkB, rpoF or rpoE integrated at a remote site in the genome, using a nutritionally regulated promoter, improved the growth of ΔrpoF cells. RNAP preparations purified from ΔrpoF cells lacked subunit F and also subunit E and a transcription factor TFE that co‐purifies with RNAP from wild‐type cells, but in vitro, this mutant RNAP exhibited no discernible differences from wild‐type RNAP in promoter‐dependent transcription, abortive transcript synthesis, transcript elongation or termination.