Hyperthermophilic Archaeon Thermococcus Kodakaraensis KOD1 Research Articles

Structural study reveals the temperature-dependent conformational flexibility of Tk-PTP, a protein tyrosine phosphatase from Thermococcus kodakaraensis KOD1.

Protein tyrosine phosphatases (PTPs) originating from eukaryotes or bacteria have been under intensive structural and biochemical investigation, whereas archaeal PTP proteins have not been investigated extensively; therefore, they are poorly understood. Here, we present the crystal structures of Tk-PTP derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, in both the active and inactive forms. Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and α4−α5 loop regions, switching between the inactive and active forms. Through comprehensive analyses of Tk-PTP, including additional structural determination of the G95A mutant form, enzymatic activity assays, and structural comparison with the other archaeal PTP, it was revealed that the presence of the GG motif in the P-loop is necessary but not sufficient for the structural flexibility of Tk-PTP. It was also proven that Tk-PTP contains dual general acid/base residues unlike most of the other DUSP proteins, and that both the residues are critical in its phosphatase activity. This work provides the basis for expanding our understanding of the previously uncharacterized PTP proteins from archaea, the third domain of living organisms.

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.

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 (Deltatgr) 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 Deltatgr mutant even under gluconeogenic conditions. The Deltatgr 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 Ribulose Monophosphate Pathway Substitutes for the Missing Pentose Phosphate Pathway in the Archaeon Thermococcus kodakaraensis

The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Delta hps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.

Ca 2+ -Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis : the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80 degrees C. This maturation process was completed within 30 min at 80 degrees C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80 degrees C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80 degrees C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only approximately 5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 +/- 3.0 nM at 20 degrees C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.

Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppA(Tk)) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppA(Tk), comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (deltaN54SppA(Tk)) was found to be stable against autoproteolysis and was examined further. DeltaN54SppA(Tk) exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. DeltaN54SppA(Tk) displayed a K(m) of 240 +/- 18 microM and a V(max) of 27.8 +/- 0.7 micromol min(-1) mg(-1) towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80 degrees C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, deltaN54SppA(Tk) was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of deltaN54SppA(Tk) was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.

Characterization of an archaeal malic enzyme from the hyperthermophilic archaeonThermococcus kodakaraensisKOD1

Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Mae). The amino acid sequence of Tk-Mae was 52-58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP(+) rather than NAD(+), but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (K(m)) of Tk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence.

Genetic evidence identifying the true gluconeogenic fructose-1,6-bisphosphatase in Thermococcus kodakaraensis and other hyperthermophiles.

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Deltafbp and Deltaimp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Deltafbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbpTk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous impTk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that impTk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the FbpTk ortholog, not the IMPase/FBPase ortholog.

Concerted Action of Diacetylchitobiose Deacetylase and Exo-β-D-glucosaminidase in a Novel Chitinolytic Pathway in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

The hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 possesses chitinase (Tk-ChiA) and exo-beta-D-glucosaminidase (Tk-GlmA) for chitin degradation; the former produces diacetylchitobiose (GlcNAc2) from chitin, and the latter hydrolyzes chitobiose (GlcN2) to glucosamine (GlcN). To identify the enzyme that physiologically links these two activities, here we focused on the deacetylase that provides the substrate for Tk-GlmA from GlcNAc2. The deacetylase could be detected in and partially purified from T. kodakaraensis cells, and the corresponding gene (Tk-dac) was identified on the genome. The deduced amino acid sequence was classified into the LmbE protein family including N-acetylglucosaminylphosphatidylinositol de-N-acetylases and 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase. Recombinant Tk-Dac showed deacetylase activity toward N-acetylchitooligosaccharides (GlcNAc(2-5)), and the deacetylation site was revealed to be specific at the nonreducing GlcNAc residue. The enzyme also deacetylated GlcNAc monomer. In T. kodakaraensis cells, the transcription of Tk-dac, Tk-glmA, Tk-chiA, and the clustered genes were induced by GlcNAc2, suggesting the function of this gene cluster in chitin catabolism in vivo. These results have revealed a unique chitin catabolic pathway in T. kodakaraensis, in which GlcNAc2 produced from chitin is degraded by the concerted action of Tk-Dac and Tk-GlmA. That is, GlcNAc2 is site-specifically deacetylated to GlcN-GlcNAc by Tk-Dac and then hydrolyzed to GlcN and GlcNAc by Tk-GlmA followed by a second deacetylation step of the remaining GlcNAc by Tk-Dac to form GlcN. This is the first elucidation of an archaeal chitin catabolic pathway and defines a novel mechanism for dimer processing using a combination of deacetylation and cleavage, distinct from any previously known pathway.

Genetic, Enzymatic, and Structural Analyses of Phenylalanyl-tRNA Synthetase from Thermococcus kodakaraensis KOD1

Phenylalanyl-tRNA synthetase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-PheRS) was cloned. The open reading frames for both the alpha-subunit (Tk-pheRSA) and beta-subunit (Tk-pheRSB) genes were 1,503 bp (501 amino acids) and 1,722 bp (574 amino acids), respectively. Tk-pheRSB located 879 bp downstream from Tk-pheRSA with a putative TATA box, suggesting that these two subunits are transcribed and regulated independently in KOD1 cells. Tk-PheRS and its respective subunits were expressed in Escherichia coli cells and the proteins were purified. Tk-PheRS showed an optimum enzymatic activity at around 95 degrees C and retained its tertiary structure at 98 degrees C. The estimated isoelectric point (pI) for the alpha-subunit is 9.4 and that for the beta-subunit is 4.6, the largest difference among the 12 kinds of PheRSs reported. The considerable thermostability of Tk-PheRS may be responsible for the electrostatic interaction between the alpha- and beta-subunits.

Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.

We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc(2)) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc(2). Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative beta-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the beta-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to beta-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-beta-D-glucosaminidase (GlcNase) activity but not beta-galactosidase activity. This GlcNase (GlmA(Tk)), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80 degrees C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA(Tk) was induced by GlcNAc(2) and chitin, strongly suggesting that GlmA(Tk) is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc(2) from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.

Crystallization and preliminary X-ray analysis of TBP-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1.

The 26 kDa TBP (TATA-binding protein) interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-TIP26) is a possible transcriptional regulatory protein in Thermococcales. Here, the crystallization of both the native and selenomethionine-substituted proteins and data collection are described. The native crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 73.83, c = 86.41 A, and diffract to 2.2 A using synchrotron radiation. MAD data was collected and a Bijvoet difference Patterson map showed strong peaks sufficient to determine the positions of the Se atoms.

Gene cloning and characterization of fructose-1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

The fructose-1,6-bisphosphate (FBP) aldolase gene from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned. The gene encoding FBP aldolase ( Tk-Fba) was expressed in Escherichia coli and the purified recombinant protein was characterized at high temperature. Tk-Fba is a homodecamer with a subunit molecular mass of 31,283 Da. The amino acid sequence, decameric conformation, formation of a Schiff-base intermediate, and stimulation (286%) of FBP cleavage activity by citrate suggested that Tk-Fba belonged to Class IA, a subtype of the classical Class I aldolases. The specific activity for the FBP cleavage reaction was 18.9 U/mg, which was much higher than those of other Class IA type FBP aldolases. Tk-Fba was extremely thermostable since the optimum temperature seemed to be above 100°C. The optimum pH for Tk-Fba was determined to be 5.0 in the absence of citrate, while it shifted to around 7.0 in the presence of citrate. Tk-Fba accepted FBP and fructose-1-phosphate as substrates and K m values were determined to be 0.063 mM and 4.37 mM, respectively. In addition to citrate, phosphoenolpyruvate and pyrophosphate were also found to be potent activators of Tk-Fba, enhancing activities up to 346% and 201%, respectively. Erythrose-4-phosphate acted as an inhibitor and caused a decrease in the activity to 49%. Tk-Fba also catalyzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage. However, none of the above compounds seemed to have a significant effect on the condensation reaction by Tk-Fba. These results suggest a regulatory function of Tk-Fba toward the catabolic direction of sugar metabolism in T. kodakaraensis KOD1.

Tk-PTP, protein tyrosine/serine phosphatase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1: enzymatic characteristics and identification of its substrate proteins

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein ( Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit β-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.

Gene Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

The fructose-1,6-bisphosphate (FBP) aldolase gene from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned. The gene encoding FBP aldolase (Tk-Fba) was expressed in Escherichia coli and the purified recombinant protein was characterized at high temperature. Tk-Fba is a homodecamer with a subunit molecular mass of 31,283 Da. The amino acid sequence, decameric conformation, formation of a Schiff-base intermediate, and stimulation (286%) of FBP cleavage activity by citrate suggested that Tk-Fba belonged to Class IA, a subtype of the classical Class I aldolases. The specific activity for the FBP cleavage reaction was 18.9 U/mg, which was much higher than those of other Class IA type FBP aldolases. Tk-Fba was extremely thermostable since the optimum temperature seemed to be above 100 degrees C. The optimum pH for Tk-Fba was determined to be 5.0 in the absence of citrate, while it shifted to around 7.0 in the presence of citrate. Tk-Fba accepted FBP and fructose-1-phosphate as substrates and K(m) values were determined to be 0.063 mM and 4.37 mM, respectively. In addition to citrate, phosphoenolpyruvate and pyrophosphate were also found to be potent activators of Tk-Fba, enhancing activities up to 346% and 201%, respectively. Erythrose-4-phosphate acted as an inhibitor and caused a decrease in the activity to 49%. Tk-Fba also catalyzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage. However, none of the above compounds seemed to have a significant effect on the condensation reaction by Tk-Fba. These results suggest a regulatory function of Tk-Fba toward the catabolic direction of sugar metabolism in T. kodakaraensis KOD1.

Different Cleavage Specificities of the Dual Catalytic Domains in Chitinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

The chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, Tk-ChiA, has an interesting multidomain structure containing dual catalytic domains and triple chitin-binding domains. To determine the biochemical properties of each domain, we constructed deletion mutant genes corresponding to the individual catalytic domains and purified the recombinant proteins. A synergistic effect was observed when chitin was degraded in the presence of both catalytic domains, suggesting different cleavage specificity of these domains. Analyses of degradation products from N-acetyl-chitooligosaccharides and their chromogenic derivatives with thin layer chromatography indicated that the N-terminal catalytic domain mainly hydrolyzed the second glycosidic bond from the nonreducing end of the oligomers, whereas the C-terminal domain randomly hydrolyzed glycosidic bonds other than the first bond from the nonreducing end. Both catalytic domains formed diacetyl-chitobiose as a major end product and possessed transglycosylation activity. Further analysis of degradation products from colloidal chitin with high performance liquid chromatography showed that the N-terminal catalytic domain exclusively liberated diacetyl-chitobiose, whereas reactions with the C-terminal domain led to N-acetyl-chitooligosaccharides of various lengths. These results demonstrated that the N-terminal and C-terminal catalytic domains functioned as exo- and endochitinases, respectively. The biochemical results provide a physiological explanation for the presence of two catalytic domains with different specificity and suggest a cooperative function between the two on a single polypeptide in the degradation of chitin.

Interaction of TIP26 from a hyperthermophilic archaeon with TFB/TBP/DNA ternary complex.

Interactions of TBP-interacting protein (TIP26), TBP, and TFB from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with TATA-DNA were examined by electrophoretic mobility shift assay. Tk-TFB formed a ternary complex with Tk-TBP and TATA-DNA. Tk-TIP26 did not inhibit the formation of this ternary complex, but interacted with it to form a TIP26/TFB/TBP/DNA quaternary complex. This interaction is rather weak, and a large excess of Tk-TIP26 over Tk-TBP is required to fully convert the TFB/TBP/DNA ternary complex to the quaternary complex. However, determination of the concentration of Tk-TIP26 and Tk-TBP in KOD1 cells by Western blotting analysis indicated that the concentration of Tk-TIP26 is approximately ten times that of Tk-TBP, suggesting that the quaternary complex might also form in vivo.

Crystal Structure of a Novel-Type Archaeal Rubisco with Pentagonal Symmetry

Background: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of the Calvin-Benson cycle and catalyzes the primary reaction of CO 2 fixation in plants, algae, and bacteria. Rubiscos have been so far classified into two types. Type I is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L 8S 8), but type II is usually composed only of two L subunits (L 2). Recently, some genuinely active Rubiscos of unknown physiological function have been reported from archaea. Results: The crystal structure of Rubisco from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 ( Tk-Rubisco) was determined at 2.8 Å resolution. The enzyme is composed only of L subunits and showed a novel (L 2) 5 decameric structure. Compared to previously known type I enzymes, each L 2 dimer is inclined approximately 16° to form a toroid-shaped decamer with its unique L 2-L 2 interfaces. Differential scanning calorimetry (DSC), circular dichroism (CD), and gel permeation chromatography (GPC) showed that Tk-Rubisco maintains its secondary structure and decameric assembly even at high temperatures. Conclusions: The present study provides the first structure of an archaeal Rubisco, an unprecedented (L 2) 5 decamer. Biochemical studies indicate that Tk-Rubisco maintains its decameric structure at high temperatures. The structure is distinct from type I and type II Rubiscos and strongly supports that Tk-Rubisco should be classified as a novel type III Rubisco.

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.

Anthranilate Synthase without an LLES Motif from a Hyperthermophilic Archaeon Is Inhibited by Tryptophan

Tk-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensisKOD1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk–AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (α subunit) and Tk-TrpG (β subunit), at 85°C for 10 min. Further purification of Tk–AS complex was carried out by anion-exchange chromatography followed by gel-filtration. Molecular mass estimations from gel-filtration chromatography indicated that Tk–AS complex was a heterodimer (αβ). The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor. The optimal pH was pH 10.0 and the optimal temperature was 85°C in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75°C, the Km values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 μM, respectively. The Km value of Mg2+ was 20.5 μM. The Km values of glutamine and NH4Cl were 88 μM and 5.6 mM, respectively. Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by l-tryptophan were not present in Tk-TrpE. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S. typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE. Nevertheless, significant inhibition by l-tryptophan was observed, with Ki values of 5.25 and 74 μM for ammonia and glutamine-dependent activities, respectively. The inhibition was competitive with respect to chorismate. The results suggest that the amino acid residues involved in the feedback inhibition by l-tryptophan in the case of Tk–AS complex are distinct from previously reported anthranilate synthases.