Improvement of the Catalytic Activity of Thermoacidophilic Pullulan Hydrolase Type III by Error-Prone PCR Technology

Applied Biochemistry and Microbiology - Thermoacidophilic pullulan hydrolase type III from Thermococcus kodakarensis (TK-PUL) completely hydrolyzes starch under liquefaction conditions. It has...     

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. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complexes with substrates, ATP, and ATP and 4‐phosphopantoate. PPS forms an asymmetric homodimer, in which two monomers composing a dimer, deviated from the exact twofold symmetry, displaying 4°–13° distortion. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on these structures, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate, which is thought to be a reaction intermediate. Proteins 2014; 82:1924–1936. © 2014 Wiley Periodicals, Inc.     

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. Based on the structure of the active site that contains a glycerol molecule, the pantoate binding site and the roles of the highly conserved residues are suggested.     

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer that had sarcosine oxidase and l-proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αβ)₄ form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and β subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αβ)₄ complex may separately exist as a function enzyme in different conditions.     

Biochemical characterization of deblocking aminopeptidases from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.     

Biochemical Characterization of Deblocking Aminopeptidases from the Hyperthermophilic Archaeon Thermococcus kodakarensis KOD1

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.     

Thermococcus kodakarensis encodes three MCM homologs but only one is essential

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukaryotes. In eukaryotes, this complex is an assembly of six different but related polypeptides (MCM2-7) but, in most archaea, one MCM protein assembles to form a homohexameric complex. Atypically, the Thermococcus kodakarensis genome encodes three archaeal MCM homologs, here designated MCM1-3, although MCM1 and MCM2 are unusual in having long and unique N-terminal extensions. The results reported establish that MCM2 and MCM3 assemble into homohexamers and exhibit DNA binding, helicase and ATPase activities in vitro typical of archaeal MCMs. In contrast, MCM1 does not form homohexamers and although MCM1 binds DNA and has ATPase activity, it has only minimal helicase activity in vitro. Removal of the N-terminal extension had no detectable effects on MCM1 but increased the helicase activity of MCM2. A T. kodakarensis strain with the genes TK0096 (MCM1) and TK1361 (MCM2) deleted has been constructed that exhibits no detectable defects in growth or viability, but all attempts to delete TK1620 (MCM3) have been unsuccessful arguing that that MCM3 is essential and is likely the replicative helicase in T. kodakarensis. The origins and possible function(s) of the three MCM proteins are discussed.     

Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis

Phytoene (C₄₀H₆₄) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L⁻¹ medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

     

High level expression and characterization of a thermostable lysophospholipase from Thermococcus kodakarensis KOD1

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783?bp encodes a 260-amino acid protein with a molecular mass of 29?kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent K (m) value for p-nitrophenyl butyrate was 607.1?μM with V (max) values of 95.5?U/mg. The enzyme was active at a broad range of pH (5-8) and temperatures (70-95?°C) with the optimum pH and temperature being 8.0 and 85?°C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.     

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N⁴-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N⁴-bis(aminopropyl)-N¹-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N⁴-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.     

Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1

The abnormal prion protein (scrapie-associated prion protein, PrP^Sc) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP^Sc is highly resistant to normal sterilization procedures, the decontamination of PrP^Sc is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP^Sc. Although PrP^Sc is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP^Sc under extreme conditions. The level of PrP^Sc in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP^Sc at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP^Sc in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP^Sc have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP^Sc degradation.     

A linear pathway for mevalonate production supports growth of Thermococcus kodakarensis

Extremophiles - The sole unifying feature of Archaea is the use of isoprenoid-based glycerol lipid ethers to compose cellular membranes. The branched hydrocarbon tails of archaeal lipids are...     

Requirement of Ca(2+) Ions for the Hyperthermostability of Tk-Subtilisin from Thermococcus kodakarensis

Tk-subtilisin, a hyperthermostable subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca(2+) ions. Four of them (Ca2-Ca5) are responsible for folding of Tk-subtilisin. In this study, to clarify the role of the other three Ca(2+) ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/ΔCa1), Ca6 ion (Pro-TKS/ΔCa6), and Ca7 ion (Pro-TKS/ΔCa7), and their active site mutants (Pro-S324A/ΔCa1, Pro-S324A/ΔCa6, and Pro-S324A/ΔCa7, respectively). Pro-TKS/ΔCa6 and Pro-TKS/ΔCa7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/ΔCa1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/ΔCa1 was determined to be 50% of that of Tk-subtilisin using the refolded protein. This result suggests that the Ca1 ion is required for the maximal activity of Tk-subtilisin. The refolding rates of all Pro-S324A derivatives were comparable to that of Pro-S324A (active site mutant of Pro-TKS), indicating that these Ca(2+) ions are not needed for folding of Tk-subtilisin. The stabilities of Pro-S324A/ΔCa1 and Pro-S324A/ΔCa6 were decreased by 26.6 and 11.7 °C, respectively, in T(m) compared to that of Pro-S324A. The half-lives of Tk-subtilisin/ΔCa6 and Tk-subtilisin/ΔCa7 at 95 °C were 8- and 4-fold lower than that of Tk-subtilisin, respectively. These results suggest that the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin.     

Crystal structure of a [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1

A [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1 (TkHybD) is involved in the cleavage of the C‐terminal residues of [NiFe] hydrogenase large subunits by Ni recognition. Here, we report the crystal structure of TkHybD at 1.82 Å resolution to better understand this process. TkHybD exhibits an α/β/α sandwich fold with conserved residues responsible for the Ni recognition. Comparisons of TkHybD with homologous proteins also reveal that they share a common overall architecture, suggesting that they have similar catalytic functions. Our results including metal binding site prediction provide insight into the substrate recognition and catalysis mechanism of TkHybD. Proteins 2016; 84:1321–1327. © 2016 Wiley Periodicals, Inc.     

Architecture and characterization of a thermostable MoxR family AAA+ ATPase from Thermococcus kodakarensis KOD1

AAA⁺ ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.