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ABSTRACT: Enzymes from many archaea colonizing extreme environments are of great interest because of their potential for various biotechnological processes and scientific value of evolution. Many enzymes from archaea have been reported to catalyze promiscuous reactions or moonlight in different functions. Here, we summarize known archaeal enzymes of both groups that include different kinds of proteins. Knowledge of their biochemical properties and three-dimensional structures has proved invaluable in understanding mechanism, application, and evolutionary implications of this manifestation. In addition, the review also summarizes the methods to unravel the extra function which almost was discovered serendipitously. The study of these amazing enzymes will provide clues to optimize protein engineering applications and how enzymes might have evolved on Earth.
Extremophiles 01/2013; · 2.94 Impact Factor
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ABSTRACT: 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.
Extremophiles 05/2012; 16(4):619-25. · 2.94 Impact Factor
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ABSTRACT: 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.
Extremophiles 11/2011; 16(1):87-93. · 2.94 Impact Factor
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ABSTRACT: 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.
Bioscience Biotechnology and Biochemistry 06/2011; 75(6):1160-6. · 1.28 Impact Factor
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ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80-90°C. The apparent K (m) values for NAD(+) and D-G3P were 77.8 ± 7.5 μM and 49.3 ± 3.0 μM, respectively, with V (max) values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.
Extremophiles 03/2011; 15(3):337-46. · 2.94 Impact Factor
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ABSTRACT: NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.
The Protein Journal 11/2010; 29(8):609-16. · 1.04 Impact Factor
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ABSTRACT: Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.
Plant Molecular Biology 04/2010; 72(6):569-83. · 4.15 Impact Factor
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ABSTRACT: NADH oxidases (NOXs) catalyze the two-electron reduction of oxygen to H2O2 or four-electron reduction of oxygen to H2O. In this report, we show that an NADH oxidase from Thermococcus profundus (NOXtp) displays two forms: a native dimeric protein under physiological conditions and an oxidized hexameric form under oxidative stress. Native NOXtp displays high NADH oxidase activity, and oxidized NOXtp can accelerate the aggregation of partially unfolded proteins. The aggregates formed by NOXtp have characteristics similar to beta-amyloid and Lewy bodies in neurodegenerative diseases, including an increase of beta-sheet content. Oxidized NOXtp can also bind nucleic acids and cause their degradation by oxidizing NADH to produce H2O2. Furthermore, Escherichia coli cells expressing NOXtp are less viable than cells not expressing NOXtp after treatment with H2O2. As NOXtp shares similar features with eukaryotic cell death isozymes and life may have originated from hyperthermophiles, we suggest that NOXtp may be an ancestor of cell death proteins.
Molecules and Cells 03/2010; 29(4):363-71. · 2.18 Impact Factor
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Jung Ro Lee,
Seung Sik Lee,
Ho Hee Jang,
Young Mee Lee,
Jin Ho Park,
Seong-Cheol Park,
Jeong Chan Moon,
Soo Kwon Park,
Sun Young Kim,
Sun Yong Lee,
Ho Byoung Chae,
Young Jun Jung,
Woe Yeon Kim,
Mi Rim Shin, Gang-Won Cheong,
Min Gab Kim,
Kee Ryeon Kang,
Kyun Oh Lee,
Dae-Jin Yun,
Sang Yeol Lee
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ABSTRACT: We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes. Because deletion of the TPR domains results in a significant enhancement of AtTDX disulfide reductase activity and complete loss of the holdase chaperone function, our data suggest that the TPR domains of AtTDX block the active site of Trx and play a critical role in promoting the holdase chaperone function. The oligomerization status of AtTDX is reversibly regulated by heat shock, which causes a transition from LMW to HMW complexes with concomitant functional switching from a disulfide reductase and foldase chaperone to a holdase chaperone. Overexpression of AtTDX in Arabidopsis conferred enhanced heat shock resistance to plants, primarily via its holdase chaperone activity.
Proceedings of the National Academy of Sciences 05/2009; 106(14):5978-83. · 9.68 Impact Factor
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Baolei Jia,
Seong-Cheol Park,
Sangmin Lee,
Bang P Pham,
Rui Yu,
Thuy L Le,
Sang Woo Han,
Jae-Kyung Yang,
Myung-Suk Choi,
Wolfgang Baumeister, Gang-Won Cheong
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ABSTRACT: An NADH oxidase (NOX) was cloned from the genome of Thermococcus profundus (NOXtp) by genome walking, and the encoded protein was purified to homogeneity after expression in Escherichia coli. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 49 kDa that exists as a hexamer with a native molecular mass of 300 kDa. A ring-shaped hexameric form was revealed by electron microscopic and image processing analyses. NOXtp catalyzed the oxidization of NADH and NADPH and predominantly converted O(2) to H(2)O, but not to H(2)O(2), as in the case of most other NOX enzymes. To our knowledge, this is the first example of a NOX that can produce H(2)O predominantly in a thermophilic organism. As an enzyme with two cysteine residues, NOXtp contains a cysteinyl redox center at Cys45 in addition to FAD. Mutant analysis suggests that Cys45 in NOXtp plays a key role in the four-electron reduction of O(2) to H(2)O, but not in the two-electron reduction of O(2) to H(2)O(2).
FEBS Journal 12/2008; 275(21):5355-66. · 3.79 Impact Factor
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ABSTRACT: Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.
Biochimica et Biophysica Acta 06/2008; 1784(5):783-8. · 4.66 Impact Factor
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ABSTRACT: Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.
Molecules and Cells 05/2007; 23(2):252-7. · 2.18 Impact Factor
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ABSTRACT: The essential oil obtained from the leaves of Chamaecyparis obtusa was analyzed by GC and GC-MS. alpha-Terpinyl acetate, sabinene, isobornyl acetate and limonene were found to be the major components. The oil showed relatively strong antibacterial activities against Gram (+) bacteria and some fungi.
Fitoterapia 03/2007; 78(2):149-52. · 1.85 Impact Factor
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ABSTRACT: An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The N-terminal amino acid sequence of the protein was highly homologous to that of plant cyclophilins and consequently the protein was denoted as C-CyP. To understand the antifungal activity of C-CyP, we isolated a cDNA encoding its gene from a Chinese cabbage leaf cDNA library. The Chinese cabbage genome bears more than one C-CyP gene copy and C-CyP mRNA is highly expressed in all tissues except the seeds. Recombinant C-CyP catalyzed the cis-trans inter-conversion of the Ala-Pro bond of the substrate, which indicates this protein has peptidyl-prolyl cis-trans isomerase activity. It also inhibited the growth of several fungal pathogens.
Biochemical and Biophysical Research Communications 03/2007; 353(3):672-8. · 2.48 Impact Factor
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ABSTRACT: We studied the effects of melittin on various cell wall components and vesicles of various lipid compositions. To interact with the cytoplasmic membrane, melittin must traverse the cell wall, which is composed of oligosaccharides. Here, we found that melittin had a strong affinity for chitin, peptidoglycan, and lipopolysaccharide. We further examined the influence of lipid compositions on the lysis of the membranes by melittin. The result showed that melittin bound better to negatively charged than to zwitterionic lipid vesicles but was more potent at inducing leakage from zwitterionic lipid vesicles. Our studies further indicated that the oligomeric state of melittin varied between tetramers and octamers during the formation of toroidal pores. Dextran leakage experiments confirmed the formation and dimension of these toroidal pores. Finally, transmission electron microscopy revealed that melittin formed pores via peptide oligomerization by the toroidal pore-forming mechanism. The toroidal pores composed of 7-8 nm diameter rings that encircled 3.5-4.5 nm diameter cavities on zwitterionic lipid vesicles.
Biochemical and Biophysical Research Communications 05/2006; 343(1):222-8. · 2.48 Impact Factor
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ABSTRACT: Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of Mg(2+), but dissociated into monomers and/or dimers in its absence. Moreover, Mg(2+)-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires Mg(2+), but not ATP.
Molecules and Cells 03/2006; 21(1):129-34. · 2.18 Impact Factor
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Ho Hee Jang,
Kyun Oh Lee,
Yong Hun Chi,
Bae Gyo Jung,
Soo Kwon Park,
Jin Ho Park,
Jung Ro Lee,
Seung Sik Lee,
Jeong Chan Moon,
Jeong Won Yun,
Yeon Ok Choi,
Woe Yeon Kim,
Ji Seoun Kang, Gang-Won Cheong,
Dae-Jin Yun,
Sue Goo Rhee,
Moo Je Cho,
Sang Yeol Lee
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ABSTRACT: Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.
Cell 06/2004; 117(5):625-35. · 32.40 Impact Factor
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Min Suk Kang,
Soon Rae Kim,
Pyeongsu Kwack,
Byung Kook Lim,
Sung Won Ahn,
Young Min Rho,
Ihn Sik Seong,
Seong-Chul Park,
Soo Hyun Eom, Gang-Won Cheong,
Chin Ha Chung
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ABSTRACT: CodWX in Bacillus subtilis is an ATP-dependent, N-terminal serine protease, consisting of CodW peptidase and CodX ATPase. Here we show that CodWX is an alkaline protease and has a distinct molecular architecture. ATP hydrolysis is required for the formation of the CodWX complex and thus for its proteolytic function. Remarkably, CodX has a 'spool-like' structure that is formed by interaction of the intermediate domains of two hexameric or heptameric rings. In the CodWX complex, CodW consisting of two stacked hexameric rings (WW) binds to either or both ends of a CodX double ring (XX), forming asymmetric (WWXX) or symmetric cylindrical particles (WWXXWW). CodWX can also form an elongated particle, in which an additional CodX double ring is bound to the symmetric particle (WWXXWWXX). In addition, CodWX is capable of degrading EzrA, an inhibitor of FtsZ ring formation, implicating it in the regulation of cell division. Thus, CodWX appears to constitute a new type of protease that is distinct from other ATP-dependent proteases in its structure and proteolytic mechanism.
The EMBO Journal 07/2003; 22(12):2893-902. · 9.20 Impact Factor
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ABSTRACT: The purification and characterization of thermostable chaperonin of the thermosome family from hyperthermophilic archaeon Thermococcus profunds are described. The purified thermosome is a homooligomeric complex and an ATPase with maximal activity at 80 degrees C. The electron micrographs obtained from negatively stained as well as frozen-hydrated specimen showed an eight-fold symmetry of chaperonin. They were about 15 nm height and 16 nm in diameter with a central cavity of 5 nm. In order to understand the ATPase cycling of thermosome, we analyzed the oligomeric structure of thermosome treated with several nucleotides.
Molecules and Cells 09/2002; 14(1):85-92. · 2.18 Impact Factor
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ABSTRACT: C60-mediated self-assembly of gold nanoparticles at the liquid/liquid interface in the form of a stable nanocomposite film is repoted. The metallic luster of the interfacial film results from the electronic coupling of Au nanoparticles, suggesting the formation of closely packed nanoparticle thin films. The interfacial film of nanoparticles could be transferred to mica substrates and carbon-coated transmission electron microscopy (TEM) grids, and then studied by UV–vis, Raman spectroscopy and TEM.
Colloids and Surfaces A: Physicochemical and Engineering Aspects.
