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Tairo Oshima - One of the best experts on this subject based on the ideXlab platform.
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Structural analysis of the plasmid pTA1 isolated from the Thermoacidophilic Archaeon Thermoplasma acidophilum.
Extremophiles : life under extreme conditions, 2006Co-Authors: Kan Yamashiro, Tairo Oshima, Shin-ichi Yokobori, Akihiko YamagishiAbstract:Thermoplasma acidophilum is a Thermoacidophilic Archaeon that grows optimally at pH1.8 and 56°C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.
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Characterization of the precursor of tetraether lipid biosynthesis in the Thermoacidophilic Archaeon Thermoplasma acidophilum.
Extremophiles : life under extreme conditions, 2003Co-Authors: Naoki Nemoto, Tairo Oshima, Yasuo Shida, Haruo Shimada, Akihiko YamagishiAbstract:Polar lipid biosynthesis in the Thermoacidophilic Archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [14C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [14C]mevalonic acid, [14C]glycerol, and [32P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and β-l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.
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effects of a squalene epoxidase inhibitor terbinafine on ether lipid biosyntheses in a Thermoacidophilic Archaeon thermoplasma acidophilum
Journal of Bacteriology, 2002Co-Authors: Takahide Kon, Tairo Oshima, Naoki Nemoto, Akihiko YamagishiAbstract:The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the Thermoacidophilic Archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.
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cloning of the gene for inorganic pyrophosphatase from a Thermoacidophilic Archaeon sulfolobus sp strain 7 and overproduction of the enzyme by coexpression of trna for arginine rare codon
Bioscience Biotechnology and Biochemistry, 1998Co-Authors: Takayoshi Wakagi, Tairo Oshima, Hiromi Imamura, Hiroshi MatsuzawaAbstract:The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a Thermoacidophilic Archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNAArg, cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obatined after removal of heat denatured E. coli proteins.
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crystallization and preliminary x ray studies on the hyperstable 3 isopropylmalate dehydrogenase from the Thermoacidophilic Archaeon sulfolobus sp strain 7
Acta Crystallographica Section D-biological Crystallography, 1998Co-Authors: Toshiharu Suzuki, Raita Hirose, Hideaki Moriyama, Masahiro Sakurai, Nobuo Tanaka, Tairo OshimaAbstract:3-Isopropylmalate dehydrogenase from the Thermoacidophilic Archaeon, Sulfolobus sp. strain 7, has been crystallized by the vapor-diffusion method. The crystals were grown from a solution containing ammonium sulfate, 2-methyl-2,4-pentanediol and magnesium chloride. The crystallization requires 2-methyl-2,4-pentanediol to avoid twinning of the crystals. The crystal belongs to the orthorhombic system with the space group P2221 and unit-cell dimensions a = 67.9, b = 93.3 and c = 134.1 A.
Robert M. Kelly - One of the best experts on this subject based on the ideXlab platform.
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Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal "Shock" Reveal Generic and Specific Metal Responses.
Applied and environmental microbiology, 2016Co-Authors: Garrett H. Wheaton, Arpan Mukherjee, Robert M. KellyAbstract:ABSTRACT The extremely Thermoacidophilic Archaeon Metallosphaera sedula mobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by “shocking” M. sedula with representative metals (Co2+, Cu2+, Ni2+, UO22+, Zn2+) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu2+ (259 ORFs, 106 Cu2+-specific ORFs) and Zn2+ (262 ORFs, 131 Zn2+-specific ORFs) triggered the largest responses, followed by UO22+ (187 ORFs, 91 UO22+-specific ORFs), Ni2+ (93 ORFs, 25 Ni2+-specific ORFs), and Co2+ (61 ORFs, 1 Co2+-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu2+ (6-fold) but also in response to UO22+ (4-fold) and Zn2+ (9-fold). Cu2+ challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu2+ resistance or issues in sulfur metabolism. The results indicate that M. sedula employs a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions. IMPORTANCE The mechanisms by which extremely Thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.
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Impact of molecular hydrogen on chalcopyrite bioleaching by the extremely Thermoacidophilic Archaeon Metallosphaera sedula.
Applied and environmental microbiology, 2010Co-Authors: Kathryne S. Auernik, Robert M. KellyAbstract:Hydrogen served as a competitive inorganic energy source, impacting the CuFeS(2) bioleaching efficiency of the extremely Thermoacidophilic Archaeon Metallosphaera sedula. Open reading frames encoding key terminal oxidase and electron transport chain components were triggered by CuFeS(2). Evidence of heterotrophic metabolism was noted after extended periods of bioleaching, presumably related to cell lysis.
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the genome sequence of the metal mobilizing extremely Thermoacidophilic Archaeon metallosphaera sedula provides insights into bioleaching associated metabolism
Applied and Environmental Microbiology, 2008Co-Authors: Kathryne S. Auernik, Yukari Maezato, Paul H Blum, Robert M. KellyAbstract:Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely Thermoacidophilic Archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ∼2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the Thermoacidophilic Archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.
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Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely Thermoacidophilic Archaeon sulfolobus solfataricus P1
Biotechnology and bioengineering, 2002Co-Authors: Amitabh C. Sehgal, Richele Tompson, John Cavanagh, Robert M. KellyAbstract:The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely Thermoacidophilic Archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.
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Enantiomeric resolution of 2-aryl propionic esters with hyperthermophilic and mesophilic esterases: contrasting thermodynamic mechanisms.
Journal of the American Chemical Society, 2002Co-Authors: Amitabh C. Sehgal, Robert M. KellyAbstract:The enantiomeric resolution of 2-aryl propionic esters by hyperthermophilic and mesophilic esterases was found to be governed by contrasting thermodynamic mechanisms. Entropic contributions predominated for mesophilic esterases from Candida rugosa and Rhizomucor miehei, while enthalpic forces controlled this resolution by the esterase from the extremely Thermoacidophilic Archaeon, Sulfolobus solfataricus P1. This disparity in thermodynamic mechanism can be attributed to the differences in conformational flexibility of mesophilic and thermophilic enzymes as they relate to the temperature range (4−70 °C) examined.
Takayoshi Wakagi - One of the best experts on this subject based on the ideXlab platform.
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Archaeal Aldehyde Dehydrogenase ST0064 from Sulfolobus tokodaii, a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase, Is a Succinate Semialdehyde Dehydrogenase
Bioscience Biotechnology and Biochemistry, 2013Co-Authors: Fumiaki Ito, Hidehiro Chishiki, Shinya Fushinobu, Takayoshi WakagiAbstract:Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a Thermoacidophilic Archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (K m of 0.0029 mM and k cat of 30.0 s−1) with no allosteric regulation. Activity toward GAP was lower (K m of 4.6 mM and k cat of 4.77 s−1), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.
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Comparative analysis of two glyceraldehyde-3-phosphate dehydrogenases from a Thermoacidophilic Archaeon, Sulfolobus tokodaii
FEBS letters, 2012Co-Authors: Fumiaki Ito, Hidehiro Chishiki, Shinya Fushinobu, Takayoshi WakagiAbstract:Abstract Sulfolobus tokodaii, a Thermoacidophilic Archaeon, possesses two structurally and functionally different enzymes that catalyze the oxidation of glyceraldehyde-3-phosphate (GAP): non-phosphorylating GAP dehydrogenase (St-GAPN) and phosphorylating GAP dehydrogenase (St-GAPDH). In contrast to previously characterized GAPN from Sulfolobus solfataricus, which exhibits V-type allosterism, St-GAPN showed K-type allosterism in which the positive cooperativity was abolished with concomitant activation by glucose 1-phosphate (G1P). St-GAPDH catalyzed the reversible oxidation of GAP to 1,3-bisphosphoglycerate (1,3-BPG) with high gluconeogenic activity, which was specific for NADPH, while both NAD+ and NADP+ were utilized in the glycolytic direction. Structured summary of protein interactions GAPDH and GAPDH bind by molecular sieving ( View interaction ) GAPN and GAPN bind by 2.2 molecular sieving ( View interaction ).
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Conserved residues in membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a Thermoacidophilic Archaeon
Extremophiles, 2011Co-Authors: Fumitoshi Manabe, Hirofumi Shoun, Takayoshi WakagiAbstract:A membrane-intrinsic acid pyrophosphatase (ST2226) from Sulfolobus tokodaii , a Thermoacidophilic Archaeon, is possibly involved in glycoprotein biosynthesis and belongs to the phosphatidic acid phosphatase class 2 superfamily, including both membrane-intrinsic and soluble enzymes with divergent functions ranging from dephosphorylation of undecaprenylpyrophosphate and phospho-monoesters such as glucose-6-phosphate to vanadium-containing chloroperoxidation. ST2226 is an archaeal ortholog of these enzymes sharing a common phosphatase motif. Through site-directed mutagenesis as to each of the conserved residues, the catalytic roles of the latter were deduced, as well as the transmembrane topology with all the conserved residues in close proximity to the outside of the membrane.
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Membrane-bound acid pyrophosphatase from Sulfolobus tokodaii, a Thermoacidophilic Archaeon: heterologous expression of the gene and characterization of the product
Extremophiles, 2009Co-Authors: Fumitoshi Manabe, Yuko H. Itoh, Hirofumi Shoun, Takayoshi WakagiAbstract:Membranes of Sulfolobus tokodaii , a Thermoacidophilic Archaeon that grows optimally at pH 2–3, 75–80°C, show the ability to hydrolyze PPi with an optimum pH of 2–3. This acid PPase is proposed to be a dolicholpyrophosphatase that participates in glycoprotein biosynthesis. In the present study, the archaeal membranes hydrolyzed isopentenylpyrophosphate and geranylpyrophosphate, compounds related to dolicholpyrophosphate, at pH 3. However, the dolicholpyrophosphate-binding antibiotic bacitracin failed to inhibit the acid PPase. To investigate further the function and structure of the acid PPase, the gene was cloned and heterologously expressed in Escherichia coli. The membranes from recombinant E. coli showed PPase activity with similar pH and temperature dependence, substrate specificity, and kinetic parameters to those reported for Sulfolobus membranes. The acid PPase was solubilized and purified to electrophoretic homogeneity from the recombinant E. coli . The purified enzyme showed similar K _m values for PPi, ATP, and ADP to the membrane-bound enzyme. Lipids from the Sulfolobus membranes enhanced the activity to about threefold. Studies involving deletion mutants indicated that basic amino acids in the N-terminal (Arg2 and Lys3), as well as the residues (4th–69th) possibly twice-spanning the membrane, are essential for integration of the enzyme into membranes.
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identification of the lysine residue responsible for coenzyme a binding in the heterodimeric 2 oxoacid ferredoxin oxidoreductase from sulfolobus tokodaii a Thermoacidophilic Archaeon using 4 fluoro 7 nitrobenzofurazan as an affinity label
Biochimica et Biophysica Acta, 2009Co-Authors: Jing Luo, Shinya Fushinobu, Hirofumi Shoun, Eriko Fukuda, Hirofumi Takase, Takayoshi WakagiAbstract:Abstract The heterodimeric 2-oxoacid:ferredoxin oxidoreductase (StOFOR) from Sulfolobus tokodaii , a Thermoacidophilic Archaeon, was inactivated by low concentrations of 4-fluoro-7-nitrobenzofurazan (NBD-F), with concomitant increase in fluorescence in subunit-b. The inactivation was prevented by CoA, suggesting that NBD-F covalently bound to the Lys which is responsible for CoA binding. The NBD-labeled subunit-b was isolated and digested with endoproteinase Lys-C. The resulting polypeptide mixture was separated by reverse phase HPLC and the fluorescent fraction was isolated. Amino acid sequencing of the fraction revealed that it comprised a mixture of two polypeptides containing Lys125 and Lys173, respectively. Two StOFOR mutants, K125A and K173A, were constructed, expressed and purified. K125A showed a large increase in the K m value for CoA and showed poor inactivation by NBD-F, compared with K173A and wild type StOFOR, indicating Lys125 in subunit-b is the critical residue that interacts with CoA.
Tokuzo Nishino - One of the best experts on this subject based on the ideXlab platform.
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Total Synthesis of Geranylgeranylglyceryl Phosphate Enantiomers: Substrates for Characterization of 2,3-O-Digeranylgeranylglyceryl Phosphate Synthase
Organic letters, 2006Co-Authors: Honglu Zhang, Hisashi Hemmi, Tokuzo Nishino, Kyohei Shibuya, Glenn D. PrestwichAbstract:To determine the enantioselectivity of (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase (DGGGPS) from the Thermoacidophilic Archaeon Sulfolobus solfataricus, we developed an efficient enantioselective route to the enantiomeric geranylgeranylglyceryl phosphates (R)-GGGP and (S)-GGGP. Previous routes to these substrates involved enzymatic conversions due to the lability of the polyprenyl chains toward common phosphorylation reaction conditions. The synthesis described herein employs a mild trimethyl phosphite/carbon tetrabromide oxidative phosphorylation to circumvent this problem. In contrast to previous results suggesting that only (S)-GGGP can act as the prenyl acceptor substrate, both (R)-GGGP and (S)-GGGP were found to be substrates for DGGGPS.
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s 2 3 di o geranylgeranylglyceryl phosphate synthase from the Thermoacidophilic Archaeon sulfolobus solfataricus molecular cloning and characterization of a membrane intrinsic prenyltransferase involved in the biosynthesis of archaeal ether linked me
Journal of Biological Chemistry, 2004Co-Authors: Hisashi Hemmi, Toru Nakayama, Kyohei Shibuya, Yoshihiro Takahashi, Tokuzo NishinoAbstract:The core structure of membrane lipids of archaea have some unique properties that permit archaea to be distinguished from the others, i.e. bacteria and eukaryotes. (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase, which catalyzes the transfer of a geranylgeranyl group from geranylgeranyl diphosphate to (S)-3-O-geranylgeranylglyceryl phosphate, is involved in the biosynthesis of archaeal membrane lipids. Enzymes of the UbiA prenyltransferase family are known to catalyze the transfer of a prenyl group to various acceptors with hydrophobic ring structures in the biosynthesis of respiratory quinones, hemes, chlorophylls, vitamin E, and shikonin. The Thermoacidophilic Archaeon Sulfolobus solfataricus was found to encode three homologues of UbiA prenyltransferase in its genome. One of the homologues encoded by SSO0583 was expressed in Escherichia coli, purified, and characterized. Radio-assay and mass spectrometry analysis data indicated that the enzyme specifically catalyzes the biosynthesis of (S)-2,3-di-O-geranylgeranylglyceryl phosphate. The fact that the orthologues of the enzyme are encoded in almost all archaeal genomes clearly indicates the importance of their functions. A phylogenetic tree constructed using the amino acid sequences of some typical members of the UbiA prenyltransferase family and their homologues from S. solfataricus suggests that the two other S. solfataricus homologues, excluding the (S)-2,3-di-O-geranylgeranylglyceryl phosphate synthase, are involved in the production of respiratory quinone and heme, respectively. We propose here that archaeal prenyltransferases involved in membrane lipid biosynthesis might be prototypes of the protein family and that archaea might have played an important role in the molecular evolution of prenyltransferases.
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Type 2 isopentenyl diphosphate isomerase from a Thermoacidophilic Archaeon Sulfolobus shibatae
European journal of biochemistry, 2004Co-Authors: Satoshi Yamashita, Hisashi Hemmi, Toru Nakayama, Yosuke Ikeda, Tokuzo NishinoAbstract:Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a Thermoacidophilic Archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.
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Fusion-type lycopene β-cyclase from a Thermoacidophilic Archaeon Sulfolobus solfataricus
Biochemical and biophysical research communications, 2003Co-Authors: Hisashi Hemmi, Toru Nakayama, Satoru Ikejiri, Tokuzo NishinoAbstract:Examination of the sequence of a hypothetical gene with an unknown function included in the carotenogenic gene cluster in the genome of a Thermoacidophilic Archaeon Sulfolobus solfataricus led to the prediction that the gene encodes a novel-type lycopene β-cyclase, whose N- and C-terminal halves are homologous to the subunits of the bacterial heterodimeric enzymes. The recombinant expression of the gene in lycopene-producing Escherichia coli resulted in the accumulation of β-carotene in the cells, which verifies the function of the gene. Homologues of the archaeal lycopene β-cyclase from various organisms such as bacteria, archaea, and fungi have been reported. Although their primary structures are clearly specific to the biological taxa, a phylogenetic analysis revealed the unexpected complicity of the evolutional route of these enzymes.
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Change of product specificity of hexaprenyl diphosphate synthase from Sulfolobus solfataricus by introducing mimetic mutations
Biochemical and biophysical research communications, 2002Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, Tokuzo NishinoAbstract:The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) prenyl diphosphate synthase, hexaprenyl diphosphate synthase from a Thermoacidophilic Archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes.
Toshio Iwasaki - One of the best experts on this subject based on the ideXlab platform.
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Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp. strain 7
FEMS microbiology letters, 1996Co-Authors: Toshio Iwasaki, Tairo OshimaAbstract:Abstract The role of cytochrome b 562 , a fragile constituent of the respiratory terminal oxidase supercomplex of the Thermoacidophilic Archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b 562 did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form ( b 562 ) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b 562 was upstream of both the ‘ g y = 1.89’ Rieske FeS cluster and the a -type cytochromes. These results show that the b -type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.
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Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7: III. THE ARCHAEAL NOVEL RESPIRATORY COMPLEX II (SUCCINATE:CALDARIELLAQUINONE OXIDOREDUCTASE COMPLEX) INHERENTLY LACKS HEME GROUP
The Journal of biological chemistry, 1995Co-Authors: Toshio Iwasaki, Takayoshi Wakagi, Tairo OshimaAbstract:An active respiratory complex II (succinate:quinone oxidoreductase) has been purified from tetraether lipid membranes of the Thermoacidophilic Archaeon, Sulfolobus sp. strain 7. It consists of four different subunits with apparent molecular masses of 66, 37, 33, and 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 66-kDa subunit contains a covalently bound flavin, the 37-kDa subunit is a possible iron-sulfur protein carrying three distinct types of EPR-visible FeS cluster, and the 33- and 12-kDa subunits are putative membrane-anchor subunits, respectively. While no heme group is detected in the purified complex II, it catalyzes succinate-dependent reduction of ubiquinone-1 and 2,6-dichlorophenolindophenol in the absence of phenazine methosulfate. The respiratory complex II of Sulfolobus sp. strain 7 appears to be novel in that it functions as a true succinate:caldariellaquinone oxidoreductase, although inherently lacking any heme group. This further indicates that the heme group of several respiratory complexes II may not be involved in the redox intermediates of the electron transfer from succinate to quinone.
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Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7: II. CHARACTERIZATION OF THE ARCHAEAL TERMINAL OXIDASE SUBCOMPLEXES AND IMPLICATION FOR THE INTRAMOLECULAR ELECTRON TRANSFER
The Journal of biological chemistry, 1995Co-Authors: Toshio Iwasaki, Takayoshi Wakagi, Yasuhiro Isogai, Tetsutaro Iizuka, Tairo OshimaAbstract:Abstract The terminal segment of the aerobic respiratory chain of the Thermoacidophilic Archaeon Sulfolobus sp. strain 7 is an unusual caldariellaquinol oxidase supercomplex, which contains at least one b-type and three spectroscopically distinguishable a-type cytochromes, one copper, and a Rieske-type FeS center. In this paper, we report the purification and characterization of two different forms of the archaeal a-type cytochromes, namely, a three-subunit cytochrome a-aa3 subcomplex and a single-subunit cytochrome aa3 derived from the cytochrome subcomplex, in order to facilitate further studies on the terminal oxidase segment of Sulfolobus. The optical and EPR spectroscopic analyses suggest the presence of two different low-spin heme centers and one high-spin heme center in the purified cytochrome a-aa3 subcomplex, and one low-spin and one high-spin hemes in cytochrome aa3, respectively. The Rieske-type FeS center detected in the purified cytochrome supercomplex was absent in two forms of the a-type cytochrome oxidase, indicating its association with cytochrome b. The crystal field parameters of the low-spin heme a center indicate that its axial ligands may be similar to those of cytochromes c, rather than conventional bis-histidine ligation. In spite of the absence of any c-type cytochrome, a ferrocytochrome c oxidase activity was detected in the archaeal purified cytochrome a-aa3 subcomplex with no quinol oxidase activity, but not in the purified cytochrome oxidase supercomplex, which has been tentatively interpreted as a representative of electron transfer from the Rieske FeS center to cytochrome ain vivo. Thus, our results indicate the following scheme for the intramolecular electron transfer of the terminal oxidase supercomplex from Sulfolobus sp. strain 7: [caldariellaquinol ] b Rieske FeS center aaa3 molecular oxygen.
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Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7: I. THE ARCHAEAL TERMINAL OXIDASE SUPERCOMPLEX IS A FUNCTIONAL FUSION OF RESPIRATORY COMPLEXES III AND IV WITH NO c-TYPE CYTOCHROMES
The Journal of biological chemistry, 1995Co-Authors: Toshio Iwasaki, Katsumi Matsuura, Tairo OshimaAbstract:Abstract The aerobic respiratory system of the Thermoacidophilic Archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a- and b-type cytochromes but no c-type cytochromes. In previous studies, a novel cytochrome oxidase a-aa3 subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Muller, M., Azzi, A., and Sone, N.(1989) Biochem. Biophys. Res. Commun. 165, 1110-1114). We show here that the cytochrome subcomplex could be copurified with a non-CO-reactive cytochrome b as a novel terminal oxidase “supercomplex,” which also contained a Rieske-type FeS cluster at g = 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectrophotometry and the potentiometric titration: cytochromes b (+146 mV), a (+270 mV), and aa3 (+117 and +325 mV). The presence of one copper atom indicates that it contains the conventional heme a3-Cu binuclear center for reducing molecular oxygen. In conjunction with the presence of a Rieske-type FeS center, inhibitor studies suggest that the terminal oxidase segment of the respiratory chain of Sulfolobus sp. strain 7 is a functional fusion of respiratory complexes III and IV, where cytochrome b and the Rieske-type FeS center probably play a central role in the oxidation of caldariellaquinol. This archaeal terminal oxidase supercomplex reconstitutes the in vitro succinate oxidase respiratory chain for the first time together with caldariellaquinone and the purified cognate succinate:caldariellaquinone oxidoreductase complex. The reconstitution system requires caldariellaquinone for the activity, and is highly sensitive to cyanide and 2-heptyl-4-hydroxy-quinoline-N-oxide. These results are also discussed in terms of the evolutionary considerations.
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sulredoxin a novel iron sulfur protein of the Thermoacidophilic Archaeon sulfolobus sp strain 7 with a rieske type 2fe 2s center
Journal of Bacteriology, 1995Co-Authors: Toshio Iwasaki, Y Isogai, T Iizuka, Tairo OshimaAbstract:A novel pink [2Fe-2S] protein has been purified from the cytosol fraction of the Thermoacidophilic Archaeon Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius 7) and called ‘‘sulredoxin.’’ Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type [2Fe-2S] cluster (g-factors of 2.01, 1.91, and 1.79; averageg- factor [gav] 51.90) which is remarkably similar to that ofThermus thermophilusrespiratory Rieske FeS protein (J. A. Fee, K. L. Findling, T. Yoshida, R. Hille,
