Tomohiko Kuwabara - Academia.edu (original) (raw)

Papers by Tomohiko Kuwabara

Plant and Cell Physiology, 1994

An endoproteinase, which specifically cleaves the Pro12-Leu13 bond of the extrinsic 18-kDa protei... more An endoproteinase, which specifically cleaves the Pro12-Leu13 bond of the extrinsic 18-kDa protein of PSII, was purified from PSII membranes of spinach. The presence of 0.05% (w/v) Tween 20 and 1 M NaCl was essential for maintenance of proteolytic activity during the purification. The molecular mass of the enzyme was estimated to be 95 kDa by gel-filtration chromatography. Active fractions contained a polypeptide of 165 kDa that was converted into diffusely stained polypeptides of 54 kDa upon reduction with dithiothreitol. The Km of the 18-kDa protein in the proteolytic reaction was 0.3 microM. Inhibition of the proteolysis by compounds that contain prolyl bonds revealed that both a prolyl bond and a positive charge are necessary for interaction with the proteinase, but some other structural factor(s) must also be involved in the high-affinity interaction between the proteinase and the 18-kDa protein. Reconstitution of NaCl-treated PSII membranes with the 23-kDa protein and/or the 18-kDa protein revealed that the 18-kDa protein was not cleaved by the proteinase when the substrate protein was functionally associated with the membranes. A comparison of the properties of the proteinase with those of a proline-specific endopeptidase from Flavobacterium suggests that these enzymes are quite different in terms of substrate specificity.

Photosynthesis Research, 1986

International Journal of Systematic and Evolutionary Microbiology, 2007

A fast-growing and cell-fusing hyperthermophilic archaeon was isolated from a hydrothermal vent a... more A fast-growing and cell-fusing hyperthermophilic archaeon was isolated from a hydrothermal vent at Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean. Strain TS2T is an irregular, motile coccus that is generally 0.7–1.5 μm in diameter and possesses a polar tuft of flagella. In the mid-exponential phase of growth, cells that appeared black under phase-contrast microscopy fused at room temperature in the presence of a DNA-intercalating dye, as previously observed in Thermococcus coalescens. Cell fusion was not observed in later growth phases. Transmission electron microscopy revealed that the cells in the mid-exponential phase had a 5 nm-thick, electron-dense cell envelope that appeared to associate loosely with the cytoplasmic membrane. As the growth stage progressed, a surface layer developed on the membrane under the envelope and the envelope eventually peeled off. These observations suggest that the surface layer prevents the fusion of cells. Cells of strain TS2T grew at 50–85 °...

FEBS Letters, 1986

The amino acid sequence of the 33 kDa protein isolated as an extrinsic protein from spinach photo... more The amino acid sequence of the 33 kDa protein isolated as an extrinsic protein from spinach photosystem II particles was determined by using solid-phase sequencing and conventional procedures. The 33 kDa protein was found to be composed of 248 amino acid residues, to lack hi&dine and to have a molecular mass of 26 663 Da, which was considerably smaller than the value deduced from SDS-poiy~rylamide gel electrophoresis. The sequence of the 33 kDa protein was compared with those of the bacterial superoxide dismutases (SOD) with Mn atoms at an active site. A part of the sequence of the 33 kDa protein was similar to a region in Mn-SODS from Bacillus stearothermophilus and Escherichia coli, which was expected to be the Mn-binding site. 33 kDa protein Photosynthesis Oxygen evolution Amino acid sequence manganese-beading site

Journal of Theoretical Biology, 2020

How eukaryotes were generated is an enigma of evolutionary biology. Widely accepted archaeal-orig... more How eukaryotes were generated is an enigma of evolutionary biology. Widely accepted archaeal-origin eukaryogenesis scenarios, based on similarities of genes and related characteristics between archaea and eukaryotes, cannot explain several eukaryote-specific features of the last eukaryotic common ancestor, such as glycerol-3-phosphate-type membrane lipids, large cells and genomes, and endomembrane formation. Thermotogales spheroids, having multicopy-integrated large nucleoids and producing progeny in periplasm, may explain all of these features as well as endoplasmic reticulum-type signal cleavage sites, although they cannot divide. We hypothesize that the progeny chromosome is formed by random joining small DNAs in immature progeny, followed by reorganization by mechanisms including homologous recombination enabled with multicopy-integrated large genome (MILG). We propose that Thermotogales ancestor spheroids came to divide owing to the archaeal cell division genes horizontally transferred via virus-related particles, forming the first eukaryotic common ancestor (FECA). Referring to the hypothesis, the archaeal information-processing system would have been established in FECA by random joining DNAs excised from the MILG, which contained horizontally transferred archaeal and bacterial DNAs, followed by reorganization by the MILG-enabled homologous recombination. Thus, the large genome may have been a prerequisite, but not a consequence, of eukaryogenesis. The random joining of DNAs likely provided the basic mechanisms for eukaryotic evolution: producing the diversity by the formations of supergroups, novel genes, and introns that are involved in exon shuffling.

Bioresource technology, 2016

The demand for precious metals has increased in recent years. However, low concentrations of prec... more The demand for precious metals has increased in recent years. However, low concentrations of precious metals dissolved in wastewater are yet to be recovered because of high operation costs and technical problems. The unicellular red alga, Galdieria sulphuraria, efficiently absorbs precious metals through biosorption. In this study, over 90% of gold and palladium could be selectively recovered from aqua regia-based metal wastewater by using G. sulphuraria. These metals were eluted from the cells into ammonium solutions containing 0.2M ammonium salts without other contaminating metals. The use of G. sulphuraria is an eco-friendly and cost-effective way of recovering low concentrations of gold and palladium discarded in metal wastewater.

Plant and Cell Physiology, Mar 1, 1999

Plant and Cell Physiology, Mar 1, 1997

Proceedings of the National Academy of Sciences, 1987

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is inv... more The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amin...

Plant and Cell Physiology, 1999

Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional ... more Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional column chromatography in the presence of Tween 20. The neutral detergent was necessary to prevent non-specific interaction of VDE with column resins. In anion-exchange chromatography on Mono Q, VDE appeared in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully reduced with 5 raM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on the concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptides moved down to the position of 40 kDa, and then up to the position of 41 kDa, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and takes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive to pepstatin A, a specific inhibitor of aspartic protease. This finding suggests that the reaction center of VDE contains a reactive aspartic acid residue(s).

Plant and Cell Physiology, 1999

Plant polyphenol oxidase (PPO) is apt to degrade during and even after purification. We developed... more Plant polyphenol oxidase (PPO) is apt to degrade during and even after purification. We developed a method to stabilize PPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethylene glycol at pH 6.5. The protein slowly degraded by itself when the stabilizing reagents were removed. Ascorbate and/or H 2 O 2 accelerated the degradation. The ascorbate-induced degradation was inhibited by catalase, suggesting that H 2 O 2 is generated through reduction of PPO by ascorbate. It is likely that dissolved oxygen is converted to peroxide through two-electron reduction by the reaction center of PPO, binuclear Cu site, and a Fenton-type reaction occurred on it. This understanding was supported by the finding that the H 2 O 2-induced degradation was inhibited by metal-chelators as well as by polyphenolic substrate of PPO. Considering the postulated mechanism of the self-degradation of PPO, we reexamined the degradation of the 23-kDa protein of PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38: 179]. The obtained results suggested that the 23-kDa protein triggers the active oxygen production by the binuclear Cu site, probably as reductant, and receives the radical species preferentially to the polypeptide moiety of PPO.

Plant and Cell Physiology, 1998

PSII membranes were used as a substrate for violaxanthin de-epoxidase (VDE) that had been solubil... more PSII membranes were used as a substrate for violaxanthin de-epoxidase (VDE) that had been solubilized from spinach thylakoids by sonication. Inclusion of Tween 20 in the assay mixture was essential, although the detergent apparently inhibited the activity in the conventional assay with purified violaxanthin and lipid as substrate. The maximum enhancing effect of the detergent was observed near its critical micellar concentration. It is likely that the monomer of the detergent helped VDE react with the substrate in the membranes. Dependence of the activity on the substrate concentration suggested that VDE functions at least at two sites in the membranes, probably on both their lumenal and stromal surfaces. The ability of the enzyme to function on the stromal surface in in vitro assays was demonstrated by using intact thylakoids as the substrate. Under such conditions where the endogenous VDE was functioning in the lumen, the exogenously added VDE converted antheraxanthin to zeaxanthin in the absence of Tween 20. This result suggests that, in the reaction with PSII membranes, the detergent was required for VDE to react with violaxanthin but not with antheraxanthin. Otherwise, the detergent was necessary for the reaction on the lumenal surface.

FEBS Letters, 1992

A prolyl cndopcptidase (PEPasc, EC X4.21.26) that specifictally clcavcs 1hc IS-kDa protein ofphot... more A prolyl cndopcptidase (PEPasc, EC X4.21.26) that specifictally clcavcs 1hc IS-kDa protein ofphotosystcm ii was cxtracthti from photosystem II mcmbrancs with 1 M NaCI. Protcolytic activity mcasurcd with anificial substrates was ICW 1han u quarter of that with the protein. Studies on ~nhibitiou of the protcolysis by an artificial substrate suggested lhat the protestsc rceognizcs the scissi~c prolyt bond. The protcasc aas inhibited by CuCl?. but not by diisopropyl fluorophosphdlc or p-chloromcrcuriphmylsulforGc acid. Thcsc findings suggest that the protea= rcprc=ts a new &ass of PEPaxc. The specificity of the cnzymc is discussed in relation 10 the structure of 1h1: IX-k& protein.

Archives of Biochemistry and Biophysics, 1986

Geochimica et Cosmochimica Acta, 2016

Greigite, a ferrimagnetic iron sulfide Fe(II)Fe(III) 2 S 4 , is thought to have played an essenti... more Greigite, a ferrimagnetic iron sulfide Fe(II)Fe(III) 2 S 4 , is thought to have played an essential role in chemical evolution leading to the origin of life. Greigite contains a [4Fe-4S] cluster-like structure and has been synthesized in the laboratory by liquid-state reactions. However, it is unclear how greigite can be synthesized in nature. Herein, we show that greigite is synthesized by the solid-gas reaction of Fe(III)-oxide-hydroxides and H 2 S. We discovered that the hyperthermophilic hydrogenotrophic methanogen Methanocaldococcus jannaschii reduced elemental sulfur, and the resulting sulfide generated greigite from hematite. The time course and pH dependence of the reaction respectively indicated the involvement of amorphous FeS and H 2 S as reaction intermediates. An abiotic solid-gas reaction of hematite and H 2 S (g) under strictly anaerobic conditions was developed. The solid-gas reaction fully converted hematite to greigite/pyrite at 40-120°C within 12 h and was unaffected by the bulk gas phase. Similar abiotic reactions occurred, but relatively slowly, with aqueous H 2 S in acidulous liquids using hematite, magnetite, or amorphous FeO(OH) as starting materials, suggesting that greigite was extensively produced in the Hadean Eon as these Fe(III)-oxide-hydroxides were shown to be present or routinely produced during that era. Surprisingly, the obtained greigite induced methanogenesis and growth of hydrogenotrophic methanogens, suggesting that the external greigite crystals enhanced reactions that would otherwise require enzymes, such as [4Fe-4S] cluster-harboring membranebound hydrogenases. These data suggested that the greigite produced by the solid-gas and solid-dissolved gas reactions was bioactive.

Plant and Cell Physiology, 1990

ABSTRACT

Science Access, 2001

In search of cyanobacterial extracellular Cu-chelator, we found that Synechocystis sp. PCC6803 se... more In search of cyanobacterial extracellular Cu-chelator, we found that Synechocystis sp. PCC6803 secretes a homolog of hemolysin (toxin that lyses red blood cells of animals). The cyanobacterium was exposed to a Cu stress by culturing in BG-11 medium containing 3 µM Cu2+. When the culture medium was collected at the late log phase and subjected to hydroxyapatite chromatography, an elution peak of Cu was associated with a polypeptide of 173 kDa. The N-terminal amino acid sequence of the polypeptide was determined to be ALSPNVIAALQIMYTGRGVS. In a genome DNA database, this sequence had been registered as that of ?hemolysin? with the N-terminal methionine. This fact indicated that there is no cleavable N-terminal signal peptide in the protein, as in hemolysin (HlyA) of Escherichia coli that has been shown to be secreted from the cells by an ABC transporter. The full amino acid sequence of the cyanobacterial hemolysin deduced from the DNA sequence indicated that the protein contains a numb...

Plant and Cell Physiology, 1994

An endoproteinase, which specifically cleaves the Pro12-Leu13 bond of the extrinsic 18-kDa protei... more An endoproteinase, which specifically cleaves the Pro12-Leu13 bond of the extrinsic 18-kDa protein of PSII, was purified from PSII membranes of spinach. The presence of 0.05% (w/v) Tween 20 and 1 M NaCl was essential for maintenance of proteolytic activity during the purification. The molecular mass of the enzyme was estimated to be 95 kDa by gel-filtration chromatography. Active fractions contained a polypeptide of 165 kDa that was converted into diffusely stained polypeptides of 54 kDa upon reduction with dithiothreitol. The Km of the 18-kDa protein in the proteolytic reaction was 0.3 microM. Inhibition of the proteolysis by compounds that contain prolyl bonds revealed that both a prolyl bond and a positive charge are necessary for interaction with the proteinase, but some other structural factor(s) must also be involved in the high-affinity interaction between the proteinase and the 18-kDa protein. Reconstitution of NaCl-treated PSII membranes with the 23-kDa protein and/or the 18-kDa protein revealed that the 18-kDa protein was not cleaved by the proteinase when the substrate protein was functionally associated with the membranes. A comparison of the properties of the proteinase with those of a proline-specific endopeptidase from Flavobacterium suggests that these enzymes are quite different in terms of substrate specificity.

Photosynthesis Research, 1986

International Journal of Systematic and Evolutionary Microbiology, 2007

A fast-growing and cell-fusing hyperthermophilic archaeon was isolated from a hydrothermal vent a... more A fast-growing and cell-fusing hyperthermophilic archaeon was isolated from a hydrothermal vent at Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean. Strain TS2T is an irregular, motile coccus that is generally 0.7–1.5 μm in diameter and possesses a polar tuft of flagella. In the mid-exponential phase of growth, cells that appeared black under phase-contrast microscopy fused at room temperature in the presence of a DNA-intercalating dye, as previously observed in Thermococcus coalescens. Cell fusion was not observed in later growth phases. Transmission electron microscopy revealed that the cells in the mid-exponential phase had a 5 nm-thick, electron-dense cell envelope that appeared to associate loosely with the cytoplasmic membrane. As the growth stage progressed, a surface layer developed on the membrane under the envelope and the envelope eventually peeled off. These observations suggest that the surface layer prevents the fusion of cells. Cells of strain TS2T grew at 50–85 °...

FEBS Letters, 1986

The amino acid sequence of the 33 kDa protein isolated as an extrinsic protein from spinach photo... more The amino acid sequence of the 33 kDa protein isolated as an extrinsic protein from spinach photosystem II particles was determined by using solid-phase sequencing and conventional procedures. The 33 kDa protein was found to be composed of 248 amino acid residues, to lack hi&dine and to have a molecular mass of 26 663 Da, which was considerably smaller than the value deduced from SDS-poiy~rylamide gel electrophoresis. The sequence of the 33 kDa protein was compared with those of the bacterial superoxide dismutases (SOD) with Mn atoms at an active site. A part of the sequence of the 33 kDa protein was similar to a region in Mn-SODS from Bacillus stearothermophilus and Escherichia coli, which was expected to be the Mn-binding site. 33 kDa protein Photosynthesis Oxygen evolution Amino acid sequence manganese-beading site

Journal of Theoretical Biology, 2020

How eukaryotes were generated is an enigma of evolutionary biology. Widely accepted archaeal-orig... more How eukaryotes were generated is an enigma of evolutionary biology. Widely accepted archaeal-origin eukaryogenesis scenarios, based on similarities of genes and related characteristics between archaea and eukaryotes, cannot explain several eukaryote-specific features of the last eukaryotic common ancestor, such as glycerol-3-phosphate-type membrane lipids, large cells and genomes, and endomembrane formation. Thermotogales spheroids, having multicopy-integrated large nucleoids and producing progeny in periplasm, may explain all of these features as well as endoplasmic reticulum-type signal cleavage sites, although they cannot divide. We hypothesize that the progeny chromosome is formed by random joining small DNAs in immature progeny, followed by reorganization by mechanisms including homologous recombination enabled with multicopy-integrated large genome (MILG). We propose that Thermotogales ancestor spheroids came to divide owing to the archaeal cell division genes horizontally transferred via virus-related particles, forming the first eukaryotic common ancestor (FECA). Referring to the hypothesis, the archaeal information-processing system would have been established in FECA by random joining DNAs excised from the MILG, which contained horizontally transferred archaeal and bacterial DNAs, followed by reorganization by the MILG-enabled homologous recombination. Thus, the large genome may have been a prerequisite, but not a consequence, of eukaryogenesis. The random joining of DNAs likely provided the basic mechanisms for eukaryotic evolution: producing the diversity by the formations of supergroups, novel genes, and introns that are involved in exon shuffling.

Bioresource technology, 2016

The demand for precious metals has increased in recent years. However, low concentrations of prec... more The demand for precious metals has increased in recent years. However, low concentrations of precious metals dissolved in wastewater are yet to be recovered because of high operation costs and technical problems. The unicellular red alga, Galdieria sulphuraria, efficiently absorbs precious metals through biosorption. In this study, over 90% of gold and palladium could be selectively recovered from aqua regia-based metal wastewater by using G. sulphuraria. These metals were eluted from the cells into ammonium solutions containing 0.2M ammonium salts without other contaminating metals. The use of G. sulphuraria is an eco-friendly and cost-effective way of recovering low concentrations of gold and palladium discarded in metal wastewater.

Plant and Cell Physiology, Mar 1, 1999

Plant and Cell Physiology, Mar 1, 1997

Proceedings of the National Academy of Sciences, 1987

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is inv... more The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amin...

Plant and Cell Physiology, 1999

Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional ... more Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional column chromatography in the presence of Tween 20. The neutral detergent was necessary to prevent non-specific interaction of VDE with column resins. In anion-exchange chromatography on Mono Q, VDE appeared in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully reduced with 5 raM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on the concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptides moved down to the position of 40 kDa, and then up to the position of 41 kDa, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and takes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive to pepstatin A, a specific inhibitor of aspartic protease. This finding suggests that the reaction center of VDE contains a reactive aspartic acid residue(s).

Plant and Cell Physiology, 1999

Plant polyphenol oxidase (PPO) is apt to degrade during and even after purification. We developed... more Plant polyphenol oxidase (PPO) is apt to degrade during and even after purification. We developed a method to stabilize PPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethylene glycol at pH 6.5. The protein slowly degraded by itself when the stabilizing reagents were removed. Ascorbate and/or H 2 O 2 accelerated the degradation. The ascorbate-induced degradation was inhibited by catalase, suggesting that H 2 O 2 is generated through reduction of PPO by ascorbate. It is likely that dissolved oxygen is converted to peroxide through two-electron reduction by the reaction center of PPO, binuclear Cu site, and a Fenton-type reaction occurred on it. This understanding was supported by the finding that the H 2 O 2-induced degradation was inhibited by metal-chelators as well as by polyphenolic substrate of PPO. Considering the postulated mechanism of the self-degradation of PPO, we reexamined the degradation of the 23-kDa protein of PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38: 179]. The obtained results suggested that the 23-kDa protein triggers the active oxygen production by the binuclear Cu site, probably as reductant, and receives the radical species preferentially to the polypeptide moiety of PPO.

Plant and Cell Physiology, 1998

PSII membranes were used as a substrate for violaxanthin de-epoxidase (VDE) that had been solubil... more PSII membranes were used as a substrate for violaxanthin de-epoxidase (VDE) that had been solubilized from spinach thylakoids by sonication. Inclusion of Tween 20 in the assay mixture was essential, although the detergent apparently inhibited the activity in the conventional assay with purified violaxanthin and lipid as substrate. The maximum enhancing effect of the detergent was observed near its critical micellar concentration. It is likely that the monomer of the detergent helped VDE react with the substrate in the membranes. Dependence of the activity on the substrate concentration suggested that VDE functions at least at two sites in the membranes, probably on both their lumenal and stromal surfaces. The ability of the enzyme to function on the stromal surface in in vitro assays was demonstrated by using intact thylakoids as the substrate. Under such conditions where the endogenous VDE was functioning in the lumen, the exogenously added VDE converted antheraxanthin to zeaxanthin in the absence of Tween 20. This result suggests that, in the reaction with PSII membranes, the detergent was required for VDE to react with violaxanthin but not with antheraxanthin. Otherwise, the detergent was necessary for the reaction on the lumenal surface.

FEBS Letters, 1992

A prolyl cndopcptidase (PEPasc, EC X4.21.26) that specifictally clcavcs 1hc IS-kDa protein ofphot... more A prolyl cndopcptidase (PEPasc, EC X4.21.26) that specifictally clcavcs 1hc IS-kDa protein ofphotosystcm ii was cxtracthti from photosystem II mcmbrancs with 1 M NaCI. Protcolytic activity mcasurcd with anificial substrates was ICW 1han u quarter of that with the protein. Studies on ~nhibitiou of the protcolysis by an artificial substrate suggested lhat the protestsc rceognizcs the scissi~c prolyt bond. The protcasc aas inhibited by CuCl?. but not by diisopropyl fluorophosphdlc or p-chloromcrcuriphmylsulforGc acid. Thcsc findings suggest that the protea= rcprc=ts a new &ass of PEPaxc. The specificity of the cnzymc is discussed in relation 10 the structure of 1h1: IX-k& protein.

Archives of Biochemistry and Biophysics, 1986

Geochimica et Cosmochimica Acta, 2016

Greigite, a ferrimagnetic iron sulfide Fe(II)Fe(III) 2 S 4 , is thought to have played an essenti... more Greigite, a ferrimagnetic iron sulfide Fe(II)Fe(III) 2 S 4 , is thought to have played an essential role in chemical evolution leading to the origin of life. Greigite contains a [4Fe-4S] cluster-like structure and has been synthesized in the laboratory by liquid-state reactions. However, it is unclear how greigite can be synthesized in nature. Herein, we show that greigite is synthesized by the solid-gas reaction of Fe(III)-oxide-hydroxides and H 2 S. We discovered that the hyperthermophilic hydrogenotrophic methanogen Methanocaldococcus jannaschii reduced elemental sulfur, and the resulting sulfide generated greigite from hematite. The time course and pH dependence of the reaction respectively indicated the involvement of amorphous FeS and H 2 S as reaction intermediates. An abiotic solid-gas reaction of hematite and H 2 S (g) under strictly anaerobic conditions was developed. The solid-gas reaction fully converted hematite to greigite/pyrite at 40-120°C within 12 h and was unaffected by the bulk gas phase. Similar abiotic reactions occurred, but relatively slowly, with aqueous H 2 S in acidulous liquids using hematite, magnetite, or amorphous FeO(OH) as starting materials, suggesting that greigite was extensively produced in the Hadean Eon as these Fe(III)-oxide-hydroxides were shown to be present or routinely produced during that era. Surprisingly, the obtained greigite induced methanogenesis and growth of hydrogenotrophic methanogens, suggesting that the external greigite crystals enhanced reactions that would otherwise require enzymes, such as [4Fe-4S] cluster-harboring membranebound hydrogenases. These data suggested that the greigite produced by the solid-gas and solid-dissolved gas reactions was bioactive.

Plant and Cell Physiology, 1990

ABSTRACT

Science Access, 2001

In search of cyanobacterial extracellular Cu-chelator, we found that Synechocystis sp. PCC6803 se... more In search of cyanobacterial extracellular Cu-chelator, we found that Synechocystis sp. PCC6803 secretes a homolog of hemolysin (toxin that lyses red blood cells of animals). The cyanobacterium was exposed to a Cu stress by culturing in BG-11 medium containing 3 µM Cu2+. When the culture medium was collected at the late log phase and subjected to hydroxyapatite chromatography, an elution peak of Cu was associated with a polypeptide of 173 kDa. The N-terminal amino acid sequence of the polypeptide was determined to be ALSPNVIAALQIMYTGRGVS. In a genome DNA database, this sequence had been registered as that of ?hemolysin? with the N-terminal methionine. This fact indicated that there is no cleavable N-terminal signal peptide in the protein, as in hemolysin (HlyA) of Escherichia coli that has been shown to be secreted from the cells by an ABC transporter. The full amino acid sequence of the cyanobacterial hemolysin deduced from the DNA sequence indicated that the protein contains a numb...