Publications (38)132.26 Total impact
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Article: Creation of a thermostable NADP⁺-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis.
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ABSTRACT: A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.Biotechnology Letters 05/2012; 34(9):1693-9. · 1.68 Impact Factor -
Article: Identification of an entire set of tRNA molecules and characterization of cleavage sites of the intron-containing tRNA precursors in acidothermophilic crenarchaeon Sulfolobus tokodaii strain7.
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ABSTRACT: The acidothermophilic crenarchaeon, Sulfolobus tokodaii strain7, was isolated from a hot spring in Beppu, Kyushu, Japan. Whole genomic data of this microorganism indicated that among 46 putative tRNA genes identified, 24 were interrupted tRNA genes containing an intron. A sequence comparison between the cDNA sequences for unspliced and spliced tRNAs indicated that all predicted tRNAs were expressed and all intron portions were spliced in this microorganism. However, the actual cleavage site in the splicing process was not determined for 13 interrupted tRNAs because of the presence of the same nucleotides at both 5' and 3' border regions of each intron. The cleavage sites for all the introns, which were determined by an in vitro cleavage experiment with recombinant splicing endonuclease as well as cDNA sequencing of the spliced tRNAs, indicated that non-canonical BHB structure motifs were also recognized and processed by the splicing machinery in this organism. This is the first report to empirically determine the actual cleavage and splice sites of introns in the whole set of archaeal tRNA genes, and reassigns the exon-intron borders with a novel and more plausible non-canonical BHB structure.Gene 08/2011; 489(2):103-10. · 2.34 Impact Factor -
Article: Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus.
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ABSTRACT: Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5'-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes. In order to further understand the structural basis of the DNA recognition, binding and cleavage mechanism of FEN1, the structure of FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus (DaFEN1) was determined at 2.00 Å resolution. The overall fold of DaFEN1 was similar to those of other archaeal FEN1 proteins; however, the helical clamp and the flexible loop exhibited a putative substrate-binding pocket with a unique conformation.Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2011; 67(Pt 2):209-13. · 0.51 Impact Factor -
Article: Identification of novel acetyltransferase activity on the thermostable protein ST0452 from Sulfolobus tokodaii strain 7.
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ABSTRACT: A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.Journal of bacteriology 07/2010; 192(13):3287-93. · 3.94 Impact Factor -
Article: Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticus.
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ABSTRACT: Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5'-overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00 A resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58 A. The crystal contained one molecule in the asymmetric unit.Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2009; 65(Pt 9):923-5. · 0.51 Impact Factor -
Article: Abundance of Zetaproteobacteria within crustal fluids in back-arc hydrothermal fields of the Southern Mariana Trough.
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ABSTRACT: To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.Environmental Microbiology 09/2009; 11(12):3210-22. · 5.84 Impact Factor -
Article: Crystal structure of STS042, a stand-alone RAM module protein, from hyperthermophilic archaeon Sulfolobus tokodaii strain 7.
Proteins Structure Function and Bioinformatics 06/2008; 71(3):1557-62. · 3.39 Impact Factor -
Article: Crystal structure of an archaeal homologue of multidrug resistance repressor protein, EmrR, from hyperthermophilic archaea Sulfolobus tokodaii strain 7.
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ABSTRACT: MarR family proteins, MarR, MexR, and EmrR, are known as bacterial regulators for a phenotype resistant to multiple antibiotic drugs. Genomic data have indicated the presence of bacterial-type transcriptional regulators, including MarR family proteins in archaea, though the archaeal transcription system is close to that of eukaryote. To elucidate the structural basis of the transcriptional regulation mechanism of archaeal MarR family proteins, the crystal structure of the ST1710 protein, which was identified as an archaeal EmrR homologue, StEmrR, from hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was determined at 1.45-A resolution. The protein was composed of two N- and C-terminal dimerization domains, and the DNA-binding domain consisted of a winged helix motif, as in the case of bacterial MarR family proteins. Despite the relatively low overall structural similarity between StEmrR and bacterial MarR family proteins, the structure of the DNA-binding domain displayed high structural similarity. A comparison with the crystal structures of bacterial MarR family proteins revealed that structural variation was mainly due to the different orientation of the two helices at the N- and C-termini. Our results indicated that the distance between the two DNA-binding domains of MarR family proteins would be changed by the rotation of the two terminal helices to interact with the target DNA.Proteins Structure Function and Bioinformatics 07/2007; 67(4):1138-46. · 3.39 Impact Factor -
Article: Increasing in archaeal GlcNAc-1-P uridyltransferase activity by targeted mutagenesis while retaining its extreme thermostability.
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ABSTRACT: UDP-GlcNAc, an activated and essential form of GlcNAc which is an important component in the polysaccharide structure of most organisms, is synthesized from GlcNAc-1-P and UTP by GlcNAc-1-P UTase. We previously reported the identification of the extremely thermostable ST0452 protein, which has dual sugar-1-P NTase activities (Glc-1-P TTase and GlcNAc-1-P UTase activities) from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Detailed analyses of the protein indicated that the activity is slightly lower than that of bacteria. For industrial applications, activity needs to be increased without decreasing thermostability. Therefore, to enhance this activity, we introduced mutations into the amino acid residues located within the predicted reaction centre by targeted mutagenesis. All 12 mutant ST0452 proteins showed no decrease in thermostability. Among them, six mutant proteins were found to have increased GlcNAc-1-P UTase activity under optimal reaction conditions with sufficient substrates or an appropriate metal ion. Our results indicate that targeted mutagenesis is a powerful technique for in vitro production of a thermostable enzyme with enhanced activity. The results of this study also indicate that the space for the metal ion is important for selecting the type of metal ion and also affects the rate of the reaction.Journal of Biochemistry 05/2007; 141(4):553-62. · 2.37 Impact Factor -
Article: Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor.
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ABSTRACT: UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.Journal of Molecular Biology 02/2007; 365(4):903-10. · 4.00 Impact Factor -
Article: Identification of sn-glycerol-1-phosphate dehydrogenase activity from genomic information on a hyperthermophilic archaeon, Sulfolobus tokodaii strain 7.
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ABSTRACT: sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.Bioscience Biotechnology and Biochemistry 02/2006; 70(1):282-5. · 1.28 Impact Factor -
Article: Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.
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ABSTRACT: The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.Journal of Biochemistry 09/2005; 138(2):159-66. · 2.37 Impact Factor -
Article: L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization.
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ABSTRACT: A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100 degrees C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL-threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn(2+), Mn(2+) and Co(2+) for the activity, and contained one zinc ion/subunit. The K(m) values for L-threonine and NAD at 50 degrees C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain.Extremophiles 09/2005; 9(4):317-24. · 2.94 Impact Factor -
Article: The archaeon Pyrococcus horikoshii possesses a bifunctional enzyme for formaldehyde fixation via the ribulose monophosphate pathway.
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ABSTRACT: Pyrococcus horikoshii OT3, a hyperthermophilic and anaerobic archaeon, was found to have an open reading frame (PH1938) whose deduced amino acid sequence of the N-terminal and C-terminal halves showed significant similarity to two key enzymes of the ribulose monophosphate pathway for formaldehyde fixation in methylotrophic bacteria, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), respectively. The organism constitutively produced the encoded protein and exhibited activity of the sequential HPS- and PHI-mediated reactions in a particulate fraction. The full-length gene encoding the hybrid enzyme, the sequence corresponding to the HPS region, and the sequence corresponding to the PHI region were expressed in Escherichia coli and were found to produce active enzymes, rHps-Phi, rHps, or rPhi, respectively. Purified rHps-Phi and rHps were found to be active at the growth temperatures of the parent strain, but purified rPhi exhibited significant susceptibility to heat, suggesting that thermostability of the PHI moiety of the bifunctional enzyme (rHps-Phi) resulted from fusion with HPS. The bifunctional enzyme catalyzed the sequential reaction much more efficiently than a mixture of rHps and rPhi. These and other biochemical characterizations of the PH1938 gene product suggest that the ribulose monophosphate pathway plays a significant role in the archaeon under extreme environmental conditions.Journal of Bacteriology 07/2005; 187(11):3636-42. · 3.83 Impact Factor -
Article: The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.
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ABSTRACT: Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene was cloned and expressed, and the product was mainly obtained as inactive inclusion body in Escherichia coli. The inclusion body was dissolved in 6 M guanidine-HCl and successively refolded to active enzyme by the dilution of the denaturant. The enzyme exclusively catalyzed the hydrolysis of agmatine, but not arginine. This indicates that PH0083 codes agmatinase. The enzyme required divalent cations such as Co(2+), Ca(2+) and Mn(2+) for the activity. The highest activity was observed under fairly alkaline conditions, like pH 11. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of 110-145 kDa. The enzyme was extremely thermostable: the full activity was retained on heating at 80 degrees C for 10 min, and a half of the activity was retained by incubation at 90 degrees C for 10 min. From a typical Michaelis-Menten type kinetics, an apparent K(m) value for agmatine was determined to be 0.53 mM. Phylogenic analysis revealed that the agmatinase from P. horikoshii does not belong to any clusters of enzymes found in bacteria and eukarya. This is the first description of the presence of archaeal agmatinase and its characteristics.Biochimica et Biophysica Acta 05/2005; 1748(1):110-5. · 4.66 Impact Factor -
Article: Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7.
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ABSTRACT: L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 min at 95 degrees C and 180 min at 80 degrees C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing alpha-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not alpha-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.Journal of Biological Chemistry 04/2005; 280(10):9698-705. · 4.77 Impact Factor -
Article: Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria.
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ABSTRACT: In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis-Menten constants or K(m) value between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the K(m) values of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis-Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.Metabolic Engineering 04/2005; 7(2):59-69. · 5.61 Impact Factor -
Article: Characterization of a whole set of tRNA molecules in an aerobic hyper-thermophilic Crenarchaeon, Aeropyrum pernix K1.
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ABSTRACT: The tRNA molecule has an important role in translation, the function of which is to carry amino acids to the ribosomes. It is known that tRNA is transcribed from tRNA genes, some of which, in Eukarya and Archaea, contain introns. A computational analysis of the complete genome of Aeropyrum pernix K1 predicted the presence of 14 intron-containing tRNA genes. To elucidate whether these introns are actually processed in living cells and what mechanism detects the intron regions, cDNAs for premature and mature forms of the tRNA molecules transcribed from the intron-containing tRNA genes in the model aerobic acidothermophilic crenarchaeon, A. pernix K1 were identified and analyzed. A comparison between the nucleotide sequences of these two types of cDNAs indicated that the intron regions of the tRNA molecules were indeed processed in A. pernix K1 living cells. Some cDNA clones showed that the actual splicing positions were different from those predicted by computational analysis. However, the bulge-helix-bulge structure, which has been previously identified in exon-intron boundaries of archaeal tRNA genes, was evident in all boundary regions confirmed in this work. These results indicate that the generally described mechanism for tRNA processing in Archaea is utilized for processing the intron region of the tRNA molecules in A. pernix K1.DNA Research 02/2005; 12(6):403-16. · 5.16 Impact Factor -
Article: Oxidative stress response in an anaerobic hyperthermophilic archaeon: presence of a functional peroxiredoxin in Pyrococcus horikoshii.
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ABSTRACT: Oxidative stress response in an anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was analyzed by two-dimensional gel electrophoresis. When P. horikoshii was grown on medium supplemented with air, a marked increase in the level of a 25-kDa protein was observed in comparison with cells grown under anaerobic conditions. The N-terminal amino acid sequence of the protein was determined to be VVIGEKFPEVEVKTTHGVIKLPDYF, which coincides with that of the putative alkyl hydroperoxide reductase that has been predicted in the genome database of P. horikoshii. The gene (PH1217) encoding the protein was cloned and expressed in Escherichia coli. The produced enzyme was a hyperthermostable peroxiredoxin whose activity was not lost after incubation at 90 degrees C for 20 min. The enzyme catalyzes the reduction of cumene hydroperoxide and hydrogen peroxide using dithiothreitol as an electron donor. Northern blot analysis revealed that the transcription of the gene increased by the addition of exogenous oxygen and by the addition of an oxidative stress-inducing reagent, and reached maximum within 30 min. These results suggest that the peroxiredoxin plays an important role in the peroxide-scavenging system in an anaerobic archaeon P. horikoshii.Journal of Biochemistry 11/2004; 136(4):541-7. · 2.37 Impact Factor -
Article: Evolutionary process of amino acid biosynthesis in Corynebacterium at the whole genome level.
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ABSTRACT: Corynebacterium glutamicum, which is the closest relative of Corynebacterium efficiens, is widely used for the large scale production of many kinds of amino acids, particularly glutamic acid and lysine, by fermentation. Corynebacterium diphtheriae, which is well known as a human pathogen, is also closely related to these two species of Corynebacteria, but it lacks such productivity of amino acids. It is an important and interesting question to ask how those closely related bacterial species have undergone such significant functional differentiation in amino acid biosynthesis. The main purpose of the present study is to clarify the evolutionary process of functional differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome sequences. When Mycobacterium and Streptomyces were used as out groups, our comparative study suggested that the common ancestor of Corynebacteria already possessed almost all of the gene sets necessary for amino acid production. However, C. diphtheriae was found to have lost the genes responsible for amino acid production. Moreover, we found that the common ancestor of C. efficiens and C. glutamicum have acquired some of genes responsible for amino acid production by horizontal gene transfer. Thus, we conclude that the evolutionary events of gene loss and horizontal gene transfer must have been responsible for functional differentiation in amino acid biosynthesis of the three species of Corynebacteria.Molecular Biology and Evolution 10/2004; 21(9):1683-91. · 5.55 Impact Factor
Top co-authors
Top Journals
Institutions
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2012
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Kyushu University
- Faculty of Agriculture
Fukuoka-shi, Fukuoka-ken, Japan
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2007–2011
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The University of Tokyo
- Department of Applied Biological Chemistry
Tokyo, Tokyo-to, Japan
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1998–2011
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National Institute of Technology and Evaluation
Tokyo, Tokyo-to, Japan
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2005–2010
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National Institute of Advanced Industrial Science and Technology
- Research Center for Stem Cell Engineering
Amagasaki, Hyogo-ken, Japan
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2006
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University of Occupational and Environmental Health
- Department of Chemistry
Kitakyūshū, Fukuoka-ken, Japan
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2002–2005
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The University of Tokushima
- Department of Biological Science and Technology
Tokushima-shi, Tokushima-ken, Japan
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2004
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Tokyo University of Agriculture and Technology
- Division of Biotechnology and Life Science
Tokyo, Tokyo-to, Japan
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