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Yoshihiko Sako - One of the best experts on this subject based on the ideXlab platform.
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provirus induction in hyperthermophilic archaea characterization of Aeropyrum Pernix spindle shaped virus 1 and Aeropyrum Pernix ovoid virus 1
Journal of Bacteriology, 2011Co-Authors: Tomohiro Mochizuki, Yoshihiko Sako, David PrangishviliAbstract:By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum Pernix, and under special conditions of A. Pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum Pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum Pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.
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Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum Pernix spindle-shaped virus 1 and Aeropyrum Pernix ovoid virus 1
2011Co-Authors: Tomohiro Mochizuki, Yoshihiko Sako, David PrangishviliAbstract:By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum Pernix, and under special conditions of A. Pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum Pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum Pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae. Sequencing of diverse bacterial and archaeal genomes has revealed the presence of integrated putative viral genomes, their fragments, and viral genes in cellular chromosomes (3, 8, 14–16, 28, 32). These results served as a basis for speculations on virus-host relationships and the nature of presumed provi
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Diversity of viruses of the hyperthermophilic archaeal genus Aeropyrum, and isolation of the Aeropyrum Pernix bacilliform virus 1, APBV1, the first representative of the family Clavaviridae.
Virology, 2010Co-Authors: Tomohiro Mochizuki, Yoshihiko Sako, Patrick Forterre, Takashi Yoshida, Reiji Tanaka, David PrangishviliAbstract:We have surveyed the morphological diversity of viruses infecting the archaeon Aeropyrum Pernix, the most thermophilic species among aerobic organisms, growing optimally at 90 degrees C, and isolated and characterized a novel virus, Aeropyrum Pernix bacilliform virus 1, APBV1. This is the first virus to be described of the genus Aeropyrum and the archaeal order Desulfurococcales. The virion of APBV1 has rigid bacilliform morphology, about 140x20nm, with one end pointed and the other rounded. It contains highly glycosylated single major protein and three minor proteins. The circular, double-stranded DNA genome comprising 5278bp is the smallest for known archaeal viruses. None of the 14 putative genes, all on the same DNA strand, shows significant similarity to sequences in the public databases. The APBV1 infection caused neither retardation of host growth nor lysis of host cells, and integration of the viral genome into the host chromosome was not detected. On the basis of unusual morphological and genomic properties, we propose to consider APBV1 as the first representative of a new viral family, the Clavaviridae.
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i apei a novel intron encoded laglidadg homing endonuclease from the archaeon Aeropyrum Pernix k1
Nucleic Acids Research, 2005Co-Authors: Norimichi Nomura, Yayoi Morinaga, Nobuaki Shirai, Yoshihiko SakoAbstract:Over 50 introns have been reported in archaeal rRNA genes (rDNAs), a subset of which nests putative homing endonuclease (HEase) genes. Here, we report the identification and characterization of a novel archaeal LAGLIDADG-type HEase, I-ApeI, encoded by the ApeK1.S908 intron within the 16S rDNA of Aeropyrum Pernix K1. I-ApeI consists of 222 amino acids and harbors two LAGLIDADG-like sequences. It recognizes the 20 bp non-palindromic sequence 5′-GCAAGGCTGAAAC↓TTAAAGG and cleaves target DNA to produce protruding tetranucleotide 3′ ends. Either Mn2+ or Co2+ can be substituted for Mg2+ as a cofactor in the cleavage reaction. Of the 20 bases within the minimal recognition site, 7 are essential for cleavage and are located at positions proximal to the cleavage sites.
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molecular recognition of threonine trna by threonyl trna synthetase from an extreme thermophilic archaeon Aeropyrum Pernix k1
Nucleic acids research. Supplement (2001), 2002Co-Authors: Yoshiyuki Nagaoka, Jun Iwaki, Junji Yokozawa, Yoshinori Koyama, Takuya Umehara, Yutaka Kawarabayasi, Yoshihiko Sako, Koji Okamoto, Takayoshi Wakagi, Atsushi KunoAbstract:To investigate the recognition sites of tRNA 71" for threonyl-tRNA synthetase (ThrRS) from an extreme thermophilic and aerobic archaeon, Aeropyrum Pernix Kl, threonylation experiments using various in vitro mutant transcripts of tRNA 11"" were examined. The results indicated that A. Pernix ThrRS did recognize the first three base pairs of acceptor stem in addition to the second and the third letters of anticodon of tRNAThr, in spite of its N-terminal truncated unique structure. Discriminator base was not involved in recognition by A Pernix ThRS. These determinants were confirmed by the identity switching experiments from the in vitro mutants of A. Pernix tRNA1*™ and *
Nataša Poklar Ulrih - One of the best experts on this subject based on the ideXlab platform.
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insights into the maturation of pernisine a subtilisin like protease from the hyperthermophilic archaeon Aeropyrum Pernix
Applied and Environmental Microbiology, 2020Co-Authors: Miha Bahun, Marko Snajder, Dusan Turk, Nataša Poklar UlrihAbstract:Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum Pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. Pernix Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum Pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.
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molecular adaptation to high temperatures pernisine from the archaeon Aeropyrum Pernix k1
Biologia Serbica, 2020Co-Authors: Kevin Hartman, Miha Bahun, Marko Snajder, Nataša Poklar UlrihAbstract:Pernisine is a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum Pernix . Due to its high thermal stability and its activity in the presence of denaturants, pernisine represents a promising enzyme for use in various industrial applications. Another potentially applicable characteristic of this protease is its ability to degrade infectious prion aggregates. Production of pernisine in A . Pernix does not provide sufficient yield for its commercial use, and alternative production strategies are hence needed. This review summarizes the biochemical and biophysical characteristics of pernisine and progress that has been made toward production of recombinant pernisine using Escherichia coli and Streptomyces rimosus as expression systems.
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Optimization of growth for the hyperthermophilic archaeon Aeropyrum Pernix on a small-batch
2020Co-Authors: Igor Milek, Blañ Cigi, Mihaela Skrt, Gönül Kaletunç, Nataša Poklar UlrihAbstract:Abstract: Growth of Aeropyrum Pernix, the first reported aerobic neutrophilic hyperthermophilic archaeon, was investigated under different cultivation parameters. Different sources of seawater, pH, and the cultivation methods were tested with the aim to improve the biomass production. A 1-L glass flask fitted with a condenser and air diffuser was used as a bioreactor. The optimum conditions for maximizing A. Pernix biomass were obtained when Na 2 S 2 O 3 ·5H 2 O (1 g/L) with added marine broth 2216 at pH 7.0 (20 mmol HEPES buffer/L) was used as a growing medium in a 1-L flask. The biomass production was 0.45 g dry cell mass/L in 40 h under the optimum conditions, which is more than the 0.42 g dry cell mass/L in 60 h previously obtained. Key words: Aeropyrum Pernix, hyperthermophilic archaeon, biomass production, simple glass flask based system. Mots clés : Aeropyrum Pernix, archéen hyperthermophile, production de biomasse, système simple reposant sur un flacon de verre
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antioxidative activity of methanolic and water extracts from the hyperthermophilic archaeon Aeropyrum Pernix k1
Acta Chimica Slovenica, 2018Co-Authors: Mihaela Skrt, Polona Jamnik, Nataša Poklar UlrihAbstract:The hyperthermophilic archaeon Aeropyrum Pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. Pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. Pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.
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bending elasticity modulus of giant vesicles composed of Aeropyrum Pernix k1 archaeal lipid
Life, 2015Co-Authors: J Genova, Nataša Poklar Ulrih, Veronika Kraljiglic, Ales Iglic, I BivasAbstract:Abstract: Thermally induced shape fluctuations were used to study elastic properties of giant vesicles composed of archaeal lipids C 25,25 -archetidyl (glucosyl) inositol and C 25,25 -archetidylinositol isol ated from lyophilised Aeropyrum Pernix K1 cells. Giant vesicles were created by electroformation in pure water environment. Stroboscopic illumination using a xenon flash lamp was implemented to remove the blur effect due to the finite integration time of the camera and to obtain an instant picture of the fluctuating vesicle shape. The mean weighted value of the bending elasticity modulus k c of the archaeal membrane determined from the measurements meeting the entire set of qualification criteria was (1.89 ± 0.18) × 10 −19 J, which is similar to the values obtained for a membrane composed of the eukaryotic phospholipi ds SOPC (1.88 ± 0.17) × 10 −19 J and POPC (2.00 ± 0.21) 10 −19 J. We conclude that membranes composed of archaeal lipids isolated from Aeropyrum Pernix K1 cells have similar elastic properties as membranes composed of eukaryotic lipids. This fact, together with the importa nce of the elastic properties fo r the normal circulation through
David Prangishvili - One of the best experts on this subject based on the ideXlab platform.
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provirus induction in hyperthermophilic archaea characterization of Aeropyrum Pernix spindle shaped virus 1 and Aeropyrum Pernix ovoid virus 1
Journal of Bacteriology, 2011Co-Authors: Tomohiro Mochizuki, Yoshihiko Sako, David PrangishviliAbstract:By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum Pernix, and under special conditions of A. Pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum Pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum Pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.
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Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum Pernix spindle-shaped virus 1 and Aeropyrum Pernix ovoid virus 1
2011Co-Authors: Tomohiro Mochizuki, Yoshihiko Sako, David PrangishviliAbstract:By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum Pernix, and under special conditions of A. Pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum Pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum Pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae. Sequencing of diverse bacterial and archaeal genomes has revealed the presence of integrated putative viral genomes, their fragments, and viral genes in cellular chromosomes (3, 8, 14–16, 28, 32). These results served as a basis for speculations on virus-host relationships and the nature of presumed provi
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Diversity of viruses of the hyperthermophilic archaeal genus Aeropyrum, and isolation of the Aeropyrum Pernix bacilliform virus 1, APBV1, the first representative of the family Clavaviridae.
Virology, 2010Co-Authors: Tomohiro Mochizuki, Yoshihiko Sako, Patrick Forterre, Takashi Yoshida, Reiji Tanaka, David PrangishviliAbstract:We have surveyed the morphological diversity of viruses infecting the archaeon Aeropyrum Pernix, the most thermophilic species among aerobic organisms, growing optimally at 90 degrees C, and isolated and characterized a novel virus, Aeropyrum Pernix bacilliform virus 1, APBV1. This is the first virus to be described of the genus Aeropyrum and the archaeal order Desulfurococcales. The virion of APBV1 has rigid bacilliform morphology, about 140x20nm, with one end pointed and the other rounded. It contains highly glycosylated single major protein and three minor proteins. The circular, double-stranded DNA genome comprising 5278bp is the smallest for known archaeal viruses. None of the 14 putative genes, all on the same DNA strand, shows significant similarity to sequences in the public databases. The APBV1 infection caused neither retardation of host growth nor lysis of host cells, and integration of the viral genome into the host chromosome was not detected. On the basis of unusual morphological and genomic properties, we propose to consider APBV1 as the first representative of a new viral family, the Clavaviridae.
Yutaka Kawarabayasi - One of the best experts on this subject based on the ideXlab platform.
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specific interactions of three proliferating cell nuclear antigens with replication related proteins in Aeropyrum Pernix
Molecular Microbiology, 2007Co-Authors: Kaori Imamura, Yutaka Kawarabayasi, Kenzo Fukunaga, Yoshizumi IshinoAbstract:Proliferating cell nuclear antigen (PCNA) is a well-known multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms. We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum Pernix. PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.
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characterization of a whole set of trna molecules in an aerobic hyper thermophilic crenarchaeon Aeropyrum Pernix k1
DNA Research, 2006Co-Authors: Syuji Yamazaki, Hisashi Kikuchi, Yutaka KawarabayasiAbstract: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.
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crystallization of the xeroderma pigmentosum group f endonuclease from Aeropyrum Pernix
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: John Lally, Yutaka Kawarabayasi, Matthew Newman, Judith Murrayrust, Andrew J Fadden, Neil Q McdonaldAbstract:The xeroderma pigmentosa group F protein (XPF) is a founding member of a family of 3′-flap endonucleases that play an essential role in nucleotide-excision repair, DNA replication and recombination. The XPF gene has been cloned from Aeropyrum Pernix, encoding a 254-residue protein (apXPF). Recombinant protein was produced in Escherichia coli and purified by three chromatographic steps. Three different crystal forms of apXPF were grown in trigonal, monoclinic and triclinic systems. The trigonal crystals diffracted to 2.8 A and were grown in the presence of double-stranded DNA. Monoclinic crystals were grown without DNA and diffracted to 3.2 A. Triclinic crystals were grown from a truncated apXPF protein lacking the tandem helix–hairpin–helix motifs and diffracted to 2.1 A.
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Cloning, expression, and characterization of the first archaeal ATP-dependent glucokinase from aerobic hyperthermophilic archaeon Aeropyrum Pernix.
Journal of biochemistry, 2003Co-Authors: Haruhiko Sakuraba, Yutaka Kawarabayasi, Yuri Mitani, Shuichiro Goda, Toshihisa OhshimaAbstract:The gene encoding the ATP-dependent glucokinase of hyperthermophilic archaeon Aeropyrum Pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed 40% identity to that of the putative glucokinase from hyperthermophilic archaeon Pyrobacurum aerophilum. The purified recombinant enzyme was a monomer with a molecular mass of 35 kDa. The enzyme retained its full activity on heating at 70 degrees C for 10 min and retained 65% of the activity after 10-min incubation at 100 degrees C. The enzyme exclusively catalyzed the phosphorylation of D-glucose using ATP as a phosphoryl donor. ITP was accepted in addition to ATP. The rate dependence with both glucose and ATP followed Michaelis-Menten kinetics, with apparent K(m) values of 0.054 and 0.50 mM, respectively. The enzyme activity required divalent cations; Mg(2+), which was most effective, could partially be replaced by Mn(2+) or Ca(2+). Phylogenetic analysis revealed that the glucokinase from A. Pernix does not belong to the clusters of enzymes found in bacteria and eukarya. This is the first description of the characteristics of an ATP-dependent glucokinase from an archaeon.
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molecular recognition of threonine trna by threonyl trna synthetase from an extreme thermophilic archaeon Aeropyrum Pernix k1
Nucleic acids research. Supplement (2001), 2002Co-Authors: Yoshiyuki Nagaoka, Jun Iwaki, Junji Yokozawa, Yoshinori Koyama, Takuya Umehara, Yutaka Kawarabayasi, Yoshihiko Sako, Koji Okamoto, Takayoshi Wakagi, Atsushi KunoAbstract:To investigate the recognition sites of tRNA 71" for threonyl-tRNA synthetase (ThrRS) from an extreme thermophilic and aerobic archaeon, Aeropyrum Pernix Kl, threonylation experiments using various in vitro mutant transcripts of tRNA 11"" were examined. The results indicated that A. Pernix ThrRS did recognize the first three base pairs of acceptor stem in addition to the second and the third letters of anticodon of tRNAThr, in spite of its N-terminal truncated unique structure. Discriminator base was not involved in recognition by A Pernix ThRS. These determinants were confirmed by the identity switching experiments from the in vitro mutants of A. Pernix tRNA1*™ and *
Kazuhiko Ishikawa - One of the best experts on this subject based on the ideXlab platform.
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Role of F225 in O-phosphoserine sulfhydrylase from Aeropyrum Pernix K1
Extremophiles, 2016Co-Authors: Emi Takeda, Kazuhiko Ishikawa, Kohei Kunimoto, Yoshito Kawai, Misumi Kataoka, Takashi NakamuraAbstract:O -Phosphoserine sulfhydrylase (OPSS) synthesizes cysteine from O -phospho- l -serine (OPS) and sulfide. We have determined the three-dimensional structures of OPSS from hyperthermophilic archaeon Aeropyrum Pernix K1 (ApOPSS) in complex with aminoacrylate intermediate (AA) formed from pyridoxal 5′-phosphate with OPS or in complex with cysteine and compared them with that of ApOPSS. We found an orientational change of F225 at the active-site entrance and constructed an F225A mutant to examine its activities and AA stability and clarify the role of F225 in ApOPSS. The OPS and O -acetyl- l -serine (OAS) sulfhydrylase activities of the F225A mutant decreased by 4.2- and 15-fold compared to those of the wild-type (wt) ApOPSS, respectively. The ability of OPS and OAS to form AA also decreased by 12- and 27-fold, respectively. AA was less stable in the F225A mutant than in the wt ApOPSS. Simulated docking showed that leaving groups, such as phosphate and acetate, were oriented to the inside of the active site in the F225A mutant, whereas they were oriented to the entrance in the wt ApOPSS. These results suggest that F225 in ApOPSS plays important roles in maintaining the hydrophobic environment of AA from solvent water and in controlling the orientation of leaving groups.
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thermostability and reactivity in organic solvent of o phospho l serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum Pernix k1
Bioscience Biotechnology and Biochemistry, 2015Co-Authors: Takashi Nakamura, Kohei Kunimoto, Shinji Asai, Kaori Nakata, Masateru Oguri, Kazuhiko IshikawaAbstract:O-phospho-l-serine sulfhydrylase (OPSS) from archaeon Aeropyrum Pernix K1 is able to synthesize l-cysteine even at 80 °C. In this article, we compared thermal stability and reactivity in organic solvent of OPSS with those of O-acetyl-l-serine sulfhydrylase B (OASS-B) from Escherichia coli. As a result, the thermostability of OPSS was much higher than that of OASS-B. Moreover, the activity of OPSS increased in the reaction mixture containing the organic solvent, such as N, N′-dimethyl formamide and 1,4-dioxane, whereas that of OASS-B gradually decreased as the content of organic solvent increased. From the crystal structural analysis, the intramolecular electrostatic interactions of N-terminal domain in OPSS seemed to be correlated with the tolerance of OPSS to high temperature and organic solvent. These results indicate that OPSS is more superior to OASS-B for the industrial production of l-cysteine and unnatural amino acids that are useful pharmaceuticals in the presence of organic solvent.
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crystal structure of peroxiredoxin from Aeropyrum Pernix k1 complexed with its substrate hydrogen peroxide
Journal of Biochemistry, 2010Co-Authors: Tsutomu Nakamura, Kazuhiko Ishikawa, Hiroyoshi Matsumura, Yuji Kado, Takafumi Yamaguchi, Tsuyoshi InoueAbstract:Peroxiredoxin (Prx) reduces hydrogen peroxide and alkyl peroxides to water and corresponding alcohols, respectively. The reaction is dependent on a peroxidatic cysteine, whose sulphur atom nucleophilically attacks one of the oxygen atoms of the peroxide substrate. In spite of the many structural studies that have been carried out on this reaction, the tertiary structure of the hydrogen peroxide-bound form of Prx has not been elucidated. In this paper, we report the crystal structure of Prx from Aeropyrum Pernix K1 in the peroxide-bound form. The conformation of the polypeptide chain is the same as that in the reduced apo-form. The hydrogen peroxide molecule is in close contact with the peroxidatic Cys50 and the neighbouring Thr47 and Arg126 side chain atoms, as well as with the main chain nitrogen atoms of Val49 and Cys50. Bound peroxide was also observed in the mutant C50S, in which the peroxidatic cysteine was replaced by serine. Therefore, the sulphur atom of the peroxidatic cysteine is not essential for peroxide binding, although it enhances the binding affinity. Hydrogen peroxide binds to the protein so that it fills the active site pocket. This study provides insight into the early stage of the Prx reaction.
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crystallization and preliminary x ray diffraction analysis of thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum Pernix k1
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2005Co-Authors: Tsutomu Nakamura, Mitsuo Ataka, Hiroyoshi Matsumura, Tsuyoshi Inoue, Yasushi Kai, Koichi Uegaki, Yoshihisa Hagihara, Kazuhiko IshikawaAbstract:Thioredoxin peroxidase is a member of the peroxiredoxin family and plays a dominant role in a hydrogen peroxide metabolism. A recombinant form of the hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum Pernix K1, a polypeptide consisting of 250 amino acids, was purified. The C207S mutant protein was crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as the precipitant at 298 K. Diffraction data were collected and processed to 2.7 A resolution. The crystal belongs to space group P1, with unit-cell parameters a = 126.2, b = 126.3, c = 213.7 A, α = 80.4, β = 80.3, γ = 70.7°. Calculation of the self-rotation function showed that the protein quaternary structure includes a fivefold axis and five twofold axes.
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cloning purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum Pernix k1
Journal of Molecular Catalysis B-enzymatic, 2003Co-Authors: Yan Feng, Renjun Gao, Kazuhiko Ishikawa, Hiroyasu Ishida, Susumu Ando, Yoshitsugu Kosugi, Shugui CaoAbstract:Abstract The gene APE1547 of the aerobic thermophilic Aeropyrum Pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli. BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. Pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90 °C and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p-nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90 °C was over 160 h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications.