Frontiers MediaActa Medica Okayama1664-462X152024Illumina-based transcriptomic analysis of the fast-growing leguminous tree Acacia crassicarpa: functional gene annotation and identification of novel SSR-markers1339958ENShougoIshioTsukuba Research Institute, Sumitomo Forestry Co. Ltd.KazutakaKusunokiTsukuba Research Institute, Sumitomo Forestry Co. Ltd.MichikoNemotoGraduate School of Environment, Life, Natural Science and Technology, Okayama UniversityTadayoshiKanaoGraduate School of Environment, Life, Natural Science and Technology, Okayama UniversityTakashiTamuraInstitute of Global Human Resource Development, Okayama UniversityAcacia crassicarpa is a fast-growing leguminous tree that is widely cultivated in tropical areas such as Indonesia, Malaysia, Australia, and southern China. This tree has versatile utility in timber, furniture, and pulp production. Illumina sequencing of A. crassicarpa was conducted, and the raw data of 124,410,892 reads were filtered and assembled de novo into 93,317 unigenes, with a total of 84,411,793 bases. Blast2GO annotation, Benchmark Universal Single-Copy Ortholog evaluation, and GO-term classification produced a catalogue of unigenes for studying primary metabolism, phytohormone signaling, and transcription factors. Massive transcriptomic analysis has identified microsatellites composed of simple sequence repeat (SSR) loci representing di-, tri-, and tetranucleotide repeat units in the predicted open reading frames. Polymorphism was induced by PCR amplification of microsatellite loci located in several genes encoding auxin response factors and other transcription factors, which successfully distinguished 16 local trees of A. crassicarpa tested, representing potentially exploitable molecular markers for efficient tree breeding for plantation and biomass exploitation.No potential conflict of interest relevant to this article was reported.Springer Science and Business Media LLCActa Medica Okayama1436-22282562023Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation12081219ENKokiOkadaGraduate School of Environmental and Life Science, Okayama UniversityYuMorimotoGraduate School of Environmental and Life Science, Okayama UniversityYukineShiraishiGraduate School of Environmental and Life Science, Okayama UniversityTakashiTamuraFaculty of Environmental, Life, Natural Science and Technology, Okayama UniversityShigekiMayamaThe Advanced Support Center for Science Teachers, Tokyo Gakugei UniversityTakashiKadonoFaculty of Agriculture and Marine Science, Kochi UniversityMasaoAdachiFaculty of Agriculture and Marine Science, Kochi UniversityKentaroIfukuGraduate School of Agriculture, Kyoto UniversityMichikoNemotoFaculty of Environmental, Life, Natural Science and Technology, Okayama UniversityNitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 μg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.No potential conflict of interest relevant to this article was reported.Nature PortfolioActa Medica Okayama2041-17231412023Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota1730ENTitouanJaunet-LaharyResearch Center for Computational Science, Institute for Molecular Science, National Institutes of Natural SciencesTatsuroShimamuraGraduate School of Medicine, Kyoto UniversityMasahiroHayashiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityNorimichiNomuraGraduate School of Medicine, Kyoto UniversityKoutaHirasawaGraduate School of Medicine, Kyoto UniversityTetsuyaShimizuRIKEN SPring-8 CenterMasaoYamashitaRIKEN SPring-8 CenterNaotakaTsutsumiGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYutaSuehiroSchool of Pharmaceutical Sciences, Okayama UniversityKeiichiKojimaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityYukiSudoGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityTakashiTamuraGraduate School of Environmental and Life Sciences, Okayama UniversityHirokoIwanariResearch Center for Advanced Science and Technology, The University of TokyoTakaoHamakuboResearch Center for Advanced Science and Technology, The University of TokyoSoIwataGraduate School of Medicine, Kyoto UniversityKei-IchiOkazakiResearch Center for Computational Science, Institute for Molecular Science, National Institutes of Natural SciencesTeruhisaHiraiRIKEN SPring-8 CenterAtsukoYamashitaGraduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityAn oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.No potential conflict of interest relevant to this article was reported.ElsevierActa Medica Okayama2590152452021Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride100044ENMasakiKitagawaDepartment of Macromolecular Science, Graduate School of Science, Osaka UniversityNanakoItoDepartment of Macromolecular Science, Graduate School of Science, Osaka UniversityYuyaMatsumotoDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama UniversityMasayaSaitoDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama UniversityTakashiTamuraDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama UniversityHitoshiKusakabeEnzyme Sensor Co., Ltd.KenjiInagakiDepartment of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama UniversityKatsumiImadaDepartment of Macromolecular Science, Graduate School of Science, Osaka UniversityHarmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Å resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.No potential conflict of interest relevant to this article was reported. SpringerActa Medica Okayama1436-22282020Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein MethyltransferasesENMichikoNemotoGraduate School of Environmental and Life Science, Okayama UniversitySayakoIwakiGraduate School of Environmental and Life Science, Okayama UniversityHisaoMoriyaGraduate School of Environmental and Life Science, Okayama UniversityYukiMondenGraduate School of Environmental and Life Science, Okayama UniversityTakashiTamuraGraduate School of Environmental and Life Science, Okayama UniversityKenjiInagakiGraduate School of Environmental and Life Science, Okayama UniversityShigekiMayamaDepartment of Biology, Tokyo Gakugei UniversityKioriObuseGraduate School of Environmental and Life Science, Okayama UniversitySilica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551092020Marinomonas mediterranea由来キノン含有新規グリシンオキシダーゼの大腸菌発現系の確立と性質検討16ENYukiKajiyamaGraduate School of Environmental and Life Science, Okayama UniversitySatsukiMizobataGraduate School of Environmental and Life Science, Okayama UniversityShusakuAkajiGraduate School of Environmental and Life Science, Okayama UniversityMichikoNemotoGraduate School of Environmental and Life Science, Okayama UniversityTakashiTamuraGraduate School of Environmental and Life Science, Okayama UniversityKenjiInagakiGraduate School of Environmental and Life Science, Okayama University Novel glycine oxidase (GlyOX) from Marinomonas mediterranea depends on cysteine tryptophilquinone (CTQ) and catalyzes the oxidative deamination of glycine to produce a glyoxylate, ammonia, and hydrogen peroxide. M. mediterranea GlyOX genes (goxA and goxB) were cloned and recombinant GlyOX was heterologously expressed by E. coli. The purification of recombinant GlyOX was carried out by metal affinity and DEAE-Toyopearl 650M column chromatographies. M. mediterranea GlyOX was homotetramic with a molecular mass of 76kDa and showed optimum activity around 30°C and at pH 5.0, and stability below 50°C and between pH 5.0 to 9.0. M. mediterranea GlyOX shows a strict substrate specificity toward glycine, and the Michaelis constant for glycine was 0.5mM. M. mediterranea GlyOX could determine the quantity of glycine in human serum and human blood plasma with high sensitivity. This study revealed the catalytic and structural properties of M. mediterranea GlyOX with high substrate specificity.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551052016大学生の朝食欠食習慣の統計解析と改善への新指針15ENT.TamuraT.IbiK.InagakiY.KuboK.OkudaThis study investigated the current status and causes underneath the life of university students who tend to lack breakfast at a relatively high frequency, and statistical analysis on consequences leading to such lack of well-nourished eating habitat in their university life. In October 2014, self-assessed questionnaires were administered to over 150 faculty students. It contained questions about breakfast habits, time allowance for the morning class, and lunchtime setting in their high school timetable. Breakfast states were clearly separated in three groups : 68% of students regularly have breakfast throughout the weekdays, 21% students skipping the breakfast occasionally, and 11% student no habit for breakfast at all. The survey on the high school lives revealed that 70% students used to have lunch 30 min later than the lunchtime set in the university timetable, 7% of them had the lunch time even more than 1 h later. Lunchtime varies among high schools, and statistical significance was revealed (p<0.01) that schools with higher deviation scores tend have late lunch beyond 12: 30. Accordingly, university students were given directions to prepare for the timetable reform on postulation of having lunch time over one o’clock. After continuous survey on the breakfast habits during the second semester, more than 90% of students established the habit of breakfast regularly in their university lives with the improved consciousness toward well-balanced healthy breakfast contents for their higher level of education quality.No potential conflict of interest relevant to this article was reported.Nature Publishing GroupActa Medica Okayama2045-232262016Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico19742ENTakashiTamuraNaokiTsunekawaMichikoNemotoKenjiInagakiToshiyukiHiranoFumitoshiSatoOxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni–Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.No potential conflict of interest relevant to this article was reported.the American Society for MicrobiologyActa Medica Okayama2169-8287342015Draft Genome Sequence of Streptomyces incarnatus NRRL8089, which Produces the Nucleoside Antibiotic Sinefungine00715-15e00715-15ENKenshiroOshimaMasahiraHattoriHitomiShimizuKojiFukudaMichikoNemotoKenjiInagakiTakashiTamuraA draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551032014高基質特異性L-グルタミン酸オキシダーゼより作成した基質特異性改変酵素L-アルギニンオキシダーゼの性質検討59ENRyuichiroNakaiShihokoFujinoTomohiroUtsumiTakashiTamuraHitoshiKusakabeaKenjiInagaki L‒Glutamate oxidase (LGOX) from Streptomyces sp. X‒119‒6 has strict substrate specificity toward
L‒glutamate. Recently, we solved the X‒ray crystal structure of LGOX and this revealed that Arg305 in
the active site is the key residue involved in substrate recognition. Therefore, we created 19 mutant
enzymes of R305X‒LGOX by saturation mutagenesis. One of them R305D‒LGOX, Arg305 substituted
with Asp exhibited oxidase activity for L‒Arg. Optimum pH of R305D‒LGOX mutant enzyme was pH
8.5. Interestingly, the activity of R305D‒LGOX toward L‒Arg was inhibited by phosphate. And furthermore,
the substrate specificity of R305D‒LGOX was affected by using buffer. The results of inhibition
analysis suggest, that phosphate is a competitive inhibitor of R305D‒LGOX when L‒Arg is used as
substrate. Kinetic analysis of R305D‒LGOX showed that Km value and kcat value of R305D‒LGOX toward
l-Arg were 0.68 mM and 6.7 s-1 respectively. In this study, we showed that R305D‒LGOX mutant
enzyme is a novel l-arginine oxidase and useful for l-arginine biosensor.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama2186-77551012012好熱性細菌Thermus sp.O-3-1由来耐熱性アミダーゼの精製及び性質検討16ENFumiakiKobayashiHirokiAomineWataruMizunashiFujioYuTakashiTamuraKenjiInagakiThe gene encoding a thermostable amidase (EC 3.5.1.4) from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally,
as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 1002011放線菌Streptomyces sp.590由来l-メチオニン脱炭酸酵素の精製および性質検討37ENTomomiMaemuraKumikoUchitomiChikaKusakaJunkoInagakiTakashiTamuraKenjiSodaKenjiInagakiL-Methionine decarboxylase [EC 4.1.1.57] catalyzes the decarboxylation of L-methionine and is a pyridoxal 5’-phosohate(PLP)-dependent enzyme. L-Methionine decarboxylase has been purified 630-fold by DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M and Sephacryl S-300 column chromatographies from Streptomyces sp.590. The enzyme has a dimeric structure with identical subunits of Mr 60,000. This enzyme shows optimum activity at pH7.0 and 45°C, and is stable between pH5.7 and pH9.0. L-Methionine decarboxylase has antitumor activity against RERF-LC-AI and HeLa cells. Ten N-terminal amino acid sequence of L-methionine decarboxylase was determined, and the sequence showed no homology with other reported proteins.No potential conflict of interest relevant to this article was reported.Acta Medica Okayama14342008Selenite Assimilation into Formate Dehydrogenase H Depends on Thioredoxin Reductase in Escherichia coli467473ENMuneakiTakahataTakashiTamuraKatsumasaAbeHisaakiMiharaSuguruKurokawaYoshihiroYamamotoRyuheiNakanoNobuyoshiEsakiKenjiInagaki<p>Escherichia coli growing under anaerobic conditions produce H-2 and CO2 by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDHH), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma(54)-dependent transcriptional activator Fh1A that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDHH activity, we have identified a low-salt nutrient medium containing <0.1% NaCl enabled constitutive, high expression of FDHH even without formate and D-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDHH activity and also reductive assimilation of selenite (SeO32-) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDHH production and for labelling the FDHH polypeptide with Se-75-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.</p>
No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 9912010好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニン γ-シンターゼの精製及び性質検討712ENMaiShinozakiMasahikoYanagitaniShouichirouKanedaDaizouKudouYuuichiEndouTakashiTamuraSeikiKuramitsuKenjiInagakiThe gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase ［EC 2. 5. 1. 48］ from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 9912010シネフンギン生産菌Streptomyces incarnatus NRRL 8089由来アルギニン変換化合物「オルニチンラクタム」の同定15ENKojiFukudaTakashiTamuraKenjiInagakiSinefungin is a nucleoside antibiotic, in which a molecule of L-ornithine is linked to the 5' end of adenosine through a C-C bond. The antibiotic was isolated from the culture broth of Streptomyces incarnatus. For the purpose of detecting intermediate of sinefungin biosynthesis, resting cell suspensions were incubated with supplemental L-arginine, and L-ornithine. 50mM Arginine was converted to a compound X that has low polarity. 50mM ornithine was not converted and remained in reaction solution. Compound X was purified using HPLC, and analyzed using (1)H-NMR and FAB-MS. These analyses showed that a compound X is "ornithine-lactam" (Mw＝114), which has a structure of circularized ornithine. These results indicated that S. incarnatus has an enzyme that converts arginine to ornithine-lactam. Such an enzyme has never been reported, and suggested that it may be relevant to sinefungin biosynthesis.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 9812009高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼの大腸菌での発現，精製及び諸性質の検討17ENMasahikoYanagitaniSatoshiUemaeTomoyukiShiragaTakashiTamuraKenjiInagakiAn alanine racemase (EC 5.1.1.1) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 amino
acid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0∼10.0 and an optimum temperature of 55°C∼60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 9712008海産性好熱性細菌 Rhodothermus marinus 由来イソアミラーゼ
の精製，性質検討及びＸ線結晶構造解析17ENAkikoTachibanaTakashiTamuraKatsumiImadaMikiKinoshitaKeiichiNambaNorikoTsutsumiMiyokoHashidaHiromichiSakaguchiKenjiInagakiThe isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coli
Top 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimum
pH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinus
isoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. These
results suggest that R. marinus isoamylase has high thermostability. The crystallization and crystal
structure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9Å
resolution was determined in complex with the panose. R. marinus isoamylase is composed of three
domains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of the
R. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-site
consensus regions characteristic of the α-amylase family. And the essential residue of the α-amylase
family (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loops
than P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These results
are thought to be factors of thermostability of R. marinus isoamylase.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548411995セレノシステイン含有ペプチドの合成と生化学的機能の研究6972ENTakashiTamuraSelenium belongs to the Ⅵb group of the periodic table,and possesses both metallic and nonmetallic characteristics. Physicochemical properties of selenium resemble more or less those of sulfur,and selenium may be indiscriminately incorporated in place of sulfur in cellular constituents and disturb metabolism. Alkali disease and blind stagger disease of livestock are caused by selenium-polluted grass. Carcinogenic effect is also one of the marked biological properties of selenium. In spite of the toxic and carcinogenic effects,selenium is actually an essential trace element for bacteria,fish,and mammals. Life has exploied the high reactivity and unique characteristics of organoselenium compounds,especially in the form of selenoenzymes. Mammalian glutathione peroxidase(EC 1.11.1.9) has selenocysteine residue at the active site, and the enzyme plays a central role in the biological defense system against oxidative challenge by activated oxygens and radicals. The author has studied the low redox potential and high reactivity of selenium-containing compounds, and developed glutathione peroxidase mimics. Glutaselenone, a selenium analog of glutaheione, catalyzes a glutathione peroxidase like reaction in vitro. Studies on the mechanisum of glutaselenone-catalyzed reaction revealed that glutaselenone is converted to a selenosulfide conjugate with glutathione in its catalysis. Thioredoxins contain a conserved sequence,Cys-Gly-Pro-Cys, which is known to form intramolecular disulfide bond with consecutive β-turn conformations. THe peptide is expected to serve as an template for intramolecular selenosulfide bond formation when either the cysteine is replaced by selenocystein. A tetrapeptide,Secys-Gly-Pro-Cys, was synthesized chemically. It showed glutathione peroxidase-like activity three times as high as glutaselenone. The high catalytic activity is ascribed to an intramolecular selenosulfide bond formation in the catalytic reaction.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548611997Alanine Racemase from an Acidophilic Bacterium, Acidiphilium organovorum1319ENTeck KeongSeowKenjiInagakiTakashiTamuraHidehikoTanakaAlanine racemase(EC 5.1.1.1)was screened from several acidophilic bacteria.Acidiphilium organovorum 13H showed the highest activity and was chosen as the representative source to study alaine racemase from acidphlic bacteria.The enzyme was found to be localised in the cytoplasm of the bacteria.Relative molecular mass syudies indicated that it had a dimeric native structure with identical subunits of about 34 kDa each.Maximum activity was observed between 50 and 60℃and at pH9.There was no loss of enzyme activity even after incubation at 65℃.The loss of activity upon dialysis against pyridoxal 5'-phosphate-free buffer containing hydroxylamine,and its partial recovery upon subsequent dialysis against buffer containing phyridoxal 5'-phosphate suggested that the enzyme required piridoxal 5'-phosphate as a co-factor for its catalytic activity.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548711998Pseudomonas putida のＬ－メチオニン分解系オペロンの解析5358ENHiroyukiInoueTakashiTamuraKenjiInagakiHidehikoTanakaThe mde operon and an upstream regulatory gene (mdeR) have been cloned and sequenced from Pseudomonas putida chromosomal DNA. The mde operon contains two structural genes involved in L-methionine degradative metabolism, which are mdeA (L-methionine γ-lyase gene) and mdeB (a gene encoding a homologous protein to the E1 component of pyruvate dehydrogenase complex). A rho-independent terminator was present just downstream of mdeB and open reading frames corresponding to other components of α-keto acid dehydrogenase complex were not found. When the mdeB gene product was overproduced in Escherichia coli, the E1 activity of the cell extract showed high specificity for α-ketobutyrate rather than pyruvate. these results suggest that mdeB encodes a novel E1 component, α-ketobutyrate dehydrogenase E1 component, and plays an important role in the metabolism of α-ketobutyrate produced by L-methionine γ-lyase from L-methionine. In addition,we found that mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been idetified as a member of the leucine responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02548711998染料脱色微生物の検索，単離及び脱色機構の解析4751ENKenjiInagakiMasakazuKawaguchiTakaakiTaguchiTakashiTamuraHidehikoTanakaA yeast, Candida sp. MK-1, newly isolated from activated sludge as a dye decolorizing microorganism, decolorized Color lndex (C.l.) Reactive orange 16 and C.l. Reactive red 21 on the solid medium. Both azo dyes were also decolorized even in the liquid medium. The decolorizing activity by Candida sp. MK-1 depends on glucose concentration. Textile stained by Reactive orange 16 or Reactive red 21 was decolorized by Candida sp. MK-1. There results suggest that Candida sp. MK-1 has potential applications for the decolorization of textile and for the bioremediation of dye-contaminated wastewater.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549112002Ｌ－グルタミン酸オキシダーゼを用いたグルタミン酸センサーの開発及びＧＯＴ/ＧＰＴセンシングへの応用1522ENJiroArimaTakashiTamuraHiroakiShinoharaHitoshiKusakabeHidehikoTanakaKenjiInagakiL-Glutamate measurement and GOT/GPT assay was successful by H2O2 measurement using the L-glutamate oxidase with 4-aminoantipyrine / phenol method. But, in examination of oxigen electrode, immobilized L-glutamate oxidase at the cellulose to L-glutamate and GOT-GPT sensor, Lpglutamate measurement was used for the amperometric determination with non-fixed enzyme. On examination of electron mediator, response for L-glutamate was observed with each of the compounds ferricyane, ferrocene-COOH, ferrocene-MeOH, and benzoquinone. L-Glutamate was measured by carbon printed tip electrode the L-glutamate oxidase and ferricyane based on the principle of chronoamperometry. A linear calibration graph was obtained between 1mM and 30mM. These results suggest that L-glutamate oxidase is able to utilize to L-glutamate sensor, and that there is a strong possibility to put this sensor to sensing for GOT/GPT activity.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549112002好酸性細菌Acidocella Facilis由来菌体外酸性エステラーゼ遺伝子のクローニング713ENTatsuyaTakahashiNoboruTanakaTakashiTamuraChikaMukaiKimiyasuIsobeNorioWakaoHidehikoTanakaKenjiInagakiAn acidophilic heterotrophic bacterium, Acidocella facilis sp. AIU409 produces an extracellular acid esterase when grown in a medium containing sorbitan mono ester, Tween 80. The estA gene encoding for the acid esterase was isolated from the genomic library of A. facilis AIU409, cloned into Escherichia coli MV1184, and the nucleotide sequence of the gene was determined. The structural gene of estA was found to be 1881bp. The open reading frame of estA encodes 627 amino acid residues (calculated molecular mass, 64,140 daltons). A rho-independent terminator was present just downstream of the termination codon, TGA. The deduced N-terminal amino acid showed that the presursor of the acid esterase had a signal peptide composed of 23 amino acids and a consensus sequence of lipase, G-X-S-X-S. The molecular mass excluding the signal peptide calculated from the deduced amino acid sequence if the acid esterase is 61,846. This is lower than the molecular mass, 64kDa estimated by gel electrophoresis. The predicated amino acid sequence of the acid esterase has high similarity to the acyltransferase from Aeromonas hydrophila and the lipase from Xenorhadbus luminescens.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549112002Improvement of Sinefungin-Producing Strain of Streptomyces incarnatus by Conferring Rifampicin-Resistance through Ultraviolet Light Irradiation and Protoplast Regeneration15ENTakashiTamuraYin ShuLiHidehikoTanakaKenjiInagakiSecondary metabolite production by gram-positive bacteria is strictly regulated at the transcription of the biosynthetic genes to mRNA in response to certain stringent conditions. Therefore, some mutational disruption of regulatory domains of the bacterial RNA polymerase might increase the production of the antibiotics. In this study, we have attempted to improve the sinefungin-producing strain of Streptomyces incarnatus NRRL 8057 by irradiating ultraviolet light on the protoplast, and selecting mutants that acquired the resistance to rifampicin, the antibiotic which specifically binds to the β-subunit of bacterial RNA polymerase. After three rounds of mutation, 10 strains were obtained with varied resistance to rifampicin. A mutant which showed the highest resistance was found to have the highest sinefungin production, which was 2.4 times higher(0.45±0.11μg/ml)than the wild type strain (0.19±0.07μg/ml). The breeding approach by rifampicin-resistance may be advantageous over the classical random screening since it requires much smaller number of candidates to be examined.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549212003Semiempirical Molecular Orbital Calculation for the Redox Property of C-Terminal Active Site Sequence of Human Thioredoxin Reductase1720ENTakashiTamuraHidehikoTanakaKenjiInagakiSemiempirical molecular orbital calculation (MOPAC) was used to estimate the enthalpy difference (ΔH) between the reduce and oxidized states of the C-terminal rebox center of human thioredoxin reductase. Heat of formation was computed by WinMOPAC 3.5Pro for the model peptides, N-Acetyl-Ser-Ile-Leu-Gly-X1-X2-Gly, whose-X1-X2-sequence was-Cys SeCys-(natural sequence), -SeCys-Cys-(reverse sequence), -Cys-Cys, and-SeCys-SeCys-. Calculation by Hamiltonian AM1 and PM3 agreed that the oxidized state with selenosulfide bonds and a diselenide bond were more favoralbe than their reduced states. Only the peptide that contained-Cys-Cys-sequence was shown to have lower enthalpy when the two Cys were in the reduced form. It has been reported that substitution of SeCys498 to Cys results in the mujtant TrxRs retaining only about 1% of the enzyme activity. The results of computational estimation supported the experimental hypothesis that the inactivation by SeCys498Cys mutation was due to the unfavorable formation of disulfide bond between Cys497-Cys498.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549212003好酸性細菌Acidocella facilis 22Mの制限修飾系遺伝子のクローニングと塩基配列決定915ENSeijiYamaokaTakashiTamuraHisanoriTakenobuToshiyukiKojoHidehikoTanakaKenjiInagakiThe gene encoding the Afa22MI restriction-modification system recognizing the sequence 5'-CGATCG-3' was cloned from Acidocella facilis 22M and sequenced. The cloned DNA fragment contained three genes encoding the Afa22MI methylase (M.Afa22MI) , the putative restriction endonuclease Afa22MI (R.Afa22MI) and a very short patch repair endonuclease (Afa22MI vsr) . M. Afa22MI gene has the conserved motifs of C5-cytosine methyltransferase. Afa22MI vsr gene was localized upstream of M. Afa22MI gene in opposite orientation, and an open reading frame of R. Afa22MI has about 53% sequence similarity to the amino acid sequence for the variable region of M.XorⅡ. Afa22MI vsr has about 66% sequence similarity to the amino acid sequence of XorII vsr which was associated M. XorII.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549312004Saccharomyces cerevisiae 由来 D-アミノ酸アセチルトランスフェラーゼの精製と性質1318ENYurikoTashiro (Yamada)TakashiTamuraHidehikoTanakaKenjiInagakiSome properties of D-amino scid acetyltransferase purified from Saccharomyces cerevisiae were investigated. The enzyme was purified to homogeneity by ammonium sulfate fractionation, column chromatographies on DEAE-Toyopearl 650M, Sephacryl S-200, QAE-Toyopearl 550C and affinity chromatography with D-glutamate as a ligand. The molecular weight was estimated to be about 53,000 by gel filtration. Relative molecular mass studies indicated that the enzyme was a monomer structure. The purified enzyme had an optimum pH of 8.4 and an optimum temperature of 40C. The Km values of the purified enzyme determined with tryptophan and acetyl-CoA were 4.5 * 10 -3M, respectively. The 20 residues of N-terminal amino acid sequence were analyzed.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-0254 9612007The C-Terminal Tetrapeptide Sequence of Drosophila Thioredoxin Reductase Does not Function as a Redox-active Motif in the Human Lung Counterpart15ENMitsuhikoKuwaharaTakashiTamuraKentaroKawamuraKenjiInagakiThe isozymes of mammalian thioredoxin reductase (TrxR) contain the penultimate selenocysteineresidue (SeCys) in the redox-active C-terminal tetrapeptide, -Gly-Cys-SeCys-Gly (end). Amutant form of the mammalian enzyme TrxR-X498C in which SeCys is replaced with Cys showsa dramatically decreased catalytic activity, suggesting that SeCys residue plays an integral role inthe catalysis. In contrast, TrxR of the fruit fly, Drosophila melanogaster, has no selenium in the corresponding C-terminal redox sequence, which instead of SeCys has flanking serine residues in the terminal sequence, -Ser-Cys-Cys-Ser (end). Because the catalytic activity of Dm-TrxR is comparable to that of the mammalian selenoenzyme, we introduced the serine residues at the corresponding positions of the recombinant TrxR-X498C and mimicked the redox center of the fruit fly TrxR. However, the catalysis remained as low as the Cys mutant of the selenoenzyme,
suggesting that the additional structural features are still required for the tetrapeptide to function as a redox center. MOPAC calculation suggested that the complete motif might involve the hexapeptide sequence, which includes a proline residue, -Pro-X-Ser-Cys-Cys-Ser (end). The proline-containing motif is conserved among other insect TrxRs such as those of honeybee and fruit fly.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549512006染料脱色微生物による染料脱色機構の解析15ENNaokoBabaHiromichiSakaguchiTakaakiTaguchiNobukiHayaseTakashiTamuraKenjiInagakiThe mehcanism of azo dyes decolorization by Candida sp. MK-1, Aeromonas sp. B-5 and Actinobacillus sp. B-11 were analyzed. The maximal decolorization activity was observed at pH 7.5 and 30℃ on Candida sp. MK-1, at alkaline and at 35℃ on Aeromonas sp. B-5 and Actinobacillus sp. B-11. The HPLC analysis of the supernatant of the Acid Red 27 detected in the blank. The retention time of this peak matched that of a reference standard compound of 4-amino-1-naphthalenesulfonate, produced by reductive cleavage of Acid Red 27. The decolorization of azo dyes with cell free extract of Candida sp. MK-a was promoted by the addition of several coenzymes or lawsone. The remarkable promotion of decolorization was observed by the addition of glutamate dehydrogenase with α-ketoglutarate and NH4+. Therefore, it was suggested that Candida sp. MK-1 azoreductase catalyzed decolorization of azo dye by NADPH dependent reductive cleavage.No potential conflict of interest relevant to this article was reported.岡山大学農学部Acta Medica Okayama0474-02549412005化学合成独立栄養細菌 Acidithiobacillus thiooxidans 由来アコニターゼの遺伝子解析と大腸菌での発現17ENYoheiKanaharaTakashiTamuraChizukaTokudaAtsuoNakamuraHirokazuMatsukawaKenjiInagakiAn aconitase from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The cloned aconitase gene (acn) was expressed in Escherichia coli JM 109; aconitase activity was found in the cell extarct. The acn gene encodes a 646-amino acid polypeptide and is located upstream of the isocitrate dehydrogenase gene (icd). A. thiooxidans aconitase showes high sequence similar to pig heart aconitase and E.coli aconitase B. Twenty-five of twenty-seven active site residues assigned in pig heart aconitase are conserved in A. thiooxidans aconitase. The enzyme was purified by DEAE-Toyopearl 650M column chromatogrophy. The purified enzyme had an optimum pH of 7.5 and an optimum temperature of 60 C. Thermal inactivation studies of the purified enzyme revealed the enzyme activity to be uninfluenced after one hour incubation at 40 c. Enzyme activity was retained 100% after incubation of the enzyme at pH 6.0-9.0 for 60min. The A. thiooxidans aconitase was composed of a single polypeptide chain with a molecular mass of 66 kDa.No potential conflict of interest relevant to this article was reported.