Research activity information

• May 2018 日本NO学会, 第18回日本NO学会Young Investigator Awards優秀賞, 酵母における銅代謝関連転写因子 Mac1 を介した NO による高温ストレス耐性機 構

  那須野 亮


• Jul. 2017 The Japanese Biochemical Society, JB Award, Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast

  R. Nasuno, S. Hirase, S. Norifune, D. Watanabe, H. Takagi


• Dec. 2012 日本生化学会, 鈴木紘一メモリアル賞, 酵母の酸化ストレス耐性に関与するN-アセチルトランスフェラーゼMpr1の構造機能解析

  那須野 亮

• Metallothionein Cup1 attenuates nitrosative stress in the yeast Saccharomyces cerevisiae.

  Yuki Yoshikawa, Ryo Nasuno, Naoki Takaya, Hiroshi Takagi

  Metallothionein (MT), which is a small metal-binding protein with cysteine-rich motifs, functions in the detoxification of heavy metals in a variety of organisms. Even though previous studies suggest that MT is involved in the tolerance mechanisms against nitrosative stress induced by toxic levels of nitric oxide (NO) in mammalian cells, the physiological functions of MT in relation to NO have not been fully understood. In this study, we analyzed the functions of MT in nitrosative stress tolerance in the yeast Saccharomyces cerevisiae. Our phenotypic analyses showed that deletion or overexpression of the MT-encoding gene, CUP1, led to higher sensitivity or tolerance to nitrosative stress in S. cerevisiae cells, respectively. We further examined whether the yeast MT Cup1 in the cell-free lysate scavenges NO. These results showed that the cell-free lysate containing a higher level of Cup1 degraded NO more efficiently. On the other hand, the transcription level of CUP1 was not affected by nitrosative stress treatment. Our findings suggest that the yeast MT Cup1 contributes to nitrosative stress tolerance, possibly as a constitutive rather than an inducible defense mechanism.

  Aug. 2023, Microbial cell (Graz, Austria), 10(8) (8), 170 - 177, English, International magazine

  Scientific journal


• S-glutathionylation of fructose-1,6-bisphosphate aldolase confers nitrosative stress tolerance on yeast cells via a metabolic switch.

  Seiya Shino, Ryo Nasuno, Hiroshi Takagi

  Nitric oxide as a signaling molecule exerts cytotoxicity known as nitrosative stress at its excess concentrations. In the yeast Saccharomyces cerevisiae, the cellular responses to nitrosative stress and their molecular mechanisms are not fully understood. Here, focusing on the posttranslational modifications that are associated with nitrosative stress response, we show that nitrosative stress increased the protein S-glutathionylation level in yeast cells. Our proteomic and immunochemical analyses demonstrated that the fructose-1,6-bisphosphate aldolase Fba1 underwent S-glutathionylation at Cys112 in response to nitrosative stress. The enzyme assay using a recombinant Fba1 demonstrated that S-glutathionylation at Cys112 inhibited the Fba1 activity. Moreover, we revealed that the cytosolic glutaredoxin Grx1 reduced S-glutathionylation of Fba1 and then recovered its activity. The intracellular contents of fructose-1,6-bisphosphate and 6-phosphogluconate, which are a substrate of Fba1 and an intermediate of the pentose phosphate pathway (PPP), respectively, were increased in response to nitrosative stress, suggesting that the metabolic flow was switched from glycolysis to PPP. The cellular level of NADPH, which is produced in PPP and functions as a reducing force for nitric oxide detoxifying enzymes, was also elevated under nitrosative stress conditions, but this increase was canceled by the amino acid substitution of Cys112 to Ser in Fba1. Furthermore, the viability of yeast cells expressing Cys112Ser-Fba1 was significantly lower than that of the wild-type cells under nitrosative stress conditions. These results indicate that the inhibition of Fba1 by its S-glutathionylation changes metabolism from glycolysis to PPP to increase NADPH production, leading to nitrosative stress tolerance in yeast cells.

  Nov. 2022, Free radical biology & medicine, 193(Pt 1) (Pt 1), 319 - 329, English, International magazine

  Scientific journal


• Intracellular production of reactive oxygen species and a DAF-FM-related compound in Aspergillus fumigatus in response to antifungal agent exposure.

  Sayoko Oiki, Ryo Nasuno, Syun-Ichi Urayama, Hiroshi Takagi, Daisuke Hagiwara

  Fungi are ubiquitously present in our living environment and are responsible for crop and infectious diseases. Developing new antifungal agents is constantly needed for their effective control. Here, we investigated fungal cellular responses to an array of antifungal compounds, including plant- and bacteria-derived antifungal compounds. The pathogenic fungus Aspergillus fumigatus generated reactive oxygen species in its hyphae after exposure to the antifungal compounds thymol, farnesol, citral, nerol, salicylic acid, phenazine-1-carbonic acid, and pyocyanin, as well as under oxidative and high-temperature stress conditions. The production of nitric oxide (NO) was determined using diaminofluorescein-FM diacetate (DAF-FM DA) and occurred in response to antifungal compounds and stress conditions. The application of reactive oxygen species or NO scavengers partly suppressed the inhibitory effects of farnesol on germination. However, NO production was not detected in the hyphae using the Greiss method. An LC/MS analysis also failed to detect DAF-FM-T, a theoretical product derived from DAF-FM DA and NO, in the hyphae after antifungal treatments. Thus, the cellular state after exposure to antifungal agents may be more complex than previously believed, and the role of NO in fungal cells needs to be investigated further.

  Aug. 2022, Scientific reports, 12(1) (1), 13516 - 13516, English, International magazine

  Scientific journal


• Acetaldehyde reacts with a fluorescent nitric oxide probe harboring an o-phenylenediamine structure that interferes with fluorometry.

  Ryo Nasuno, Yuki Yoshikawa, Hiroshi Takagi

  Nitric oxide (NO) is a ubiquitous signaling molecule, and thus a variety of methods have been developed for its detection and quantification. Fluorometric analyses using a fluorescent NO probe harboring an o-phenylenediamine (OPD) structure are widely used for NO analyses in various organisms, including yeast. Here, we discovered that an NO-independent fluorophore (UNK436) was generated from a fluorescent NO probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), which has an OPD structure, in yeast cells. The molecules responsible for this undesirable fluorescence and their reaction mechanisms were analyzed. Our mass spectrometric analysis showed that two carbon atoms from glucose were incorporated into UNK436. Subsequent analyses indicated that a non-proteinous small compound leads to the synthesis of UNK436 through an oxidative reaction. Furthermore, our LC/MS/MS analysis of the reaction mixture of DAF-FM with acetaldehyde in combination with stable isotope labeling demonstrated that acetaldehyde reacts with DAF-FM oxidatively, generating UNK436. Another NO probe with an OPD structure, diaminorhodamine-4M, reacted with acetaldehyde in the same way to emit fluorescence. Based on our findings, we recommend that in researches using OPD-based fluorescent NO probes, alternative analyses also be performed to identify the reaction products of the probes with NO to avoid false-positives.

  Jul. 2022, Free radical biology & medicine, 187, 29 - 37, English, International magazine

  Scientific journal


• Development of a microtiter plate-based analysis method of nitric oxide dioxygenase activity.

  Ryo Nasuno, Nozomi Iwai, Hiroshi Takagi

  Nitric oxide (NO) functions in cell protection or cell death, depending on its concentration. Therefore, regulation of the intracellular concentrations of NO by its degradation systems is important for cellular functions. One of the NO degrading enzymes, flavohemoglobin (FHb), which has NO dioxygenase (NOD) activity, is a promising target for antibiotics, based on the finding that FHb-deficient pathogens exhibited reduced host toxicity. Here, we developed a high-throughput method to measure the NOD activity. Our newly developed method could contribute to the screening of potential antibiotics with NOD inhibitory activity.

  Mar. 2022, The Journal of general and applied microbiology, 68(1) (1), 38 - 41, English, Domestic magazine

  Scientific journal


• Molecular mechanism of ethanol fermentation inhibition via protein tyrosine nitration of pyruvate decarboxylase by reactive nitrogen species in yeast.

  Supapid Eknikom, Ryo Nasuno, Hiroshi Takagi

  Protein tyrosine nitration (PTN), in which tyrosine (Tyr) residues on proteins are converted into 3-nitrotyrosine (NT), is one of the post-translational modifications mediated by reactive nitrogen species (RNS). Many recent studies have reported that PTN contributed to signaling systems by altering the structures and/or functions of proteins. This study aimed to investigate connections between PTN and the inhibitory effect of nitrite-derived RNS on fermentation ability using the yeast Saccharomyces cerevisiae. The results indicated that RNS inhibited the ethanol production of yeast cells with increased intracellular pyruvate content. We also found that RNS decreased the activities of pyruvate decarboxylase (PDC) as a critical enzyme involved in ethanol production. Our proteomic analysis revealed that the main PDC isozyme Pdc1 underwent the PTN modification at Tyr38, Tyr157, and Tyr344. The biochemical analysis using the recombinant purified Pdc1 enzyme indicated that PTN at Tyr157 or Tyr344 significantly reduced the Pdc1 activity. Interestingly, the substitution of Tyr157 or Tyr344 to phenylalanine, which is no longer converted into NT, recovered the ethanol production under the RNS treatment conditions. These findings suggest that nitrite impairs the fermentation ability of yeast by inhibiting the Pdc1 activity via its PTN modification at Tyr157 and Tyr344 of Pdc1.

  Mar. 2022, Scientific reports, 12(1) (1), 4664 - 4664, English, International magazine

  Scientific journal


• Identification and Functional Analysis of GTP Cyclohydrolase II in Candida glabrata in Response to Nitrosative Stress.

  Ryo Nasuno, Soma Suzuki, Sayoko Oiki, Daisuke Hagiwara, Hiroshi Takagi

  Reactive nitrogen species (RNS) are signal molecules involved in various biological events; however, excess levels of RNS cause nitrosative stress, leading to cell death and/or cellular dysfunction. During the process of infection, pathogens are exposed to nitrosative stress induced by host-derived RNS. Therefore, the nitrosative stress resistance mechanisms of pathogenic microorganisms are important for their infection and pathogenicity, and could be promising targets for antibiotics. Previously, we demonstrated that the RIB1 gene encoding GTP cyclohydrolase II (GCH2), which catalyzes the first step of the riboflavin biosynthesis pathway, is important for nitrosative stress resistance in the yeast Saccharomyces cerevisiae. Here, we identified and characterized the RIB1 gene in the opportunistic pathogenic yeast Candida glabrata. Our genetic and biochemical analyses indicated that the open reading frame of CAGL0F04279g functions as RIB1 in C. glabrata (CgRIB1). Subsequently, we analyzed the effect of CgRIB1 on nitrosative stress resistance by a growth test in the presence of RNS. Overexpression or deletion of CgRIB1 increased or decreased the nitrosative stress resistance of C. glabrata, respectively, indicating that GCH2 confers nitrosative stress resistance on yeast cells. Moreover, we showed that the proliferation of C. glabrata in cultures of macrophage-like cells required the GCH2-dependent nitrosative stress detoxifying mechanism. Additionally, an infection assay using silkworms as model host organisms indicated that CgRIB1 is indispensable for the virulence of C. glabrata. Our findings suggest that the GCH2-dependent nitrosative stress detoxifying mechanism is a promising target for the development of novel antibiotics.

  2022, Frontiers in microbiology, 13, 825121 - 825121, English, International magazine

  Scientific journal


• Cysteine residues in the fourth zinc finger are important for activation of the nitric oxide‐inducible transcription factor Fzf1 in the yeast Saccharomyces cerevisiae

  Ryo Nasuno, Natsuko Yoshioka, Yuki Yoshikawa, Hiroshi Takagi

  Nitric oxide (NO) is a ubiquitous signaling molecule in various organisms. In the yeast Saccharomyces cerevisiae, NO functions in both cell protection and cell death, depending on its concentration. Thus, it is important for yeast cells to strictly regulate NO concentration. The transcription factor Fzf1, containing five zinc fingers, is reportedly important for NO homeostasis by regulating the expression of the YHB1 gene, which encodes NO dioxygenase. However, the mechanism by which NO activates Fzf1 is still unclear. In this study, we showed that NO activated Fzf1 specifically at the protein level by RT-qPCR and Western blotting. Our further transcriptional analyses indicated that cysteine residues in the fourth zinc finger (ZF4) are required for the NO-responsive activation of Fzf1. Additionally, the present results suggest that ZF4 is important for the protein stability of Fzf1. From these results, we proposed possible mechanisms underlying Fzf1 activation.

  Wiley, Jul. 2021, Genes to Cells, 26(10) (10), 823 - 829, English, International magazine

  Scientific journal


• An NADPH‐independent mechanism enhances oxidative and nitrosative stress tolerance in yeast cells lacking glucose‐6‐phosphate dehydrogenase activity

  Yuki Yoshikawa, Ryo Nasuno, Hiroshi Takagi

  The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), which is required for various redox systems involving antioxidative stress enzymes, is thus important for stress tolerance mechanisms. Here, we analyzed the stress response of the NADPH-depleted cells of Saccharomyces cerevisiae. A cell viability assay showed that the NADPH depletion induced by disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase, which is the major determinant of the intracellular NADPH/NADP+ ratio, enhanced the tolerance of S. cerevisiae to both oxidative and nitrosative stresses. The subsequent analyses demonstrated that the antioxidative transcriptional factor Yap1 was activated and the cytosolic catalase Ctt1, whose expression is regulated by Yap1, was upregulated in zwf1Δ cells irrespective of the presence or absence of stress stimuli. Moreover, deletion of the YAP1 or CTT1 gene inhibited the increased stress tolerance of zwf1Δ cells, indicating that Ctt1 dominantly contributed to the higher stress tolerance of zwf1Δ cells. Our findings suggest that an NADPH-independent mechanism enhances oxidative and nitrosative stress tolerance in ZWF1-lacking yeast cells.

  Wiley, Jul. 2021, Yeast, 38(7) (7), 414 - 423, English, International magazine

  Scientific journal


• NADPH is important for isobutanol tolerance in a minimal medium of Saccharomyces cerevisiae

  Yuki Yoshikawa, Ryo Nasuno, Hiroshi Takagi

  We showed that the isobutanol sensitivity in glucose-6-phosphate dehydrogenase-deficient cells of the yeast Saccharomyces cerevisiae was rescued by an alternative NADPH producer, acetaldehyde dehydrogenase, but not in the cells lacking 6-phosphogluconate dehydrogenase. This phenotype correlated with the intracellular NADPH/NADP+ ratio in yeast strains. Our findings indicate the importance of NADPH for the isobutanol tolerance of yeast cells.

  Oxford University Press (OUP), Jun. 2021, Bioscience, Biotechnology, and Biochemistry, 85(9) (9), 2084 - 2088, English, International magazine

  Scientific journal


• The analytical method to identify the nitrogen source for nitric oxide synthesis

  Ryo Nasuno, Yuki Yoshikawa, Hiroshi Takagi

  Nitric oxide (NO) is a ubiquitous signaling molecule synthesized from various nitrogen sources. An analytical method to identify a nitrogen source for NO generation was developed using liquid chromatography with tandem mass spectrometry in combination with stable isotope labeling. Our method successfully detected the 15N-labeled NO-containing compound generated from 15N-labeled substrate nitrite in vitro and in vivo.

  Oxford University Press (OUP), Feb. 2021, Bioscience, Biotechnology, and Biochemistry, 85(2) (2), 211 - 214, English, International magazine

  Scientific journal


• High-level production of ornithine by expression of the feedback inhibition-insensitive N-acetyl glutamate kinase in the sake yeast Saccharomyces cerevisiae.

  Masataka Ohashi, Ryo Nasuno, Shota Isogai, Hiroshi Takagi

  We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4-5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.

  Nov. 2020, Metabolic engineering, 62, 1 - 9, English, International magazine

  Scientific journal


• Detection system of the intracellular nitric oxide in yeast by HPLC with a fluorescence detector

  Ryo Nasuno, Seiya Shino, Yuki Yoshikawa, Natsuko Yoshioka, Yuichi Sato, Kohei Kamiya, Hiroshi Takagi

  Nitric oxide (NO) is an important signaling molecule involved in various biological phenomena in many organisms. The physiological functions and metabolism of NO in yeast, a unicellular microorganism, are still unknown, mainly because it is difficult to analyze the intracellular NO levels accurately. Here, we developed a new method of more accurately measuring NO content in yeast cells with the detection limit of 6 nM, by treating the cells with an NO-specific fluorescence probe followed by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). This approach successfully detected and quantified the NO content inside yeast cells treated with an NO donor. Moreover, the HPLC/FLD analysis indicates that the fluorescence induced under some environmental stress conditions, such as ethanol, vanillin, and heat-shock, was not derived from NO. The HPLC/FLD method developed in this study provides a new strategy for measuring the intracellular NO concentration with higher accuracy.

  Elsevier BV, Jun. 2020, Analytical Biochemistry, 598, 113707 - 113707, English, International magazine

  Scientific journal


• A Novel Mechanism for Nitrosative Stress Tolerance Dependent on GTP Cyclohydrolase II Activity Involved in Riboflavin Synthesis of Yeast.

  Khairul Anam, Ryo Nasuno, Hiroshi Takagi

  The biological functions of nitric oxide (NO) depend on its concentration, and excessive levels of NO induce various harmful situations known as nitrosative stress. Therefore, organisms possess many kinds of strategies to regulate the intracellular NO concentration and/or to detoxify excess NO. Here, we used genetic screening to identify a novel nitrosative stress tolerance gene, RIB1, encoding GTP cyclohydrolase II (GTPCH2), which catalyzes the first step in riboflavin biosynthesis. Our further analyses demonstrated that the GTPCH2 enzymatic activity of Rib1 is essential for RIB1-dependent nitrosative stress tolerance, but that riboflavin itself is not required for this tolerance. Furthermore, the reaction mixture of a recombinant purified Rib1 was shown to quench NO or its derivatives, even though formate or pyrophosphate, which are byproducts of the Rib1 reaction, did not, suggesting that the reaction product of Rib1, 2,5-diamino-6-(5-phospo-D-ribosylamino)-pyrimidin-4(3 H)-one (DARP), scavenges NO or its derivatives. Finally, it was revealed that 2,4,5-triamino-1H-pyrimidin-6-one, which is identical to a pyrimidine moiety of DARP, scavenged NO or its derivatives, suggesting that DARP reacts with N2O3 generated via its pyrimidine moiety.

  Apr. 2020, Scientific reports, 10(1) (1), 6015 - 6015, English, International magazine

  Scientific journal


• Stable N-acetyltransferase Mpr1 improves ethanol productivity in the sake yeast Saccharomyces cerevisiae.

  Masataka Ohashi, Ryo Nasuno, Daisuke Watanabe, Hiroshi Takagi

  N-Acetyltransferase Mpr1 was originally discovered as an enzyme that detoxifies L-azetidine-2-carboxylate through its N-acetylation in the yeast Saccharomyces cerevisiae Σ1278b. Mpr1 protects yeast cells from oxidative stresses possibly by activating a novel L-arginine biosynthesis. We recently constructed a stable variant of Mpr1 (N203K) by a rational design based on the structure of the wild-type Mpr1 (WT). Here, we examined the effects of N203K on ethanol fermentation of the sake yeast S. cerevisiae strain lacking the MPR1 gene. When N203K was expressed in the diploid Japanese sake strain, its fermentation performance was improved compared to WT. In a laboratory-scale brewing, a sake strain expressing N203K produced more ethanol than WT. N203K also affected the contents of flavor compounds and organic acids. These results suggest that the stable Mpr1 variant contributes to the construction of new industrial yeast strains with improved fermentation ability and diversity of taste and flavor.

  Jul. 2019, Journal of industrial microbiology & biotechnology, 46(7) (7), 1039 - 1045, English, International magazine

  [Refereed]

  Scientific journal


• Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells.

  Nishimura A, Nasuno R, Yoshikawa Y, Jung M, Ida T, Matsunaga T, Morita M, Takagi H, Motohashi H, Akaike T

  Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.

  Jul. 2019, The Journal of biological chemistry, 294(37) (37), 13781 - 13788, English, International magazine

  [Refereed]

  Scientific journal


• Characterization of a New Saccharomyces cerevisiae Isolated From Hibiscus Flower and Its Mutant With L-Leucine Accumulation for Awamori Brewing.

  Takayuki Abe, Yoichi Toyokawa, Yukiko Sugimoto, Haruna Azuma, Keiko Tsukahara, Ryo Nasuno, Daisuke Watanabe, Masatoshi Tsukahara, Hiroshi Takagi

  Since flavors of alcoholic beverages produced in fermentation process are affected mainly by yeast metabolism, the isolation and breeding of yeasts have contributed to the alcoholic beverage industry. To produce awamori, a traditional spirit (distilled alcoholic beverage) with unique flavors made from steamed rice in Okinawa, Japan, it is necessary to optimize yeast strains for a diversity of tastes and flavors with established qualities. Two categories of flavors are characteristic of awamori; initial scented fruity flavors and sweet flavors that arise with aging. Here we isolated a novel strain of Saccharomyces cerevisiae from hibiscus flowers in Okinawa, HC02-5-2, that produces high levels of alcohol. The whole-genome information revealed that strain HC02-5-2 is contiguous to wine yeast strains in a phylogenic tree. This strain also exhibited a high productivity of 4-vinyl guaiacol (4-VG), which is a precursor of vanillin known as a key flavor of aged awamori. Although conventional awamori yeast strain 101-18, which possesses the FDC1 pseudogene does not produce 4-VG, strain HC02-5-2, which has the intact PAD1 and FDC1 genes, has an advantage for use in a novel kind of awamori. To increase the contents of initial scented fruity flavors, such as isoamyl alcohol and isoamyl acetate, we attempted to breed strain HC02-5-2 targeting the L-leucine synthetic pathway by conventional mutagenesis. In mutant strain T25 with L-leucine accumulation, we found a hetero allelic mutation in the LEU4 gene encoding the Gly516Ser variant α-isopropylmalate synthase (IPMS). IPMS activity of the Gly516Ser variant was less sensitive to feedback inhibition by L-leucine, leading to intracellular L-leucine accumulation. In a laboratory-scale test, awamori brewed with strain T25 showed higher concentrations of isoamyl alcohol and isoamyl acetate than that brewed with strain HC02-5-2. Such a combinatorial approach to yeast isolation, with whole-genome analysis and metabolism-focused breeding, has the potentials to vary the quality of alcoholic beverages.

  2019, Frontiers in genetics, 10, 490 - 490, English, International magazine

  [Refereed]

  Scientific journal


• Nitric Oxide Signalling in Yeast

  Rika I. Astuti, Ryo Nasuno, Hiroshi Takagi

  Academic Press, Jan. 2018, Advances in Microbial Physiology, 72, 29 - 63, English, International magazine

  [Refereed]

  In book


• Nitric oxide signaling in yeast

  Rika Indri Astuti, Ryo Nasuno, Hiroshi Takagi

  Nov. 2016, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100(22) (22), 9483 - 9497, English, International magazine

  [Refereed]

  Scientific journal


• Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast

  Yuki Yoshikawa, Ryo Nasuno, Nobuhiro Kawahara, Akira Nishimura, Daisuke Watanabe, Hiroshi Takagi

  Jul. 2016, NITRIC OXIDE-BIOLOGY AND CHEMISTRY, 57, 85 - 91, English, International magazine

  [Refereed]

  Scientific journal


• Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast

  Ryo Nasuno, Saeka Hirase, Saki Norifune, Daisuke Watanabe, Hiroshi Takagi

  Feb. 2016, JOURNAL OF BIOCHEMISTRY, 159(2) (2), 271 - 277, English, International magazine

  [Refereed]

  Scientific journal


• Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol

  Hiroshi Takagi, Keisuke Hashida, Daisuke Watanabe, Ryo Nasuno, Masataka Ohashi, Tomoya Iha, Maiko Nezuo, Masatoshi Tsukahara

  Feb. 2015, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 119(2) (2), 140 - 147, English, Domestic magazine

  [Refereed]

  Scientific journal


• A Novel Antioxidative Enzyme "N-Acetyltransferase Mpr1" Found in Yeast

  TAKAGI Hiroshi, NASUNO Ryo

  微生物から高等生物まで広く存在する「N-アセチルトランスフェラーゼ」は，さまざまな基質をアセチル化することで，多くの重要な細胞機能の制御に関与している．筆者らは，環状の二級アミンであるプロリンアナログ（L-アゼチジン-2-カルボン酸，シス-4-ヒドロキシ-L-プロリン）を基質とする新規のN-アセチルトランスフェラーゼMpr1を酵母Saccharomyces cerevisiaeに見いだした．また，Mpr1がアルギニン合成を亢進することで一酸化窒素の生成を誘導し，酵母の酸化ストレス耐性に寄与する新しいタイプの「抗酸化酵素」であることを明らかにした．さらに，X線結晶構造解析により，Mpr1のユニークな立体構造と反応機構の解明にも成功した．本稿では，Mpr1の分子構造と生理的役割について概説する．また，Mpr1の酵素特性や生理機能に基づく応用研究の成果も紹介する．

  Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2015, KAGAKU TO SEIBUTSU, 53(3) (3), 148 - 155, Japanese


• Nitric Oxide-Mediated Antioxidative Mechanism in Yeast through the Activation of the Transcription Factor Mac1

  Ryo Nasuno, Miho Aitoku, Yuki Manago, Akira Nishimura, Yu Sasano, Hiroshi Takagi

  Nov. 2014, PLOS ONE, 9(11) (11), e113788, English, International magazine

  [Refereed]

  Scientific journal


• Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae.

  Daisuke Watanabe, Rie Kikushima, Miho Aitoku, Akira Nishimura, Iwao Ohtsu, Ryo Nasuno, Hiroshi Takagi

  The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1, which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.

  Jul. 2014, Microbial cell (Graz, Austria), 1(7) (7), 241 - 246, English, International magazine

  [Refereed]

  Scientific journal


• Structural and functional analysis of the yeast n-Acetyltransferase mpr1 involved in oxidative stress tolerance via proline metabolism

  Ryo Nasuno, Yoshinori Hirano, Takafumi Itoh, Toshio Hakoshima, Takao Hibi, Hiroshi Takagi

  29, Jul. 2013, Proceedings of the National Academy of Sciences of the United States of America, 110(29) (29), 11821 - 11826, English, International magazine

  [Refereed]

  Scientific journal


• Production of N-acetyl cis-4-hydroxy-L-proline by the yeast N-acetyltransferase Mpr1

  Bach Thi Mai Hoa, Takao Hibi, Ryo Nasuno, Goh Matsuo, Yu Sasano, Hiroshi Takagi

  Aug. 2012, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 114(2) (2), 160 - 165, English, Domestic magazine

  [Refereed]

  Scientific journal


• The proline metabolism intermediate Delta(1)-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast

  Akira Nishimura, Ryo Nasuno, Hiroshi Takagi

  Jul. 2012, FEBS LETTERS, 586(16) (16), 2411 - 2416, English, International magazine

  [Refereed]

  Scientific journal

• Regulatory Mechanism of Nitric Oxide Synthesis and Its Physiological Function in Yeast : How to Use Nitric Oxide, Which Can Become both Medicine and Poison

  那須野 亮, 吉川 雄樹, 高木 博史

  日本農芸化学会 ; 1962-, Sep. 2017, 化学と生物 : 日本農芸化学会会誌 : 生命・食・環境, 55(9) (9), 617 - 623, Japanese


• 酵母における活性イオウ分子種:システインパースルフィドの生理的役割の解明

  西村明, 吉川雄樹, 那須野亮, 松永哲郎, 井田智章, 守田匡伸, 藤井重元, 高木博史, 赤池孝章

  2017, 日本生化学会大会(Web), 90th


• 酵母における活性イオウ分子種:システインパースルフィドの産生とその生理的役割

  西村明, 那須野亮, 松永哲郎, 井田智章, 笠松真吾, 守田匡伸, 藤井重元, 高木博史, 赤池孝章

  2016, 日本酸化ストレス学会学術集会プログラム・抄録集, 69th


• Regulatory mechanism and physiological role of the flavoprotein Tah18-dependent Nitric Oxide synthesis in yeast

  Yuki Yoshikawa, Ryo Nasuno, Hiroshi Takagi

  Sep. 2015, YEAST, 32, S131 - S132, English

  Summary international conference


• Nitric oxide-mediated antioxidative mechanism in yeast: The transcription factor Mac1-dependent activation of the superoxide dismutase Sod1

  Ryo Nasuno, Hiroshi Takagi

  Nov. 2014, NITRIC OXIDE-BIOLOGY AND CHEMISTRY, 42, 134 - 135, English

  Summary international conference


• 1P-175 Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol

  Takagi,Hiroshi, Hashida,Keisuke, Watanabe,Daisuke, Nasuno,Ryo, Ohashi,Masataka, Iha,Tomoya, Nezuo,Maiko, Tsukahara,Masatoshi

  日本生物工学会, 05 Aug. 2014, 日本生物工学会大会講演要旨集, 66, 61, Japanese

• THE MOLECULAR BIOLOGY SOCIETY OF JAPAN


• 酵母遺伝学フォーラム


• JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY

• 酵母におけるニトロ化タンパク質還元酵素の同定と機能解析

  那須野 亮

  日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 奈良先端科学技術大学院大学, 01 Apr. 2022 - 31 Mar. 2025


• Integrated understanding of nitric oxide in yeasts and fungi and its application to microbial

  高木 博史, 那須野 亮, 西村 明, 渡辺 大輔, 高谷 直樹, 萩原 大祐

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S), Grant-in-Aid for Scientific Research (S), Nara Institute of Science and Technology, 26 Jun. 2019 - 31 Mar. 2024

  １．酵母におけるNOの分子機能の解明 酵母NOS様活性の責任分子と考えられる酸化酵素Oxyを同定する目的で、モノオキシゲナーゼ活性を有する酵母タンパク質を解析した。3種類のP450酵素の遺伝子破壊・発現抑制株を作製し、過酸化水素処理条件下におけるNO合成について、NO反応性蛍光プローブを用いたフローサイトメトリーにより評価した。その結果、ラノステロール-14α-デメチラーゼをコードするERG11の発現抑制株において、NO由来の蛍光が減少した。また、Erg11阻害剤を処理した場合も同様の現象が観察された。以上のことから、ERG11が酵母NOS様活性のOxyをコードする可能性が示唆された。酵母のNO耐性に寄与する新たな遺伝子を探索した結果、リボフラビン合成の初発酵素であるGTP cyclohydrolase II（GCH2）をコードするRIB1遺伝子の過剰発現が、NOドナー処理時の細胞内NO濃度を低下させるとともに、細胞生存率の低下を抑制することを見出し、RIB1を新規なNO耐性遺伝子として同定した。また、組換え酵素を用いた生化学的解析等の結果、GCH2の活性によって生成する代謝中間体（DARP）がNOを消去することを明らかにした。 ２．糸状菌におけるNOの分子機能の解明 病原性糸状菌A. fumigatusを対象に、各種ストレス処理時におけるNO産生を解析した。その結果、植物や微生物が産生する天然抗菌物質（ファルネソール、チモール、ピオシアニン等）の処理に対して細胞内にNOが産生されることが明らかになった。麹菌A. oryzaeの転写因子を対象に、NOドナーに対して生育が低下する株を探索した。その結果、エルゴステロール合成系の制御や低酸素応答に重要な転写因子、亜鉛ホメオスタシスやプロリン代謝への関与が予想される転写因子などが候補として見出された。


• Elucidation of multifunctional proline metabolizing enzymes found in yeast and its application to improvement of cellular functions

  Takagi Hiroshi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Exploratory), Nara Institute of Science and Technology, 28 Jun. 2019 - 31 Mar. 2022

  First, we examined the effect of proline on cellular longevity. The results showed that the replicative lifespan was independent of proline content, but was significantly shortened by deletion of the gene encoding the proline oxidase Put1, which oxidatively degrades proline and transfers electrons and protons to the mitochondrial electron transport system, indicating that Put1-mediated energy production may be involved in the regulation of the replicative lifespan in yeast. We next analyzed the inhibitory mechanism of proline utilization. We found that arginine suppressed proline utilization via endocytosis of the proline transporter Put4 in the arginine transporter Can1-dependent manner. The results also suggest that Can1 is a transceptor with both transporter and receptor functions.


• Understanding of reactive nitrogen species-dependent signal transduction in yeast by comprehensive and quantitative analysis methods

  Nasuno Ryo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Early-Career Scientists, Nara Institute of Science and Technology, Apr. 2019 - 31 Mar. 2022, Principal investigator

  My proteomic and biochemical analyses identified pyruvate decarboxylase Pdc1 as a nitrated protein. Nitration at Tyr157 and Tyr344 decreased the enzymatic activity of Pdc1. Further analyses showed that reactive nitrogen species (RNS) suppress the fermentation efficiency through the inhibition of Pdc1 by its nitration at Tyr157 and Try344. I also demonstrated that fructose-1,6-bisphosphate aldolase Fba1 was S-glutathionylated at Cys112, which inhibits the enzymatic activity of Fba1, in response to RNS. My metabolite quantification suggested that S-glutathionylation of Fba1 increased the intracellular NADPH, via the metabolic shift from glycolysis to pentose phosphate pathway. Furthermore, My cell viability assay showed that S-glutathionylation of Fba1 contributed to RNS tolerance in yeast. These results suggest that yeast protects cells from RNS stress by the increased NADPH via the metabolic shift induced by S-glutathionylation of Fba1.

  Competitive research funding


• ニトロ化タンパク質の脱ニトロ化・還元酵素の同定と機能解析

  公益財団法人 日本応用酵素協会, 酵素研究助成, 2022, Principal investigator


• 機能性アミノ酸高含有酵母の育種技術を活用した発酵・醸造食品の高付加価値化および海外ブランド化

  高木 博史, 那須野 亮, 西村 明, 仲原 丈晴, 光永 均, 小高 敦史, 村上 直之, 五味 勝也, 新谷 尚弘

  国立研究開発法人農業・食品産業技術総合研究機構, イノベーション創出強化研究推進事業, Apr. 2018 - Mar. 2021, Coinvestigator


• ニトロ化タンパク質の脱ニトロ化・還元酵素の探索・同定・機能解析

  那須野 亮

  公益財団法人 日本応用酵素協会, 酵素研究助成, 2020, Principal investigator


• 真菌の一酸化窒素耐性に関与する酵素GTP cyclohydrolase II を標的とした新規抗生物質の探索と開発

  那須野 亮

  公益財団法人 持田記念医学薬学振興財団, 研究助成, 2020, Principal investigator


• Synthetic regulatory mechanism and physiological function of nitric oxidce in yeasts and fungi in

  TAKAGI Hiroshi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), Nara Institute of Science and Technology, 01 Apr. 2016 - 31 Mar. 2019

  We found a candidate gene encoding an enzyme involved in NADPH synthesis, which is required for the NOS reaction. We also showed that an NADPH-dependent diflavin oxidoreductase 1 (NDOR1) and its interaction partner protein CIAPIN1, which are homologous to Tah18 and Dre2, respectively, are involved in the NOS activity in a manner similar to Tah18-Dre2, suggesting that the Tah18-dependent NO synthesis and its regulatory system are conserved in eukaryotic cells from yeasts to mammals. To identify S-nitrosylated proteins in yeast, we developed the biotin switch method combined with LC-MS. Using cell extract treated with a NO donor S-nitrosoglutathione, we identified many S-nitrosylated proteins. In research of pathogenic yeasts and fungi, we found that some of multidrug-resistant strains of Candida species and clinical strains of Aspergillus fumigatus had lower levels of NO or less sensitivity to NO than those of other strains, suggesting that NO is related to pathogenicity.


• Screening, identification, and functional analyses of protein denitrase

  Nasuno Ryo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Nara Institute of Science and Technology, Apr. 2015 - Mar. 2019, Principal investigator

  We tried to identify Denitrase which decreases the protein tyrosine (Tyr) nitration-dependent signal. Using the yeast glutamine synthetase Gln1 as a model substrate, we detected the clear denitrase activity against the nitrated Gln1 from the yeast lysate. Our proteomic analyses showed that many metabolic enzymes are nitrated under nitrite treatment.

  Competitive research funding


• Synthetic mechanism and physiological role of nitric oxidce in yeasts

  Takagi Hiroshi, KAWAMOTO Susumu, CHIBANA Hiroji, WATANABE Daisuke, NASUNO Ryo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), Nara Institute of Science and Technology, 21 Oct. 2013 - 31 Mar. 2016

  The flavoprotein Tah18 is involved in NO synthase (NOS)-like activity and Tah18-dependent NO synthesis confers high-temperature stress tolerance on the budding yeast Saccharomyces cerevisiae. We showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to oxidative stresses. We also studied the antioxidative mechanism by NO. NO increased the transcription of the CTR1 gene encoding copper transporter, the intracellular copper content, the activity of superoxide dismutase Sod1, and the cell viability in a manner dependent on Mac1. Thus, Tah18-dependent NO synthesis exhibits dual effects, cell protection and death, in yeast. Our results also suggest that NO is involved in the growth, infection and pathogenicity of the pathogenic yeasts and fungi, Candida glabrata, Cryptococcus neoformans and Aspergillus fumigatus.


• New regulatory mechanism of intracellular antioxidative system by the yeast acetyltransferase Mpr1

  Takagi Hiroshi, OHTSU Iwao, NASUNO Ryo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for Challenging Exploratory Research, Nara Institute of Science and Technology, 01 Apr. 2013 - 31 Mar. 2016

  We constructed stable Mpr1 variants by a rational design based on its crystal structure. Substitution of Asn203 to a Lys was suggested to stabilize α-helix 2, which is important for the whole structure of Mpr1, probably by neutralizing its dipole moment, leading to an increase in the thermostability of Mpr1. Expression of the Mpr1 variant enhanced the arginine biosynthesis in yeast. This finding will lead to the construction of new yeast strains with increased arginine synthetic activity and fermentation rate. We also attempted to clarify the Mpr1-dependent arginine synthetic pathway in yeast including identification of the cellular substrate of Mpr1 and its catalytic reaction. As a result, N-acetyl proline, which is converted from proline catalyzed by Mpr1, was suggested to function as the intermediate for Mpr1-dependent ariginine synthesis or the regulator for the arginine metabolic enzyme.


• 酵母のN-アセチルトランスフェラーゼMpr1依存的な新規アルギニン代謝制御機構の解析

  那須野 亮

  公益財団法人 野田産業科学研究所, 奨励研究助成, 2016, Principal investigator