RESUMO
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.
Assuntos
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cisteína , Regulação Fúngica da Expressão Gênica , Óxido Nítrico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
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.
Assuntos
NADP/metabolismo , Estresse Nitrosativo , Estresse Oxidativo , Saccharomyces cerevisiae/enzimologia , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Butanóis/metabolismo , NADP/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetaldeído/metabolismo , Aldeído Oxirredutases/metabolismo , Meios de Cultura , Fermentação , Glucose-6-Fosfato/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Saccharomyces cerevisiae/enzimologiaRESUMO
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.
Assuntos
Cromatografia Líquida/métodos , Óxido Nítrico/síntese química , Nitrogênio/química , Espectrometria de Massas em Tandem/métodos , Óxido Nítrico/químicaRESUMO
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.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Sequência de Bases , Códon/metabolismo , Códon de Iniciação/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Metabolismo Energético , Mitocôndrias/genética , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Bebidas Alcoólicas/análise , Retroalimentação , Ornitina , Fosfotransferases (Aceptor do Grupo Carboxila) , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Óxido Nítrico/análise , Saccharomyces cerevisiae/química , Cromatografia Líquida de Alta Pressão , Citometria de Fluxo , Óxido Nítrico/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Acetiltransferases/metabolismo , Etanol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Fermentação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes.
Assuntos
Óxido Nítrico/biossíntese , Oxirredutases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Morte Celular , Peróxido de Hidrogênio/farmacologia , Proteínas Ferro-Enxofre/fisiologia , Complexos Multiproteicos/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
As a cellular signaling molecule, nitric oxide (NO) is widely conserved from microorganisms, such as bacteria, yeasts, and fungi, to higher eukaryotes including plants and mammals. NO is mainly produced by NO synthase (NOS) or nitrite reductase (NIR) activity. There are several NO detoxification systems, including NO dioxygenase (NOD) and S-nitrosoglutathione reductase (GSNOR). NO homeostasis based on the balance between NO synthesis and degradation is important for the regulation of its physiological functions because an excess level of NO causes nitrosative stress due to the high reactivity of NO and NO-derived compounds. In yeast, NO may be involved in stress responses, but NO and its signaling have been poorly understood due to the lack of mammalian NOS orthologs in the genome. Even though the activities of NOS and NIR have been observed in yeast cells, the gene encoding NOS and the NO production mechanism catalyzed by NIR remain unclear. On the other hand, yeast cells employ NOD and GSNOR to maintain an intracellular redox balance following endogenous NO production, exogenous NO treatment, or environmental stresses. This article reviews NO metabolism (synthesis, degradation) and its regulation in yeast. The physiological roles of NO in yeast, including the oxidative stress response, are also discussed here. Such investigations into NO signaling are essential for understanding the NO-dependent genetic and physiological modulations. In addition to being responsible for the pathology and pharmacology of various degenerative diseases, NO signaling may be a potential target for the construction and engineering of industrial yeast strains.
Assuntos
Homeostase , Óxido Nítrico/metabolismo , Transdução de Sinais , Leveduras/metabolismo , Estresse Oxidativo , Estresse FisiológicoRESUMO
Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ(1)-pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/ß-structure with eight-stranded mixed ß-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ(1)-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.
Assuntos
Acetiltransferases/química , Acetiltransferases/genética , Hidroxiprolina/química , Modelos Moleculares , Estresse Oxidativo/genética , Prolina/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Hidroxiprolina/metabolismo , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
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.
RESUMO
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.
Assuntos
Frutose-Bifosfato Aldolase , Proteínas de Saccharomyces cerevisiae , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Glutarredoxinas/metabolismo , NADP/metabolismo , Óxido Nítrico , Estresse Nitrosativo , Proteômica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Piruvato Descarboxilase , Saccharomyces cerevisiae , Etanol/metabolismo , Fermentação , Nitritos/metabolismo , Proteômica , Piruvato Descarboxilase/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Tirosina/metabolismoRESUMO
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.
Assuntos
Óxido Nítrico , Oxigenases , Antibacterianos/farmacologia , Óxido Nítrico/metabolismo , Oxigenases/metabolismoRESUMO
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.
Assuntos
Corantes Fluorescentes , Óxido Nítrico , Acetaldeído , Fluoresceínas/química , Corantes Fluorescentes/química , Fluorometria , Óxido Nítrico/química , Fenilenodiaminas , Saccharomyces cerevisiae , Espectrometria de Massas em TandemRESUMO
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.
RESUMO
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.
Assuntos
Antifúngicos , Aspergillus fumigatus , Antifúngicos/farmacologia , Farneseno Álcool/farmacologia , Hifas , Espécies Reativas de Oxigênio/farmacologiaRESUMO
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.
Assuntos
GTP Cicloidrolase/metabolismo , Estresse Nitrosativo , Riboflavina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas , GTP Cicloidrolase/genética , Genes Fúngicos , Riboflavina/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.