RESUMEN
A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2- mutants produced in this study will be deposited in several repository organizations. KEY POINTS: ⢠All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains ⢠Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains ⢠The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Mutación , Plásmidos , Poliploidía , Plásmidos/genética , Edición Génica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces/genética , Saccharomyces cerevisiae/genética , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Genoma Fúngico/genéticaRESUMEN
DNA double-strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with the recombinational repair, we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription and need to be removed to allow recombinational repair, rather than playing a positive role.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN , ARN , Reparación del ADN por Recombinación , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Reparación del ADN , Replicación del ADN , Regulación Fúngica de la Expresión Génica , Hibridación de Ácido Nucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/metabolismo , Metabolismo de los Lípidos/fisiología , Lipogénesis/fisiología , Mortierella/enzimología , Mortierella/metabolismo , 3-Isopropilmalato Deshidrogenasa/genética , Acetilcoenzima A , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Ácidos Grasos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Cetoácidos/metabolismo , Metabolismo de los Lípidos/genética , Lipogénesis/genética , Metabolómica , Mortierella/genética , Mucor/metabolismo , Alineación de SecuenciaRESUMEN
Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.
Asunto(s)
Secuencia de Aminoácidos , Catálisis , Enzimas/metabolismo , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos/genética , Secuencia de Aminoácidos/fisiología , Frío , Estabilidad de Enzimas/genética , Enzimas/genética , Cinética , Filogenia , Alineación de Secuencia , Temperatura , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
Mucor circinelloides is a valuable oleaginous filamentous fungus rich in γ-linolenic acid (GLA, 18:3; n-6), which is beneficial for human health. Our previous comparative proteomic analysis between high lipid-producing M. circinelloides WJ11 and low lipid-producing M. circinelloides CBS 277.49 indicated that glucose 6-phosphate dehydrogenase (G6PDH) and ß-isopropylmalate dehydrogenase (IPMDH) were closely involved in lipid accumulation. Transcription analysis suggested that in the strain WJ11, g6pdh1 and g6pdh2, which encode G6PDH, and leuB, which encodes IPMDH, could be the key genes regulating lipid accumulation. To further analyze the effects of these three genes (i.e., g6pdh1, g6pdh2, and leuB) on lipid accumulation, we respectively overexpressed these genes from M. circinelloides WJ11 in defective CBS 277.49 strains in this study. The results showed that overexpression of g6pdh1 and g6pdh2 genes from strain WJ11 increased the fatty acid content of cell dry weight by 23-38 and 41-47%, respectively, compared with the control strain. Furthermore, overexpression of the leuB gene from strain WJ11 increased the fatty acid content of cell dry weight by up to 67-73%. These results suggest that g6pdh1, g6pdh2, and especially leuB genes play important roles in regulating fatty acid synthesis in M. circinelloides.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Mucor/genética , Ácido gammalinolénico/metabolismo , 3-Isopropilmalato Deshidrogenasa/genética , Secuencia de Bases , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/genética , Genoma Microbiano , Glucosafosfato Deshidrogenasa/genética , Metabolismo de los Lípidos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genéticaRESUMEN
Recursive elongation pathways produce compounds of increasing carbon-chain length with each iterative cycle. Of particular interest are 2-ketoacids derived from recursive elongation, which serve as precursors to a valuable class of advanced biofuels known as branched-chain higher alcohols (BCHAs). Protein engineering has been used to increase the number of iterative elongation cycles completed, yet specific production of longer-chain 2-ketoacids remains difficult to achieve. Here, we show that mitochondrial compartmentalization is an effective strategy to increase specificity of recursive pathways to favor longer-chain products. Using 2-ketoacid elongation as a proof of concept, we show that overexpression of the three elongation enzymes-LEU4, LEU1, and LEU2-in mitochondria of an isobutanol production strain results in a 2.3-fold increase in the isopentanol to isobutanol product ratio relative to overexpressing the same elongation enzymes in the cytosol, and a 31-fold increase relative to wild-type enzyme expression. Reducing the loss of intermediates allows us to further boost isopentanol production to 1.24 ± 0.06 g/L of isopentanol. In this strain, isopentanol accounts for 86% of the total BCHAs produced, while achieving the highest isopentanol titer reported for Saccharomyces cerevisiae. Localizing the elongation enzymes in mitochondria enables the development of strains in which isopentanol constitutes as much as 93% of BCHA production. This work establishes mitochondrial compartmentalization as a new approach to favor high titers and product specificities of larger products from recursive pathways.
Asunto(s)
Ingeniería Metabólica/métodos , Mitocondrias/metabolismo , Pentanoles/metabolismo , Saccharomyces cerevisiae/metabolismo , 2-Isopropilmalato Sintasa/genética , 2-Isopropilmalato Sintasa/metabolismo , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Butanoles/metabolismo , Compartimento Celular , Citosol/metabolismo , Regulación Fúngica de la Expresión Génica , Hemiterpenos/metabolismo , Hidroliasas/genética , Hidroliasas/metabolismo , Cetoácidos/metabolismo , Leucina/biosíntesis , Malatos/metabolismo , Microorganismos Modificados Genéticamente , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Proteinaceous infectious particles causing mammalian transmissible spongiform encephalopathies or prions are being extensively studied. However due to their hazardous nature, the initial screening of potential anti-prion drugs is often made in a yeast-based screening system utilizing a well-characterized [PSI+] prion (amyloid formed by the translation termination factor Sup35p). In the [PSI+] prion screening system (white/red colony assay), the prion phenotype yields white colonies while addition of an anti-prion drug will yield red colonies. However, this system has some limitations. It is difficult to quantify the effectiveness of the anti-prion compound, the diffusion of the studied compound may affect the result, and the deficiency of glutathione in cells may prevent the formation of red pigment in cured cells. Therefore, alternative yeast prion screening systems are still needed. This article aims to present an alternative yeast-based system to evaluate anti-prion activity of chemical compounds. The method that was used is based on an artificial [LEU2+] prion created by fusing Leu2p with the prion-forming domain of Sup35p in Saccharomyces cerevisiae. Phenotypic analysis and semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) confirmed the presence of the artificial [LEU2+] prion in yeast cells. This screening system verified the anti-prion activity of 3 drugs that were found to have been active in the white/red colony assay, while one compound (6-chlorotacrine) that was active in the white/red colony assay was found to be inactive in the [LEU2+] system. This new system also appears to be more sensitive than the white/red colony assay.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/genética , Evaluación Preclínica de Medicamentos/métodos , Priones/efectos de los fármacos , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Guanabenzo/farmacología , Factores de Terminación de Péptidos/genética , Fenantridinas/farmacología , Fenotipo , Tacrina/análogos & derivados , Tacrina/síntesis química , Tacrina/farmacologíaRESUMEN
DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Sitios Genéticos/fisiología , Genoma Fúngico/fisiología , Saccharomyces cerevisiae , 3-Isopropilmalato Deshidrogenasa/biosíntesis , 3-Isopropilmalato Deshidrogenasa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
For nearly a century adaptive landscapes have provided overviews of the evolutionary process and yet they remain metaphors. We redefine adaptive landscapes in terms of biological processes rather than descriptive phenomenology. We focus on the underlying mechanisms that generate emergent properties such as epistasis, dominance, trade-offs and adaptive peaks. We illustrate the utility of landscapes in predicting the course of adaptation and the distribution of fitness effects. We abandon aged arguments concerning landscape ruggedness in favor of empirically determining landscape architecture. In so doing, we transform the landscape metaphor into a scientific framework within which causal hypotheses can be tested.
Asunto(s)
Adaptación Biológica , Evolución Biológica , Modelos Genéticos , 3-Isopropilmalato Deshidrogenasa/genética , Animales , Quimiotaxis , Escherichia coli , Aptitud Genética , Genotipo , Operón Lac , Represoras Lac/genética , Metanol/metabolismo , Opsinas/genética , Biología SintéticaRESUMEN
We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100â¯MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pHâ¯7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200â¯MPa, employing a midpoint pressure of 245.3⯱â¯0.1â¯MPa and a volume change of -225⯱â¯24â¯mLâ¯mol-1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 andâ¯-30â¯mLâ¯mol-1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/química , Proteínas Bacterianas/química , Shewanella/enzimología , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Adaptación Fisiológica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Presión Hidrostática , Modelos Químicos , Modelos Moleculares , Mutación , Conformación Proteica , Desplegamiento Proteico , Shewanella/clasificación , Shewanella/genética , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Urea/químicaRESUMEN
Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded ß-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.
Asunto(s)
Bebidas Alcohólicas/microbiología , Ciclo del Carbono/genética , Etanol/metabolismo , Microbiología de Alimentos/métodos , Hidroliasas/genética , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Treonina Deshidratasa/genética , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , China , Fermentación , Eliminación de Gen , Humanos , Hidroliasas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina Deshidratasa/metabolismoRESUMEN
The centromere is the chromosomal locus at which the kinetochore is assembled to direct chromosome segregation. The histone H3 variant, centromere protein A (CENP-A), is known to epigenetically mark active centromeres, but the mechanism by which CENP-A propagates at the centromere, replacing histone H3, remains poorly understood. Using fission yeast, here we show that the Ino80 adenosine triphosphate (ATP)-dependent chromatin-remodeling complex, which removes histone H3-containing nucleosomes from associated chromatin, promotes CENP-ACnp1 chromatin assembly at the centromere in a redundant manner with another chromatin-remodeling factor Chd1Hrp1. CENP-ACnp1 chromatin actively recruits the Ino80 complex to centromeres to elicit eviction of histone H3-containing nucleosomes. Artificial targeting of Ino80 subunits to a non-centromeric DNA sequence placed in a native centromere enhances the spreading of CENP-ACnp1 chromatin into the non-centromeric DNA. Based on these results, we propose that CENP-ACnp1 chromatin employs the Ino80 complex to mediate the replacement of histone H3 with CENP-ACnp1, and thereby reinforces itself.The histone variant CENP-A marks active centromeres and replaces H3 at centromeres through a poorly understood mechanism. Here, the authors provide evidence that the chromatin remodeller Ino80 promotes CENP-A chromatin assembly at the centromere in fission yeast.
Asunto(s)
Centrómero/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , 3-Isopropilmalato Deshidrogenasa/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/genética , Ensamble y Desensamble de Cromatina/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Epigénesis Genética , Histonas/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
3-isopropylmalate dehydrogenase (IPMD) encoded by LEU2 is a key enzyme in leucine (Leu) biosynthetic pathway. Analysis of the genome sequence of Fusarium graminearum revealed two paralogous LEU2 genes (designated as FgLEU2A and FgLEU2B) in this fungus and the deduced amino acid sequences of FgLeu2A and FgLeu2B share 45% identity. Targeted disruption of individual FgLEU2A/B gene in F. graminearum assigned a more crucial role of FgLeu2A in Leu biosynthesis as disruption of FgLEU2A resulted in mutant (ΔFgLeu2A-10) that was Leu-auxotrophic and could not grow in minimal medium limited for amino acids, whereas FgLEU2B deletion mutant ΔFgLeu2B-2 was morphologically indistinguishable from the wild type strain PH-1. The growth defects of ΔFgLeu2A-10 could be overcome by exogenous addition of Leu at 0.25 mM. Double deletion of FgLEU2A and FgLEU2B (ΔFgLeu2AB-8) caused a more severe Leu-auxotrophic phenotype as the concentration of Leu exogenously added to medium to rescue the growth defect of ΔFgLeu2AB-8 should be raised to 1.25 mM, indicating a less important but nonnegligible role of FgLeu2B in Leu biosynthesis. Disturb of Leu biosynthesis caused by FgLEU2A deletion leads to slower growth rate, reduced aerial hyphal formation and red pigmentation on PDA plates and completely blocked conidial production and germination. All of the defects above could be overcome by Leu addition or complementation of the full-length FgLEU2A gene. ΔFgLeu2A-10 also showed significantly increased sensitivity to osmotic and oxidative stresses. Pathogenicity assay results showed that virulence of mutants lacking FgLEU2A were dramatically impaired on wheat heads and non-host cherry tomatoes. Additionally, a low level of deoxynivalenol (DON) production of ΔFgLeu2A-10 and ΔFgLeu2AB-8 in wheat kernels was also detected. Taken together, results of this study indicated a crucial role of FgLeu2A and a less important role of FgLeu2B in Leu biosynthesis and fungal infection-related morphogenesis in F. graminearum and FgLeu2A may serve as a potential target for novel antifungal development.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/genética , Proteínas Fúngicas/genética , Fusarium/enzimología , Regulación Fúngica de la Expresión Génica , Leucina/biosíntesis , 3-Isopropilmalato Deshidrogenasa/metabolismo , Adaptación Fisiológica/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/crecimiento & desarrollo , Fusarium/patogenicidad , Interacciones Huésped-Patógeno , Hifa/enzimología , Hifa/genética , Hifa/crecimiento & desarrollo , Hifa/patogenicidad , Isoenzimas/genética , Isoenzimas/metabolismo , Solanum lycopersicum/microbiología , Presión Osmótica , Estrés Oxidativo , Esporas Fúngicas/enzimología , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/patogenicidad , Triticum/microbiologíaRESUMEN
Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and ß-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/metabolismo , Luciérnagas/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Luciferasas de Luciérnaga/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , 3-Isopropilmalato Deshidrogenasa/genética , Animales , Luciérnagas/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Luciferasas de Luciérnaga/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two ß-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg2+, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/genética , Proteínas Bacterianas/genética , Isocitrato Deshidrogenasa/genética , Sulfolobus acidocaldarius/enzimología , 3-Isopropilmalato Deshidrogenasa/metabolismo , Proteínas Bacterianas/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Isocitratos/metabolismo , Magnesio/metabolismo , Malatos/metabolismo , Unión Proteica , Sulfolobus acidocaldarius/genéticaRESUMEN
Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different ß-hydroxyacids in the leucine- and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here, we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg(2+) or NAD(+) These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys-232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys-232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the ß-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Glucosinolatos/biosíntesis , Leucina/biosíntesis , 3-Isopropilmalato Deshidrogenasa/genética , 3-Isopropilmalato Deshidrogenasa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Glucosinolatos/química , Glucosinolatos/genética , Leucina/química , Leucina/genética , Relación Estructura-ActividadRESUMEN
3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/metabolismo , Aclimatación/genética , Proteínas Bacterianas/metabolismo , Shewanella/enzimología , 3-Isopropilmalato Deshidrogenasa/química , 3-Isopropilmalato Deshidrogenasa/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , PresiónRESUMEN
The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/química , Proteínas Bacterianas/química , Thermus thermophilus/enzimología , 3-Isopropilmalato Deshidrogenasa/genética , Sustitución de Aminoácidos , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Mutación , Estructura Terciaria de Proteína , Teoría Cuántica , Dispersión del Ángulo Pequeño , Rayos XRESUMEN
Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 µs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies.
Asunto(s)
3-Isopropilmalato Deshidrogenasa/química , 3-Isopropilmalato Deshidrogenasa/genética , Simulación de Dinámica Molecular , Mutación Puntual/genética , Temperatura , Thermus thermophilus/enzimología , Estabilidad de Enzimas , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Mutantes/química , Proteínas Mutantes/genética , Solventes , Factores de TiempoRESUMEN
Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)--a process that is mediated by the conserved DNA damage response kinase, Tel1(ATM). The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1(ATM) prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.