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1.
Plant J ; 119(4): 2033-2044, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38949911

RESUMEN

Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle is frequently upregulated in both fungi and their host plants. Here, we examined the response of the glyoxylate cycle in bread wheat (Triticum aestivum) to infection by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Gene expression analysis revealed that wheat genes encoding the two unique enzymes of the glyoxylate cycle, isocitrate lyase (TaICL) and malate synthase, diverged in their expression between susceptible and resistant Pst interactions. Focusing on TaICL, we determined that the TaICL B homoeolog is specifically upregulated during early stages of a successful Pst infection. Furthermore, disruption of the B homoeolog alone was sufficient to significantly perturb Pst disease progression. Indeed, Pst infection of the TaICL-B disruption mutant (TaICL-BY400*) was inhibited early during initial penetration, with the TaICL-BY400* line also accumulating high levels of malic acid, citric acid, and aconitic acid. Exogenous application of malic acid or aconitic acid also suppressed Pst infection, with trans-aconitic acid treatment having the most pronounced effect by decreasing fungal biomass 15-fold. Thus, enhanced TaICL-B expression during Pst infection may lower accumulation of malic acid and aconitic acid to promote Pst proliferation. As exogenous application of aconitic acid and malic acid has previously been shown to inhibit other critical pests and pathogens, we propose TaICL as a potential target for disruption in resistance breeding that could have wide-reaching protective benefits for wheat and beyond.


Asunto(s)
Glioxilatos , Isocitratoliasa , Malato Sintasa , Enfermedades de las Plantas , Puccinia , Triticum , Triticum/microbiología , Triticum/genética , Triticum/metabolismo , Triticum/enzimología , Isocitratoliasa/metabolismo , Isocitratoliasa/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Glioxilatos/metabolismo , Malato Sintasa/metabolismo , Malato Sintasa/genética , Puccinia/fisiología , Puccinia/patogenicidad , Regulación de la Expresión Génica de las Plantas , Resistencia a la Enfermedad/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
2.
Nat Commun ; 15(1): 5073, 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38871714

RESUMEN

Methyl-TROSY nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterising large biomolecules in solution. However, preparing samples for these experiments is demanding and entails deuteration, limiting its use. Here we demonstrate that NMR spectra recorded on protonated, uniformly 13C labelled samples can be processed using deep neural networks to yield spectra that are of similar quality to typical deuterated methyl-TROSY spectra, potentially providing information for proteins that cannot be produced in bacterial systems. We validate the methodology experimentally on three proteins with molecular weights in the range 42-360 kDa. We further demonstrate the applicability of our methodology to 3D NOESY spectra of Escherichia coli Malate Synthase G (81 kDa), where observed NOE cross-peaks are in good agreement with the available structure. The method represents an advance in the field of using deep learning to analyse complex magnetic resonance data and could have an impact on the study of large biomolecules in years to come.


Asunto(s)
Escherichia coli , Escherichia coli/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Aprendizaje Profundo , Malato Sintasa/química , Malato Sintasa/metabolismo , Redes Neurales de la Computación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Isótopos de Carbono/química , Proteínas/química , Proteínas/metabolismo
3.
Chem Biol Drug Des ; 103(4): e14512, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38570316

RESUMEN

A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1H NMR, 13C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 µM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC99 2.55 µg/mL whereas compounds 5d and 5j showed activity with MIC99 18.91 µg/mL and 25.07 µg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC99 value of 2.55 µg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Antituberculosos , Relación Estructura-Actividad , Malato Sintasa , Morfolinos , Simulación del Acoplamiento Molecular , Pruebas de Sensibilidad Microbiana
4.
Arch Microbiol ; 206(3): 126, 2024 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-38411730

RESUMEN

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the ß-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in ß galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.


Asunto(s)
Escherichia coli , Salmonella typhimurium , Salmonella typhimurium/genética , Malato Sintasa , Operón , Estrés Oxidativo/genética , Acetatos , Carbono , Nutrientes
5.
Yeast ; 40(7): 265-275, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37170862

RESUMEN

Debaryomyces hansenii is a halotolerant/halophilic yeast usually found in salty environments. The yeast accumulated sodium at high concentrations, which improved growth in salty media. In contrast, lithium was toxic even at low concentrations and its presence prevented cell proliferation. To analyse the responses to both cations, metabolite levels, enzymatic activities and gene expression were determined, showing that NaCl and LiCl trigger different cellular responses. At high concentrations of NaCl (0.5 or 1.5 M) cells accumulated higher amounts of the intermediate metabolites glyoxylate and malate and, at the same time, the levels of intracellular oxoglutarate decreased. Additionally, 0.5 M NaCl increased the activity of the enzymes isocitrate lyase and malate synthase involved in the synthesis of glyoxylate and malate respectively and decreased the activity of isocitrate dehydrogenase. Moreover, transcription of the genes coding for isocitrate lyase and malate synthase was activated by NaCl. Also, cells accumulated phosphate upon NaCl exposure. None of these effects was provoked when LiCl (0.1 or 0.3 M) was used instead of NaCl. Lithium induced accumulation of higher amounts of oxoglutarate and decreased the concentrations of glyoxylate and malate to non-detectable levels. Cells incubated with lithium also showed higher activity of the isocitrate dehydrogenase and neither increased isocitrate lyase and malate synthase activities nor the transcription of the corresponding genes. In summary, we show that sodium, but not lithium, up regulates the shunt of the glyoxylic acid in D. hansenii and we propose that this is an important metabolic adaptation to thrive in salty environments.


Asunto(s)
Debaryomyces , Sodio , Cloruro de Sodio/farmacología , Malato Sintasa/genética , Malato Sintasa/metabolismo , Isocitratoliasa/genética , Isocitratoliasa/metabolismo , Malatos , Debaryomyces/metabolismo , Saccharomyces cerevisiae/metabolismo , Isocitrato Deshidrogenasa/genética , Carbono , Ácidos Cetoglutáricos , Glioxilatos/metabolismo
6.
J Struct Biol ; 215(2): 107958, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36997036

RESUMEN

Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/ß domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy.


Asunto(s)
Escherichia coli , Malato Sintasa , Microscopía por Crioelectrón/métodos , Conformación Molecular , Cristalografía por Rayos X
7.
J Plant Physiol ; 280: 153863, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36423447

RESUMEN

Expression of rice malate synthase (OsMS), one of the two key genes in the glyoxylate cycle, is highly upregulated under salt stress. In this study, we aimed to investigate the role of OsMS in salt stress responses using the Arabidopsis T-DNA insertional mutant line of malate synthase (AtMS), an OsMS orthologous gene, for ectopic expression. Germination of the Atms mutant under salt stress was lower than that of Arabidopsis Col-0 wildtype (WT); furthermore, the two Atms mutant lines ectopically expressing OsMS reversed the salt-sensitive phenotype. Atms mutants salt-treated for 3 days exhibited higher electrolyte leakage, higher Na+/K+ ratio, lower expression of stress-responsive genes, and lower soluble sugar content than WT and the two OsMS-expressing Atms mutant lines. Consistently, Atms mutants salt-treated for 3 days followed by a 5-day recovery period displayed greater fresh-weight reduction. Notably, leaf greenness and chlorophyll and total carotenoid contents were higher in the Atms mutant lines than in the WT under stress. OsMS-expressing Atms mutants exhibited a change in pigment content closer to that of WT. During dark-induced senescence, an Atms mutant, Aticl, mutant (the other key gene in the glyoxylate cycle), and three double mutant lines of Atms and Aticl exhibited lower decreases in leaf greenness than the WT and OsMS-expressing Atms mutant lines. Furthermore, SAG12 expression levels in the Atms mutant, Aticl mutant, and three double mutant lines were lower than those in OsMS-expressing Atms mutant lines. Altogether, our data indicate that OsMS likely plays a key role in salt stress responses, possibly through the induction of leaf senescence.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Oryza , Arabidopsis/metabolismo , Oryza/metabolismo , Malato Sintasa/genética , Malato Sintasa/metabolismo , Expresión Génica Ectópica , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Estrés Salino/genética , Glioxilatos , Regulación de la Expresión Génica de las Plantas , Estrés Fisiológico/genética , Proteínas de Arabidopsis/metabolismo
8.
Int J Mol Sci ; 25(1)2023 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-38203573

RESUMEN

Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.


Asunto(s)
Arthrodermataceae , Factores de Transcripción , Humanos , Isocitratoliasa/genética , Malato Sintasa/genética , Gluconeogénesis/genética , Empalme Alternativo , Carbono , Glucosa , Queratinas , Glioxilatos
9.
Sci Rep ; 12(1): 15979, 2022 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-36155623

RESUMEN

To survive and replicate in the host, S. Typhimurium have evolved several metabolic pathways. The glyoxylate shunt is one such pathway that can utilize acetate for the synthesis of glucose and other biomolecules. This pathway is a bypass of the TCA cycle in which CO2 generating steps are omitted. Two enzymes involved in the glyoxylate cycle are isocitrate lyase (ICL) and malate synthase (MS). We determined the contribution of MS in the survival of S. Typhimurium under carbon limiting and oxidative stress conditions. The ms gene deletion strain (∆ms strain) grew normally in LB media but failed to grow in M9 minimal media supplemented with acetate as a sole carbon source. However, the ∆ms strain showed hypersensitivity (p < 0.05) to hypochlorite. Further, ∆ms strain has been significantly more susceptible to neutrophils. Interestingly, several folds induction of ms gene was observed following incubation of S. Typhimurium with neutrophils. Further, ∆ms strain showed defective colonization in poultry spleen and liver. In short, our data demonstrate that the MS contributes to the virulence of S. Typhimurium by aiding its survival under carbon starvation and oxidative stress conditions.


Asunto(s)
Isocitratoliasa , Malato Sintasa , Acetatos/metabolismo , Carbono/metabolismo , Dióxido de Carbono , Glucosa , Glioxilatos/metabolismo , Ácido Hipocloroso , Isocitratoliasa/genética , Isocitratoliasa/metabolismo , Malato Sintasa/genética , Malato Sintasa/metabolismo , Nutrientes , Estrés Oxidativo , Salmonella typhimurium/metabolismo
10.
J Appl Microbiol ; 133(5): 2760-2778, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-35665578

RESUMEN

AIMS: Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. METHODS AND RESULTS: Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2 •- ) and hydrogen peroxide (H2 O2 ) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. CONCLUSIONS: TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.


Asunto(s)
Glycine max , Deficiencias de Hierro , Glycine max/metabolismo , Malatos/metabolismo , Antioxidantes/metabolismo , Peróxido de Hidrógeno/metabolismo , Glutatión Reductasa/metabolismo , Raíces de Plantas/metabolismo , Superóxidos/metabolismo , Citrato (si)-Sintasa/metabolismo , Malato Sintasa/metabolismo , Clorofila/metabolismo , Hierro/metabolismo , Glutatión/metabolismo , Fenoles/metabolismo , Suelo , Citratos , Metionina/metabolismo , ARN Mensajero/metabolismo
11.
J Biomol Struct Dyn ; 39(8): 2945-2958, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-32306850

RESUMEN

Mycobacterium tuberculosis remains one of the most successful bacterial pathogens worldwide. The development of drug-resistant strains and the ability of the bacteria to persist in a latent form in the host are major problems for tuberculosis (TB) control. Glyoxylate shunt is a metabolic bypass of the Krebs cycle and is the key for M. tuberculosis to survive under latent conditions. Malate synthase (MtbMS) catalyzes the second step of the glyoxylate cycle and converts glyoxylate into malate. Phenyl-diketo acid (PDKA) is a potent inhibitor of MtbMS, and its efficacy is validated in a mouse model of TB. To identify novel PDKA analogs as anti-TB compounds, PDKA analogs that obeyed the Lipinski rules (n = 5473) were analyzed and docked with MtbMS structure in three sequential modes. These compounds were then assessed for ADMET parameters. Of the compounds examined, 19 were found to fit well for redocking studies. After optimization, four prospective inhibitors were identified, that along with the reference compound PDKA were subjected to 50 ns molecular dynamics simulation and binding-free energy analyses to evaluate the complex dynamics after ligand binding, the stability of the bound complexes, and the intermolecular interactions between the complexes. The MtbMS-PDKA complex showed the binding free energy of -57.16 kJ·mol-1. After a thorough analysis, our results suggested that three compounds which had binding-free energy of -127.96, -97.60, and -83.98 kJ·mol-1, with PubChem IDs 91937661, 14016246, and 126487337, respectively, have the potential to inhibit MtbMS and can be taken as lead compounds for drug discovery against TB.Communicated by Ramaswamy H. Sarma.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Animales , Antituberculosos/farmacología , Malato Sintasa/metabolismo , Malatos , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/metabolismo , Estudios Prospectivos
12.
Mol Biochem Parasitol ; 240: 111335, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33058935

RESUMEN

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min-1. mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01µM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.


Asunto(s)
Proteínas del Helminto/química , Malato Sintasa/química , Modelos Moleculares , Trichostrongyloidea/enzimología , Secuencia de Aminoácidos , Animales , Activación Enzimática , Glioxilatos/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Concentración de Iones de Hidrógeno , Malato Sintasa/genética , Malato Sintasa/inmunología , Malato Sintasa/metabolismo , Peso Molecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Trichostrongyloidea/genética
13.
Proc Natl Acad Sci U S A ; 117(36): 22452-22461, 2020 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-32820073

RESUMEN

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2 We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2 We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.


Asunto(s)
Crecimiento Quimioautotrófico/fisiología , Glicolatos/metabolismo , Fotosíntesis/fisiología , Acetilcoenzima A/metabolismo , Proteínas Bacterianas/metabolismo , Ciclo del Carbono/fisiología , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Malato Sintasa/metabolismo , Malatos/metabolismo , Oxidación-Reducción
14.
Microb Cell Fact ; 19(1): 144, 2020 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-32677952

RESUMEN

BACKGROUND: Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. RESULTS: The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. CONCLUSIONS: The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.


Asunto(s)
Beijerinckiaceae/crecimiento & desarrollo , Beijerinckiaceae/genética , Isocitratoliasa/genética , Malato Sintasa/genética , Modelos Biológicos , Propano/metabolismo , Carbono/metabolismo , Etanol/metabolismo , Genes Bacterianos , Ingeniería Genética , Glioxilatos/metabolismo , Microbiología Industrial , Redes y Vías Metabólicas/genética , Metano/metabolismo , Mutación , Proteómica
15.
Sci Rep ; 10(1): 10123, 2020 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-32572104

RESUMEN

Orchid (Orchidaceae) is one of the largest families in angiosperms and presents exceptional diversity in lifestyle. Their unique reproductive characteristics of orchid are attracted by scientist for centuries. One of the synapomorphies of orchid plants is that their seeds do not contain endosperm. Lipids are used as major energy storage in orchid seeds. However, regulation and mobilization of lipid usage during early seedling (protocorm) stage of orchid is not understood. In this study, we compared transcriptomes from developing Phalaenopsis aphrodite protocorms grown on 1/2-strength MS medium with sucrose. The expression of P. aphrodite MALATE SYNTHASE (PaMLS), involved in the glyoxylate cycle, was significantly decreased from 4 days after incubation (DAI) to 7 DAI. On real-time RT-PCR, both P. aphrodite ISOCITRATE LYASE (PaICL) and PaMLS were down-regulated during protocorm development and suppressed by sucrose treatment. In addition, several genes encoding transcription factors regulating PaMLS expression were identified. A gene encoding homeobox transcription factor (named PaHB5) was involved in positive regulation of PaMLS. This study showed that sucrose regulates the glyoxylate cycle during orchid protocorm development in asymbiotic germination and provides new insights into the transcription factors involved in the regulation of malate synthase expression.


Asunto(s)
Malato Sintasa/genética , Malato Sintasa/metabolismo , Orchidaceae/genética , Metabolismo de los Hidratos de Carbono , Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Germinación , Glioxilatos/metabolismo , Orchidaceae/metabolismo , Plantones/crecimiento & desarrollo , Semillas/fisiología , Simbiosis , Factores de Transcripción/genética , Transcriptoma
16.
Int J Mol Sci ; 21(7)2020 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-32260167

RESUMEN

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.


Asunto(s)
Aminopiridinas/farmacología , Antibacterianos/farmacología , Isocitratoliasa/antagonistas & inhibidores , Malato Sintasa/antagonistas & inhibidores , Pseudomonas aeruginosa/crecimiento & desarrollo , Aminopiridinas/química , Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Calorimetría , Línea Celular , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glioxilatos/metabolismo , Humanos , Isocitratoliasa/química , Malato Sintasa/química , Estructura Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología
17.
Cells ; 9(1)2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31906273

RESUMEN

Autophagy is a universal self-degradation process involved in the removal and recycling of cellular constituents and organelles; however, little is known about its possible role in fruit ripening, in which the oxidation of lipids and proteins and changes in the metabolism of different cellular organelles occur. In this work, we analyzed several markers of autophagy in two critical maturation stages of pepper (Capsicum annuum L.) fruits where variations due to ripening become clearly visible. Using two commercial varieties that ripen to yellow and red fruits respectively, we studied changes in the gene expression and protein content of several autophagy (ATG) components, ATG4 activity, as well as the autophagy receptor NBR1 and the proteases LON1 and LON2. Additionally, the presence of intravacuolar vesicles was analyzed by electron microscopy. Altogether, our data reveal that autophagy plays a role in the metabolic changes which occur during ripening in the two studied varieties, suggesting that this process may be critical to acquiring final optimal quality of pepper fruits.


Asunto(s)
Autofagia , Capsicum/citología , Capsicum/crecimiento & desarrollo , Frutas/citología , Frutas/crecimiento & desarrollo , Biomarcadores/metabolismo , Capsicum/genética , Citocromos c/genética , Citocromos c/metabolismo , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Humanos , Peroxidación de Lípido , Malato Sintasa/metabolismo , Estrés Oxidativo , Extractos Vegetales/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vacuolas/metabolismo , Vacuolas/ultraestructura
18.
FEBS J ; 287(7): 1359-1368, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31623016

RESUMEN

Specialized metabolites are structurally diverse and cell- or tissue-specific molecules produced in restricted plant lineages. In contrast, primary metabolic pathways are highly conserved in plants and produce metabolites essential for all of life, such as amino acids and nucleotides. Substrate promiscuity - the capacity to accept non-native substrates - is a common characteristic of enzymes, and its impact is especially apparent in generating specialized metabolite variation. However, promiscuity only leads to metabolic diversity when alternative substrates are available; thus, enzyme cellular and subcellular localization directly influence chemical phenotypes. We review a variety of mechanisms that modulate substrate availability for promiscuous plant enzymes. We focus on examples where evolution led to modification of the 'cellular context' through changes in cell-type expression, subcellular relocalization, pathway sequestration, and cellular mixing via tissue damage. These varied mechanisms contributed to the emergence of structurally diverse plant specialized metabolites and inform future metabolic engineering approaches.


Asunto(s)
Hidroliasas/metabolismo , Malato Sintasa/metabolismo , Oxidorreductasas de Alcohol Dependientes de NAD (+) y NADP (+)/metabolismo , Plantas/enzimología , Plantas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Hidroliasas/química , Malato Sintasa/química , Ingeniería Metabólica , Oxidorreductasas de Alcohol Dependientes de NAD (+) y NADP (+)/química , Nucleótidos/biosíntesis , Nucleótidos/química
19.
Chemphyschem ; 21(1): 13-19, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31703148

RESUMEN

Optimized NMR experiments are developed for isolating magnetization belonging to the I=1/2 manifolds of 13 CH3 methyl groups in proteins, enabling the manipulation of the magnetization of a 13 CH3 moiety as if it were an AX (1 H-13 C) spin-system. These experiments result in the same 'simplification' of a 13 CH3 spin-system that would be obtained from the production of {13 CHD2 }-methyl-labeled protein samples. The sensitivity of I=1/2 manifold-selection experiments is a factor of approximately 2 less than that of the corresponding experiments acquired on {13 CHD2 }-labeled methyl groups. The methodology described here is primarily intended for small-to-medium sized proteins, where the losses in sensitivity associated with the isolation of I=1/2 manifold transitions can be tolerated. Several NMR applications that benefit from simplification of the 13 CH3 (AX3 ) spin-systems are described, with an emphasis on the measurements of methyl 1 H-13 C residual dipolar couplings in a {13 CH3 }-methyl-labeled deletion mutant of the human chaperone DNAJB6b, where modulation of NMR signal intensities due to evolution of methyl 1 H-13 C scalar and dipolar couplings follows a simple cosine function characteristic of an AX (1 H-13 C) spin-system, significantly simplifying data analysis.


Asunto(s)
Malato Sintasa/química , Resonancia Magnética Nuclear Biomolecular , Ubiquitina/química , Humanos , Malato Sintasa/metabolismo
20.
Biochim Biophys Acta Gen Subj ; 1864(2): 129439, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31593752

RESUMEN

BACKGROUND: The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. METHODS: The 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. RESULTS: The MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. CONCLUSIONS: We demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. GENERAL SIGNIFICANCE: The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.


Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Malato Sintasa/química , Mutación , Proteínas/química , Isótopos de Carbono , Escherichia coli/enzimología , Sustancias Macromoleculares , Péptidos/química , Fenilalanina/química , Estructura Secundaria de Proteína , Protones , Triptófano/química
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