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1.
Molecules ; 26(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34500562

RESUMO

Gluconeogenesis is a key interface between organic acid/amino acid/lipid and sugar metabolism. The aims of this article are four-fold. First, to provide a concise overview of plant gluconeogenesis. Second, to emphasise the widespread occurrence of gluconeogenesis and its utilisation in diverse processes. Third, to stress the importance of the vacuolar storage and release of Krebs cycle acids/nitrogenous compounds, and of the role of gluconeogenesis and malic enzyme in this process. Fourth, to outline the contribution of fine control of enzyme activity to the coordinate-regulation of gluconeogenesis and malate metabolism, and the importance of cytosolic pH in this.


Assuntos
Aminoácidos/metabolismo , Gluconeogênese/fisiologia , Lipídeos/fisiologia , Plantas/metabolismo , Açúcares/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Malato Desidrogenase/metabolismo , Nitrogênio/metabolismo
2.
Commun Biol ; 4(1): 949, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376783

RESUMO

Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.


Assuntos
Malato Desidrogenase/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sítios de Ligação , Domínio Catalítico , Malato Desidrogenase/química , Modelos Moleculares , Plasmodium falciparum/química , Proteínas de Protozoários/química
3.
J Plant Physiol ; 264: 153482, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34330009

RESUMO

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.


Assuntos
Genes de Plantas/genética , Malato Desidrogenase/genética , Oryza/genética , Proteínas de Plantas/genética , Piruvato Ortofosfato Diquinase/genética , Setaria (Planta)/genética , Clorofila/metabolismo , Clonagem Molecular , Malato Desidrogenase/metabolismo , Oryza/anatomia & histologia , Oryza/enzimologia , Oryza/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/anatomia & histologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Setaria (Planta)/enzimologia , Setaria (Planta)/metabolismo
4.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202757

RESUMO

The aim of this study was to investigate the effect of the application of homocysteine as well as its effect under the condition of aerobic physical activity on the activities of matrix metalloproteinases (MMP), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in cardiac tissue and on hepato-renal biochemical parameters in sera of rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C: 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.); H: homocysteine 0.45 µmol/g b.w./day s.c.; CPA saline (0.9% NaCl 0.2 mL/day s.c.) and a program of physical activity on a treadmill; and HPA homocysteine (0.45 µmol/g b.w./day s.c.) and a program of physical activity on a treadmill. Subcutaneous injection of substances was applied 2 times a day at intervals of 8 h during the first two weeks of experimental protocol. Hcy level in serum was significantly higher in the HPA group compared to the CPA group (p < 0.05). Levels of glucose, proteins, albumin, and hepatorenal biomarkers were higher in active groups compared with the sedentary group. It was demonstrated that the increased activities of LDH (mainly caused by higher activity of isoform LDH2) and mMDH were found under the condition of homocysteine-treated rats plus aerobic physical activity. Independent application of homocysteine did not lead to these changes. Physical activity leads to activation of MMP-2 isoform and to increased activity of MMP-9 isoform in both homocysteine-treated and control rats.


Assuntos
Hiper-Homocisteinemia/metabolismo , Rim/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Malato Desidrogenase/metabolismo , Metaloproteinases da Matriz/metabolismo , Miocárdio/metabolismo , Condicionamento Físico Animal , Animais , Biomarcadores , Pesos e Medidas Corporais , Ativação Enzimática , Hiper-Homocisteinemia/etiologia , Miocárdio/enzimologia , Especificidade de Órgãos , Ratos , Fatores de Tempo
5.
Artigo em Inglês | MEDLINE | ID: mdl-34298180

RESUMO

In the last decade, there has been an increase in the study of the ecology of deep-sea organisms. One way to understand an organism's ecology is the study of its metabolism. According to literature, deep-sea sharks possess a lower anaerobic enzyme activity than their shallow-water counterparts, but no difference has been observed regarding their aerobic enzyme activities. These studies have suggested deep-sea sharks should be slow and listless swimmers. However, other studies based on video observations have revealed differences in cruise swimming speed between different species. The present study examined muscles of squaliform sharks, including both luminous and non-luminous species. We combined measurements of the relative amounts of red and white muscle with assays of enzymes that are used as markers for aerobic (citrate synthase, malate dehydrogenase) and anaerobic (lactate dehydrogenase) metabolism, searching for a relationship with cruising speeds. Non-luminous deep-sea species displayed lower aerobic enzyme activities but similar anaerobic enzyme activities than the benthic shallow-water counterpart (Squalus acanthias). Conversely, luminous Etmopteridae species were found to have similar aerobic enzyme activities to S. acanthias but displayed lower anaerobic enzyme activities. Analyses revealed that red muscle proportion and aerobic enzyme activities were positively related to the cruise swimming speed. In contrast, Dalatias licha, which swims at the slowest cruise swimming speed ever recorded, presented a very low aerobic metabolic phenotype (lower aerobic marker enzymes and less red muscle). Finally, the values obtained for white muscle proportion and anaerobic metabolic phenotype suggested a high burst capacity for D. licha and non-luminous sharks.


Assuntos
Citrato (si)-Sintase/metabolismo , Proteínas de Peixes/metabolismo , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Músculo Esquelético/metabolismo , Tubarões/metabolismo , Animais , Citrato (si)-Sintase/genética , Proteínas de Peixes/genética , L-Lactato Desidrogenase/genética , Malato Desidrogenase/genética , Músculo Esquelético/crescimento & desenvolvimento , Tubarões/genética , Tubarões/crescimento & desenvolvimento , Natação
6.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063066

RESUMO

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.


Assuntos
Proteínas Fúngicas/metabolismo , Malato Desidrogenase/metabolismo , Ácido Oleico/metabolismo , Peroxissomos/metabolismo , Saccharomycetales/enzimologia , Carbono/farmacologia , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , NAD/metabolismo , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos Testes
7.
Biochem Biophys Res Commun ; 562: 1-8, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34030039

RESUMO

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes.


Assuntos
Aeromonas hydrophila/enzimologia , Regulação Enzimológica da Expressão Gênica , Lisina/metabolismo , Propionatos/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/farmacologia , Glucose/farmacologia , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo
8.
Biochem Biophys Res Commun ; 557: 288-293, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33894416

RESUMO

Glycosomal malate dehydrogenase from Trypanosoma cruzi (tcgMDH) catalyzes the oxidation/reduction of malate/oxaloacetate, a crucial step of the glycolytic process occurring in the glycosome of the human parasite. Inhibition of tcgMDH is considered a druggable trait for the development of trypanocidal drugs. Sequence comparison of MDHs from different organisms revealed a distinct insertion of a prolin rich 9-mer (62-KLPPVPRDP-70) in tcgMDH as compared to other eukaryotic MDHs. Crystal structure of tcgMDH is solved here at 2.6 Å resolution with Rwork/Rfree values of 0.206/0.216. The tcgMDH forms homo-dimer with the solvation free energy (ΔGo) gain of -9.77 kcal/mol. The dimeric form is also confirmed in solution by biochemical assays, chemical-crosslinking and dynamic light scattering. The inserted 9-mer adopts a structure of a solvent accessible loop in the vicinity of NAD+ binding site. The distinct sequence and structural feature of tcgMDH, revealed in the present report, provides an anchor point for the development of inhibitors specific for tcgMDH, possible trypanocidal agents of the future.


Assuntos
Malato Desidrogenase/química , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Difusão Dinâmica da Luz , Escherichia/metabolismo , Malato Desidrogenase/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes , Alinhamento de Sequência , Trypanosoma cruzi/química , Trypanosoma cruzi/enzimologia
9.
mBio ; 12(2)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824210

RESUMO

The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance.IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Malato Desidrogenase/metabolismo , NADP/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Glucose/metabolismo , Malato Desidrogenase/genética , Malatos/metabolismo , Oxirredução
10.
J Biochem ; 170(1): 97-105, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-33723609

RESUMO

Malate dehydrogenase (MDH) catalyzes the reversible reduction of oxaloacetate (OAA) to L-malate using nicotinamide adenine dinucleotide hydrogen. MDH has two characteristic loops, the mobile loop and the catalytic loop, in the active site. On binding to the substrate, the enzyme undergoes a structural change from the open-form, with an open conformation of the mobile loop, to the closed-form, with the loop in a closed conformation. In this study, three crystals of MDH from a moderate thermophile, Geobacillus stearothermophilus (gs-MDH) were used to determine four different enzyme structures (resolutions, 1.95-2.20 Å), each of which was correspondingly assigned to its four catalytic states. Two OAA-unbound structures exhibited the open-form, while the other two OAA-bound structures exhibited both the open- and closed-form. The structural analysis suggested that the binding of OAA to the open-form gs-MDH promotes conformational change in the mobile loop and simultaneously activates the catalytic loop. The mutations on the key amino acid residues involving the proposed catalytic mechanism significantly affected the gs-MDH activity, supporting our hypothesis. These findings contribute to the elucidation of the detailed molecular mechanism underlying the substrate recognition and structural switching during the MDH catalytic cycle.


Assuntos
Geobacillus stearothermophilus/enzimologia , Malato Desidrogenase/metabolismo , Biocatálise , Malato Desidrogenase/química , Malato Desidrogenase/isolamento & purificação , Modelos Moleculares , Conformação Proteica
11.
Bioorg Chem ; 110: 104779, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33689977

RESUMO

Hypoxia inducible factor-1 (HIF-1) is a pivotal transcription factor, which is strongly correlated with the induction of angiogenesis, tumor survival, metastasis, and cell proliferation, making it a pivotal therapeutic target for solid tumor therapeutic agents. Herein, a new series of multi-functional chemical probes were designed including principal groups, viz. adamantyl and indene, at various locations of the parent compound LW6. Molecular docking studies were performed on the designed compounds and their relationship with HIF-1α and malate dehydrogenase 2 (MDH2). Inhibition of MDH2 by our compounds was expected to decrease the NADH level. Indeed, treatment of the breast cancer cell line 4T1 led to a strong reduction of the NADH concentration. The greatest reduction in NADH production in mitochondria was observed with (E)-3-(4-((3r, 5r, 7r)-adamantan-1-yl) phenoxy)-N-(5-(piperidine-1-carbonyl)-1, 4-dihydroindeno [1, 2-c] pyrazol-3-yl) acrylamide (18: IC50 = 59 nM), and has the best inhibitory potential under hypoxic conditions (MCF-7: IC50 = 57 nM). This compound also gave one of the highest docking "higher than the score obtained with LW6 in parallel (-31.63 kcal/mol) in the initial docking runs (PDB Code: 4WLO). Other related compounds with good yields were also synthesized from docking results, and all the synthesized compounds (14, 18, 22, 26, 29, 30) were evaluated in vitro on human adenocarcinoma cell lines.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indenos/farmacologia , Malato Desidrogenase/antagonistas & inibidores , Simulação de Acoplamento Molecular , Pirazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indenos/síntese química , Indenos/química , Malato Desidrogenase/metabolismo , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade
12.
J Med Chem ; 64(7): 4109-4116, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33761256

RESUMO

Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two mechanisms might explain this: low concentrations of the colloid and enzyme might mean low encounter rates, or colloid-based protein denaturation might impose a kinetic barrier. These two mechanisms should have different concentration dependencies. Perplexingly, when enzyme concentration was increased, incubation times actually lengthened, inconsistent with both models and with classical chemical kinetics of solution species. We therefore considered molecular crowding, where colloids with lower protein surface density demand a shorter incubation time than more crowded colloids. To test this, we grew and shrank colloid surface area. As the surface area shrank, the incubation time lengthened, while as it increased, the converse was true. These observations support a crowding effect on protein binding to colloidal aggregates. Implications for drug delivery and for detecting aggregation-based inhibition will be discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Coloides/metabolismo , Malato Desidrogenase/metabolismo , beta-Lactamases/metabolismo , Adsorção , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Coloides/química , Ensaios Enzimáticos , Fulvestranto/química , Cinética , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/química , Ligação Proteica , Sorafenibe/química , beta-Lactamases/química
13.
DNA Cell Biol ; 40(5): 694-705, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33751897

RESUMO

Several studies have reported that miR-885-5p was dysregulated in a variety of cancers. However, there are few studies on the biological function of miR-885-5p in gastric cancer (GC). In this study, we investigated the biological function and underlying mechanism of miR-885-5p in GC. Quantitative real-time PCR was used to examine the expression of miR-885-5p in GC. Bioinformatics analysis was used to predict the target of miR-885-5p and confirmed using the luciferase reporter assay. Wound-healing and Transwell assay were conducted to evaluate the biological function of miR-885-5p and malic enzyme 1 (ME1). Western blotting was used to assess molecular changes. Hepatic and lung metastasis models were constructed and used to verify the role of miR-885-5p. We found that the expression of miR-885-5p was significantly downregulated in GC. Overexpression of miR-885-5p inhibited invasion and metastasis of GC in vivo and in vitro, while inhibition of miR-885-5p has the opposite result in vitro. ME1 is a direct target of miR-885-5p, overexpressed in GC, associated with poor prognosis. Overexpression of miR-885-5p negatively regulates ME1 and causes changes in downstream molecules Vimentin and Fibronectin. Our research found that miR-885-5p plays a tumor suppressor gene and could potentially serve as a biomarker and therapeutic target in GC.


Assuntos
Malato Desidrogenase/metabolismo , MicroRNAs/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo/genética , Feminino , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Regulação para Cima/genética , Vimentina/metabolismo
14.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668723

RESUMO

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glicerol/metabolismo , Malatos/metabolismo , Aerobiose , Bicarbonatos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Cinética , Malato Desidrogenase/metabolismo , Mutação/genética , Fosfoenolpiruvato Carboxilase/metabolismo
15.
Plant Physiol Biochem ; 162: 69-73, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33667968

RESUMO

It is well known that the photosynthetic performance of a leaf is highly dependent on the systemic regulation from distal parts within a plant under light heterogeneity. However, there are few studies focusing on C4-specific processes. In the present study, two cultivars of maize (Zea mays L.), 'Rongyu 1210' (RY) and 'Zhongdan 808' (ZD), were treated with heterogeneous light (HL). The net photosynthetic rate (Pn) of newly developed leaves was found to increase in HL-treated RY, while it decreased in HL-treated ZD. Result also showed a negative correlation between the Pn and the content of malate, a key metabolite in C4 photosynthesis, in these two cultivars. In HL-treated ZD, malate content increased with a decline in the abundance of NADP-malic enzyme (EC 1.1.1.40), suggesting that less malate was decarboxylated. Moreover, a restriction of malate diffusion is proposed in HL-treated ZD, since the interface length between mesophyll cells (MC) and bundle sheath cells (BSC) decreased. In contrast, malate diffusion and subsequent decarboxylation in HL-treated RY should be stimulated, due to an increase in the abundance of NADP-malate dehydrogenase (EC 1.1.1.82) and a decline in the content of malate. In this case, malate diffusion from MC to BSC should be systemically stimulated, thereby facilitating C4 photosynthesis of a maize leaf in heterogeneous light. While if it is systemically restricted, C4 photosynthesis would be suppressed.


Assuntos
Malatos , Zea mays , Luz , Malato Desidrogenase/metabolismo , Células do Mesofilo/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Zea mays/metabolismo
16.
Oxid Med Cell Longev ; 2021: 6626286, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33763170

RESUMO

Photobiomodulation with 808 nm laser light electively stimulates Complexes III and IV of the mitochondrial respiratory chain, while Complexes I and II are not affected. At the wavelength of 1064 nm, Complexes I, III, and IV are excited, while Complex II and some mitochondrial matrix enzymes seem to be not receptive to photons at that wavelength. Complex IV was also activated by 633 nm. The mechanism of action of wavelengths in the range 900-1000 nm on mitochondria is less understood or not described. Oxidative stress from reactive oxygen species (ROS) generated by mitochondrial activity is an inescapable consequence of aerobic metabolism. The antioxidant enzyme system for ROS scavenging can keep them under control. However, alterations in mitochondrial activity can cause an increment of ROS production. ROS and ATP can play a role in cell death, cell proliferation, and cell cycle arrest. In our work, bovine liver isolated mitochondria were irradiated for 60 sec, in continuous wave mode with 980 nm and powers from 0.1 to 1.4 W (0.1 W increment at every step) to generate energies from 6 to 84 J, fluences from 7.7 to 107.7 J/cm2, power densities from 0.13 to 1.79 W/cm2, and spot size 0.78 cm2. The control was equal to 0 W. The activity of the mitochondria's complexes, Krebs cycle enzymes, ATP production, oxygen consumption, generation of ROS, and oxidative stress were detected. Lower powers (0.1-0.2 W) showed an inhibitory effect; those that were intermediate (0.3-0.7 W) did not display an effect, and the higher powers (0.8-1.1 W) induced an increment of ATP synthesis. Increasing the power (1.2-1.4 W) recovered the ATP production to the control level. The interaction occurred on Complexes III and IV, as well as ATP production and oxygen consumption. Results showed that 0.1 W uncoupled the respiratory chain and induced higher oxidative stress and drastic inhibition of ATP production. Conversely, 0.8 W kept mitochondria coupled and induced an increase of ATP production by increments of Complex III and IV activities. An augmentation of oxidative stress was also observed, probably as a consequence of the increased oxygen consumption and mitochondrial isolation experimental conditions. No effect was observed using 0.5 W, and no effect was observed on the enzymes of the Krebs cycle.


Assuntos
Lasers Semicondutores , Terapia com Luz de Baixa Intensidade , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Bovinos , Respiração Celular/efeitos da radiação , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Isocitrato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos da radiação , Malato Desidrogenase/metabolismo , Masculino , Fosforilação Oxidativa/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , ATPases Translocadoras de Prótons/metabolismo , Superóxidos/metabolismo , Temperatura
17.
J Sci Food Agric ; 101(12): 5116-5123, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33583040

RESUMO

BACKGROUND: Ferulic acid (FA) is a common polyphenolic compound. The purpose of this study was to explore the effect of dietary FA supplementation on growth performance and muscle fiber type conversion in weaned piglets. In this study, eighteen 21-day-old DLY (Duroc × Landrace × Yorkshire) weaned piglets were randomly divided into control, 0.05% FA, and 0.45% FA groups. RESULTS: Our study showed that dietary FA supplementation had no effect on growth performance, but it could upregulate the expression of slow myosin heavy chain (MyHC) protein, increase the activities of succinic dehydrogenase and malate dehydrogenase, and downregulate the expression of fast MyHC protein. Dietary FA supplementation also increased the expression levels of phosphorylated AMP-activated protein kinase, sirtuin 1 (Sirt1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), myocyte enhancer factor 2C, and troponin I-SS, increased the proportion of slow-twitch fiber, and decreased the proportion of fast-twitch fiber. In addition, our results showed that dietary FA supplementation increased the messenger RNA abundance of mitochondrial nuclear transcription genes, including ATP synthase membrane subunit c locus 1, cytochrome oxidase subunit 1, nuclear respiratory factor 1, mitochondrial transcription factor A, mitochondrial transcription factor B1, and cytochrome c. CONCLUSION: We provided the first evidence that FA could promote muscle fiber type conversion from fast-twitch to slow-twitch via the Sirt1/AMP-activated protein kinase/PGC-1α signaling pathway and could improve the mitochondrial function in weaned piglets. This means that FA can be used as a dietary supplement to improve the quality of pork. © 2021 Society of Chemical Industry.


Assuntos
Ácidos Cumáricos/administração & dosagem , Suplementos Nutricionais/análise , Fibras Musculares Esqueléticas/efeitos dos fármacos , Suínos/crescimento & desenvolvimento , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Suínos/genética , Suínos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Desmame
18.
Nat Commun ; 12(1): 1228, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623032

RESUMO

Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.


Assuntos
Bdellovibrio bacteriovorus/enzimologia , Malato Desidrogenase/metabolismo , Acetilcoenzima A/metabolismo , Regulação Alostérica , Apoproteínas/química , Sítios de Ligação , Biocatálise , Cinética , Malato Desidrogenase/química , Modelos Moleculares , Movimento (Física) , Domínios Proteicos
19.
BMC Plant Biol ; 21(1): 48, 2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33461504

RESUMO

BACKGROUND: Despite significant limitations of growth medium reuse, a large amount of organic substrate is reused in soilless cultivation of horticultural crops in China. Arbuscular mycorrhizal fungi (AMF) can promote nutrient absorption and improve plant tolerance to biotic and abiotic stresses. However, the mechanisms governing the effects of AMF on crop growth in organic continuous cropping substrates have not been elucidated. RESULTS: In this study, we showed that the inoculation of AMF in continuous cropping substrates promoted growth and root development, and increased the root and NADP-malic enzyme (NADP-ME) activity of tomato seedlings. Root transcriptome analysis demonstrated that the plant hormone signal transduction pathway was highly enriched, and 109 genes that positively correlated with the AMF-inoculated plant phenotype were obtained by gene set enrichment analysis (GSEA), which identified 9 genes related to indole acetic acid (IAA). Importantly, the levels of endogenous IAA in tomato seedlings significantly increased after AMF inoculation. Furthermore, the application of AMF significantly increased the expression levels of NADP-ME1 and NADP-ME2, as well as the activity of NADP-ME, and enhanced the root activity of tomato seedlings in comparison to that observed without inoculation of AMF. However, these effects were blocked in plants treated with 2,3,5-triiodobenzoic acid (TIBA), a polar transport inhibitor of IAA. CONCLUSIONS: These results suggest that IAA mediates the AMF-promoted tomato growth and expression of NADP-MEs in continuous cropping substrates. The study provides convincing evidence for the reuse of continuous cropping substrates by adding AMF as an amendment.


Assuntos
Ácidos Indolacéticos/metabolismo , Lycopersicon esculentum/crescimento & desenvolvimento , Malato Desidrogenase/metabolismo , Micorrizas/fisiologia , Raízes de Plantas/fisiologia , Agricultura/métodos , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Produtos Agrícolas/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Ácidos Indolacéticos/farmacologia , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/microbiologia , Fotossíntese , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plântula/crescimento & desenvolvimento , Plântula/microbiologia
20.
Photosynth Res ; 147(2): 211-227, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393063

RESUMO

C4-like plants represent the penultimate stage of evolution from C3 to C4 plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C4 plant, its leaf anatomy and gas exchange traits suggest that it may be a C4-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C4 Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C4-like intermediate plants rather than C4 plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO2. These data demonstrate that C. prionitis is not a true C4 plant but should be considered as a C4-like plant.


Assuntos
Dióxido de Carbono/metabolismo , Fotossíntese , Poaceae/fisiologia , Cloroplastos/enzimologia , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Glicina Desidrogenase (Descarboxilante)/metabolismo , Malato Desidrogenase/metabolismo , Células do Mesofilo/enzimologia , Células do Mesofilo/fisiologia , Células do Mesofilo/ultraestrutura , Fenótipo , Fosfoenolpiruvato Carboxilase/antagonistas & inibidores , Fosfoenolpiruvato Carboxilase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Folhas de Planta/ultraestrutura , Proteínas de Plantas/metabolismo , Poaceae/enzimologia , Poaceae/ultraestrutura , Ribulose-Bifosfato Carboxilase/metabolismo
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