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1.
Appl Microbiol Biotechnol ; 104(5): 2051-2066, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31930452

RESUMO

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,ß-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.


Assuntos
Extremófilos/enzimologia , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Alcenos/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cianobactérias/enzimologia , Cianobactérias/genética , Cianobactérias/metabolismo , Bases de Dados Genéticas , Estabilidade Enzimática , Extremófilos/genética , Extremófilos/metabolismo , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , NADP/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/isolamento & purificação , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodófitas/enzimologia , Rodófitas/genética , Especificidade por Substrato
2.
Adv Exp Med Biol ; 1131: 371-394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646518

RESUMO

Ca2+ signals are probably the most common intracellular signaling cellular events, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca2+ signals by mobilizing Ca2+ from intracellular stores. Inositol trisphosphate (IP3) was the first messenger shown to link events at the plasma membrane to release Ca2+ from the endoplasmic reticulum (ER), through the activation of IP3-gated Ca2+ release channels (IP3 receptors). Subsequently, two additional Ca2+ mobilizing messengers were discovered, cADPR and NAADP. Both are metabolites of pyridine nucleotides, and may be produced by the same class of enzymes, ADP-ribosyl cyclases, such as CD38. Whilst cADPR mobilizes Ca2+ from the ER by activation of ryanodine receptors (RyRs), NAADP releases Ca2+ from acidic stores by a mechanism involving the activation of two pore channels (TPCs). In addition, other pyridine nucleotides have emerged as intracellular messengers. ADP-ribose and 2'-deoxy-ADPR both activate TRPM2 channels which are expressed at the plasma membrane and in lysosomes.


Assuntos
Cálcio , ADP-Ribose Cíclica , Piridinas , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Espaço Intracelular/metabolismo , NADP/metabolismo , Piridinas/química , Piridinas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
3.
Endocrinology ; 160(12): 2825-2836, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31580427

RESUMO

The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1ß (ARNT/HIF1ß) plays a key role in maintaining ß-cell function and has been shown to be one of the most downregulated transcription factors in islets from patients with type 2 diabetes. We have shown a role for ARNT/HIF1ß in glucose sensing and insulin secretion in vitro and no defects in in vivo glucose homeostasis. To gain a better understanding of the role of ARNT/HIF1ß in the development of diabetes, we placed control (+/+/Cre) and ß-cell-specific ARNT/HIF1ß knockout (fl/fl/Cre) mice on a high-fat diet (HFD). Unlike the control (+/+/Cre) mice, HFD-fed fl/fl/Cre mice had no impairment in in vivo glucose tolerance. The lack of impairment in HFD-fed fl/fl/Cre mice was partly due to an improved islet glucose-stimulated NADPH/NADP+ ratio and glucose-stimulated insulin secretion. The effects of the HFD-rescued insulin secretion in fl/fl/Cre islets could be reproduced by treating low-fat diet (LFD)-fed fl/fl/Cre islets with the lipid signaling molecule 1-monoacylglcyerol. This suggests that the defects seen in LFD-fed fl/fl/Cre islet insulin secretion involve lipid signaling molecules. Overall, mice lacking ARNT/HIF1ß in ß-cells have altered lipid signaling in vivo and are resistant to an HFD's ability to induce diabetes.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Diabetes Mellitus Experimental/metabolismo , Células Secretoras de Insulina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Diabetes Mellitus Experimental/etiologia , Dieta Hiperlipídica , Diglicerídeos , Glucose/metabolismo , Homeostase , Secreção de Insulina , Masculino , Camundongos Knockout , NADP/metabolismo
4.
Biochim Biophys Acta Bioenerg ; 1860(11): 148081, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520615

RESUMO

Cylic electron flow (CEF) around Photosystem I in photosynthetic eukaryotes is likely to be necessary to augment ATP production, rapidly- and precisely balancing the plastid ATP/NADPH energy budget to meet the demands of downstream metabolism. Many regulatory aspects of this process are unclear. Here we demonstrate that the higher plant plastid NADH/Fd:plastoquinone reductase (NDH) and proposed PGR5/PGRL1 ferredoxin:plastoquinone reductase (FQR) pathways of CEF are strongly, rapidly and reversibly inhibited in vitro by ATP with Ki values of 670 µM and 240 µM respectively, within the range of physiological changes in ATP concentrations. Control experiments ruled out effects on secondary reactions, e.g. FNR- and cytochrome b6f activity, nonphotochemical quenching of chlorophyll fluorescence etc., supporting the view that ATP is an inhibitor of CEF and its associated pmf generation and subsequent ATP production. The effects are specific to ATP, with the ATP analog AMP-PNP showing little inhibitory effect, and ADP inhibiting only at higher concentrations. For the FQR pathway, inhibition was found to be classically competitive with Fd, and the NDH pathway showing partial competition with Fd. We propose a straightforward model for regulation of CEF in plants in which CEF is activated under conditions when stromal ATP low, but is downregulated as ATP levels build up, allowing for effective ATP homeostasis. The differences in Ki values suggest a two-tiered regulatory system, where the highly efficient proton pumping NDH is activated with moderate decreases in ATP, with the less energetically-efficient FQR pathway being activated under more severe ATP depletion.


Assuntos
Trifosfato de Adenosina/metabolismo , Cloroplastos/metabolismo , Elétrons , Fotossíntese/fisiologia , Difosfato de Adenosina/metabolismo , Amaranthus , Arabidopsis , NADH Desidrogenase/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Spinacia oleracea
5.
Microb Cell Fact ; 18(1): 161, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547820

RESUMO

BACKGROUND: Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. RESULTS: In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. CONCLUSIONS: The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.


Assuntos
Dióxido de Carbono/metabolismo , Diatomáceas/crescimento & desenvolvimento , Diatomáceas/genética , Ácidos Graxos/metabolismo , Glucosefosfato Desidrogenase/genética , Dióxido de Carbono/análise , Diatomáceas/metabolismo , Engenharia Genética , Glucosefosfato Desidrogenase/metabolismo , Microalgas/genética , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fotossíntese
6.
J Microbiol Biotechnol ; 29(8): 1288-1298, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370116

RESUMO

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the Δcg1360 mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the Δcg1360 mutant were increased by 29% and 26%, respectively. However, the Δcg1361 mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the V-10-Δcg1360 mutant reached 9.2 ± 0.3 g/l in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.


Assuntos
Trifosfato de Adenosina/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Valina/biossíntese , Ácido Acético/metabolismo , Adenosina Trifosfatases , Carbono/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Metabolismo Energético , Fermentação , Ordem dos Genes , Glucose/metabolismo , NAD/metabolismo , NADP/metabolismo , Alinhamento de Sequência
7.
J Microbiol ; 57(10): 884-892, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31376105

RESUMO

The yeast Saccharomyces cerevisiae has two isoforms of NADP+-dependent glutamate dehydrogenase (Gdh1 and Gdh3) that catalyze the synthesis of glutamate from α-ketoglutarate and NH4+. In the present study, we confirmed that Gdh3, but not Gdh1, mainly contributes to the oxidative stress resistance of stationary-phase cells and found evidence suggesting that the insignificance of Gdh1 to stress resistance is possibly resulted from conditional and reversible aggregation of Gdh1 into punctuate foci initiated in parallel with post-diauxic growth. Altered localization to the mitochondria or peroxisomes prevented Gdh1, which was originally localized in the cytoplasm, from stationary phase-specific aggregation, suggesting that some cytosolic factors are involved in the process of Gdh1 aggregation. Glucose starvation triggered the transition of the soluble form of Gdh1 into the insoluble aggregate form, which could be redissolved by replenishing glucose, without any requirement for protein synthesis. Mutational analysis showed that the N-terminal proximal region of Gdh1 (NTP1, aa 21-26, TLFEQH) is essential for glucose starvation-induced aggregation. We also found that the substitution of NTP1 with the corresponding region of Gdh3 (NTP3) significantly increased the contribution of the mutant Gdh1 to the stress resistance of stationary-phase cells. Thus, this suggests that NTP1 is responsible for the negligible role of Gdh1 in maintaining the oxidative stress resistance of stationary-phase cells and the stationary phase-specific stresssensitive phenotype of the mutants lacking Gdh3.


Assuntos
Desidrogenase de Glutamato (NADP+)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Desidrogenase de Glutamato (NADP+)/genética , NADP/metabolismo , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
8.
Biomed Pharmacother ; 117: 109184, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31387167

RESUMO

With the elderly population rapidly growing, the prevalence of Parkinson's disease (PD) is quickly increasing because neurodegenerative disorders are usually late-onset. Herbal medicines and formula are adjuvant therapies of conventional PD agents, which result in serious side effects with long-term use. This study evaluated the neuroprotective effects of DA-9805, a standardized herbal formula that consists of an ethanolic extract of Moutan Cortex Radix, Angelica Dahuricae Radix, and Bupleuri Radix against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in vitro and in vivo. In PC12 cells, DA-9805 at concentrations of 1 and 10 µg/mL ameliorated cell viability, which was reduced by 6-OHDA. In addition, DA-9805 activated the extracellular-regulated kinase-nuclear transcription factor-erythroid 2-related factor 2 pathway, subsequently stimulating antioxidative enzymes such as NAD(P)H:quinone oxidoreductase 1 and catalase and suppressing apoptosis. Furthermore, DA-9805 prevented 6-OHDA-induced movement impairment, as well as a decrease of dopaminergic neurons and dopamine transmission in rodents. Taken together, these results suggest that the mixed herbal formula DA-9805 may be a pharmaceutical agent for preventing or improving PD.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/tratamento farmacológico , Oxidopamina/farmacologia , Doença de Parkinson/tratamento farmacológico , Preparações de Plantas/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NADP/metabolismo , Síndromes Neurotóxicas/metabolismo , Células PC12 , Extratos Vegetais/farmacologia , Ratos
9.
Chemosphere ; 237: 124461, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31374395

RESUMO

Due to the detoxification of tetrabromobisphenol A (TBBPA) varies from different bacterial strains and depends on their specific enzymatic machinery, it is necessary to understand them for potential in situ bioremediation application. The special ability of our previously isolated Ochrobactrum sp. T to simultaneously debrominate and aerobic mineralize TBBPA urgent us to continuously study its degradation molecular mechanism. Herein, the purification and characterization of the dehalogenase which can debrominate TBBPA was investigated based on its corresponding encoding gene tbbpaA. Results showed that an enzyme with molecular mass of 117 kDa, Km of 26.6 µM and Vmax of 0.133 µM min-1 mg-1 was purified and designated as bromophenol dehalogenase. It was the only detected dehalogenase which exhibited TBBPA degradation ability (78%). Moreover, its activity was significantly enhanced by adding NADPH or methyl viologen to the reaction solution. The high similarity of substrate spectrum between the dehalogenase from the recombinant strain and the wild strain further indicated that it was the main dehalogenase responsible for the debromination in wild strain. Based on three identified metabolites, a metabolic pathway of TBBPA by purified enzyme under oxic condition was proposed. This study provides an excellent dehalogenase candidate for mechanistic study of aerobic dehalogenation of brominated aromatic compound.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Ochrobactrum/metabolismo , Bifenil Polibromatos/metabolismo , Aerobiose , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Poluentes Ambientais/metabolismo , Peso Molecular , NADP/metabolismo , Ochrobactrum/enzimologia , Paraquat/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
10.
Nature ; 572(7768): 249-253, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367038

RESUMO

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.


Assuntos
Antioxidantes/metabolismo , Lisina/metabolismo , Poliaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters/metabolismo , Cadaverina/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , NADP/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Oxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Dokl Biochem Biophys ; 486(1): 213-215, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367824

RESUMO

The functioning of NAD(P)H:FMN­oxidoreductase (Red) from Vibrio fischeri under conditions of macromolecular crowding (MMC) simulated in vitro by adding biopolymers (starch and gelatin) was studied. The dissociation rate constants and the activation energies of dissociation of Red to the subunits were calculated, and the process of denaturation of Red was analyzed. It is shown that the functioning of Red both under conditions of MMC and in diluted solutions is the same. This result refutes the common belief that the native conformation of enzymes in vivo is stabilized due to MMC as compared to the in vitro conditions.


Assuntos
FMN Redutase/metabolismo , Modelos Moleculares , NADP/metabolismo , Aliivibrio fischeri/enzimologia
12.
J Agric Food Chem ; 67(31): 8527-8535, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298526

RESUMO

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.


Assuntos
Proteínas de Bactérias/química , Corynebacterium glutamicum/enzimologia , Cetol-Ácido Redutoisomerase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Cristalografia por Raios X , Cetol-Ácido Redutoisomerase/genética , Cetol-Ácido Redutoisomerase/metabolismo , Metais/química , Metais/metabolismo , Simulação de Acoplamento Molecular , NADP/química , NADP/metabolismo
13.
Biochim Biophys Acta Bioenerg ; 1860(9): 689-698, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336103

RESUMO

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 µM and ∆H of -20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ∆CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Å2. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.


Assuntos
Arabidopsis/metabolismo , Chlamydomonas reinhardtii/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , NADP/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Cristalografia por Raios X , Cianobactérias/metabolismo , Transporte de Elétrons , Ferredoxina-NADP Redutase/química , Ferredoxinas/química , Luz , Complexos de Proteínas Captadores de Luz , NADP/química , Complexo de Proteína do Fotossistema I/química , Conformação Proteica
14.
Nat Commun ; 10(1): 3337, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350399

RESUMO

Various biosynthetic pathways have been designed to explore sustainable production of glutarate, an attractive C5 building block of polyesters and polyamides. However, its efficient production has not been achieved in Escherichia coli. Here, we use E. coli native lysine catabolic machinery for glutarate biosynthesis. This endogenous genes-only design can generate strong metabolic driving force to maximize carbon flux toward glutarate biosynthesis by replenishing glutamate and NAD(P)H for lysine biosynthesis, releasing lysine feedback inhibition, and boosting oxaloacetate supply. We use native transporters to overcome extracellular accumulation of cadaverine and 5-aminovalerate. With these efforts, both high titer (54.5 g L-1) and high yield (0.54 mol mol-1 glucose) of glutarate production are achieved under fed-batch conditions. This work demonstrates the power of redirecting carbon flux and the role of transporters to decrease intermediate accumulation.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Glutaratos/metabolismo , Lisina/metabolismo , Vias Biossintéticas , Ácido Glutâmico/metabolismo , Engenharia Metabólica , NADP/metabolismo
15.
Pol J Vet Sci ; 22(2): 271-278, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31269353

RESUMO

This study aimed to determine the levels of milk cell total protein (TP), reduced nicotinamide adenine dinucleotide phosphate (NADPH), total glutathione (tGSH), activities of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase (GPx) in subclinical mastitic cows. Milk from each udder was collected and grouped by the California Mastitis Test. Then, a somatic cell count (SCC) was performed, and the groups were re-scored as control (5-87 × 103 cells), 1st group (154-381 × 103 cells), 2nd group (418-851 × 103 cells), 3rd group (914-1958 × 103 cells), and 4th group (2275-8528 × 103 cells). Milk cell TP, NADPH, tGSH levels, G6PD, and GPx activities were assessed. Microbiological diagnosis and aerobic mesophyle general organism (AMG, cfu/g) were also conducted. In mastitic milk, TP, NADPH, and tGSH levels, and G6PD and GPx activities were significantly reduced per cell (in samples of 106 cells). In addition, milk SCC was positively correlated with AMG (r=0.561, p⟨0.001), NADPH (r=0.380, p⟨0.01), TP (r=0.347, p⟨0.01) and G6PD (r=0.540, p⟨0.001). There was also positive correlation between NADPH (r=0.428, p⟨0.01), TP (r=0.638, p⟨0.001) and AMG. NADPH was positively correlated with TP (r=0.239, p⟨0.05), GPx (r=0.265, p⟨0.05) and G6PD (r=0.248, p=0.056). Total protein was positively correlated with tGSH (r=0.354, p⟨0.01) and G6PD (r=0.643, p⟨0.001). There was a negative correlation between tGSH and GPx activity (r=-0.306, p⟨0.05). The microbiological analysis showed the following ratio of pathogens: Coagulase-Negative Staphylococci 66.6%, Streptococcus spp 9.5%, Bacillus spp 9.5%, yeast 4.8%, and mixed infections 9.5%. As a conclusion, when evaluating the enzyme and oxidative stress parameters in milk, it is more suitable to assign values based on cell count rather than ml of milk. The linear correlation between the SCC and AMG, milk cell NADPH, TP and G6PD suggests that these parameters could be used as markers of mastitis.


Assuntos
Glucosefosfato Desidrogenase/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa/metabolismo , Mastite Bovina/patologia , Leite/citologia , NADP/metabolismo , Animais , Bovinos , Contagem de Células/veterinária , Feminino , Regulação Enzimológica da Expressão Gênica , Glucosefosfato Desidrogenase/química , Glutationa/química , Glutationa Peroxidase/genética , NADP/química
16.
J Agric Food Chem ; 67(30): 8382-8392, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31271032

RESUMO

Staphylococcus aureus is a common pathogen that can cause life-threatening infections. Treatment of antibiotic-resistant S. aureus infection needs effective antibacterial agents. Thymol, a generally recognized safe natural compound, has potential as an alternative to treat S. aureus infections. However, the targets and mechanisms of action of thymol were not fully understood. Bioinformatics analysis showed that IolS, a predicted aldo-keto reductase (AKR) in S. aureus, could be a potential target of thymol. Isothermal titration calorimetry (ITC) analysis demonstrated that thymol directly binds IolS and amino acid residues (Y30 and L33) are essential for such binding. Deletion of IolS or mutation of Y30A and L33A reduced the bactericidal activity of thymol at the concentration of 200 µg/mL, suggesting that thymol mediates bactericidal activity via binding with IolS. Biochemical analysis showed that addition of thymol significantly increased AKR activity of IolS from 1.6 ± 0.1 to 2.4 ± 0.2 U (p < 0.05). The content of NADPH within S. aureus cells decreased significantly from 105 ± 5 to 72 ± 3 pmol/108 cells (p < 0.05) following thymol treatment at the concentration of 200 µg/mL. Importantly, addition of NADPH could alleviate the bactericidal effect of thymol on S. aureus, indicating that the depletion of NADPH is responsible for thymol-mediated bactericidal activity. Overall, these results demonstrated that thymol could directly bind IolS and increase its AKR activity, leading to the depletion of NADPH and bactericidal effect. AKR activity of IolS could be a promising target for the development of new antimicrobials.


Assuntos
Aldo-Ceto Redutases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , NADP/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Timol/farmacologia , Aldo-Ceto Redutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
17.
J Biotechnol ; 303: 53-64, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31325477

RESUMO

Carboxylic acids (CAs) are widespread in Nature. A prominent example is fatty acids, a major constituent of lipids. CAs are potentially economical precursors for bio-based products such as bio-aldehydes and bio-alcohols. However, carboxylate reduction is a challenging chemical transformation due to the thermodynamic stability of carboxylate. Carboxylic acid reductases (CARs), found in bacteria and fungi, offer a good solution to this challenge. These enzymes catalyse the NADPH- and ATP-dependent reduction of aliphatic and aromatic CAs. This review summarised all the protein engineering work that has been done on these versatile biocatalysts to date. The intricate catalytic mechanism and structure of CARs prompted us to first examine their domain architecture to facilitate the subsequent discussion of various protein engineering strategies. This then led to a survey of assays to detect aldehyde formation and to monitor aldenylation activity. Strategies for NADPH and ATP regeneration were also incorporated, as they are deemed vital to developing preparative-scale biocatalytic process and high-throughput screening systems. The objectives of the review are to consolidate CAR engineering research, stimulate interest, discussion or debate, and advance the field of bioreduction.


Assuntos
Ácidos Carboxílicos/metabolismo , Oxirredutases/genética , Engenharia de Proteínas/métodos , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , NADP/metabolismo , Oxirredutases/metabolismo
18.
Oxid Med Cell Longev ; 2019: 9464608, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183001

RESUMO

Traditional remedies prepared from Lannea microcarpa leaves, barks, roots, and fruits are used to treat many diseases including hypertension. This study investigated whether oral administration of the ethyl acetate fraction of Lannea microcarpa trunk barks (LMAE) corrects angiotensin (Ang) II-induced hypertension in mice. Its effects on vascular function were specifically investigated. Experiments explored hemodynamic and echocardiographic parameters in vivo and vascular reactivity to acetylcholine (ACh) and CaCl2 ex vivo on isolated aortas. Mice received LMAE for 3 weeks (50 mg/kg/day) by oral gavage. In the last two weeks of treatment, mice were implanted with osmotic minipumps delivering NaCl (0.9%) or Ang II (0.5 mg/kg/day). LMAE completely prevented the increase in systolic and diastolic blood pressure induced by Ang II. Echocardiographic and kidney parameters were not affected by the different conditions. LMAE abrogated Ang II-induced impairment of ACh-induced relaxation without affecting that of sodium nitroprusside. LMAE also completely prevented CaCl2-induced contraction in KCl-exposed aorta ex vivo. The extract alone did not modify superoxide (O2 -) and nitric oxide (NO·) production in femoral arteries from control mice but significantly limited Ang II-induced O2 - production. These effects were associated with reduced expression of inducible isoform of cyclooxygenase- (COX-) 2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform NOX-2 in aortas. Finally, phytochemical analysis showed that LMAE contains sterols, triterpenes, coumarins, and anthraquinone. These results showed that LMAE prevents Ang II-induced hypertension and vascular dysfunction through a reduction of oxidative stress linked to COX-2 and NOX-2 pathway and inhibition of calcium entry. This study provides pharmacological basis of the empirical use of Lannea microcarpa trunk bark extract against hypertension.


Assuntos
Acetatos/química , Anacardiaceae/química , Angiotensina II/farmacologia , Hipertensão/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/induzido quimicamente , Camundongos , NADP/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Superóxidos/metabolismo
19.
Biochemistry (Mosc) ; 84(4): 390-397, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228930

RESUMO

Recombinant malic enzyme from the aerobic methanotroph Methylosinus trichosporium was obtained by heterologous expression in Escherichia coli and purified by affinity metal-chelating chromatography. The homohexameric enzyme of 6×80 kDa catalyzed the reversible reaction of oxidative decarboxylation of malate to pyruvate in the presence of mono- and divalent cations and NADP+ as a cofactor. The kcat/Km ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction. Analysis of the protein sequence revealed that the C-region of the enzyme contains a large domain homologous to phosphoacetyltransferase, but no phosphoacetyltransferase activity was detected either for a full chimeric malic enzyme or for the C-end fragment obtained as a separate protein. This C-end domain promoted activity of the malic enzyme.


Assuntos
Malato Desidrogenase/metabolismo , Methylosinus trichosporium/enzimologia , Biocatálise , Descarboxilação , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/genética , NADP/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
20.
Oxid Med Cell Longev ; 2019: 9192413, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049140

RESUMO

Oxidative stress induced by long-term glucocorticoid (GC) use weakens the repair capacity of bone tissue. Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase (NOX) is a superoxide-generating enzyme that plays an important role in regulating bone metabolism. To clarify the role of nonphagocytic NOX isoforms in osteoblast reactive oxygen species (ROS) generation and apoptosis, dexamethasone was used to establish a high-dose GC environment in vitro. A dose-dependent increase in intracellular ROS generation was demonstrated, which was accompanied by increased osteoblastic MC3T3-E1 cell apoptosis. Addition of the ROS inhibitor NAC (N-acetyl-L-cysteine) or NOX inhibitor DPI (diphenyleneiodonium) reversed this effect, indicating that NOX-derived ROS can induce osteoblast apoptosis under high-dose dexamethasone stimulation. NOX1, NOX2, and NOX4 are NOX homologs recently identified in bone tissue. To clarify the NOX isoforms that play a role in osteoblast ROS generation, Nox1, Nox2, and Nox4 mRNA expression and NOX2 and NOX4 protein expression were analyzed. Nox1 and Nox4 mRNA expression was elevated in a dose-dependent manner after culture in 100 nM, 250 nM, 500 nM, or 1000 nM dexamethasone, and the increased expression of NOX1 mRNA was more significant compared with NOX4 mRNA. Small interfering RNAs (siRNAs) were used to confirm the role of NOX1 and NOX4 in ROS generation. To clarify the signaling pathway in ROS-induced osteoblast apoptosis, mitogen-activated protein kinase (MAPK) signaling molecules were analyzed. Phosphorylated ASK1 and p38 levels were significantly higher in the 1000 nM dexamethasone group, which NAC or DPI markedly attenuated. However, the total mRNA and protein levels of ASK1 and p38 between the dexamethasone group and control were not significantly different. This is related to ROS regulating the posttranslational modification of ASK1 and p38 in MC3T3-E1 cell apoptosis. Altogether, NOX1- and NOX4-derived ROS plays a pivotal role in high-dose dexamethasone-induced preosteoblast apoptosis by increasing phosphorylated ASK1 and p38 and may be an important mechanism in steroid-induced avascular necrosis of the femoral head (SANFH).


Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoblastos/enzimologia , Animais , Linhagem Celular , Dexametasona/efeitos adversos , Isoenzimas/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Camundongos , NADP/metabolismo , Osteoblastos/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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