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1.
Biomed Khim ; 67(3): 300-305, 2021 May.
Artigo em Russo | MEDLINE | ID: mdl-34142538

RESUMO

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.


Assuntos
Hidrolases de Éster Carboxílico , Compostos Cromogênicos , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Humanos , Hidrólise , Cinética , Simulação de Acoplamento Molecular , Especificidade por Substrato
2.
J Agric Food Chem ; 69(21): 6064-6072, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-33979121

RESUMO

The human gut microbiota regulates nutritional metabolism, especially by encoding specific ferulic acid esterases (FAEs) to release functional ferulic acid (FA) from dietary fiber. In our previous study, we observed seven upregulated FAE genes during in vitro fecal slurry fermentation using wheat bran. Here, a 29 kDa FAE (AsFAE) from Alistipes shahii of Bacteroides was characterized and identified as the type-A FAE. The X-ray structure of AsFAE has been determined, revealing a unique α-helical domain comprising five α-helices, which was first characterized in FAEs from the gut microbiota. Further molecular docking analysis and biochemical studies revealed that Tyr100, Thr122, Tyr219, and Ile220 are essential for substrate binding and catalytic efficiency. Additionally, Glu129 and Lys130 in the cap domain shaped the substrate-binding pocket and affected the substrate preference. This is the first report on A. shahii FAE, providing a theoretical basis for the dietary metabolism in the human gut.


Assuntos
Hidrolases de Éster Carboxílico , Bacteroidetes , Hidrolases de Éster Carboxílico/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica em alfa-Hélice , Especificidade por Substrato
3.
Bioresour Technol ; 332: 124967, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33845316

RESUMO

Producing ferulic acid (FA) from the natural substrate with feruloyl esterase is promising in industries, screening and engineering new enzymes with high efficiency to increase the FA yield is of great concern. Here, the feruloyl esterase of Lactobacillus acidophilus (FAELac) was heterologous expressed and the FAELac with different oligomerization states was separated. Interestingly, the activity of dimer was 37-fold higher than high-polymer. To further enhance the efficiency of FAELac, eight mutants were generated based on the simulated structure, of which Q198A, Q134T enhanced the catalytic efficiency by 5.4- and 4.3-fold in comparison with the wild type. Moreover, higher yields of FA (2.21, 6.60, and 1.67 mg/g substrate, respectively) were released by the mutants from de-starched wheat bran, insoluble wheat arabinoxylan, and steam-exploded corn stover. These results indicated that improving the purification process, engineering new FAELac and substrates bias studies hold great potential for increasing FA production yield.


Assuntos
Hidrolases de Éster Carboxílico , Lactobacillus acidophilus , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Ácidos Cumáricos , Lactobacillus acidophilus/metabolismo , Especificidade por Substrato
4.
Molecules ; 26(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920799

RESUMO

With the relentless development of drug resistance and re-emergence of many pathogenic bacteria, the need for new antibiotics and new antibiotic targets is urgent and growing. Bacterial peptidyl-tRNA hydrolase, Pth1, is emerging as a promising new target for antibiotic development. From the conserved core and high degree of structural similarity, broad-spectrum inhibition is postulated. However, Pth1 small-molecule inhibition is still in the earliest stages. Focusing on pathogenic bacteria, herein we report the phylogenetic classification of Pth1 and natural product inhibition spanning phylogenetic space. While broad-spectrum inhibition is found, narrow-spectrum and even potentially clade-specific inhibition is more frequently observed. Additionally reported are enzyme kinetics and general in vitro Pth1 solubility that follow phylogenetic boundaries along with identification of key residues in the gate loop region that appear to govern both. The studies presented here demonstrate the sizeable potential for small-molecule inhibition of Pth1, improve understanding of Pth enzymes, and advance Pth1 as a much-needed novel antibiotic target.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Antibacterianos/farmacologia , Bactérias/enzimologia , Cinética , Filogenia , Solubilidade , Especificidade por Substrato
5.
Mar Drugs ; 19(3)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808933

RESUMO

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract.


Assuntos
Antioxidantes/farmacologia , Camellia sinensis , Hidrolases de Éster Carboxílico/metabolismo , Quitosana/química , Portadores de Fármacos , Inibidores de Glicosídeo Hidrolases/farmacologia , Iridoides/química , Extratos Vegetais/farmacologia , alfa-Amilases/antagonistas & inibidores , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Camellia sinensis/metabolismo , Hidrolases de Éster Carboxílico/química , Composição de Medicamentos , Estabilidade Enzimática , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Inibidores de Glicosídeo Hidrolases/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Temperatura , Fatores de Tempo , alfa-Amilases/metabolismo
6.
Food Chem ; 356: 129704, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33831827

RESUMO

The postharvest senescence accompanied by yellowing limited the shelf-life of broccoli. In this study, we developed a novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil and applied it to broccoli for preservation. Results showed that double emulsion prepared by whey protein concentrate-high methoxyl pectin (1:3) exhibited best storage stability with largest particle size (581.30 nm), lowest PDI (0.23) and zeta potential (-40.31 mV). This double emulsion also exhibited highest encapsulation efficiency of brassinolide (92%) and cinnamon essential oil (88%). The broccoli coated with double emulsion maintained higher chlorophyll contents and activities of chlorophyllase and magnesium-dechelatase were reduced by 9% and 24%, respectively. The energy metabolic enzymes (SDH, CCO, H+-ATPase, Ca2+-ATPase) were also activated, inducing higher level of ATP and energy charge. These results demonstrated W/O/W double emulsion co-delivering brassinolide and cinnamon essential delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.


Assuntos
Brassica/metabolismo , Brassinosteroides/química , Clorofila/metabolismo , Emulsões/química , Metabolismo Energético , Óleos Voláteis/química , Esteroides Heterocíclicos/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Brassica/efeitos dos fármacos , Brassinosteroides/metabolismo , Brassinosteroides/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Cinnamomum zeylanicum/metabolismo , Emulsões/metabolismo , Metabolismo Energético/efeitos dos fármacos , Enzimas/química , Armazenamento de Alimentos/métodos , Óleos Voláteis/metabolismo , Óleos Voláteis/farmacologia , Tamanho da Partícula , Esteroides Heterocíclicos/metabolismo , Esteroides Heterocíclicos/farmacologia , Viscosidade
7.
Molecules ; 26(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923656

RESUMO

Oxidative stress is involved in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). However, reliable biomarkers of NAFLD in relation to oxidative stress are not available. While paraoxonase 1 (PON1) is an antioxidant biomarker, there appears to be mixed data on PON-1 in patients with NAFLD. The aim of this meta-analysis was to assess the current data on PON1 activity (i.e., paraoxonase and arylesterase) in patients with NAFLD. A PubMed, CENTRAL, and Embase search identified 12 eligible articles. In the meta-analysis, the paraoxonase activity was low in patients with NAFLD (mean difference (MD) -27.17 U/L; 95% confidence interval (CI) -37.31 to -17.03). No difference was noted in the arylesterase activity (MD 2.45 U/L; 95% CI -39.83 to 44.74). In a subgroup analysis, the paraoxonase activity was low in biopsy-proven nonalcoholic steatohepatitis (MD -92.11 U/L; 95% CI -115.11 to -69.11), while the activity in NAFLD as diagnosed by ultrasonography or laboratory data was similar (MD -2.91 U/L; 95% CI -11.63 to 5.80) to that of non-NAFLD. In summary, the PON1, especially paraoxonase, activity could be a useful biomarker of NAFLD. Further studies are warranted to ascertain the relevance of PON1 measurements in patients with NAFLD.


Assuntos
Arildialquilfosfatase/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Arildialquilfosfatase/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia
8.
J Food Sci ; 86(4): 1335-1342, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33682147

RESUMO

In the orange juice industry, pulp (ruptured juice sacs) is separated from the juice after extraction and pasteurized separately before blending back with juice or sold for other food applications. However, pulp is not always pasteurized immediately after extraction and the flow behavior is affected by endogenous pectinmethylesterase (PME). This is particularly important because of the high power required to pump orange pulp in industrial pasteurizers. This study characterized the effect of PME on the flow behavior of citrus pulp during storage at 4 °C using rotational rheometry and during pulp storage at 22 °C using capillary rheometry. PME activity was 0.011 ± 0.001 PEU for the pulp used in rotational rheometry and 0.030 ± 0.002 PEU for the pulp used in capillary rheometry. Rotational rheometry was used to assess the effect of PME on the Power Law parameters at shear rates ( γ ̇ ) below those that cause slip. There were no significant differences among storage time on the flow behavior index (n). However, the consistency coefficient (K) increased with storage time. Significant differences were found after 12-hr storage increasing from 120 ± 30 Pa·sn to 160 ± 15 Pa·sn (16.7%) after 24 hr. Capillary rheomtery was used to assess the effect of PME on the pressure drop in a flow system. Significant differences were found at or after 12-hr storage. The mean pressure drop increased by 34% after 24 hr, for a flow rate of 50 × 10-6 m3 /s (0.8 GPM) in an 8.9-m long (29.2 ft), 0.022-m (1-in) diameter pipe. PRACTICAL APPLICATION: Design and optimization of processing equipment and industrial handling systems of orange pulp require detailed knowledge of its rheological (flow) properties. Citrus pulp is rich in pectinmethylesterase, an enzyme that causes thickening of the product when not inactivated during pasteurization. Understanding how fast PME affects the flow properties of orange pulp is important for citrus processors to decide how long pulp can be stored before pasteurizing it.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Citrus sinensis/química , Manipulação de Alimentos/métodos , Sucos de Frutas e Vegetais/análise , Pasteurização/métodos , Temperatura Alta , Reologia
9.
Nat Plants ; 7(3): 353-364, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33686223

RESUMO

Plant roots acquire nutrients and water while managing interactions with the soil microbiota. The root endodermis provides an extracellular diffusion barrier through a network of lignified cell walls called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses. Although suberin is a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes have key roles in suberization, driving root suberin plasticity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Lipídeos , Domínios Proteicos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Conjuntos de Dados como Assunto , Endoderma/metabolismo , Técnicas de Inativação de Genes , Ácidos Indolacéticos/metabolismo , Lipídeos/genética , Células Vegetais/metabolismo , Raízes de Plantas/metabolismo , Polimerização , Proteólise
10.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33673023

RESUMO

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846. The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea, and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana. These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.


Assuntos
Ascomicetos/genética , Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Malus/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Ascomicetos/patogenicidade , Hidrolases de Éster Carboxílico/classificação , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , Malus/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Virulência/genética
11.
Nat Commun ; 12(1): 1732, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741980

RESUMO

Macrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides' macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.


Assuntos
Antibacterianos/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Esterases/química , Esterases/genética , Macrolídeos/química , Antibacterianos/farmacologia , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Eritromicina/química , Genes Bacterianos , Macrolídeos/farmacologia , Simulação de Acoplamento Molecular , Conformação Proteica , Difração de Raios X
12.
Int J Mol Sci ; 22(2)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33450841

RESUMO

Endothelial lipase (EL) is a strong modulator of the high-density lipoprotein (HDL) structure, composition, and function. Here, we examined the impact of EL on HDL paraoxonase 1 (PON1) content and arylesterase (AE) activity in vitro and in vivo. The incubation of HDL with EL-overexpressing HepG2 cells decreased HDL size, PON1 content, and AE activity. The EL modification of HDL did not diminish the capacity of HDL to associate with PON1 when EL-modified HDL was incubated with PON1-overexpressing cells. The overexpression of EL in mice significantly decreased HDL serum levels but unexpectedly increased HDL PON1 content and HDL AE activity. Enzymatically inactive EL had no effect on the PON1 content of HDL in mice. In healthy subjects, EL serum levels were not significantly correlated with HDL levels. However, HDL PON1 content was positively associated with EL serum levels. The EL-induced changes in the HDL-lipid composition were not linked to the HDL PON1 content. We conclude that primarily, the interaction of enzymatically active EL with HDL, rather than EL-induced alterations in HDL size and composition, causes PON1 displacement from HDL in vitro. In vivo, the EL-mediated reduction of HDL serum levels and the consequently increased PON1-to-HDL ratio in serum increase HDL PON1 content and AE activity in mice. In humans, additional mechanisms appear to underlie the association of EL serum levels and HDL PON1 content.


Assuntos
Arildialquilfosfatase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Endotélio/enzimologia , Lipase/metabolismo , Lipoproteínas HDL/metabolismo , Arildialquilfosfatase/química , Hidrolases de Éster Carboxílico/química , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Lipase/sangue , Lipase/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ligação Proteica
13.
Arch Microbiol ; 203(4): 1731-1742, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33459813

RESUMO

Study of carbohydrate-active enzymes (CAZymes) can reveal information about the lifestyle and behavior of an organism. Rhodococcus species is well known for xenobiotic metabolism; however, their carbohydrate utilization ability has been less discussed till date. This study aimed to present the CAZyme analysis of two Rhodococcus strains, PAMC28705 and PAMC28707, isolated from lichens in Antarctica, and compare them with other Rhodococcus, Mycobacterium, and Corynebacterium strains. Genome-wide computational analysis was performed using various tools. Results showed similarities in CAZymes across all the studied genera. All three genera showed potential for significant polysaccharide utilization, including starch, cellulose, and pectin referring their biotechnological potential. Keeping in mind the pathogenic strains listed across all three genera, CAZymes associated to pathogenicity were analyzed too. Cutinase enzyme, which has been associated with phytopathogenicity, was abundant in all the studied organisms. CAZyme gene cluster of Rhodococcus sp. PAMC28705 and Rhodococcus sp. PAMC28707 showed the insertion of cutinase in the cluster, further supporting their possible phytopathogenic properties.


Assuntos
Celulose/metabolismo , Genoma Bacteriano/genética , Polissacarídeos/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Regiões Antárticas , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Líquens/microbiologia , Pectinas/metabolismo , Rhodococcus/isolamento & purificação , Sequenciamento Completo do Genoma
14.
Toxicol Appl Pharmacol ; 412: 115388, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33383043

RESUMO

Phytosterol diosgenin (DG) exhibits cholesterol-lowering properties. Few studies focused on the underlying mechanism of DG attenuation of hypercholesterolemia by promoting cholesterol metabolism. To investigate the roles of SRB1/CES-1/CYP7A1/FXR pathways in accelerating cholesterol elimination and alleviating hypercholesterolemia, a rat model of hypercholesterolemia was induced by providing a high-fat diet (HFD). Experimental rat models were randomly divided into a normal control (Con) group, HFD group, low-dose DG (LDG) group (150 mg/kg/d), high-dose DG (HDG) group (300 mg/kg) and Simvastatin (Sim) group (4 mg/kg/d). Body weights, serum and hepatic lipid parameters of rats were tested. The expression levels of scavenger receptor class B type I (SRB1), carboxylesterase-1 (CES-1), cholesterol7α- hydroxylase (CYP7A1), and farnesoid X receptor (FXR) were determined. The results showed that DG reduced weight and lowered lipid levels in HFD-fed rats. Pathological morphology analyses revealed that DG notably improved hepatic steatosis and intestinal structure. Further studies showed the increased hepatic SRB1, CES-1, CYP7A1 and inhibited FXR-mediated signaling in DG-fed rats, which contributing to the decrease of hepatic cholesterol. DG also increased intestinal SRB1 and CES-1, inhibiting cholesterol absorption and promoting RCT. The expression levels of these receptors in the HDG group were higher than LDG and Sim groups. These data suggested that DG accelerated reverse cholesterol transport (RCT) and enhanced cholesterol elimination via SRB1/CES-1/CYP7A1/FXR pathway, and DG might be a new candidate for the alleviation of hypercholesterolemia.


Assuntos
Anticolesterolemiantes/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Colesterol/sangue , Diosgenina/farmacologia , Hipercolesterolemia/prevenção & controle , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Biomarcadores/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/sangue , Hipercolesterolemia/enzimologia , Hipercolesterolemia/patologia , Fígado/enzimologia , Fígado/patologia , Masculino , Ratos Sprague-Dawley , Transdução de Sinais , Sinvastatina/farmacologia
15.
Food Chem ; 339: 128096, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979713

RESUMO

The changes of texture and cell wall characteristics of apricot were investigated in ten clones at two maturity stages. Fruit firmness, cell wall composition and enzyme activity of three apricot flesh zones were analysed. The AIS (alcohol-insoluble solids) were characterised by high amounts of uronic acid (179-300 mg g-1 AIS) and relatively high amounts of cellulosic glucose (118-214 mg g-1 AIS). The methylesterification degree varied significantly among the different clones ranging from 58 to 97 in Ab 5 and Mans 15 respectively. Conversely to zones firmness, enzymatic activity was higher in pistil followed by equatorial and peduncle zones. The ripening effect has been observed in firmness evolution according to enzymatic activity. This correlation allowed a classification of clones depending on softening. Among studied clones, Ab 5, Marouch 16, Mans 15 and Cg 2 were less influenced by softening and have the advantage of a technological valorisation for the processing industry.


Assuntos
Parede Celular/química , Frutas/citologia , Prunus armeniaca/química , Prunus armeniaca/citologia , Açúcares/análise , Hidrolases de Éster Carboxílico/metabolismo , Frutas/química , Humanos , Pectinas/metabolismo , Proteínas de Plantas/metabolismo , Prunus armeniaca/crescimento & desenvolvimento , Açúcares/química , beta-Galactosidase/metabolismo
16.
Food Chem ; 338: 127754, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32829296

RESUMO

The production of olive (Olea europaea L.) is very important economically in many areas of the world, and particularly in countries around the Mediterranean basin. Ripening-associated modifications in cell wall composition and structure of fruits play an important role in attributes like firmness or susceptibility to infestations, rots and mechanical damage, but limited information on these aspects is currently available for olive. In this work, cell wall metabolism was studied in fruits from nine olive cultivars ('Arbequina', 'Argudell', 'Empeltre', 'Farga', 'Manzanilla', 'Marfil', 'Morrut', 'Picual' and 'Sevillenca') picked at three maturity stages (green, turning and ripe). Yields of alcohol-insoluble residue (AIR) recovered from fruits, as well as calcium content in fruit pericarp, decreased along ripening. Cultivar-specific diversity was observed in time-course change patterns of enzyme activity, particularly for those acting on arabinosyl- and galactosyl-rich pectin side chains. Even so, fruit firmness levels were associated to higher pectin methylesterase (PME) activity and calcium contents. In turn, fruit firmness correlated inversely with ascorbate content and with α-l-arabinofuranosidase (AFase) and ß-galactosidase (ß-Gal) activities, resulting in preferential loss of neutral sugars from cell wall polymers.


Assuntos
Parede Celular/metabolismo , Frutas/crescimento & desenvolvimento , Genótipo , Olea/citologia , Olea/genética , Hidrolases de Éster Carboxílico/metabolismo , Frutas/metabolismo , Olea/crescimento & desenvolvimento
17.
Biochim Biophys Acta Proteins Proteom ; 1869(1): 140554, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33068756

RESUMO

Klebsiella pneumoniae is a member of the ESKAPE panel of pathogens that are top priority to tackle AMR. Bacterial peptidyl tRNA hydrolase (Pth), an essential, ubiquitous enzyme, hydrolyzes the peptidyl-tRNAs that accumulate in the cytoplasm because of premature termination of translation. Pth cleaves the ester bond between 2' or 3' hydroxyl of the ribose in the tRNA and C-terminal carboxylate of the peptide, thereby making free tRNA available for repeated cycles of protein synthesis and preventing cell death by alleviating tRNA starvation. Pth structures have been determined in peptide-bound or peptide-free states. In peptide-bound state, highly conserved residues F67, N69 and N115 adopt a conformation that is conducive to their interaction with peptide moiety of the substrate. While, in peptide-free state, these residues move away from the catalytic center, perhaps, in order to facilitate release of hydrolysed peptide. Here, we present a novel X-ray crystal structure of Pth from Klebsiella pneumoniae (KpPth), at 1.89 Å resolution, in which out of the two molecules in the asymmetric unit, one reflects the peptide-bound while the other reflects peptide-free conformation of the conserved catalytic site residues. Each molecule of the protein has canonical structure with seven stranded ß-sheet structure surrounded by six α-helices. MD simulations indicate that both the forms converge over 500 ns simulation to structures with wider opening of the crevice at peptide-binding end. In solution, KpPth is monomeric and its 2D-HSQC spectrum displays a single set of well dispersed peaks. Further, KpPth was demonstrated to be enzymatically active on BODIPY-Lys-tRNALys3.


Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Klebsiella pneumoniae/enzimologia , RNA de Transferência de Lisina/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos de Boro/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Klebsiella pneumoniae/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência de Lisina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
18.
PLoS One ; 15(9): e0235384, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925915

RESUMO

Interstitial cystitis/bladder pain syndrome (IC) is a debilitating condition of chronic pelvic pain with unknown etiology. Recently, we used a genetic approach in a murine model of IC to identify the lipase acyloxyacyl hydrolase (AOAH) as a modulator of pelvic pain. We found that AOAH-deficient mice have elevated pelvic pain responses, and AOAH immunoreactivity was detected along the bladder-brain axis. Lipidomic analyses identified arachidonic acid (AA) and its metabolite PGE2 as significantly elevated in the sacral spinal cord of AOAH-deficient mice, suggesting AA is a substrate for AOAH. Here, we quantified the effects of AOAH on phospholipids containing AA. Spinal cord lipidomics revealed increased AA-containing phosphatidylcholine in AOAH-deficient mice and concomitantly decreased AA-phosphatidylethanolamine, consistent with decreased CoA-independent transferase activity (CoIT). Overexpression of AOAH in cell cultures similarly altered distribution of AA in phospholipid pools, promoted AA incorporation, and resulted in decreased membrane fluidity. Finally, administration of a PGE2 receptor antagonist reduced pelvic pain in AOAH-deficient mice. Together, these findings suggest that AOAH represents a potential CoA-independent AA transferase that modulates CNS pain pathways at the level of phospholipid metabolism.


Assuntos
Ácido Araquidônico/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Cistite Intersticial/metabolismo , Dor Pélvica/metabolismo , Fosfolipídeos/metabolismo , Animais , Cistite Intersticial/complicações , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Dor Pélvica/complicações , Bexiga Urinária/metabolismo
19.
Food Chem ; 332: 127416, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32619946

RESUMO

Water bamboo shoots quickly deteriorate after harvest as a result of rapid lignification and softening. Nitric oxide (NO) has been used to extend the postharvest life of several other vegetables. Here, we examined the effect of NO on the storage of water bamboo shoots at 4℃ for 28 days. Without NO, fresh weight and firmness decreased quickly, while the cellulose and lignin contents increased sharply during storage. NO treatment delayed softening by maintaining the integrity of the cell wall and inhibiting the degradation of protopectin and the expressions of pectin methylesterase and polygalacturonase. NO treatment also delayed cellulose synthesis by increasing cellulase activity. NO treatment decreased the synthesis of lignin by inhibiting the activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, laccase and peroxidase. These results indicate that NO treatment is effective at suppressing the softening and lignification of water bamboo shoots during postharvest storage.


Assuntos
Armazenamento de Alimentos/métodos , Óxido Nítrico/farmacologia , Poaceae/efeitos dos fármacos , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Celulase/metabolismo , Celulose/metabolismo , Temperatura Baixa , Lignina/metabolismo , Microscopia Eletrônica de Varredura , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Poaceae/metabolismo , Poligalacturonase/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(27): 15895-15901, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571932

RESUMO

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, in Salmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBL was acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBL deacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Salmonella enterica/metabolismo , Sirtuínas/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Isoformas de Proteínas , Salmonella enterica/enzimologia , Espectrometria de Massas em Tandem
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