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1.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638570

RESUMO

The microbial biodegradation of new PLA and PCL materials containing birch tar (1-10% v/v) was investigated. Product of dry distillation of birch bark (Betula pendula Roth) was added to polymeric materials to obtain films with antimicrobial properties. The subject of the study was the course of enzymatic degradation of a biodegradable polymer with antibacterial properties. The results show that the type of the material, tar concentration, and the environment influenced the hydrolytic activity of potential biofilm degraders. In the presence of PCL films, the enzyme activities were higher (except for α-D-glucosidase) compared to PLA films. The highest concentration of birch tar (10% v/v) decreased the activity of hydrolases produced by microorganisms to the most significant extent; however, SEM analysis showed the presence of a biofilm even on plastics with the highest tar content. Based on the results of the biological oxygen demand (BOD), the new materials can be classified as biodegradable but, the biodegradation process was less efficient when compared to plastics without the addition of birch tar.


Assuntos
Anti-Infecciosos/química , Betula/química , Plásticos Biodegradáveis/química , Poliésteres/química , Alcatrões/química , Aminopeptidases/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Betula/microbiologia , Plásticos Biodegradáveis/farmacologia , Biofilmes , Análise da Demanda Biológica de Oxigênio , Destilação , Ensaios Enzimáticos , Esterases/metabolismo , Lipase/metabolismo , Casca de Planta/química , Casca de Planta/microbiologia , Poliésteres/metabolismo , Alcatrões/farmacologia , alfa-Glucosidases/metabolismo , beta-Glucosidase/metabolismo
2.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638934

RESUMO

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (1H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.


Assuntos
Esterases/metabolismo , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Espectroscopia de Prótons por Ressonância Magnética/métodos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Sítios de Ligação , Biocatálise , Cristalização , Hidrólise , Ligantes , Nitrofenóis/química , Nitrofenóis/metabolismo , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Ligação Proteica , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo
3.
Int J Mol Sci ; 22(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638659

RESUMO

Being one of the main proteins in the human body and many animal species, albumin plays a decisive role in the transport of various ions-electrically neutral and charged molecules-and in maintaining the colloidal osmotic pressure of the blood. Albumin is able to bind to almost all known drugs, as well as many nutraceuticals and toxic substances, largely determining their pharmaco- and toxicokinetics. Albumin of humans and respective representatives in cattle and rodents have their own structural features that determine species differences in functional properties. However, albumin is not only passive, but also an active participant of pharmacokinetic and toxicokinetic processes, possessing a number of enzymatic activities. Numerous experiments have shown esterase or pseudoesterase activity of albumin towards a number of endogeneous and exogeneous esters. Due to the free thiol group of Cys34, albumin can serve as a trap for reactive oxygen and nitrogen species, thus participating in redox processes. Glycated albumin makes a significant contribution to the pathogenesis of diabetes and other diseases. The interaction of albumin with blood cells, blood vessels and tissue cells outside the vascular bed is of great importance. Interactions with endothelial glycocalyx and vascular endothelial cells largely determine the integrative role of albumin. This review considers the esterase, antioxidant, transporting and signaling properties of albumin, as well as its structural and functional modifications and their significance in the pathogenesis of certain diseases.


Assuntos
Antioxidantes/metabolismo , Esterases/metabolismo , Transporte Proteico/fisiologia , Albumina Sérica/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Oxirredução
4.
BMC Plant Biol ; 21(1): 501, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34717531

RESUMO

BACKGROUND: GDSL esterases/lipases are a large protein subfamily defined by the distinct GDSL motif, and play important roles in plant development and stress responses. However, few studies have reported on the role of GDSLs in the growth and development of axillary buds. This work aims to identify the GDSL family members in tobacco and explore whether the NtGDSL gene contributes to development of the axillary bud in tobacco. RESULTS: One hundred fifty-nine GDSL esterase/lipase genes from cultivated tobacco (Nicotiana tabacum) were identified, and the dynamic changes in the expression levels of 93 of these genes in response to topping, as assessed using transcriptome data of topping-induced axillary shoots, were analysed. In total, 13 GDSL esterase/lipase genes responded with changes in expression level. To identify genes and promoters that drive the tissue-specific expression in tobacco apical and axillary buds, the expression patterns of these 13 genes were verified using qRT-PCR. GUS activity and a lethal gene expression pattern driven by the NtGDSL127 promoter in transgenic tobacco demonstrated that NtGDSL127 is specifically expressed in apical buds, axillary buds, and flowers. Three separate deletions in the NtGDSL127 promoter demonstrated that a minimum upstream segment of 235 bp from the translation start site can drive the tissue-specific expression in the apical meristem. Additionally, NtGDSL127 responded to phytohormones, providing strategies for improving tobacco breeding and growth. CONCLUSION: We propose that in tobacco, the NtGDSL127 promoter directs expression specifically in the apical meristem and that expression is closely correlated with axillary bud development.


Assuntos
Esterases/genética , Lipase/genética , Meristema/crescimento & desenvolvimento , Meristema/genética , Tabaco/enzimologia , Tabaco/crescimento & desenvolvimento , Tabaco/genética , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Esterases/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Estudo de Associação Genômica Ampla , Lipase/metabolismo , Filogenia , Transcriptoma
5.
Sci Rep ; 11(1): 16409, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385484

RESUMO

We recently showed that NOTUM, a liver-secreted Wnt inhibitor, can acutely promote browning of white adipose. We now report studies of chronic overexpression of NOTUM in liver indicating that it protects against diet-induced obesity and improves glucose homeostasis in mice. Adeno-associated virus (AAV) vectors were used to overexpress GFP or mouse Notum in the livers of male C57BL/6J mice and the mice were fed an obesifying diet. After 14 weeks of high fat, high sucrose diet feeding, the AAV-Notum mice exhibited decreased obesity and improved glucose tolerance compared to the AAV-GFP mice. Gene expression and immunoblotting analysis of the inguinal fat and brown fat revealed increased expression of beige/brown adipocyte markers in the AAV-Notum group, suggesting enhanced thermogenic capacity by NOTUM. A ß3 adrenergic receptor agonist-stimulated lipolysis test suggested increased lipolysis capacity by NOTUM. The levels of collagen and C-C motif chemokine ligand 2 (CCL2) in the epididymal white adipose tissue of the AAV-Notum mice were significantly reduced, suggesting decreased fibrosis and inflammation, respectively. RNA sequencing analysis of inguinal white adipose of 4-week chow diet-fed mice revealed a highly significant enrichment of extracellular matrix (ECM) functional cluster among the down-regulated genes in the AAV-Notum group, suggesting a potential mechanism contributing to improved glucose homeostasis. Our in vitro studies demonstrated that recombinant human NOTUM protein blocked the inhibitory effects of WNT3A on brown adipocyte differentiation. Furthermore, NOTUM attenuated WNT3A's effects on upregulation of TGF-ß signaling and its downstream targets. Overall, our data suggest that NOTUM modulates adipose tissue function by promoting thermogenic capacity and inhibiting fibrosis through inhibition of Wnt signaling.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Esterases/metabolismo , Obesidade/metabolismo , Termogênese/fisiologia , Adipócitos Bege/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Metabolismo Energético/fisiologia , Intolerância à Glucose/metabolismo , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
6.
Clin Biochem ; 96: 56-62, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34252447

RESUMO

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Ésteres/sangue , Guanidinas/sangue , Espectrometria de Massas em Tandem/métodos , COVID-19/sangue , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Esterases/antagonistas & inibidores , Esterases/metabolismo , Ésteres/metabolismo , Ésteres/farmacologia , Guanidinas/farmacologia , Humanos , Hidrólise/efeitos dos fármacos , Isoflurofato/química , Isoflurofato/farmacologia , Paraoxon/sangue , Paraoxon/química , Paraoxon/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/isolamento & purificação
7.
J Med Chem ; 64(15): 11354-11363, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34292747

RESUMO

The carboxylesterase Notum hydrolyzes a palmitoleate moiety from Wingless/Integrated(Wnt) ligands and deactivates Wnt signaling. Notum inhibitors can restore Wnt signaling which may be of therapeutic benefit for pathologies such as osteoporosis and Alzheimer's disease. We report the identification of a novel class of covalent Notum inhibitors, 4-(indolin-1-yl)-4-oxobutanoate esters. High-resolution crystal structures of the Notum inhibitor complexes reveal a common covalent adduct formed between the nucleophile serine-232 and hydrolyzed butyric esters. The covalent interaction in solution was confirmed by mass spectrometry analysis. Inhibitory potencies vary depending on the warheads used. Mechanistically, the resulting acyl-enzyme intermediate carbonyl atom is positioned at an unfavorable angle for the approach of the active site water, which, combined with strong hydrophobic interactions with the enzyme pocket residues, hinders the intermediate from being further processed and results in covalent inhibition. These insights into Notum catalytic inhibition may guide development of more potent Notum inhibitors.


Assuntos
Butiratos/farmacologia , Inibidores Enzimáticos/farmacologia , Esterases/antagonistas & inibidores , Ésteres/farmacologia , Indóis/farmacologia , Butiratos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Esterases/metabolismo , Ésteres/química , Humanos , Indóis/química , Estrutura Molecular , Relação Estrutura-Atividade
8.
Elife ; 102021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34279224

RESUMO

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue-specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase-specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of antistaphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Pró-Fármacos/farmacologia , Staphylococcus/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxilesterase/metabolismo , Esterases/química , Esterases/metabolismo , Ésteres/metabolismo , Humanos , Hidrólise , Camundongos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
9.
Nature ; 594(7863): 430-435, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34079124

RESUMO

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.


Assuntos
Competição entre as Células , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Esterases/metabolismo , Genes APC , Mutação , Adenoma/genética , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Competição entre as Células/genética , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Meios de Cultivo Condicionados , Progressão da Doença , Esterases/antagonistas & inibidores , Esterases/genética , Feminino , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organoides/citologia , Organoides/metabolismo , Organoides/patologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
10.
Biochemistry ; 60(27): 2206-2220, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34180241

RESUMO

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.


Assuntos
Proteínas de Bactérias/química , Caldicellulosiruptor/enzimologia , Esterases/química , Xilosidases/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Caldicellulosiruptor/química , Caldicellulosiruptor/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Esterases/metabolismo , Modelos Moleculares , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Conformação Proteica , Temperatura , Xilosidases/metabolismo
11.
J Chem Inf Model ; 61(5): 2383-2395, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33949194

RESUMO

Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible. However, examples for the opposite received much less attention. Here, we exploit comprehensive experimental information on the substrate promiscuity of 147 esterases tested against 96 esters together with computationally efficient rigidity analyses to understand the molecular origin of the observed promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly less flexible than specific ones, are significantly more thermostable, and have a significantly increased specific activity. These results may be reconciled with a model according to which structural flexibility in the case of specific esterases serves for conformational proofreading. Our results signify that an esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.


Assuntos
Esterases , Ésteres , Esterases/metabolismo , Especificidade por Substrato
12.
World J Microbiol Biotechnol ; 37(6): 106, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037848

RESUMO

A novel esterase (EstKa) from marine Klebsiella aerogenes was characterized with hydrolytic activity against p-nitrophenyl caprylate (pNPC, C8) under optimum conditions (50 °C and pH 8.5). After two rounds of mutagenesis, two highly potential mutants (I6E9 and L7B11) were obtained with prominent activity, substrate affinity and thermostability. I6E9 (L90Q/P96T) and L7B11 (A37S/Q100L/S133G/R138C/Q156R) were 1.56- and 1.65-fold higher than EstKa in relative catalytic efficiency. The influence of each amino acid on enzyme activity was explored by site-directed mutation. The mutants Pro96Thr and Gln156Arg showed 1.29- and 1.48-fold increase in catalytic efficiency (Kcat/Km) and 54.4 and 36.2% decrease in substrate affinity (Km), respectively. The compound mutant Pro96Thr/Gln156Arg exhibited 68.9% decrease in Km and 1.41-fold increase in Kcat/Km relative to EstKa. Homology model structure analysis revealed that the replacement of Gln by hydrophilic Arg on the esterase surface improved the microenvironment stability and the activity. The replacement of Pro by Thr enabled the esterase enzyme to retain 90% relative activity after 3 h incubation at 45 °C. Structural analysis confirmed that the formation of a hydrogen bond leads to a notable increase of catalytic efficiency under high temperature conditions.


Assuntos
Enterobacter aerogenes/enzimologia , Esterases/genética , Esterases/metabolismo , Mutagênese Sítio-Dirigida/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caprilatos/metabolismo , Catálise , Enterobacter aerogenes/genética , Estabilidade Enzimática , Esterases/química , Hidrólise , Homologia Estrutural de Proteína , Especificidade por Substrato
13.
Chem Biol Interact ; 345: 109524, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34022193

RESUMO

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy. The R (+)-HDCP inhibits and caused the so call "aging reaction" on inhibited-NTE. This enantiomer is not hydrolyzed by Ca(II)-dependent A-esterases in mammal tissues but is hydrolyzed by Cu(II)-dependent chicken serum albumin (CSA). With the aim of identifying HDCP hydrolysis by other vertebrate albumins, we incubated albumin with 400 µM racemic HDCP in the presence of 100 µM copper sulfate. HDCPase activity was assessed by measurement of HDCP with chiral chromatography. Human, sheep, dog, pig, lamprey or cobra serum albumin did not show a significant activity (~10%). Rabbit and bovine albumins hydrolyzed both enantiomers of HDCP (25% and 50% respectively). Turkey serum albumin had more HDCPase activity (~80 µM remaining) than the chicken albumin (~150 µM remaining). No animal albumins other than chicken showed stereoselective hydrolysis. Preincubation of chicken albumin with 1 mM the histidine modifying agents, 100 µM N-bromosuccinimide (NBS) and Zn(II), inhibited its Cu(II)-dependent R (+)-HDCPase activity, where as other mM amino acids modifiers had no inhibitory effects. . These results confirm that the stereoselective hydrolysis of (+)-HDCP is a specific A-esterase catalytic property of chicken albumin. The higher HDCPase activity by turkey albumin suggests the amino-terminal sequence of avian albumins (DAEHK) is the active center of this Cu(II)-dependent A-esterase activity.


Assuntos
Biocatálise , Cobre/metabolismo , Esterases/metabolismo , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Animais , Cães , Humanos , Hidrólise , Estereoisomerismo
14.
Sci Rep ; 11(1): 10440, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001974

RESUMO

Metagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-ß-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s-1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-ß-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.


Assuntos
Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Esterases/metabolismo , Microbioma Gastrointestinal , Rúmen/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Bovinos , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/isolamento & purificação , Ensaios Enzimáticos , Esterases/genética , Esterases/isolamento & purificação , Glucuronatos/biossíntese , Microbiologia Industrial/métodos , Lignina/metabolismo , Metagenoma , Oligossacarídeos/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Energia Renovável
15.
Life Sci ; 277: 119486, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33864822

RESUMO

AIMS: Human carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) are serine-esterase enzymes catalyzing the hydrolysis of many compounds containing esters, amides, thioesters, or acetyl groups. This study aimed to investigate the presence, kinetic parameters, and inhibition of CES1, CES2, and AADAC in A549, H460, and H727 pulmonary cells in both living cells and S9 fractions. MATERIALS AND METHODS: The p-nitrophenyl acetate (pNPA) and 4-methylumbelliferyl acetate (4-MUA) were used as non-selective esterase substrates, whereas phenacetin as selective AADAC substrate. CESs activities were also investigated in living cells by cellular bioimaging using selective fluorescent probes. KEY FINDINGS: AADAC gene was detected in A549 and H460 cells; nevertheless, arylesterase activity was not found in relative S9 fractions. Besides, CES1 and CES2 were expressed to a different extent by all lung cells, and enzymatic activities were quite overlapping each other. All enzymes exhibited a typical Michaelis-Menten saturation curve and, regarding 4-MUA, similar Km values were found in both living cells and S9 fractions. Conversely, kinetic parameters relative to the pNPA hydrolysis by S9 fractions were significantly lower than those detected in living cells. Inhibition studies revealed that 4-MUA hydrolysis was inhibited by bis-p-nitrophenyl phosphate and phenylmethanesulfonyl fluoride more than loperamide; on the contrary, pNPA hydrolysis inhibition was limited with similar inhibition profiles being obtained in both living cells and S9 fractions. The presence of carboxylesterases was definitely confirmed by cellular bioimaging. SIGNIFICANCE: These findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies.


Assuntos
Carboxilesterase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Células A549 , Carboxilesterase/análise , Hidrolases de Éster Carboxílico/análise , Linhagem Celular , Esterases/metabolismo , Esterases/farmacologia , Humanos , Hidrólise , Pulmão/metabolismo , Microssomos Hepáticos/metabolismo , Nitrofenóis , Fenacetina , Especificidade por Substrato , Umbeliferonas
16.
Molecules ; 26(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799781

RESUMO

Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.


Assuntos
Ensaios Enzimáticos/métodos , Esterases/análise , Lipase/análise , Cromatografia Líquida/métodos , Compostos Cromogênicos/química , Eletroforese em Gel de Poliacrilamida/métodos , Esterases/química , Esterases/metabolismo , Lipase/química , Lipase/metabolismo , Lipólise , Espectrometria de Massas/métodos , Especificidade por Substrato
17.
ACS Chem Biol ; 16(5): 800-805, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33877811

RESUMO

In light of the continued threat of antimicrobial-resistant bacteria, new strategies to expand the repertoire of antimicrobial compounds are necessary. Prodrugs are an underexploited strategy in this effort. Here, we report on the enhanced antimicrobial activity of a prodrug toward bacteria having an enzyme capable of its activation. A screen led us to the sulfurol ester of the antibiotic trans-3-(4-chlorobenzoyl)acrylic acid. An endogenous esterase makes Mycolycibacterium smegmatis sensitive to this prodrug. Candidate esterases were identified, and their heterologous production made Escherichia coli sensitive to the ester prodrug. Taken together, these data suggest a new approach to the development of antimicrobial compounds that takes advantage of endogenous enzymatic activities to target specific bacteria.


Assuntos
Acrilatos/química , Anti-Infecciosos/química , Ésteres/química , Pró-Fármacos/química , Acrilatos/farmacologia , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Esterases/metabolismo , Ésteres/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade
18.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802163

RESUMO

Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membrane of P. aeruginosa by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-cultures under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles' heel of P. aeruginosa in communities with bacteria producing ENT.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Esterases/metabolismo , Ferro/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Transporte/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Esterases/genética , Pseudomonas aeruginosa/genética
19.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919724

RESUMO

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.


Assuntos
Ésteres/metabolismo , Genes Fúngicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sensação , Transcriptoma/genética , Acetiltransferases/metabolismo , Adulto , Epistasia Genética , Esterases/metabolismo , Ésteres/análise , Feminino , Fermentação , Haplótipos/genética , Histonas/metabolismo , Humanos , Lipase/metabolismo , Masculino , Mutação/genética , Mapeamento de Interação de Proteínas , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/metabolismo , Volatilização , Vinho/microbiologia
20.
Future Med Chem ; 13(11): 1001-1015, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33882714

RESUMO

Notum has recently been identified as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group from Wnt proteins. There are emerging reports that Notum plays a role in human disease, with published data suggesting that targeting Notum could represent a new therapeutic approach for treating cancer, osteoporosis and neurodegenerative disorders. Complementary hit-finding strategies have been applied with successful approaches that include high-throughput screening, activity-based protein profiling, screening of fragment libraries and virtual screening campaigns. Structural studies are accelerating the discovery of new inhibitors of Notum. Three fit-for-purpose examples are LP-922056, ABC99 and ARUK3001185. The application of these small-molecule inhibitors is helping to further advance an understanding of the role Notum plays in human disease.


Assuntos
Inibidores Enzimáticos/farmacologia , Esterases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Inibidores Enzimáticos/química , Esterases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química
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