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1.
J Biosci Bioeng ; 132(4): 337-342, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34376338

RESUMO

In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillus oryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with α-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50 °C, respectively, and was stable up to 50 °C. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of α-naphthyl esters (C2-C16), was α-naphthyl propionate (C3), with an activity of 0.35 ± 0.006 units/mg protein. No significant difference of the Km value was observed between C3 (2.3 ± 0.7 mM) and C2 (1.9 ± 0.4 mM). In contrast, kcat value for C3 (18 ± 3.9 s-1) was higher compared to C2 (4.5 ± 0.7 s-1). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.


Assuntos
Aspergillus oryzae , Acetilesterase , Aspergillus oryzae/genética , Clonagem Molecular , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/genética , Saccharomycetales , Especificidade por Substrato
2.
Klin Lab Diagn ; 66(7): 389-395, 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34292679

RESUMO

Sialic acids (SA) determine the degree of molecular hydrophilia, relieve binding together and their transportation, they increase mucin viscosity, stabilize the protein and membrane structure. Apart from that, SA are structural components of gangliosides participating in the formation of the outer layer of the plasma membrane. The degree of silyliation of glycoproteins and glycolipids is an important factor of molecular recognition in the cell, between the cells, between a cell and territorial matrix, as well as between a cell and some outer pathogenic factors. They can either mask the sites of recognition or be determinants of recognition. The most well-studied enzymes taking part in the SA metabolism and sialo-containing compounds are N-acetylneuraminate, cythydiltransferase, sialyltransferase, sialydase, aldolase SA and sialyl-O-acetylesterase. Numerous investigations have shown that aberrant sialylation is a specific feature of various changes and disorders of metabolism. Besides that, sialic acids are the first point of contact for different pathogenic microorganisms and the host's body due to their presence on the external surface of the cells and tissue of the mucous membrane. That is why the study of the above-mentioned various sialic acids fractions as well as of the activity of the enzymes participating in their metabolism in the blood plasma and tissues, and of the influence on the activity of these enzymes with the help of medicine can make an essential contribution to the diagnosis and treatment of many diseases.


Assuntos
Neuraminidase , Ácidos Siálicos , Acetilesterase , Glicolipídeos
3.
Microb Cell Fact ; 20(1): 129, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238305

RESUMO

BACKGROUND: Xylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the ß-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis. RESULTS: xynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50-55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold. CONCLUSION: The supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.


Assuntos
Acetilesterase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Lignina/metabolismo , Streptomyces/enzimologia , Xilanos/metabolismo , Biomassa , Celulase/metabolismo , Clonagem Molecular , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Hidrólise , Metais/farmacologia , Proteínas Recombinantes/metabolismo , Temperatura
4.
J Agric Food Chem ; 69(23): 6665-6675, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34074097

RESUMO

Gut bacteria-derived enzymes play important roles in the metabolism of dietary fiber through enabling the hydrolysis of polysaccharides. In this study, we identified and characterized a 29 kDa novel acetyl xylan esterase, BTAxe1, from Bacteroides thetaiotaomicron VPI5482. Then, we solved the structure of BTAxe1 and performed the rational design. Mutants N65S and N65A increased the activities toward short-chain (pNPA, pNPB) to near four-fold, and gained the activities toward longer-chain substrate (pNPO). Molecular docking analysis showed that the mutant N65S had a larger substrate binding pocket than the wild type. Hydrolysis studies using natural substrates showed that either N65S or N65A showed higher activity of that of wild-type, yielding 131.31 and 136.09 mM of acetic acid from xylan. This is the first study on the rational design of gut bacteria-derived Axes with broadened substrate specificity and enhanced activity, which can be referenced by other acetyl esterases or gut-derived enzymes.


Assuntos
Bacteroides thetaiotaomicron , Acetilesterase/genética , Acetilesterase/metabolismo , Bacteroides thetaiotaomicron/genética , Simulação de Acoplamento Molecular , Especificidade por Substrato
5.
PLoS One ; 16(6): e0251556, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086701

RESUMO

A diverse range of monocot and dicot grains and their by-products are commonly used in the animal feed industry. They all come with complex and variable cell wall structures which in turn contribute significant fiber to the complete feed. The cell wall is a highly interconnected matrix of various polysaccharides, proteins and lignin and, as such, requires a collaborative effort of different enzymes for its degradation. In this regard, we investigated the potential of a commercial multicomponent carbohydrase product from a wild type fermentation of Trichoderma reesei (T. reesei) (RONOZYME® MultiGrain) in degrading cell wall components of wheat, barley, rye, de-oiled rice bran, sunflower, rapeseed and cassava. A total of thirty-one different enzyme proteins were identified in the T. Reesei carbohydrase product using liquid chromatography with tandem mass spectrometry LC-MS/MS including glycosyl hydrolases and carbohydrate esterases. As measured by in vitro incubations and non-starch polysaccharide component analysis, and visualization by immunocytochemistry and confocal microscopy imaging of immuno-labeled samples with confocal microscopy, the carbohydrase product effectively solubilized cellulolytic and hemicellulolytic polysaccharides present in the cell walls of all the feed ingredients evaluated. The T. reesei fermentation also decreased viscosity of arabinoxylan, xyloglucan, galactomannan and ß-glucan substrates. Combination of several debranching enzymes including arabinofuranosidase, xylosidase, α-galactosidase, acetyl xylan esterase, and 4-O-methyl-glucuronoyl methylesterase with both GH10 and GH11 xylanases in the carbohydrase product resulted in effective hydrolyzation of heavily branched glucuronoarabinoxylans. The different ß-glucanases (both endo-ß-1,3(4)-glucanase and endo-ß-1,3-glucanase), cellulases and a ß-glucosidase in the T. reesei fermentation effectively reduced polymerization of both ß-glucans and cellulose polysaccharides of viscous cereals grains (wheat, barley, rye and oat). Interestingly, the secretome of T. reesei contained significant amounts of an exceptional direct chain-cutting enzyme from the GH74 family (Cel74A, xyloglucan-specific ß-1,4-endoglucanase), that strictly cleaves the xyloglucan backbone at the substituted regions. Here, we demonstrated that the balance of enzymes present in the T. reesei secretome is capable of degrading various cell wall components in both monocot and dicot plant raw material used as animal feed.


Assuntos
Ração Animal/análise , Parede Celular/metabolismo , Glicosídeo Hidrolases/metabolismo , Hypocreales/metabolismo , Acetilesterase/metabolismo , Celulases/metabolismo , Cromatografia Líquida/métodos , Endo-1,4-beta-Xilanases/metabolismo , Glucanos/metabolismo , Espectrometria de Massas em Tandem/métodos , Xilanos/metabolismo , Xilosidases/metabolismo
6.
J Biol Chem ; 297(1): 100841, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34058201

RESUMO

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.


Assuntos
Acetilesterase/química , Acetilesterase/metabolismo , Adaptação Fisiológica , Temperatura Baixa , Acetilesterase/antagonistas & inibidores , Acetilesterase/genética , Sequência de Aminoácidos , Bactérias/enzimologia , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Cinética , Metais/farmacologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação/genética , Filogenia , Multimerização Proteica , Especificidade por Substrato/efeitos dos fármacos , Temperatura
7.
Cancer Sci ; 112(5): 1865-1877, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33544437

RESUMO

The histone acetyltransferase MOF (KAT8) is mainly involved in the acetylation of histone H4 at lysine 16 (H4K16) and some non-histone proteins. The MOF expression level is significantly reduced in many cancers, however the biological function of MOF and its underlying mechanism are still elusive in hepatocellular carcinoma (HCC). Estrogen receptor α (ERα) has been considered as a tumor suppressor in HCC. Here, we demonstrated that MOF expression is significantly reduced in HCC samples, and is positively correlated with that of ERα. MOF interacts with ERα, and participates in acetylation of ERα at K266, K268, K299, thereby inhibiting ERα ubiquitination to maintain the stability of ERα. In addition, MOF participates in the upregulation of ERα-mediated transactivation. Depletion of MOF significantly promotes cell growth, migration, and invasion in HCC cell lines. Taken together, our results provide new insights to understand the mechanism underlying the modulation function of MOF on ERα action in HCC, suggesting that MOF might be a potential therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Receptor alfa de Estrogênio/metabolismo , Histona Acetiltransferases/metabolismo , Neoplasias Hepáticas/metabolismo , Acetilação , Acetilesterase/metabolismo , Animais , Anticorpos/uso terapêutico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Movimento Celular , Proliferação de Células , Bases de Dados Genéticas , Regulação para Baixo , Receptor alfa de Estrogênio/genética , Feminino , Xenoenxertos , Histona Acetiltransferases/deficiência , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Lisina/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Transdução de Sinais , Ativação Transcricional , Ubiquitinação , Regulação para Cima
8.
Curr Opin Chem Biol ; 61: 9-18, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33075728

RESUMO

Deacetylation of N-acetylhexosamine residues in structural polysaccharides and glycoconjugates is catalyzed by different families of carbohydrate esterases that, despite different structural folds, share a common metal-assisted acid/base mechanism with the metal cation coordinated with a conserved Asp-His-His triad. These enzymes serve diverse biological functions in the modification of cell-surface polysaccharides in bacteria and fungi as well as in the metabolism of hexosamines in the biosynthesis of cellular glycoconjugates. Focusing on carbohydrate de-N-acetylases, this article summarizes the background of the different families from a structural and functional viewpoint and covers advances in the characterization of novel enzymes over the last 2-3 years. Current research is addressed to the identification of new deacetylases and unravel their biological functions as they are candidate targets for the design of antimicrobials against pathogenic bacteria and fungi. Likewise, some families are also used as biocatalysts for the production of defined glycostructures with diverse applications.


Assuntos
Acetilesterase/metabolismo , Carboidratos/química , Glicoconjugados/metabolismo , Polissacarídeos/metabolismo , Acetilação
9.
Int J Mol Sci ; 21(12)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604730

RESUMO

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most ß-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein-carbohydrate interaction (PCI) or lectin-carbohydrate interaction (LCI). The HE gene was transmitted to a ß-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA-specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA-ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the ß-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein-glycan interaction and PCI stereochemistry potentiate the SA-ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Evolução Molecular , Interações entre Hospedeiro e Microrganismos/fisiologia , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Internalização do Vírus , Acetilesterase/metabolismo , Animais , Betacoronavirus/genética , Sítios de Ligação , COVID-19 , Linhagem Celular , Coronavirus/genética , Esterases , Transferência Genética Horizontal , Glicosaminoglicanos/metabolismo , Hemaglutininas Virais/genética , Humanos , Lectinas/metabolismo , Pandemias , Polissacarídeos , Receptores Virais/química , SARS-CoV-2 , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Torovirus , Proteínas Virais de Fusão/genética
10.
J Microbiol Biotechnol ; 30(2): 155-162, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-31986559

RESUMO

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50°C, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana ß-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.


Assuntos
Acetilesterase/genética , Acetilesterase/metabolismo , Bacillus/enzimologia , Bacillus/genética , Expressão Gênica , Lactobacillus/enzimologia , Lactobacillus/genética , Acetilesterase/química , Acetilesterase/isolamento & purificação , Sequência de Aminoácidos , Clonagem Molecular , Ativação Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Lactobacillus/química , Lactobacillus/isolamento & purificação , Temperatura , Xilanos/metabolismo
11.
Int J Biol Macromol ; 148: 333-341, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31954783

RESUMO

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic ß-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC2 to p-NPC8. Maximum enzyme activity was recorded at pH 9.0 and 50 °C, where Km and Vmax were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 µM min-1, respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.


Assuntos
Acetilesterase/metabolismo , Alicyclobacillus/metabolismo , Cefalosporinas/metabolismo , Sequência de Aminoácidos , Clonagem Molecular/métodos , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Simulação de Acoplamento Molecular/métodos , Alinhamento de Sequência , Especificidade por Substrato
12.
Bioorg Med Chem Lett ; 30(3): 126751, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31862412

RESUMO

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20-24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.


Assuntos
Acetilesterase/antagonistas & inibidores , Amidas/química , Pirimidinas/química , Acetilesterase/metabolismo , Amidas/metabolismo , Amidas/farmacologia , Animais , Sítios de Ligação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Cães , Meia-Vida , Concentração Inibidora 50 , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Via de Sinalização Wnt/efeitos dos fármacos
13.
Yeast ; 37(1): 63-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31845370

RESUMO

ScGpi12 is a 304 amino residue long endoplasmic reticulum membrane protein, which participates in the de-N-acetylation of N-acetylglucosaminyl phosphatidylinositol to produce glucosaminyl phosphatidylinositol in the second step of GPI anchor biosynthesis pathway in Saccharomyces cerevisiae. ScGpi12 was cloned in a pMAL-c2x vector and expressed heterologously in Rosetta-gami (DE3) strain of E. coli. Affinity purification of the protein yielded low amounts of the MBP-tagged enzyme, which was active. To the best of our knowledge, this is the first successful purification of full-length Gpi12 enzyme, without the accompanying GroEL that was seen in other studies. The presence of the tag did not greatly alter the activity of the enzyme. ScGpi12 was optimally active in the pH range of 6.5-8.5 and at 30 °C. It was not sensitive to treatment with EDTA but was stimulated by multiple divalent cations. The divalent cation did not alter the pH profile of the enzyme, suggesting no role of the divalent metal in creating a nucleophile for catalysis. Divalent cations did, however, enhance the turnover number of the enzyme for its substrate, suggesting that they are probably required for the production of a catalytically competent active site by bringing the active site residues within optimum distance of the substrate for catalysis.


Assuntos
Acetilesterase/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Acetilesterase/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Vias Biossintéticas , Catálise , Clonagem Molecular , Retículo Endoplasmático/enzimologia , Escherichia coli/genética , Cinética , Fosfatidilinositóis/metabolismo , Especificidade por Substrato
14.
mBio ; 10(6)2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796537

RESUMO

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.


Assuntos
Acetilesterase/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Orthomyxoviridae/metabolismo , Células A549 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cães , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino
15.
Nat Chem Biol ; 15(12): 1183-1190, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740825

RESUMO

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.


Assuntos
Acetilesterase/química , Fatores de Despolimerização de Actina/química , Optogenética , Tubulina (Proteína)/química , Acetilação
16.
Microb Cell Fact ; 18(1): 173, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601224

RESUMO

BACKGROUND: Bioemulsifiers are surface-active compounds, which exhibit advantages including low toxicity, higher biodegradability and biocompatibility over synthetic chemical surfactants. Despite their potential benefits, some obstacles impede the practical applications of bioemulsifiers, including low yields and high purification costs. Here, we aimed to exploit a novel protein bioemulsifier with efficient emulsifying activity and low-production cost, as well as proposed a design-bioemulsifier system that meets different requirements of industrial emulsification in the most economical way. RESULTS: The esterase AXE was first reported for its efficient emulsifying activity and had been studied for possible application as a protein bioemulsifier. AXE showed an excellent emulsification effect with different hydrophobic substrates, especially short-chain aliphatic and benzene derivatives, as well as excellent stability under extreme conditions such as high temperature (85 °C) and acidic conditions. AXE also exhibited good stability over a range of NaCl, MgSO4, and CaCl2 concentrations from 0 to 1000 mM, and the emulsifying activity even showed a slight increase at salt concentrations over 500 mM. A design-bioemulsifier system was proposed that uses AXE in combination with a variety of polysaccharides to form efficient bioemulsifier, which enhanced the emulsifying activity and further lowered the concentration of AXE needed in the complex. CONCLUSIONS: AXE showed a great application potential as a novel bioemulsifier with excellent emulsifying ability. The AXE-based-designer bioemulsifier could be obtained in the most economical way and open broad new fields for low-cost, environmentally friendly bioemulsifiers.


Assuntos
Acetilesterase/química , Bacillus subtilis/metabolismo , Emulsificantes/química , Polissacarídeos/química , Acetilesterase/biossíntese , Biodegradação Ambiental
17.
Plant Physiol ; 181(4): 1704-1720, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31551361

RESUMO

The plant cell wall plays an important role in damage-associated molecular pattern-induced resistance to pathogens and herbivorous insects. Our current understanding of cell wall-mediated resistance is largely based on the degree of pectin methylesterification. However, little is known about the role of pectin acetylesterification in plant immunity. This study describes how one pectin-modifying enzyme, PECTIN ACETYLESTERASE 9 (PAE9), affects the Arabidopsis (Arabidopsis thaliana) transcriptome, secondary metabolome, and aphid performance. Electro-penetration graphs showed that Myzus persicae aphids established phloem feeding earlier on pae9 mutants. Whole-genome transcriptome analysis revealed a set of 56 differentially expressed genes (DEGs) between uninfested pae9-2 mutants and wild-type plants. The majority of the DEGs were enriched for biotic stress responses and down-regulated in the pae9-2 mutant, including PAD3 and IGMT2, involved in camalexin and indole glucosinolate biosynthesis, respectively. Relative quantification of more than 100 secondary metabolites revealed decreased levels of several compounds, including camalexin and oxylipins, in two independent pae9 mutants. In addition, absolute quantification of phytohormones showed that jasmonic acid (JA), jasmonoyl-Ile, salicylic acid, abscisic acid, and indole-3-acetic acid were compromised due to PAE9 loss of function. After aphid infestation, however, pae9 mutants increased their levels of camalexin, glucosinolates, and JA, and no long-term effects were observed on aphid fitness. Overall, these data show that PAE9 is required for constitutive up-regulation of defense-related compounds, but that it is not required for aphid-induced defenses. The signatures of phenolic antioxidants, phytoprostanes, and oxidative stress-related transcripts indicate that the processes underlying PAE9 activity involve oxidation-reduction reactions.


Assuntos
Acetilesterase/metabolismo , Afídeos/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Herbivoria/fisiologia , Metaboloma/genética , Transcriptoma/genética , Animais , Arabidopsis/parasitologia , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes Reguladores , Glucosinolatos/metabolismo , Indóis/metabolismo , Mutação/genética , Estresse Oxidativo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Metabolismo Secundário , Tiazóis/metabolismo , Fatores de Transcrição/metabolismo
18.
Microb Cell Fact ; 18(1): 122, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286972

RESUMO

BACKGROUND: Acetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica. RESULTS: The AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold. CONCLUSION: Resulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates.


Assuntos
Acetilesterase/metabolismo , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Fagus/química , Flavobacteriaceae/enzimologia , Xilanos/metabolismo , Acetilesterase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Estabilidade Enzimática , Flavobacteriaceae/genética , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial , Fases de Leitura Aberta , Água do Mar/microbiologia , Especificidade por Substrato , Temperatura
19.
Biotechnol Lett ; 41(8-9): 1059-1065, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31302814

RESUMO

OBJECTIVES: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation. RESULTS: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the α/ß hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 °C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the Kcat/Km value was 2.04 s-1 mM-1. CONCLUSIONS: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.


Assuntos
Acetilesterase/metabolismo , Cefalosporinas/metabolismo , Paenibacillus/enzimologia , Acetilesterase/genética , Acetilesterase/isolamento & purificação , Biotransformação , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Paenibacillus/genética , Temperatura
20.
Int J Biol Macromol ; 136: 1042-1051, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31229546

RESUMO

Cold-active enzymes with distinctive properties from a psychrophilic Exiguobacterium antarcticum B7 could be excellent biocatalysts in industrial and biotechnological processes. Here, the characterization, immobilization, and site-directed mutagenesis of a novel cold-active acetylesterase (EaAcE) from E. antarcticum B7 is reported. EaAcE does not belong to any currently known lipase/esterase family, although there are some sequence similarities with family III and V members. Biochemical characterization of EaAcE was carried out using activity staining, mass spectrometry analysis, circular dichroism spectra, freeze-thaw experiments, kinetic analysis, acetic acid release assays, and enantioselectivity determination. Furthermore, immobilization of EaAcE using four different approaches was explored to enhance its thermal stability and recyclability. Based on a homology model of EaAcE, four mutations (F45A, S118A, S141A, and T216A) within the substrate-binding pocket were investigated to elucidate their roles in EaAcE catalysis and substrate specificity. This work has provided invaluable information on the properties of EaAcE, which can now be used to understand the acetylesterase enzyme family.


Assuntos
Acetilesterase/química , Acetilesterase/metabolismo , Bacillaceae/enzimologia , Temperatura Baixa , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Mutagênese , Acetilesterase/genética , Sequência de Aminoácidos , Biologia Computacional , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
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