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1.
Chemosphere ; 310: 136836, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36243089

RESUMO

Peroxidase (POX) is a heme-containing oxidoreductase, its voluminous immuno-diagnostic and bioremediatory intuitions have incited optimization and large scale-generation from novel microbial repertoires. Azo dyes are the most detrimental classes of synthetic dyes and they are the common ecotoxic industrial pollutants in wastewater. In addition, azo dyes are refractory to degradation owing to their chemical nature, comprising of azoic linkages, amino moieties with recalcitrant traits. Moreover, they are major carcinogenic and mutagenic on humans and animals, whereby emphasizing the need for decolorization. In the present study, a novel POX from Streptomyces coelicolor strain SPR7 was investigated for the deterioration of ecotoxic dyestuffs. The initial medium component screening for POX production was achieved using, One Factor at a Time and Placket-Burman methodologies with starch, casein and temperature as essential parameters. In auxiliary, Response Surface Methodology (RSM) was recruited and followed by model validation using Back propagation algorithm (BPA). RSM-BPA composite approach prophesied that combination of starch, casein, and temperature at optimal values 2.5%, 0.035% and 35 °C respectively, has resulted in 7 folds enhancement of POX outturn (2.52 U/mL) compared to the unoptimized media (0.36 U/mL). The concentrated enzyme decolorized 75.4% and 90% of the two azo dyes with lignin (10 mM), respectively. Hence, this investigation confirms the potentiality of mangrove actinomycete derived POX for elimination of noxious azo dyes to overcome their carcinogenic, mutagenic and teratogenic effects on humans and aquatic organisms.


Assuntos
Compostos Azo , Peroxidases , Streptomyces coelicolor , Compostos Azo/química , Biodegradação Ambiental , Bioprospecção , Carcinógenos , Caseínas , Corantes/química , Amido , Streptomyces coelicolor/enzimologia
2.
Biochim Biophys Acta Gen Subj ; 1867(1): 130249, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36183893

RESUMO

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.


Assuntos
Formigas , Quitinases , Animais , Antifúngicos/química , Formigas/enzimologia , Formigas/genética , Formigas/metabolismo , Quitina/química , Quitinases/química , Quitinases/genética , Quitinases/farmacologia , Clonagem Molecular , Fungos/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Catálise , Domínio Catalítico
3.
World J Microbiol Biotechnol ; 38(12): 252, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36316502

RESUMO

Chitinases are a group of enzymes that catalyze chitin hydrolysis and are present in all domains of life. Chitinases belong to different glycosyl hydrolase families with great diversity in their sequences. Microorganisms such as bacteria and fungi produce chitinases for nutrition, and energy, and to parasitize the chitinous hosts. But chitinases from bacteria are of special interest due to their ubiquitous nature and ability to perform under extreme conditions. Chitinases produced by bacteria have been explored for their use in agriculture and industry. In agriculture, their main role is to control chitin-containing insect pests, fungal pathogens, and nematodes. In the seafood industry, they found their role in the management of processing wastes which are mainly chitinous substances. Chitinases are also used to synthesize low molecular weight chitooligomers which are proven bioactive compounds with activities such as anti-tumour, antimicrobial, and immunity modulation. Considering their importance in ecology and biotechnological applications, several bacterial chitinases have been studied in the last two decades. Despite their potential, bacterial chitinases have a few limitations such as low production and lack of secretion systems which make the wild-type enzymes unfit for their applications in industries and other allied sectors. This review is an attempt to collate significant works in bacterial chitinases and their application in various industries and the employment of various tools and techniques for improvement to meet industrial requirements.


Assuntos
Bactérias , Quitinases , Bactérias/enzimologia , Biotecnologia/métodos , Quitina , Quitinases/biossíntese , Hidrólise
4.
BMC Bioinformatics ; 23(1): 466, 2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36344934

RESUMO

BACKGROUND: In most parts of the world, especially in underdeveloped countries, acquired immunodeficiency syndrome (AIDS) still remains a major cause of death, disability, and unfavorable economic outcomes. This has necessitated intensive research to develop effective therapeutic agents for the treatment of human immunodeficiency virus (HIV) infection, which is responsible for AIDS. Peptide cleavage by HIV-1 protease is an essential step in the replication of HIV-1. Thus, correct and timely prediction of the cleavage site of HIV-1 protease can significantly speed up and optimize the drug discovery process of novel HIV-1 protease inhibitors. In this work, we built and compared the performance of selected machine learning models for the prediction of HIV-1 protease cleavage site utilizing a hybrid of octapeptide sequence information comprising bond composition, amino acid binary profile (AABP), and physicochemical properties as numerical descriptors serving as input variables for some selected machine learning algorithms. Our work differs from antecedent studies exploring the same subject in the combination of octapeptide descriptors and method used. Instead of using various subsets of the dataset for training and testing the models, we combined the dataset, applied a 3-way data split, and then used a "stratified" 10-fold cross-validation technique alongside the testing set to evaluate the models. RESULTS: Among the 8 models evaluated in the "stratified" 10-fold CV experiment, logistic regression, multi-layer perceptron classifier, linear discriminant analysis, gradient boosting classifier, Naive Bayes classifier, and decision tree classifier with AUC, F-score, and B. Acc. scores in the ranges of 0.91-0.96, 0.81-0.88, and 80.1-86.4%, respectively, have the closest predictive performance to the state-of-the-art model (AUC 0.96, F-score 0.80 and B. Acc. ~ 80.0%). Whereas, the perceptron classifier and the K-nearest neighbors had statistically lower performance (AUC 0.77-0.82, F-score 0.53-0.69, and B. Acc. 60.0-68.5%) at p < 0.05. On the other hand, logistic regression, and multi-layer perceptron classifier (AUC of 0.97, F-score > 0.89, and B. Acc. > 90.0%) had the best performance on further evaluation on the testing set, though linear discriminant analysis, gradient boosting classifier, and Naive Bayes classifier equally performed well (AUC > 0.94, F-score > 0.87, and B. Acc. > 86.0%). CONCLUSIONS: Logistic regression and multi-layer perceptron classifiers have comparable predictive performances to the state-of-the-art model when octapeptide sequence descriptors consisting of AABP, bond composition and standard physicochemical properties are used as input variables. In our future work, we hope to develop a standalone software for HIV-1 protease cleavage site prediction utilizing the linear regression algorithm and the aforementioned octapeptide sequence descriptors.


Assuntos
Protease de HIV , HIV-1 , Humanos , Síndrome de Imunodeficiência Adquirida , Algoritmos , Teorema de Bayes , Infecções por HIV , Protease de HIV/química , HIV-1/enzimologia , Inibidores da Protease de HIV/química
5.
Science ; 378(6620): 627-634, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36356138

RESUMO

Group II introns are ribozymes that catalyze their self-excision and function as retroelements that invade DNA. As retrotransposons, group II introns form ribonucleoprotein (RNP) complexes that roam the genome, integrating by reversal of forward splicing. Here we show that retrotransposition is achieved by a tertiary complex between a structurally elaborate ribozyme, its protein mobility factor, and a structured DNA substrate. We solved cryo-electron microscopy structures of an intact group IIC intron-maturase retroelement that was poised for integration into a DNA stem-loop motif. By visualizing the RNP before and after DNA targeting, we show that it is primed for attack and fits perfectly with its DNA target. This study reveals design principles of a prototypical retroelement and reinforces the hypothesis that group II introns are ancient elements of genetic diversification.


Assuntos
Íntrons , Splicing de RNA , RNA Catalítico , Retroelementos , Ribonucleoproteínas , Microscopia Crioeletrônica , Ribonucleoproteínas/química , RNA Catalítico/química , DNA Polimerase Dirigida por RNA/genética , Eubacterium/enzimologia , Eubacterium/genética
6.
Sci Rep ; 12(1): 18084, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302925

RESUMO

Hexokinase 2 (Hxk2) of Saccharomyces cerevisiae is a dual function hexokinase, acting as a glycolytic enzyme and being involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by phosphorylation of Hxk2 at serine 15, which has been attributed to the protein kinase Tda1. To explore the role of Tda1 beyond Hxk2 phosphorylation, the proteomic consequences of TDA1 deficiency were investigated by difference gel electrophoresis (2D-DIGE) comparing a wild type and a Δtda1 deletion mutant. To additionally address possible consequences of glucose repression/derepression, both were grown at 2% and 0.1% (w/v) glucose. A total of eight protein spots exhibiting a minimum twofold enhanced or reduced fluorescence upon TDA1 deficiency was detected and identified by mass spectrometry. Among the spot identities are-besides the expected Hxk2-two proteoforms of hexokinase 1 (Hxk1). Targeted proteomics analyses in conjunction with 2D-DIGE demonstrated that TDA1 is indispensable for Hxk2 and Hxk1 phosphorylation at serine 15. Thirty-six glucose-concentration-dependent protein spots were identified. A simple method to improve spot quantification, approximating spots as rotationally symmetric solids, is presented along with new data on the quantities of Hxk1 and Hxk2 and their serine 15 phosphorylated forms at high and low glucose growth conditions. The Δtda1 deletion mutant exhibited no altered growth under high or low glucose conditions or on alternative carbon sources. Also, invertase activity, serving as a reporter for glucose derepression, was not significantly altered. Instead, an involvement of Tda1 in oxidative stress response is suggested.


Assuntos
Hexoquinase , Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteômica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
7.
BMC Bioinformatics ; 23(1): 447, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36303135

RESUMO

BACKGROUND: The site information of substrates that can be cleaved by human immunodeficiency virus 1 proteases (HIV-1 PRs) is of great significance for designing effective inhibitors against HIV-1 viruses. A variety of machine learning-based algorithms have been developed to predict HIV-1 PR cleavage sites by extracting relevant features from substrate sequences. However, only relying on the sequence information is not sufficient to ensure a promising performance due to the uncertainty in the way of separating the datasets used for training and testing. Moreover, the existence of noisy data, i.e., false positive and false negative cleavage sites, could negatively influence the accuracy performance. RESULTS: In this work, an ensemble learning algorithm for predicting HIV-1 PR cleavage sites, namely EM-HIV, is proposed by training a set of weak learners, i.e., biased support vector machine classifiers, with the asymmetric bagging strategy. By doing so, the impact of data imbalance and noisy data can thus be alleviated. Besides, in order to make full use of substrate sequences, the features used by EM-HIV are collected from three different coding schemes, including amino acid identities, chemical properties and variable-length coevolutionary patterns, for the purpose of constructing more relevant feature vectors of octamers. Experiment results on three independent benchmark datasets demonstrate that EM-HIV outperforms state-of-the-art prediction algorithm in terms of several evaluation metrics. Hence, EM-HIV can be regarded as a useful tool to accurately predict HIV-1 PR cleavage sites.


Assuntos
Protease de HIV , HIV-1 , Algoritmos , Protease de HIV/química , HIV-1/enzimologia , Aprendizado de Máquina , Especificidade por Substrato
8.
Int J Mol Sci ; 23(19)2022 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-36232994

RESUMO

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces&nbsp;macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.


Assuntos
Paracoccus , Saccharomycetales , beta-Galactosidase , Fermentação , Galactosidases , Paracoccus/enzimologia , Saccharomycetales/metabolismo , beta-Galactosidase/biossíntese
9.
Appl Microbiol Biotechnol ; 106(21): 7063-7072, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36195703

RESUMO

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.


Assuntos
Proteínas Fúngicas , Pró-Proteína Convertases , Saccharomyces cerevisiae , Candida albicans/enzimologia , Candida albicans/genética , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Pró-Proteína Convertases/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Proteínas Fúngicas/biossíntese
10.
Nature ; 611(7934): 133-138, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289340

RESUMO

The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.


Assuntos
Adenilil Ciclases , Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Ácidos Indolacéticos , Receptores de Superfície Celular , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Mutação , Gravitropismo , Raízes de Plantas/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Sistemas do Segundo Mensageiro
11.
Nature ; 611(7934): 173-179, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289326

RESUMO

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized ß2-adrenergic receptor (ß2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that ß2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated ß2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal ß2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.


Assuntos
Adrenérgicos , Endossomos , MAP Quinases Reguladas por Sinal Extracelular , Receptores Adrenérgicos beta 2 , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Proliferação de Células , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Endossomos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes myc , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
Biochemistry ; 61(22): 2607-2620, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36314559

RESUMO

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+ was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+.


Assuntos
Acinetobacter baumannii , Oxigenases de Função Mista , Acinetobacter baumannii/enzimologia , Antibacterianos , Cadaverina , Flavinas/metabolismo , Cinética , Oxigenases de Função Mista/química , NADP/metabolismo , Ornitina/química , Putrescina , Sideróforos
13.
Antimicrob Agents Chemother ; 66(11): e0042022, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36314787

RESUMO

Malaria is a mosquito-borne fatal infectious disease that affects humans and is caused by Plasmodium parasites, primarily Plasmodium falciparum. Widespread drug resistance compels us to discover novel compounds and alternative drug discovery targets. The coenzyme A (CoA) biosynthesis pathway is essential for the malaria parasite P. falciparum. The last enzyme in CoA biosynthesis, dephospho-CoA kinase (DPCK), is essential to the major life cycle development stages but has not yet been exploited as a drug target in antimalarial drug discovery. We performed a high-throughput screen of a 210,000-compound library using recombinant P. falciparum DPCK (PfDPCK). A high-throughput enzymatic assay using a 1,536-well platform was developed to identify potential PfDPCK inhibitors. PfDPCK inhibitors also inhibited parasite growth in a P. falciparum whole-cell asexual blood-stage assay in both drug-sensitive and drug-resistant strains. Hit compounds were selected based on their potency in cell-free (PfDPCK) and whole-cell (Pf3D7 and PfDd2) assays, selectivity over the human orthologue (HsCOASY) and no cytotoxicity (HepG2). The compounds were ranked using a multiparameter optimization (MPO) scoring model, and the specific binding and the mechanism of inhibition were investigated for the most promising compounds.


Assuntos
Antimaláricos , Coenzima A , Plasmodium falciparum , Animais , Humanos , Antimaláricos/uso terapêutico , Coenzima A/antagonistas & inibidores , Coenzima A/metabolismo , Ensaios de Triagem em Larga Escala , Estágios do Ciclo de Vida , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Células Hep G2
14.
Bioorg Chem ; 129: 106148, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36244324

RESUMO

Steroid sulfatase inhibitors block the local production of estrogenic steroids and are attractive agents for the treatment of estrogen-dependent cancers. Inspiration of coumarin-based inhibitors, we synthesized thirty-two 5-oxa-1,2,3,4-tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates, focusing on the substitution derivatives on the adjacent phenyl ring and evaluated their abilities to block STS from human placenta and MCF-7 cells. SAR analysis revealed that the incorporation of chlorine at either meta and/or para position of the adjacent phenyl ring of the tricyclic skeleton enhanced STS inhibition. Di-substitutions at the adjacent phenyl ring were superior to mono and tri-substitutions. Further kinetic analysis of these compounds revealed that chloride-bearing compounds, such as 19m, 19v, and 19w, had KI of 0.02 to 0.11 nM and kinact/KI ratios of 8.8-17.5 nM-1min-1, a parameter indicated for the efficiency of irreversible inhibition. We also used the docking model to illustrate the difference in STS inhibitory potency of compounds. Finally, the safety and anti-cancer activity of selected compounds 19m, 19v, and 19w were also studied, showing the results of low cytotoxicity on NHDF cell line and being more potent than irosustat on ZR-75-1 cell, which was a hormone-dependent cancer cell line with high STS expression.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Placenta , Esteril-Sulfatase , Ácidos Sulfônicos , Feminino , Humanos , Gravidez , Inibidores Enzimáticos/farmacologia , Cinética , Esteril-Sulfatase/antagonistas & inibidores , Relação Estrutura-Atividade , Ácidos Sulfônicos/química , Ácidos Sulfônicos/farmacologia , Placenta/enzimologia , Células MCF-7
15.
Dokl Biochem Biophys ; 506(1): 191-194, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36303050

RESUMO

The effect of silicon nanoparticles (1 µg/mL) on the activity of lipid peroxidation, peroxidase, superoxide dismutase, and catalase in tomato roots invaded by root-knot nematode Meloidogyne incognita was studied. It was shown that, at the early stages of parasitization in the plants treated with Si-NPs, a low activity of PO and SOD, as well as an increased level of lipid peroxidation, are observed, which indicates the formation of free radicals (reactive oxygen species, ROS) that can inhibit nematodes and limit the formation of giant cells. During the sedentary stage, at the stages of nutrition, development, and egg production, the roots of the treated plants showed an increased activity of PO, CAT, and SOD, as well as a low activity of LPO as compared to the infested untreated plants. This makes it possible to maintain a balance between the formation and neutralization of ROS and is important not only in the protection of plant tissues from oxidative processes but also in the preservation of giant cells that feed the parasite. The presented data for the first time show the mechanism of action of Si-NPs in the development of resistance and adaptation of plants to biogenic stress, associated with the effect on various components of the antioxidant system and their functional interaction.


Assuntos
Antioxidantes , Lycopersicon esculentum , Nanopartículas , Raízes de Plantas , Silício , Tylenchoidea , Animais , Antioxidantes/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/parasitologia , Raízes de Plantas/enzimologia , Raízes de Plantas/parasitologia , Espécies Reativas de Oxigênio , Silício/farmacologia , Superóxido Dismutase
16.
Bioorg Chem ; 129: 106185, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36240541

RESUMO

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. Mpro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of Mpro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against Mpro with IC50 of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 Mpro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of Mpro, and ACF has been previously reported as a PLpro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.


Assuntos
Acriflavina , COVID-19 , Proteases 3C de Coronavírus , Inibidores de Cisteína Proteinase , Proflavina , SARS-CoV-2 , Inibidores de Protease Viral , Acriflavina/farmacologia , COVID-19/tratamento farmacológico , Proflavina/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Protease Viral/farmacologia , Mesocricetus , Animais , Cricetinae , Linhagem Celular , Replicação Viral/efeitos dos fármacos
17.
Sci Rep ; 12(1): 16991, 2022 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216916

RESUMO

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.


Assuntos
Acetilglucosamina , Fosfotransferases (Aceptor do Grupo Álcool) , Saccharomyces cerevisiae , Acetilglucosamina/metabolismo , Trifosfato de Adenosina/metabolismo , Citidina Trifosfato/metabolismo , Glucose/metabolismo , Glicoproteínas/metabolismo , Guanosina Trifosfato/metabolismo , Nucleosídeos/metabolismo , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Uridina Trifosfato/metabolismo
18.
Science ; 378(6616): 155-160, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36227987

RESUMO

The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present. Using structural and biochemical approaches, we retrace how this subunit was gained and became essential. Our work illuminates the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds light on the determinants of specificity in Rubisco.


Assuntos
Dióxido de Carbono , Domínio Catalítico , Evolução Molecular , Ribulose-Bifosfato Carboxilase , Dióxido de Carbono/química , Oxigênio/química , Fotossíntese , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética , Especificidade por Substrato , Domínio Catalítico/genética , Metagenoma , Firmicutes/enzimologia
19.
Bioorg Chem ; 129: 106198, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36265353

RESUMO

The terminase complex of human cytomegalovirus (HCMV) is required for viral genome packaging and cleavage. Critical to the terminase functions is a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We have previously reported metal-chelating N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human immunodeficiency virus 1 (HIV-1) RNase H. In the current work, we have synthesized new analogs and resynthesized known analogs of two isomeric HtPD subtypes, anti-HtPD (13), and syn-HtPD (14), and characterized them as inhibitors of pUL89-C. Remarkably, the vast majority of analogs strongly inhibited pUL89-C in the biochemical endonuclease assay, with IC50 values in the nM range. In the cell-based antiviral assay, a few analogs inhibited HCMV in low µM concentrations. Selected analogs were further characterized in a biophysical thermal shift assay (TSA) and in silico molecular docking, and the results support pUL89-C as the protein target of these inhibitors. Collectively, the biochemical, antiviral, biophysical, and in silico data reported herein indicate that the isomeric HtPD chemotypes 13-14 can serve as valuable chemical platforms for designing improved inhibitors of HCMV pUL89-C.


Assuntos
Antivirais , Citomegalovirus , Endonucleases , Proteínas Virais , Humanos , Antivirais/farmacologia , Antivirais/química , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Endonucleases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Desenho de Fármacos
20.
Biotechnol Adv ; 61: 108046, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36202263

RESUMO

Lipoxygenases (LOXs) are enzymes that catalyze dioxygenation of polyunsaturated fatty acids into fatty acid hydroperoxides. The formed fatty acid hydroperoxides are of interest as they can readily be transformed to a number of value-added compounds. LOXs are widely distributed in both eukaryotic and prokaryotic organisms, including humans, animals, plants, fungi and bacteria. Compared to eukaryotic enzymes, bacterial enzymes are typically easier to produce at industrial scale in a heterologous host. However, many bacterial LOXs were only identified relatively recently and their structure and biochemical characteristics have not been extensively studied. A better understanding of bacterial LOXs' structure and characteristics will lead to the wider application of these enzymes in industrial processes. This review focuses on recent findings on the biochemical characteristics of bacterial LOXs in relation to their molecular structure. The basis of LOX catalysis as well as emerging determinants explaining the regio- and enantioselectivity of different LOXs are also summarized and critically reviewed. Clustering and phylogenetic analyses of bacterial LOX sequences were performed. Finally, the improvement of bacterial LOXs by mutagenesis approaches and their application in chemical synthesis are discussed.


Assuntos
Bactérias , Peróxidos Lipídicos , Lipoxigenases , Bactérias/enzimologia , Lipoxigenases/química , Lipoxigenases/genética , Lipoxigenases/metabolismo , Estrutura Molecular , Filogenia
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