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1.
Food Chem ; 462: 141027, 2025 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-39213963

RESUMO

Integrating multiple functionalities into a single entity is highly important, especially when a broad spectrum of application is required. In the present work, we synthesized a novel manganese-based MOF (denoted as UoZ-6) that functions as a cold/hot-adapted and recyclable oxidase nanozyme (Km 0.085 mM) further developed for ratiometric-based colorimetric and color tonality visual-mode detection of nitrite in water and food. Nitrite ions promote the diazotization process of the oxTMB product, resulting in a decay in the absorbance signal at 652 nm and the emergence of a new signal at 461 nm. The dual-absorbance ratiometric platform for nitrite ion detection functions effectively across a wide temperature range (0 °C to 100 °C), offering a linear detection range of 5-45 µM with a detection limit of 0.15 µM using visual-mode. This approach is sensitive, reliable, and selective, making it effective for detecting nitrite ions in processed meat and water.


Assuntos
Colorimetria , Nitritos , Nitritos/análise , Colorimetria/métodos , Estruturas Metalorgânicas/química , Oxirredutases/química , Oxirredutases/metabolismo , Limite de Detecção , Temperatura Baixa , Temperatura Alta , Contaminação de Alimentos/análise , Cor
2.
Commun Biol ; 7(1): 1127, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39271811

RESUMO

Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.


Assuntos
Sistemas CRISPR-Cas , Galinhas , Criopreservação , Células Germinativas , Animais , Galinhas/genética , Células Germinativas/metabolismo , Feminino , Masculino , Oxirredutases/genética , Oxirredutases/metabolismo , Edição de Genes/métodos , Regeneração/genética , Animais Geneticamente Modificados , Quimera/genética , Técnicas de Inativação de Genes
3.
Int J Mol Sci ; 25(17)2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39273329

RESUMO

The alternative oxidase (AOX), a common terminal oxidase in the electron transfer chain (ETC) of plants, plays a crucial role in stress resilience and plant growth and development. Oat (Avena sativa), an important crop with high nutritional value, has not been comprehensively studied regarding the AsAOX gene family. Therefore, this study explored the responses and potential functions of the AsAOX gene family to various abiotic stresses and their potential evolutionary pathways. Additionally, we conducted a genome-wide analysis to explore the evolutionary conservation and divergence of AOX gene families among three Avena species (Avena sativa, Avena insularis, Avena longiglumis) and four Poaceae species (Avena sativa, Oryza sativa, Triticum aestivum, and Brachypodium distachyon). We identified 12 AsAOX, 9 AiAOX, and 4 AlAOX gene family members. Phylogenetic, motif, domain, gene structure, and selective pressure analyses revealed that most AsAOXs, AiAOXs, and AlAOXs are evolutionarily conserved. We also identified 16 AsAOX segmental duplication pairs, suggesting that segmental duplication may have contributed to the expansion of the AsAOX gene family, potentially preserving these genes through subfunctionalization. Chromosome polyploidization, gene structural variations, and gene fragment recombination likely contributed to the evolution and expansion of the AsAOX gene family as well. Additionally, we hypothesize that AsAOX2 may have potential function in resisting wounding and heat stresses, while AsAOX4 could be specifically involved in mitigating wounding stress. AsAOX11 might contribute to resistance against chromium and waterlogging stresses. AsAOX8 may have potential fuction in mitigating ABA-mediated stress. AsAOX12 and AsAOX5 are most likely to have potential function in mitigating salt and drought stresses, respectively. This study elucidates the potential evolutionary pathways of the AsAOXs gene family, explores their responses and potential functions to various abiotic stresses, identifies potential candidate genes for future functional studies, and facilitates molecular breeding applications in A. sativa.


Assuntos
Avena , Evolução Molecular , Proteínas Mitocondriais , Família Multigênica , Oxirredutases , Filogenia , Proteínas de Plantas , Estresse Fisiológico , Avena/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Triticum/genética , Triticum/enzimologia , Duplicação Gênica
4.
BMC Plant Biol ; 24(1): 847, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39251901

RESUMO

BACKGROUND: Camellia nitidissima is a rare, prized camellia species with golden-yellow flowers. It has a high ornamental, medicinal, and economic value. Previous studies have shown substantial flavonol accumulation in C. nitidissima petals during flower formation. However, the mechanisms underlying the golden flower formation in C. nitidissima remain largely unknown. RESULTS: We performed an integrative analysis of the transcriptome, proteome, and metabolome of the petals at five flower developmental stages to construct the regulatory network underlying golden flower formation in C. nitidissima. Metabolome analysis revealed the presence of 323 flavonoids, and two flavonols, quercetin glycosides and kaempferol glycosides, were highly accumulated in the golden petals. Transcriptome and proteome sequencing suggested that the flavonol biosynthesis-related genes and proteins upregulated and the anthocyanin and proanthocyanidin biosynthesis-related genes and proteins downregulated in the golden petal stage. Further investigation revealed the involvement of MYBs and bHLHs in flavonoid biosynthesis. Expression analysis showed that flavonol synthase 2 (CnFLS2) was highly expressed in the petals, and its expression positively correlated with flavonol content at all flower developmental stages. Transient overexpression of CnFLS2 in the petals increased flavonol content. Furthermore, correlation analysis showed that the jasmonate (JA) pathways positively correlated with flavonol biosynthesis, and exogenous methyl jasmonate (MeJA) treatment promoted CnFLS2 expression and flavonol accumulation. CONCLUSIONS: Our findings showed that the JA-CnFLS2 module regulates flavonol biosynthesis during golden petal formation in C. nitidissima.


Assuntos
Camellia , Flavonóis , Flores , Proteínas de Plantas , Camellia/genética , Camellia/metabolismo , Camellia/crescimento & desenvolvimento , Flores/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flavonóis/metabolismo , Flavonóis/biossíntese , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Ciclopentanos/metabolismo , Transcriptoma , Pigmentação/genética , Oxilipinas/metabolismo , Acetatos/metabolismo , Acetatos/farmacologia , Proteoma/metabolismo , Metaboloma , Multiômica , Oxirredutases
5.
Methods Enzymol ; 703: 51-63, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39261003

RESUMO

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.


Assuntos
Diálise , Cromatografia Líquida de Alta Pressão/métodos , Especificidade por Substrato , Diálise/métodos , Ligação Proteica , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/instrumentação , Cinética , Lisina/metabolismo , Lisina/química , Oxirredutases/metabolismo , Oxirredutases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ferro/metabolismo , Ferro/química
6.
Methods Enzymol ; 703: 65-85, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39261004

RESUMO

Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.


Assuntos
Oxirredução , Oxigenases , Oxigenases/metabolismo , Oxigenases/química , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Rhodococcus/enzimologia , Rhodococcus/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química
7.
Int J Mycobacteriol ; 13(3): 258-264, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-39277887

RESUMO

BACKGROUND: Drug-resistant tuberculosis (DR-TB) poses a major global challenge to public health and therapeutics. It is an emerging global concern associated with increased morbidity and mortality mostly seen in the low- and middle-income countries. Molecular techniques are highly sensitive and offer timely and accurate results for TB drug resistance testing, thereby positively influencing patient management plan. METHODS: The study was carried out at the National Tuberculosis Reference Laboratory (NTRL) in Kenya in the period between January and October 2022. A total of 243 Mycobacterium tuberculosis (M.tb) clinical isolates were included in the study. These isolates comprised of 50 isolates with mutations in rpoB, 51 isolates with katG mutations, 51 isolates with mutations in inhA, and 91 M.tb isolates lacking mutations in these genes based on Genotype MTBDRplus results. DNA from the isolates was extracted using the FluoroLyse extraction kit. Real-time polymerase chain reaction targeting the rpoB, InhA, and katG genes was performed using the FluoroType MTBDR amplification mix. Isolates with discordant results between Genotype MTBDRplus and FluoroCycler® MTBDR assays underwent targeted sequencing for the respective genes, then, sequences were analyzed for mutations using Geneious version 11.0 software. RESULTS: The sensitivity of the Fluorocycler XT MTBDR assay for the detection of mutations that confer drug resistance was 86% (95% confidence interval [CI] 73.0-94.0) for rpoB, 96% (95% CI 87-100) for katG and 92% (95% CI 81-98) for inhA. The assay's specificity was 97% (95% CI 93-99) for rpoB, 98% (95% CI 96-100) for katG, and 97% (95% CI 93-99) for inhA. CONCLUSION: The diagnostic accuracy of FluoroType MTBDR for the detection of mutations conferring resistance to rifampicin and isoniazid was high compared with that of Genotype MTBDRplus and demonstrates its suitability as a replacement assay for Genotype MTBDRplus.


Assuntos
Antituberculosos , Isoniazida , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Rifampina , Tuberculose Resistente a Múltiplos Medicamentos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Humanos , Isoniazida/farmacologia , Quênia , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Mutação , Sensibilidade e Especificidade , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Catalase/genética , Genótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Oxirredutases/genética
8.
Indian J Tuberc ; 71(4): 383-388, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39278670

RESUMO

BACKGROUND: Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). The world is currently facing challenges due to the spread of anti-tuberculosis drug-resistant of M. tuberculosis. Isoniazid-resistant (INH), is one of the first-line anti-tuberculosis agents that has a high resistance case. This study used Multiplex allele-specific Polymerase Chain Reaction (MAS-PCR) to detect the most common mutations associated with isoniazid resistance on inhA, katG, and ahpC gene. METHODS: This study used samples from clinical isolates of M. tuberculosis which had been tested for their antibiotic sensitivity of first-line anti-tuberculosis drugs. The DNA extraction process was carried out using the boiling method and then amplified with specific primers for inhA, katG, and ahpC genes using the MAS-PCR method. The results are then read on the electrophoretic gel with an interpretation of the mutation gene when the target gene DNA bands were absent according to the allele-specific fragments target. RESULTS: A total of 200 isolates were tested in this study consisting of isoniazid-resistant and susceptible with the largest distribution of Multi-Drug Resistant (MDR) isolates with a total of 146 isolates (73%). The most significant gene mutation was on the ahpC gene in 61 isolates (30,5%) and the combination mutation of the katG + ahpC gene in 52 isolates (26%) with sensitivity and specificity of the test reaching 87% and 42% for the detection of INH-resistant. CONCLUSION: Mutation on the ahpC gene has the highest percentage in this study. AhpC gene can be considered one of the essential genes to be tested for the cause of isoniazid-resistant. Using MAS-PCR for detecting gene mutation in isoniazid-resistant was simple and easy, it has the potential to be widely used as a rapid screening molecular test.


Assuntos
Antituberculosos , Proteínas de Bactérias , Catalase , Isoniazida , Mutação , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Indonésia , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Catalase/genética , Oxirredutases/genética , Testes de Sensibilidade Microbiana , Feminino , Masculino , Adulto , Reação em Cadeia da Polimerase Multiplex , Farmacorresistência Bacteriana Múltipla/genética
9.
PeerJ ; 12: e18005, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39221263

RESUMO

Background: Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. Results: The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. Conclusions: The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.


Assuntos
Anti-Inflamatórios não Esteroides , Chlamydomonas reinhardtii , Diclofenaco , Potencial da Membrana Mitocondrial , Mitocôndrias , Consumo de Oxigênio , Espécies Reativas de Oxigênio , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Diclofenaco/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Anti-Inflamatórios não Esteroides/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Cianeto de Potássio/toxicidade , Oxirredutases/metabolismo , Salicilamidas , Microscopia Eletrônica de Transmissão , Proteínas de Plantas , Proteínas Mitocondriais
10.
Chin J Dent Res ; 27(3): 243-251, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39221985

RESUMO

OBJECTIVE: To investigate the antioxidant enzyme status in biological samples of patients with oral squamous cell carcinoma (OSCC) and compare them with biological samples of healthy people through a systematic review and meta-analysis. METHODS: Antioxidant enzymes of catalase (CAT), sodium dismutase (SOD) and glutathione peroxide (GPx) were included in the analysis. A literature search was conducted of the PubMed, Science Direct, Scopus, Web of Science and Wiley Online Library databases for studies published between January 1999 and December 2022. A total of 831 articles were selected, of which 131 were found to be relevant. Finally, the full texts of 12 studies were screened and included. Studies that evaluated other antioxidant enzymes were excluded. Standardised mean difference (SMD) was derived to conduct a meta-analysis using comprehensive meta-analysis v3 (Biostat, Englewood, NJ, USA). A random effects model with 95% confidence interval (CI) was used to estimate the effect size. P < 0.05 was considered significant. RESULTS: CAT levels were measured in eight studies (n = 567) and the mean values for the OSCC and control groups were 4.81 ± 2.57 and 10.02 ± 1.81, respectively (SMD 3.18, 95% CI 1.01 to 1.42; P = 0.001). SOD level was evaluated in 11 studies (n = 762) and the values for the OSCC and control groups were 3.78 ± 1.45 and 7.34 ± 1.79, respectively (SMD 3.66, 95% CI 1.51 to 1.94; P = 0.001). GPx level was evaluated in 10 studies (n = 697) and the values for the OSCC and control groups were 13.33 ± 1.42 and 16.54 ± 2.9, respectively (SMD 1.91, 95% CI 1.34 to 1.77; P = 0.001). The heterogeneity between the studies was severe (I2 ≥ 90%). The risk of bias between studies was low to moderate. CONCLUSION: Analysis revealed that the levels of antioxidant enzymes decreased in biological samples of patients with OSSC as compared to healthy controls. Understanding the pathological progress of OSCC by analysing the level of antioxidant enzymes is beneficial in formulating a personalised, targeted pro-oxidant therapy for cancer treatment.


Assuntos
Antioxidantes , Carcinoma de Células Escamosas , Neoplasias Bucais , Oxirredutases , Humanos , Antioxidantes/metabolismo , Carcinoma de Células Escamosas/patologia , Catalase/metabolismo , Glutationa Peroxidase/metabolismo , Neoplasias Bucais/patologia , Superóxido Dismutase/metabolismo
11.
Microb Cell Fact ; 23(1): 238, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39223542

RESUMO

BACKGROUND: Benzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate. RESULTS: Two biosynthetic routes based on the CoA-dependent ß-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent ß-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation. CONCLUSIONS: We were able to establish the whole pathway for benzyl acetate based on the CoA-dependent ß-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.


Assuntos
Escherichia coli , Glucose , Engenharia Metabólica , Engenharia Metabólica/métodos , Escherichia coli/metabolismo , Escherichia coli/genética , Glucose/metabolismo , Fermentação , Acetatos/metabolismo , Oxirredução , Acetilcoenzima A/metabolismo , Oxirredutases/metabolismo , Oxirredutases/genética , Compostos de Benzil/metabolismo
12.
Chem Biol Drug Des ; 104(3): e14612, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39237482

RESUMO

The Mycobacterium cell wall is a capsule-like structure comprising of various layers of biomolecules such as mycolic acid, peptidoglycans, and arabinogalactans, which provide the Mycobacteria a sort of cellular shield. Drugs like isoniazid, ethambutol, cycloserine, delamanid, and pretomanid inhibit cell wall synthesis by inhibiting one or the other enzymes involved in cell wall synthesis. Many enzymes present across these layers serve as potential targets for the design and development of newer anti-TB drugs. Some of these targets are currently being exploited as the most druggable targets like DprE1, InhA, and MmpL3. Many of the anti-TB agents present in clinical trials inhibit cell wall synthesis. The present article covers a systematic perspective of developing cell wall inhibitors targeting various enzymes involved in cell wall biosynthesis as potential drug candidates for treating Mtb infection.


Assuntos
Antituberculosos , Proteínas de Bactérias , Parede Celular , Mycobacterium tuberculosis , Parede Celular/metabolismo , Parede Celular/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Tuberculose/tratamento farmacológico , Oxirredutases/metabolismo , Oxirredutases/antagonistas & inibidores , Ácidos Micólicos/metabolismo , Oxirredutases do Álcool , Proteínas de Membrana Transportadoras
13.
Structure ; 32(9): 1296-1298, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39241761

RESUMO

Fumonisin B1 (FB1) targets sphingolipid biosynthesis, inhibiting ceramide synthases. In this issue of Structure, Zhang et al.1 determined the cryoelectron microscopic structures of yeast ceramide synthase in complex with FB1 and its acylated derivative, acyl-FB1, revealing a two-step "ping-pong" mechanism for the N-acylation of FB1 and how it inhibits ceramide synthase.


Assuntos
Microscopia Crioeletrônica , Fumonisinas , Oxirredutases , Fumonisinas/química , Fumonisinas/metabolismo , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Acilação , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Esfingolipídeos/metabolismo , Esfingolipídeos/química
14.
Org Lett ; 26(36): 7565-7570, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39230034

RESUMO

Less steric ketones exhibited low stereoselectivity toward M5 due to their difficulty in restricting the free rotation of the imine intermediate. An engineered enantio-complementary imine reductase from M5 was obtained with catalytic activity. We identified four key residues that play essential roles in controlling stereoselectivity. Two mutants, I149Y-W234L (up to 99%S ee) and L200M-F260M (up to 99%R ee), were achieved, showing excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing alkylated amphetamines.


Assuntos
Anfetaminas , Iminas , Oxirredutases , Estrutura Molecular , Estereoisomerismo , Iminas/química , Oxirredutases/metabolismo , Oxirredutases/química , Anfetaminas/química , Anfetaminas/síntese química , Alquilação , Catálise , Biocatálise
15.
Bioresour Technol ; 412: 131407, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39233185

RESUMO

Phenol, quinoline, and pyridine, commonly found in industrial wastewater, disrupt the nitrification process, leading to nitrite accumulation. This study explores the potential mechanisms through which these biotoxic organic compounds affect nitrite accumulation, using metagenomic and molecular docking analyses. Despite increasing concentrations of these compounds from 40 to 160 mg/L, ammonia nitrogen removal was not hindered, and stable nitrite accumulation rates exceeding 90 % were maintained. Additionally, these compounds inhibited nitrite-oxidizing bacteria (NOB) and enriched ammonia-oxidizing bacteria (AOB) in situ. As the concentration of these compounds rose, protein (PN) and polysaccharide (PS) concentrations also increased, along with a higher PN/PS ratio. Metagenomic analysis further revealed an increase in hao relative abundance, while microbial community analysis showed increased Nitrosomonas abundance, which contributed to nitrite accumulation stability. Molecular docking indicated that these compounds have lower binding energy with hydroxylamine oxidoreductase (HAO) and nitrate reductase (NAR), theoretically supporting the observed sustained nitrite accumulation.


Assuntos
Metagenômica , Simulação de Acoplamento Molecular , Nitrificação , Nitritos , Piridinas , Quinolinas , Nitritos/metabolismo , Quinolinas/farmacologia , Metagenômica/métodos , Piridinas/farmacologia , Piridinas/metabolismo , Fenol , Bactérias/metabolismo , Bactérias/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Águas Residuárias , Oxirredutases/metabolismo , Amônia/metabolismo
16.
Sci Rep ; 14(1): 18093, 2024 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-39103552

RESUMO

12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer-dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis.


Assuntos
Multimerização Proteica , Fosforilação , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , Oxirredutases/metabolismo , Oxirredutases/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Cristalografia por Raios X , Solanum lycopersicum/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Sulfatos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH
17.
Int J Biol Macromol ; 277(Pt 4): 134538, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39111462

RESUMO

Dihydro-ß-ionone, a high-value compound with distinctive fragrance, is widely utilized in the flavor and fragrance industries. However, its low abundance in plant sources poses a significant challenge to its application through traditional extraction methods. Development of an enzyme cascade reaction with artificial design offers a promising alternative. Herein, a short-chain dehydrogenase NaSDR, was identified from Novosphingobium aromaticivorans DSM 12444, which exhibited a high activity in converting ß-ionol to ß-ionone. A novel biosynthesis route to produce dihydro-ß-ionone from ß-ionol was developed, by utilizing alcohol dehydrogenase NaSDR and enoate reductase AaDBR1. Under the optimized conditions (0.29 mg/mL NaSDR, 0.39 mg/mL AaDBR1, 1 mM NADP+ and 2.5 mM ß-ionol at 40 °C for 2 h), a maximum yield (173.11 mg/L) of dihydro-ß-ionone was achieved with a molar conversion rate of 35.6 %, which was 2.7-fold higher than that before optimization. Additionally, this cascade reaction achieved self-sufficient NADPH regeneration through the actions of NaSDR and AaDBR1. This study offered a fresh perspective for achieving a green and sustainable synthesis of dihydro-ß-ionone and could inspire on another natural products biosynthesis.


Assuntos
Norisoprenoides , Norisoprenoides/química , Norisoprenoides/metabolismo , Redutases-Desidrogenases de Cadeia Curta/metabolismo , Redutases-Desidrogenases de Cadeia Curta/química , Sphingomonadaceae/enzimologia , NADP/metabolismo , Oxirredutases/metabolismo
18.
Plant Physiol Biochem ; 215: 109045, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39154421

RESUMO

Iron (Fe) toxicity is a major abiotic stress in lowland rice production. Breeding tolerant varieties has proven challenging due to the complex genetic architecture of Fe toxicity tolerance and the strong genotype-by-environment interactions. Additionally, conventional methods for phenotyping visible stress symptoms are often inaccurate, inconsistent, and lack reproducibility. In our previous work, we identified that ascorbate redox regulation, mediated by the activities of dehydroascorbate reductase (DHAR) and ascorbate oxidase (AO), contributed to high tolerance in an indica rice genotype across various environments. To explore whether this mechanism is common among other rice genotypes, we selected ten genotypes with contrasting stress symptoms under Fe-toxic conditions to examine the roles of DHAR and AO in regulating Fe toxicity tolerance. Additionally, we aimed to develop objective and accurate image-based phenotyping methods to replace the traditional leaf bronzing scoring method. Among the ten genotypes we tested, we found significant positive correlations between DHAR activity and stress symptoms in plants grown under both Fe toxicity and control conditions, suggesting a general link between ascorbate redox regulation and Fe toxicity tolerance. Using RGB signals from leaf images of plants exposed to 1000 mg/L Fe2+, we evaluated 36 different color indices to quantify stress symptoms. We identified the normalized green‒red difference index as most significant in quantifying stress symptoms under Fe toxicity conditions. Our findings suggest that DHAR activity could be potentially employed as a biomarker in the screening of rice germplasms and breeding tolerant cultivars to Fe toxicity.


Assuntos
Ácido Ascórbico , Ferro , Oryza , Oxirredução , Ácido Ascórbico/metabolismo , Oryza/metabolismo , Oryza/genética , Oryza/efeitos dos fármacos , Ferro/metabolismo , Oxirredutases/metabolismo , Oxirredutases/genética , Genótipo , Ascorbato Oxidase/metabolismo , Ascorbato Oxidase/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Estresse Fisiológico , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos
19.
Exp Eye Res ; 247: 110061, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39182597

RESUMO

Ceramides with varying acyl-chain lengths can have unique biological actions and hence, cellular responses to ceramides may depend not on their overall concentration but on that of individual ceramide species. The purpose of this study was to determine individual ceramide species impacting retinal ganglion cell (RGC) loss under the ocular hypertensive condition. Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human astrocytes were used to determine the effect of individual ceramide species on both RGC viability and astrocyte secretion of inflammatory cytokines in vitro. In in vivo experiments with wild-type (WT) and ceramide synthase 5 (CerS5) knockout mice, intraocular pressure was unilaterally elevated with microbead injection. Retinal function and morphology were evaluated using pattern electroretinography (pERG) and immunofluorescence, respectively. Ceramide levels were determined by LC-MS/MS analysis. Exposure to C16:0-, C18:0-, C18:1-, C20:0- and C24:0-ceramides significantly reduces RGC viability in vitro, with the very long chain C24:0-ceramide being the most neurotoxic; treatment with C18:0-, C18:1- and C24:0-ceramides stimulates an increase of TNF-α secretion by astrocytes. The retinas of CerS5 KO mice have significantly reduced levels of C16:0- and C18:1-ceramides compared to WT; ocular hypertensive eyes of these mice maintain higher pERG amplitudes and RGC numbers compared to WT. Individual ceramides with different chain lengths have different effects on RGCs and astrocytes. Our results demonstrate that suppressing C16:0- and C18:1-ceramide species effectively protects RGCs against ocular hypertensive injury. These results provide a basis for targeting specific ceramide species in the treatment of glaucoma.


Assuntos
Sobrevivência Celular , Ceramidas , Eletrorretinografia , Pressão Intraocular , Camundongos Knockout , Hipertensão Ocular , Células Ganglionares da Retina , Animais , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/metabolismo , Hipertensão Ocular/metabolismo , Camundongos , Pressão Intraocular/fisiologia , Ceramidas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Humanos , Células Cultivadas , Oxirredutases/metabolismo , Astrócitos/metabolismo , Espectrometria de Massas em Tandem , Citocinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Cromatografia Líquida
20.
J Alzheimers Dis ; 101(2): 525-539, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39213075

RESUMO

Background: Reduction of the production of amyloid-ß (Aß) species has been intensively investigated as potential therapeutic approaches for Alzheimer's disease (AD). However, the degradation of Aß species, another potential beneficial approach, has been far less explored. Objective: To investigate the potential of multi-copper oxidases (MCOs) in degrading Aß peptides and their potential benefits for AD treatment. Methods: We investigated the degradation efficiency of MCOs by using electrophoresis and validated the ceruloplasmin (CP)-Aß interaction using total internal reflection fluorescence microscopy, fluorescence photometer, and fluorescence polarization measurement. We also investigated the therapeutic effect of ascorbate oxidase (AO) by using induced pluripotent stem (iPS) neuron cells and electrophysiological analysis with brain slices. Results: We discovered that CP, an important MCO in human blood, could degrade Aß peptides. We also found that other MCOs could induce Aß degradation as well. Remarkably, we revealed that AO had the strongest degrading effect among the tested MCOs. Using iPS neuron cells, we observed that AO could rescue neuron toxicity which induced by Aß oligomers. In addition, our electrophysiological analysis with brain slices suggested that AO could prevent an Aß-induced deficit in synaptic transmission in the hippocampus. Conclusions: To the best of our knowledge, our report is the first to demonstrate that MCOs have a degrading function for peptides/proteins. Further investigations are warranted to explore the possible benefits of MCOs for future AD treatment.


Assuntos
Peptídeos beta-Amiloides , Células-Tronco Pluripotentes Induzidas , Neurônios , Oxirredutases , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Oxirredutases/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos
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