RESUMO
Methane capture via oxidation is considered one of the 'Holy Grails' of catalysis (Tucci and Rosenzweig, 2024). Methane is also a primary greenhouse gas that has to be reduced by 1.2 billion metric tonnes in 10 years to decrease global warming by only 0.23°C (He and Lidstrom, 2024); hence, new technologies are needed to reduce atmospheric methane levels. In Nature, methane is captured aerobically by methanotrophs and anaerobically by anaerobic methanotrophic archaea; however, the anaerobic process dominates. Here, we describe the history and potential of using the two remarkable enzymes that have been cloned with activity for capturing methane: aerobic capture via soluble methane monooxygenase and anaerobic capture via methyl-coenzyme M reductase. We suggest these two enzymes may play a prominent, sustainable role in addressing our current global warming crisis.
Assuntos
Metano , Oxirredutases , Oxigenases , Proteínas Recombinantes , Metano/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oxirredução , Anaerobiose , Aerobiose , Archaea/enzimologia , Archaea/genética , Archaea/metabolismoRESUMO
Production of biofuels and other value-added products from lignocellulose breakdown requires the coordinated metabolic activity of varied microorganisms. The increasing global demand for biofuels encourages the development and optimization of production strategies. Optimization in turn requires a thorough understanding of the microbial mechanisms and metabolic pathways behind the formation of each product of interest. Hydrolysis of lignocellulosic biomass is a bottleneck in its industrial use and often affects yield efficiency. The accessibility of the biomass to the microorganisms is the key to the release of sugars that are then taken up as substrates and subsequently transformed into the desired products. While the effects of different metabolic intermediates in the overall production of biofuel and other relevant products have been studied, the role of proteins and their activity under anaerobic conditions has not been widely explored. Shifts in enzyme production may inform the state of the microorganisms involved; thus, acquiring insights into the protein production and enzyme activity could be an effective resource to optimize production strategies. The application of proteomic analysis is currently a promising strategy in this area. This review deals on the aspects of enzymes and proteomics of bioprocesses of biofuels production using lignocellulosic biomass as substrate.
Assuntos
Bactérias Anaeróbias/metabolismo , Biocombustíveis/microbiologia , Biomassa , Lignina/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Anaerobiose , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/enzimologia , Celulases/metabolismo , Hidrólise , Oxigenases/metabolismoRESUMO
The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of É-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.
Assuntos
Álcool Desidrogenase , Oxigenases , Álcool Desidrogenase/genética , Estabilidade Enzimática , Oxigenases/genética , Oxigenases/metabolismoRESUMO
Flavin-containing monooxygenase 3 (FMO3) is a polymorphic drug metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped a white Argentinian 11-year-old girl by medical sensory evaluation. After pedigree analysis with her brother and parents, this proband showed to harbor a new allele p.(P73L; E158K; E308G) FMO3 in trans configuration with the second new one p.(F140S) FMO3. Recombinant FMO3 proteins of the wild-type and the novel two variants underwent kinetic analyses of their trimethylamine N-oxygenation activities. P73L; E158K; E308G and F140S FMO3 proteins exhibited moderately and severely decreased trimethylamine N-oxygenation capacities (~50% and ~10% of wild-type FMO3, respectively). Amino acids P73 and F140 were located on the outer surface region in a crystallographic structure recently reported of a FMO3 analog. Changes in these positions would indirectly impact on key FAD-binding residues. This is the first report and characterization of a patient of fish odor syndrome caused by genetic aberrations leading to impaired FMO3-dependent N-oxygenation of trimethylamine found in the Argentinian population. We found novel structural determinants of FAD-binding domains, expanding the list of known disease-causing mutations of FMO3. Our results suggest that individuals homozygous for any of these new variants would develop a severe form of this disorder.
Assuntos
Membrana Celular/enzimologia , Metilaminas/metabolismo , Oxigênio/metabolismo , Oxigenases/genética , Polimorfismo de Nucleotídeo Único/genética , Argentina , Criança , Feminino , Humanos , Erros Inatos do Metabolismo/enzimologia , Metilaminas/urina , Oxigenases/metabolismoRESUMO
There is limited information on the effect of black beans (BB) as a source of protein and resistant starch on the intestinal microbiota. The purpose of the present work was to study the effect of cooked black beans with and without high fat and sugar (HF + S) in the diet on body composition, energy expenditure, gut microbiota, short-chain fatty acids, NF-κB, occluding and insulin signaling in a rat model and the area under the curve for glucose, insulin and incretins in healthy subjects. The consumption of BB reduced the percentage of body fat, the area under the curve of glucose, serum leptin, LPS, glucose and insulin concentrations and increased energy expenditure even in the presence of HF + S. These results could be mediated in part by modification of the gut microbiota, by increasing a cluster of bacteria in the Clostridia class, mainly R. bromii, C. eutactus, R. callidus, R. flavefaciens and B. pullicaecorum and by an increase in the concentration of fecal butyrate. In conclusion, the consumption of BB can be recommended to prevent insulin resistance and metabolic endotoxemia by modifying the gut microbiota. Finally, the groups fed BB showed lower abundance of hepatic FMO-3, even with a high-fat diet protecting against the production of TMAO and obesity.
Assuntos
Clostridiales , Suplementos Nutricionais , Fabaceae , Microbioma Gastrointestinal , Resistência à Insulina , Animais , Distribuição da Gordura Corporal , Butiratos/metabolismo , Endotoxemia/prevenção & controle , Metabolismo Energético , Glucose/metabolismo , Voluntários Saudáveis , Leptina/metabolismo , Fígado/metabolismo , Masculino , Modelos Animais , Oxigenases/metabolismo , Ratos Wistar , Espondilite Anquilosante/microbiologiaRESUMO
The aim of the current work was to evaluate a potential pharmacokinetic interaction between the flukicide triclabendazole (TCBZ) and the broad-spectrum benzimidazole (BZD) anthelmintic oxfendazole (OFZ) in sheep. To this end, both an in vitro assay in microsomal fractions and an in vivo trial in lambs parasitized with Haemonchus contortus resistant to OFZ and its reduced derivative fenbendazole (FBZ) were carried out. Sheep microsomal fractions were incubated together with OFZ, FBZ, TCBZ, or a combination of either FBZ and TCBZ or OFZ and TCBZ. OFZ production was significantly diminished upon coincubation of FBZ and TCBZ, whereas neither FBZ nor OFZ affected the S-oxidation of TCBZ towards its sulfoxide and sulfone metabolites. For the in vivo trial, lambs were treated with OFZ (Vermox® oral drench at a single dose of 5â¯mg/kg PO), TCBZ (Fasinex® oral drench at a single dose of 12â¯mg/kg PO) or both compounds at a single dose of 5 (Vermox®) and 12â¯mg/kg (Fasinex®) PO. Blood samples were taken to quantify drug and metabolite concentrations, and pharmacokinetic parameters were calculated by means of non-compartmental analysis. Results showed that the pharmacokinetic parameters of active molecules and metabolites were not significantly altered upon coadministration. The sole exception was the increase in the mean residence time (MRT) of OFZ and FBZ sulfone upon coadministration, with no significant changes in the remaining pharmacokinetic parameters. This research is a further contribution to the study of metabolic drug-drug interactions that may affect anthelmintic efficacies in ruminants.
Assuntos
Anti-Helmínticos/farmacocinética , Benzimidazóis/farmacocinética , Triclabendazol/farmacocinética , Animais , Anti-Helmínticos/metabolismo , Área Sob a Curva , Benzimidazóis/metabolismo , Biotransformação , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Fenbendazol/metabolismo , Fígado/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Oxigenases/metabolismo , Ovinos , Triclabendazol/metabolismoRESUMO
Background: Flavonoids are a kind of important secondary metabolite and are commonly considered to provide protection to plants against stress and UV-B for a long time. Anthocyanidin synthase (ANS), which encodes a dioxygenase in the flavonoid pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanidins, but there is no direct evidence indicating that it provides tolerance to stress in plants. Results: To investigate whether ANS can increase tolerance to abiotic stress, MaANS was isolated from mulberry fruits and transformed into tobacco. Our results suggested that the bacterially expressed MaANS protein can convert dihydroquercetin to quercetin. Overexpression of MaANS remarkably increased the accumulation of total flavonoids in transgenic lines and anthocyanins in corollas of flowers. Transgenic lines showed higher tolerance to NaCl and mannitol stress. Conclusions: These results indicated that MaANS participates in various dioxygenase activities, and it can protect plants against abiotic stress by improving the ROS-scavenging ability. Thus, this alternative approach in crop breeding can be considered in the improvement of stress tolerance by enriching flavonoid production in plants
Assuntos
Oxigenases/metabolismo , Nicotiana , Morus/enzimologia , Oxigenases/genética , Quercetina , Estresse Fisiológico , Bactérias , Flavonoides/metabolismo , Plantas Geneticamente Modificadas , Dioxigenases/metabolismo , Expressão Ectópica do GeneRESUMO
A major pathway for the detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system to bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after the deletion of ohrR, and we show that OhrR controls the virulence of this human opportunistic pathogen. DNA microarray and Northern blot analyses of CHP-treated cells revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), the degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). Furthermore, we identified two upregulated genes (ohrA and a putative diguanylate cyclase with a GGDEF domain for cyclic di-GMP [c-di-GMP] synthesis) and three downregulated genes (hemolysin, chitinase, and collagenase) in the ohrR mutant by transcriptome analysis. Importantly, we show that OhrR directly repressed the expression of the putative diguanylate cyclase. Using a mouse infection model, we demonstrate that the ohrR mutant was attenuated for virulence and showed a decreased bacterial burden in the liver. Moreover, an ohrR-diguanylate cyclase double mutant displayed the same virulence as the wild-type strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcriptional regulator OhrR to modulate its virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Chromobacterium/genética , Chromobacterium/patogenicidade , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Quitinases/genética , Colagenases/genética , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas Hemolisinas , Humanos , Peróxido de Hidrogênio , Fígado/microbiologia , Camundongos , Oxigenases/metabolismo , Peroxidases/metabolismo , Fósforo-Oxigênio Liases/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.
Assuntos
Capsicum/enzimologia , Capsicum/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Oxigenases/genética , Filogenia , Motivos de Aminoácidos , Sequência Conservada/genética , Éxons/genética , Perfilação da Expressão Gênica , Genes de Plantas , Íntrons/genética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Oxigenases/metabolismo , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343).