RESUMO
Retinol is one of the main active forms of vitamin A, crucial for the organism's growth, development, and maintenance of eye and skin functions. It is widely used in cosmetics, pharmaceuticals, and feed additives. Although animals lack a complete pathway for synthesizing vitamin A internally, they can obtain vitamin A directly through diet or convert ß-carotene acquired from the diet. To boost the research on the biosynthesis of retinol, three different sources of alcohol dehydrogenase were firstly screened based on the ß-carotene synthesis platform CAR*1. It was determined that ybbO from Escherichia coli exhibited the highest catalytic activity,with a conversion rate of 95. 6%. To further enhance the reaction rate and yield of retinol, protein fusion technology was employed to merge two adjacent enzymes, blh and ybbO, within the retinol synthesis module. The evaluation was conducted using the high-yield engineered strain CAR*3 of ß-carotene. The optimal combination, blh-GGGS-ybbO, was obtained, with a 44. 9% increase in yield after fusion, reaching(111. 1± 3. 5) mg·L~(-1). Furthermore, through the introduction of human-derived retinol-binding protein(RBP4) and transthyretin(TTR), the process of hepatic cell secreting retinol was simulated in Saccharomyces cerevisiae, leading to an increased retinol yield of(158. 0±13. 1)mg·L~(-1). Finally, optimization strategies including overexpressing INO2 to enhance the reaction area for ß-carotene synthesis, enhancing hemoglobin VHb expression to improve oxygen supply, and strengthening PDR3m expression to facilitate retinol transport were implemented. A two-stage fermentation process resulted in the successful elevation of retinol production to(2 320. 0±26. 0)mg·L~(-1) in the fermentation tank of 5 L, which provided a significant foundation for the industrial development of retinol.
Assuntos
Fermentação , Saccharomyces cerevisiae , Vitamina A , Vitamina A/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Humanos , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , beta Caroteno/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismoRESUMO
To determine the effects of microbial proteins on Qingzhuan tea sensory quality during tea pile fermentation, tea leaf metabolomic and microorganism proteomic analyses were performed. In total, 1835 differential metabolites and 443 differentially expressed proteins of the microorganisms were identified. Correlation analysis between metabolomics and proteomics data revealed that the levels of microbial proteins EG II and CBH I cellulase may play important roles in cell wall construction and permeability, which were crucial for the interaction between tea leaves and microorganisms. Microbial proteins heat shock proteins (HSP), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and CuAO related to detoxification and stress responses showed a positive correlation with tea theanine, glutamine, γ-aminobutyric acid, glutamic acid, catechin, (-)-gallocatechin gallate, and (-)-catechin gallate, suggesting their effects on tea characteristic compound accumulation, thus affecting Qingzhuan tea sensory quality.
Assuntos
Camellia sinensis , Fermentação , Chá , Camellia sinensis/química , Camellia sinensis/metabolismo , Chá/química , Paladar , Folhas de Planta/química , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Humanos , Bactérias/metabolismo , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Catequina/metabolismo , Catequina/análise , Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteômica , GlutamatosRESUMO
Background: Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK's catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. Methods: The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. Results: The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. Conclusion: The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.
Assuntos
Álcool Desidrogenase , Escherichia coli , Ferro , Zinco , Zinco/metabolismo , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Ferro/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Sítios de Ligação , Ligação Proteica , Mutagênese Sítio-DirigidaRESUMO
1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the de novo synthesis of 1,4-butanediol was achieved after introduction of the path into Escherichia coli W3110 (K-12) chassis cells. To further improve the synthesis efficiency of this path, we deleted the genes encoding lactate dehydrogenase A (LdhA) and pyruvate formate lyase B (PflB) to block the metabolic bypass. Furthermore, the expression of citrate synthase (GltAR163L) was up-regulated to increase the α-ketoglutarate metabolic flux. In addition, we improved the synthesis of the key cofactor NADPH and up-regulated the expression of sucA, car, and yqhD by substituting with strong promoters to increase the efficiency of supplying precursors to 1,4-butanediol synthesis. Eventually, the recombinant strain produced up to 770 mg/L of 1,4-butanediol within 48 h in a shake flask, and 4.22 g/L of 1,4-butanediol within 60 h in a 5 L fermenter with a yield of 12.46 mg/g glucose. Compared with the previously reported method, the novel path designed in this study for the de novo synthesis of 1,4-butanediol does not need acetyl coenzyme A and avoids the byproduct acetate or the addition of ammonia. Therefore, the outcome is expected to provide a new idea for the metabolic engineering of microbial chassis for the production of 1,4-butanediol and its high-value derivatives.
Assuntos
Butileno Glicóis , Escherichia coli , Engenharia Metabólica , Butileno Glicóis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , OxirredutasesRESUMO
The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca2+-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K142A- and K142D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K142D-PqqT binds tightly (Kd = 0.6 ± 0.2 µM) to La3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K142A-variants or upon addition of Ca2+ to PQQ ⸦ K142D-PqqT. Addition of benzyl alcohol to La3+-bound PQQ ⸦ K142D-PqqT (but not Ca2+-bound PQQ ⸦ K142D-PqqT, or La3+-bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.
Assuntos
Methylobacterium extorquens , Methylobacterium extorquens/enzimologia , Methylobacterium extorquens/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/química , Cristalografia por Raios X , Cofator PQQ/metabolismo , Cofator PQQ/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Metais Terras Raras/química , Metais Terras Raras/metabolismo , Modelos Moleculares , Lantânio/química , Lantânio/metabolismoRESUMO
This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60-99%) and high selectivities (ee > 95%). The best bioreductions-in terms of conversion (>90%) and enantiomeric excess (>99% ee)-at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions.
Assuntos
Álcool Desidrogenase , Oxirredução , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Estereoisomerismo , Rhodococcus/enzimologia , Rhodococcus/metabolismo , Lactobacillus/metabolismo , Lactobacillus/enzimologia , Biocatálise , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , Propanóis/metabolismo , Propanóis/químicaRESUMO
2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.
Assuntos
Oxirredutases do Álcool , Butileno Glicóis , Etanol , Butileno Glicóis/metabolismo , Butileno Glicóis/química , Etanol/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/química , NADH NADPH Oxirredutases/metabolismo , NADH NADPH Oxirredutases/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/químicaRESUMO
Formaldehyde (FA) is a carcinogen that is not only widespread in the environment, but is also produced endogenously by metabolic processes. In organisms, FA is converted to formic acid in a glutathione (GSH)-dependent manner by alcohol dehydrogenase 5 (ADH5). The abnormal accumulation of FA in the body can cause a variety of diseases, especially cognitive impairment leading to Alzheimer's disease (AD). In this study, melatonin derivative 6a (MD6a) markedly improved the survival and chemotactic performance of wild-type Caenorhabditis elegans exposed to high concentrations of FA. MD6a lowered FA levels in the nematodes by enhancing the release of covalently-bound GSH from S-hydroxymethyl-GSH in an adh-5-dependent manner. In addition, MD6a protected against mitochondrial dysfunction and cognitive impairment in beta-amyloid protein (Aß) transgenic nematodes by lowering endogenous FA levels and reducing Aß aggregation in an adh-5-dependent manner. Our findings suggest that MD6a detoxifies FA via ADH5 and protects against Aß toxicity by reducing endogenous FA levels in the C. elegans AD models. Thus, ADH5 might be a potential therapeutic target for FA toxicity and AD.
Assuntos
Álcool Desidrogenase , Doença de Alzheimer , Peptídeos beta-Amiloides , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Formaldeído , Melatonina , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Melatonina/farmacologia , Formaldeído/toxicidade , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Animais Geneticamente Modificados , Glutationa/metabolismo , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Humanos , FormiatosRESUMO
OBJECTIVE: This study aims to explore ADH4 expression in hepatocellular carcinoma (HCC), its prognostic impact, and its immune correlation to provide novel insights into HCC prognostication and treatment. METHODS: HCC prognostic marker genes were rigorously selected using GEO database, Lasso regression, GEPIA, Kaplan-Meier and pROC analyses. The expression of interested markers (ADH4, DNASE1L3, RDH16, LCAT, HGFAC) in HCC and adjacent tissues was assessed by Immunohistochemistry (IHC). We observed that ADH4 exhibited low expression levels in liver cancer tissues and high expression levels in normal liver tissues. However, the remaining four genes did not manifest any statistically significant differences between hepatocellular carcinoma (HCC) tissue and adjacent non-cancerous tissue. Consequently, ADH4 became the primary focus of our research. ADH4 expression was validated by signed-rank tests and unpaired Wilcoxon rank sum tests across pan-cancer and HCC datasets. Clinical significance and associations with clinicopathological variables were determined using Kaplan-Meier, logistic regression and Cox analyses on TCGA data. The ADH4-related immune responses were explored by Spearman correlation analysis using TIMER2 data. CD68, CD4, and CD19 protein levels were confirmed by IHC in HCC and non-cancerous tissues. RESULTS: ADH4 showed significant downregulation in various cancers, particularly in HCC. Moreover, low ADH4 expression was associated with clinicopathological variables and served as an independent prognostic marker for HCC patients. Additionally, ADH4 affects a variety of biochemical functions and may influence cancer development, prognosis, and treatment by binding to immune cells. Furthermore, at the immune level, the low expression pattern of ADH4 is TME-specific, indicating that ADH4 has the potential to be used as a target for cancer immunotherapy. CONCLUSION: This study highlights the diagnostic, prognostic and immunomodulatory roles of ADH4 in HCC. ADH4 could serve as a valuable biomarker for HCC diagnosis and prognosis, as well as a potential target for immunotherapeutic interventions.
Assuntos
Álcool Desidrogenase , Biomarcadores Tumorais , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/mortalidade , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/mortalidade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Prognóstico , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Masculino , Feminino , Regulação Neoplásica da Expressão Gênica , Estimativa de Kaplan-MeierRESUMO
Hepatocellular carcinoma (HCC) is the primary malignant tumor of the liver. c-Myc is one of the most common oncogenes in clinical settings, and amplified levels of c-Myc are frequently found in HCC. Histone deacetylase inhibitors (HDACi), such as Trichostatin A (TSA), hold enormous promise for the treatment of HCC. However, the potential and mechanism of TSA in the treatment of c-Myc-induced HCC are unclear. In this study, we investigated the effects of TSA treatment on a c-Myc-induced HCC model in mice. TSA treatment delayed the development of HCC, and liver function indicators such as ALT, AST, liver weight ratio, and spleen weight ratio demonstrated the effectiveness of TSA treatment. Oil red staining further demonstrated that TSA attenuated lipid accumulation in the HCC tissues of mice. Through mRNA sequencing, we identified that TSA mainly affected cell cycle and fatty acid degradation genes, with alcohol dehydrogenase 4 (ADH4) potentially being the core molecular downstream target. QPCR, immunohistochemistry, and western blot analysis revealed that ADH4 expression was repressed by c-Myc and restored after TSA treatment both in vitro and in vivo. Furthermore, we observed that the levels of total NAD+ and NADH, NAD+, NAD+/NADH, and ATP concentration increased after c-Myc transfection in liver cells but decreased after TSA intervention. The levels of phosphorylated protein kinase B (p-AKT) and p-mTOR were identified as targets regulated by TSA, and they governed the ADH4 expression and the downstream regulation of total NAD+ and NADH, NAD+, NAD+/NADH, and ATP concentration. Overall, our study suggests that TSA has a therapeutic effect on c-Myc-induced HCC through the AKT-mTOR-ADH4 pathway. These findings provide valuable insights into the potential treatment of HCC using TSA and shed light on the underlying molecular mechanisms involved.
Assuntos
Carcinoma Hepatocelular , Ácidos Hidroxâmicos , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-myc , Animais , Camundongos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Masculino , Progressão da Doença , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by â¼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.
Assuntos
Álcool Desidrogenase , Biocatálise , Domínio Catalítico , DNA , Engenharia de Proteínas , Engenharia de Proteínas/métodos , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/química , DNA/metabolismo , DNA/química , DNA/genética , Especificidade por SubstratoRESUMO
Synthetic biohybrid systems by coupling artificial system with nature's machinery may offer a disruptive solution to address the global energy crisis. We developed a versatile electroenzymatic pathway for the continuous synthesis of valuable chemicals, facilitated by formate-driven NADH regeneration. Utilizing a bismuth electrocatalyst, we achieved stable CO2 reduction to formate with approximately 90 % Faraday efficiency at a current density of 150â mA cm-2. The generated formate acts as a mediator to regenerate NADH, which is then coupled with immobilized redox enzymes-alcohol dehydrogenase (ADH), L-lactate dehydrogenase (LDH), and L-glutamate dehydrogenase (GDH)-to produce targeted chemicals at significant rates and exceptionally high turnover numbers (1.8×106 to 3.1×106). These achievements not only underscore the efficiency of the system but also its practical applicability in industrial settings. By leveraging in situ generated formate, this innovative approach demonstrates the potential of integrating electrocatalysis with enzymatic reactions for sustainable and efficient chemical production on a practical scale.
Assuntos
Técnicas Eletroquímicas , Formiatos , NAD , NAD/química , NAD/metabolismo , Formiatos/química , Formiatos/metabolismo , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Glutamato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/química , Oxirredução , Bismuto/química , Catálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/metabolismoRESUMO
Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase enzyme, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.
Assuntos
Álcool Desidrogenase , Clostridium thermocellum , Etanol , Clostridium thermocellum/metabolismo , Clostridium thermocellum/genética , Etanol/metabolismo , Etanol/farmacologia , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Mutação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Engenharia Metabólica/métodosRESUMO
Alcohol dehydrogenase (ADH) is an important enzyme that catalyzes alcohol oxidation and/or aldehyde reduction. As one of NAD+-dependent ADH types, iron-containing/activated ADH (Fe-ADH) is ubiquitous in Bacteria, Archaea, and Eukaryotes, possessing a similar "tunnel-like" structure that is composed of a domain A in its N-terminus and a domain B in its C-terminus. A conserved "GGGS" sequence in the domain A of Fe-ADH associates with NAD+, and one conserved Asp residue and three conserved His residues in the domain B are its catalytic active sites by surrounding with Fe atom, suggesting that it might employ similar catalytic mechanism. Notably, all the biochemically characterized Fe-ADHs from hyperthermophiles that thrive in above 80 °C possess two unique characteristics that are absent in other Fe-ADHs: thermophilicity and thermostability, thereby demonstrating that they can oxidize alcohol and reduce aldehyde at high temperature. Considering these two unique characteristics, Fe-ADHs from hyperthermophiles are potentially industrial biocatalysts for alcohol and aldehyde biotransformation at high temperature. Herein, we reviewed structural and biochemical characteristics of Fe-ADHs from hyperthermophiles, focusing on similarity and difference between Fe-ADHs from hyperthermophiles and their homologs from non-hyperthermophiles, and between hyperthermophilic archaeal Fe-ADHs and bacterial homologs. Furthermore, we proposed future directions of Fe-ADHs from hyperthermophiles.
Assuntos
Álcool Desidrogenase , Estabilidade Enzimática , Ferro , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Ferro/metabolismo , Ferro/química , Archaea/enzimologia , Domínio Catalítico , Modelos Moleculares , Temperatura Alta , OxirreduçãoRESUMO
Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal-organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD+) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe3O4/NAD+ (ZIF-8@Fe3O4/NAD+) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe3O4/NAD+ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe3O4/NAD+ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe3O4/NAD+ENF after ten cycles. The magnetic metal-organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications.
Assuntos
Acetoína , Biomassa , Etanol , Estruturas Metalorgânicas , Etanol/metabolismo , Etanol/química , Estruturas Metalorgânicas/química , Acetoína/metabolismo , NAD/metabolismo , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Biocombustíveis , Álcool Desidrogenase/metabolismo , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/químicaRESUMO
Asymmetric reduction of 2-chloro-1-(6-fluorochroman-2-yl)ethan-1-one (NEB-7) into 2-chloro-1-(6-fluorochroman-2-yl)ethan-1-ol (NEB-8) is the crucial step for synthesis of liposoluble ß1 receptor blocker nebivolol. Four efficient and stereoselective alcohol dehydrogenases were identified, enabling the stereoselective synthesis of all enantiomers of NEB-8 at a substrate loading of 137 g·L-1 with ee values of >99% and high space-time yields. This study provides novel biocatalysts for the efficient synthesis of nebivolol precursors and uncovers the molecular basis for enantioselectivity manipulation by parametrization of Prelog's rule.
Assuntos
Biocatálise , Nebivolol , Nebivolol/química , Estereoisomerismo , Estrutura Molecular , Antagonistas de Receptores Adrenérgicos beta 1/química , Antagonistas de Receptores Adrenérgicos beta 1/síntese química , Álcool Desidrogenase/antagonistas & inibidores , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/químicaRESUMO
Honey bees (Apis mellifera) are one of the most crucial pollinators, providing vital ecosystem services. Their development and functioning depend on essential nutrients and substances found in the environment. While collecting nectar as a vital carbohydrate source, bees routinely encounter low doses of ethanol from yeast fermentation. Yet, the effects of repeated ethanol exposure on bees' survival and physiology remain poorly understood. Here, we investigate the impacts of constant and occasional consumption of food spiked with 1% ethanol on honey bee mortality and alcohol dehydrogenase (ADH) activity. This ethanol concentration might be tentatively judged close to that in natural conditions. We conducted an experiment in which bees were exposed to three types of long-term diets: constant sugar solution (control group that simulated conditions of no access to ethanol), sugar solution spiked with ethanol every third day (that simulated occasional, infrequent exposure to ethanol) and daily ethanol consumption (simulating constant, routine exposure to ethanol). The results revealed that both constant and occasional ethanol consumption increased the mortality of bees, but only after several days. These mortality rates rose with the frequency of ethanol intake. The ADH activity remained similar in bees from all groups. Our findings indicate that exposure of bees to ethanol carries harmful effects that accumulate over time. Further research is needed to pinpoint the exact ethanol doses ingested with food and exposure frequency in bees in natural conditions.
Assuntos
Álcool Desidrogenase , Etanol , Longevidade , Animais , Abelhas/efeitos dos fármacos , Abelhas/fisiologia , Etanol/toxicidade , Álcool Desidrogenase/metabolismo , Longevidade/efeitos dos fármacos , Dieta/veterináriaRESUMO
Cryptosporidium parvum is a waterborne and foodborne zoonotic protozoan parasite, a causative agent of moderate to severe diarrheal diseases in humans and animals. However, fully effective treatments are unavailable for medical and veterinary uses. There is a need to explore new drug targets for potential development of new therapeutics. Because C. parvum relies on anaerobic metabolism to produce ATP, fermentative enzymes in this parasite are attractive targets for exploration. In this study, we investigated the ethanol-fermentation in the parasite and characterized the basic biochemical features of a bacterial-type bifunctional aldehyde/alcohol dehydrogenase, namely CpAdhE. We also screened 3892 chemical entries from three libraries and identified 14 compounds showing >50% inhibition on the enzyme activity of CpAdhE. Intriguingly, antifungal imidazoles and unsaturated fatty acids are the two major chemical groups among the top hits. We further characterized the inhibitory kinetics of selected imidazoles and unsaturated fatty acids on CpAdhE. These compounds displayed lower micromolar activities on CpAdhE (i.e., IC50 values ranging from 0.88 to 11.02 µM for imidazoles and 8.93 to 35.33 µM for unsaturated fatty acids). Finally, we evaluated the in vitro anti-cryptosporidial efficacies and cytotoxicity of three imidazoles (i.e., tioconazole, miconazole and isoconazole). The three antifungal imidazoles exhibited lower micromolar efficacies against the growth of C. parvum in vitro (EC50 values ranging from 4.85 to 10.41 µM and selectivity indices ranging from 5.19 to 10.95). The results provide a proof-of-concept data to support that imidazoles are worth being further investigated for potential development of anti-cryptosporidial therapeutics.
Assuntos
Antifúngicos , Cryptosporidium parvum , Imidazóis , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/enzimologia , Imidazóis/farmacologia , Imidazóis/química , Antifúngicos/farmacologia , Animais , Humanos , Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Ácidos Graxos Insaturados/farmacologia , Zoonoses , Criptosporidiose/tratamento farmacológicoRESUMO
Ethylene glycol (EG) is a widely used industrial chemical with manifold applications and also generated in the degradation of plastics such as polyethylene terephthalate. Rhodococcus jostii RHA1 (RHA1), a potential biocatalytic chassis, grows on EG. Transcriptomic analyses revealed four clusters of genes potentially involved in EG catabolism: the mad locus, predicted to encode mycofactocin-dependent alcohol degradation, including the catabolism of EG to glycolate; two GCL clusters, predicted to encode glycolate and glyoxylate catabolism; and the mft genes, predicted to specify mycofactocin biosynthesis. Bioinformatic analyses further revealed that the mad and mft genes are widely distributed in mycolic acid-producing bacteria such as RHA1. Neither ΔmadA nor ΔmftC RHA1 mutant strains grew on EG but grew on acetate. In resting cell assays, the ΔmadA mutant depleted glycolaldehyde but not EG from culture media. These results indicate that madA encodes a mycofactocin-dependent alcohol dehydrogenase that initiates EG catabolism. In contrast to some mycobacterial strains, the mad genes did not appear to enable RHA1 to grow on methanol as sole substrate. Finally, a strain of RHA1 adapted to grow ~3× faster on EG contained an overexpressed gene, aldA2, predicted to encode an aldehyde dehydrogenase. When incubated with EG, this strain accumulated lower concentrations of glycolaldehyde than RHA1. Moreover, ecotopically expressed aldA2 increased RHA1's tolerance for EG further suggesting that glycolaldehyde accumulation limits growth of RHA1 on EG. Overall, this study provides insights into the bacterial catabolism of small alcohols and aldehydes and facilitates the engineering of Rhodococcus for the upgrading of plastic waste streams.IMPORTANCEEthylene glycol (EG), a two-carbon (C2) alcohol, is produced in high volumes for use in a wide variety of applications. There is burgeoning interest in understanding and engineering the bacterial catabolism of EG, in part to establish circular economic routes for its use. This study identifies an EG catabolic pathway in Rhodococcus, a genus of bacteria well suited for biocatalysis. This pathway is responsible for the catabolism of methanol, a C1 feedstock, in related bacteria. Finally, we describe strategies to increase the rate of degradation of EG by increasing the transformation of glycolaldehyde, a toxic metabolic intermediate. This work advances the development of biocatalytic strategies to transform C2 feedstocks.
Assuntos
Proteínas de Bactérias , Etilenoglicol , Rhodococcus , Rhodococcus/metabolismo , Rhodococcus/genética , Etilenoglicol/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Glicolatos/metabolismo , Glioxilatos/metabolismo , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , PeptídeosRESUMO
Alcohol dehydrogenase 1B (ADH1B) is a primate-specific enzyme which, uniquely among the ADH class 1 family, is highly expressed both in adipose tissue and liver. Its expression in adipose tissue is reduced in obesity and increased by insulin stimulation. Interference with ADH1B expression has also been reported to impair adipocyte function. To better understand the role of ADH1B in adipocytes, we used CRISPR/Cas9 to delete ADH1B in human adipose stem cells (ASC). Cells lacking ADH1B failed to differentiate into mature adipocytes manifested by minimal triglyceride accumulation and a marked reduction in expression of established adipocyte markers. As ADH1B is capable of converting retinol to retinoic acid (RA), we conducted rescue experiments. Incubation of ADH1B-deficient preadipocytes with 9-cis-RA, but not with all-transretinol, significantly rescued their ability to accumulate lipids and express markers of adipocyte differentiation. A homozygous missense variant in ADH1B (p.Arg313Cys) was found in a patient with congenital lipodystrophy of unknown cause. This variant significantly impaired the protein's dimerization, enzymatic activity, and its ability to rescue differentiation in ADH1B-deficient ASC. The allele frequency of this variant in the Middle Eastern population suggests that it is unlikely to be a fully penetrant cause of severe lipodystrophy. In conclusion, ADH1B appears to play an unexpected, crucial and cell-autonomous role in human adipocyte differentiation by serving as a necessary source of endogenous retinoic acid.