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The widespread use of plastic mulch film (PMF) has led to significant environmental pollution, with PMF residues dispersed and mixed with straw and soil, posing challenges for recycling. Here, we proposed the mobile pyrolysis facility for the cotton straw and mulch film mixture (CMM) to mitigate the collection, storage, and transportation costs, while the application of co-pyrolysis technology for CMM conversion could improve the added value of products. Additionally, centralized combustion power generation and centralized pyrolysis systems were also established to evaluate and compare their sustainability from economic and environmental perspectives. Results showed that mobile pyrolysis has better economic performance than the centralized scenarios, due to its high internal rate of return (31 %) and significant net present value (29.21 M USD). Meanwhile, the mobile pyrolysis facility achieved a GWP of -1.298 kgCO2-eq/kg, reducing emissions by 70.79 % and 38.82 % compared to the two centralized scenarios. In conclusion, mobile pyrolysis technology provides a promising solution for PMF residue recycling because of its economically competitive approach with a lower carbon footprint.
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Plásticos , Pirólisis , Reciclaje , Residuos Sólidos , China , Reciclaje/métodos , Residuos Sólidos/análisis , Gossypium , Administración de Residuos/métodos , Eliminación de Residuos/métodosRESUMEN
Monitoring intracellular pyruvate is useful for the exploration of fundamental metabolism and for guiding the construction of yeast cell factories for chemical production. Here, we employed a genetically encoded fluorescent Pyronic biosensor to light up the pyruvate metabolic state in the cytoplasm, nucleus, and mitochondria of Saccharomyces cerevisiae BY4741. A strong correlation was observed between the pyruvate fluctuation in mitochondria and cytoplasm when exposed to different metabolites. Further metabolic analysis of pyruvate uptake and glycolytic dynamics showed that glucose and fructose dose-dependently activated cytoplasmic pyruvate levels more effectively than direct exposure to pyruvate. Meanwhile, the Pyronic biosensor could visually distinguish phenotypes of the wild-type S. cerevisiae BY4741 and the pyruvate-hyperproducing S. cerevisiae TAM at a single-cell resolution, having the potential for high-throughput screening. Overall, Pyronic biosensors targeting different suborganelles contribute to mapping and studying the central carbon metabolism in-depth and guide the design and construction of yeast cell factories.
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Técnicas Biosensibles , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Glucólisis , Ácido Pirúvico/metabolismoRESUMEN
Rhodococcus biphenylivorans GA1 was successfully isolated, which can efficiently degrade alkali lignin and a variety of lignin-derived aromatic compounds as the sole carbon source. Whole genome sequencing of strain GA1 showed that it possessed G + C content of 68% with the size of 6.0 Mb and 4319 putative open reading frames (ORFs). Four replicons consisting of one circular chromosome (ChrA1) and three circular plasmids (pGA1, pGA2, pGA3) were found. Among these annotated proteins, lignin depolymerizing peroxidases (Dyp) and two lignin-derived aromatic compounds cleavage dioxygenases, protocatechuate 3,4-dioxygenase(P34D) and catechol-1,2-dioxygenase (C12D) play key roles in the catabolism of lignin.
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This paper describes an amperometric biosensor utilizing an engineered l-glutamate oxidase for glutamate monitoring in microbial fermentation processes. We designed a general immobilization strategy that utilized a chitin-binding domain (ChBD-tag) as a biotether to further immobilize l-glutamate oxidase (GLOX) in an oriented manner on a screen-printed Prussian blue nanocube microchip (PB/SPC) with the biopolymer chitosan. The improved l-glutamate biosensor exhibited an enhanced sensitivity of 53.4 µA L mmol-1 cm-2 and a linear range from 25 µmol/L to 300 µmol/L with a detection limit of 9 µmol/L, and retained 95 % of its initial activity after two weeks of usage. In addition, the as-prepared biosensor was applied for real-time monitoring of food ingredient l-glutamate concentration during the fermentation process, which was in good agreement with that of high-performance liquid chromatography (HPLC). Above all, the l-glutamate biosensor prepared by this method had high analytical performance, and could fully realize real-time and high-efficiency monitoring in glutamate fermentation.
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Técnicas Biosensibles , Ácido Glutámico , Fermentación , Enzimas Inmovilizadas/química , Técnicas Biosensibles/métodos , ElectrodosRESUMEN
As an important biosynthesis technology, electron-assisted biosynthesis (EABS) system can utilize exogenous electrons to regulate the metabolic network of microorganisms, realizing the biosynthesis of high value-added chemicals and CO2 fixation. Electrons play crucial roles as the energy carriers in the EABS process. In fact, efficient interfacial electron transfer (ET) is the decisive factor to realize the rapid energy exchange, thus stimulating the biosynthesis of target metabolic products. However, due to the interfacial resistance of ET between the abiotic solid electrode and biotic microbial cells, the low efficiency of interfacial ET has become a major bottleneck, further limiting the practical application of EABS system. As the cell membrane is insulated, even the cell membrane embedded electron conduit (no matter cytochromes or channel protein for shuttle transferring) to increase the cell membrane conductivity, the ET between membrane electron conduit and electrode surface is kinetically restricted. In this review, the pathway of bi-directional interfacial ET in EABS system was summarized. Furthermore, we reviewed representative milestones and advances in both the anode outward interfacial ET (from organism to electrode) and cathode inward interfacial ET (from electrode to organism). Here, new insights from the perspectives of material science and synthetic biology were also proposed, which were expected to provide some innovative opinions and ideas for the following in-depth studies.
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Electrones , Biología Sintética , Electrodos , Transporte de ElectrónRESUMEN
Bioconversion of acetate, a byproduct generated in industrial processes, into microbial lipids using oleaginous yeasts offers a promising alternative for the economic utilization of acetate-containing waste streams. However, high acetate concentrations will inhibit microbial growth and metabolism. In this study, the acetate utilization capability of Yarrowia lipolytica PO1f was successively improved by overexpressing the key enzyme of acetyl-CoA synthetase (ACS), which resulted in an accumulation of 9.2% microbial lipids from acetate in shake flask fermentation. By further overexpressing the second key enzymes of acetyl-CoA carboxylase (ACC1) and fatty acid synthase (FAS) in Y. lipolytica, the lipid content was increased to 25.7% from acetate. Finally, the maximum OD600 of 29.2 and a lipid content of 41.7% were obtained with the engineered strain by the adoption of cosubstrate (glycerol and acetate) fed-batch fermentation, which corresponded to an increase of 68 and 95%, respectively. These results presented a promising strategy for economic and efficient microbial lipid production from the waste acetate.
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Acetatos/metabolismo , Fermentación/genética , Lípidos/genética , Yarrowia/genética , Acetilcoenzima A/genética , Glicerol/metabolismo , Ingeniería Metabólica/métodos , Yarrowia/metabolismoRESUMEN
Microbial consortia are ubiquitous in nature and exhibit several attractive features such as sophisticated metabolic capabilities and strong environmental robustness. This study aimed to decipher the metabolic and ecological characteristics of synergistic interactions in acetamiprid-degrading consortia, suggesting an optimal scheme for bioremediation of organic pollutants. The microbial consortium ACE-3 with excellent acetamiprid-degrading ability was enriched from the soil of an acetamiprid-contaminated site and characterized using high-throughput sequencing (HTS). Consortium ACE-3 was able to completely degrade 50 mgâ L-1 acetamiprid in 144 h, and was metabolically active at a wide range of pH values (6.0-8.0) and temperatures (20-42°C). Furthermore, plausible metabolic routes of acetamiprid biodegradation by the consortium were proposed based on the identification of intermediate metabolites (Compounds I, II, III and IV). The findings indicated that the consortium ACE-3 has promising potential for the removal and detoxification of pesticides because it produces downstream metabolites (Compounds I and II) that are less toxic to mammals and insects than acetamiprid. Finally, Illumina HTS revealed that ß Proteobacteria were the dominant group, accounting for 85.61% of all sequences at the class level. Among the more than 50 genera identified in consortium ACE-3, Sphingobium, Acinetobacter, Afipia, Stenotrophomonas, and Microbacterium were dominant, respectively accounting for 3.07, 10.01, 24.45, and 49.12% of the total population.
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Lignin, one of the most abundant natural polymers, has been successfully used as an effective lubricant additive with high value. The chemical structure of lignin is very diverse and strongly affected by both the source of lignin (i.e. plant species) and the lignin extraction process. In this work, a series of lignin from different biomass sources (hard or soft wood) and extraction process (organosolv with or without acid catalyst) has been successfully incorporated into poly(ethylene glycol) (PEG) and fortified lubricating properties were achieved. The effects of different lignin on the rheological, thermal and tribological properties of the lignin/EG lubricants were systematically investigated by different characterization techniques. Lignin in PEG significantly improves the lubricating property, where a wear reduction of 93.8% was observed. The thermal and lubrication properties of the PEG lubricants filled with different kinds of lignin are tightly related to the synergistic state of hydrogen bonding and molecular weight distribution. Lignin with broader molecular weight distribution and higher hydroxyl content shows better adhesion on metal surfaces and strengthened lubricating film, which could be used as the efficient lubricating additives. This work provides a criterion for selecting appropriate lignin as the efficient lubricant additive and accelerates the application of lignin.
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Lignina/química , Lubricantes/química , Polietilenglicoles/química , Enlace de Hidrógeno , Peso Molecular , ViscosidadRESUMEN
Ethylene glycol (EG)-based lubricant was prepared with dissolved organosolv lignin from birch wood (BL) and softwood (SL) biomass. The effects of different lignin types on the rheological, thermal, and tribological properties of the lignin/EG lubricants were comprehensively investigated by various characterization techniques. Dissolving organosolv lignin in EG results in outstanding lubricating properties. Specifically, the wear volume of the disc by EG-44BL is only 8.9% of that lubricated by pure EG. The enhanced anti-wear property of the EG/lignin system could be attributed to the formation of a robust lubrication film and the strong adhesion of the lubricant on the contacting metal surface due to the presence of a dense hydrogen bonding (H-bonding) network. The lubricating performance of EG-BL outperforms EG-SL, which could be attributed to the denser H-bonding sites in BL and its broader molecular weight distribution. The disc wear loss of EG-44BL is only 45.7% of that lubricated by EG-44SL. Overall, H-bonding is the major contributor to the different tribological properties of BL and SL in EG-based lubricants.
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Glicol de Etileno/química , Lignina/química , Lubricantes/química , Madera/química , Biomasa , Peso Molecular , Solventes , Espectroscopía Infrarroja por Transformada de Fourier , ViscosidadRESUMEN
MAIN CONCLUSION: Loss of function mutation of rice OsACOS12 impairs lipid metabolism-mediated anther cuticle and pollen wall formation, and interferes with tapetum programmed cell death, leading to male sterility. Acyl-CoA Synthetase (ACOS) is one of the enzymes activating fatty acids for various metabolic functions in plants. Here, we show that OsACOS12, an orthologue of Arabidopsis ACOS5 in rice, is crucial for rice fertility. Similar to acos5, osaocs12 mutant had no mature pollen. But unlike acos5, osaocs12 produced defective anthers lacking cutin and Ubisch bodies on the epidermal and inner surfaces, respectively, and delayed programmed cell death (PCD)-induced tapetum degradation. Those phenotypic changes were evident at stage 10, during which OsACOS12 had its maximum expression in tapetal cells and microspores. Chemical analysis revealed that the levels of anther cuticular lipid components (wax and cutin monomers) were significantly reduced in osaocs12, while the expression levels of three known lipid biosynthetic genes were unchanged. Recombinant OsACOS12 enzyme was shown to catalyze the conversion of C18:1 fatty acid to C18:1 CoA in vitro. Phylogenetic analysis indicated that OsACOS12 is an ancient and conserved enzyme associated with the plant's colonization to earth. Collectively, our study suggests that OsACOS12 is an ancient enzyme participating in a conserved metabolic pathway for diversified biochemical functions to secure male reproduction in plants.
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Apoptosis/fisiología , Coenzima A Ligasas/metabolismo , Oryza/enzimología , Oryza/fisiología , Infertilidad Vegetal/fisiología , Proteínas de Plantas/metabolismo , Apoptosis/genética , Coenzima A Ligasas/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Oryza/genética , Infertilidad Vegetal/genética , Proteínas de Plantas/genéticaRESUMEN
Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ΔybgC and ΔentH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.
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Vías Biosintéticas/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Tioléster Hidrolasas/metabolismo , Vitamina K 2/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Eliminación de Gen , Naftoles/metabolismo , Especificidad por Sustrato , Tioléster Hidrolasas/genéticaRESUMEN
Contrary to the helical carbon structure from pure cotton fabrics under microwave heating and radical oxidized ignition of nanoparticles from conventional heating, magnetic carbon tubular nanocomposite fabrics decorated with uniformly dispersed Co-Co(3)O(4) nanoparticles were successfully synthesized via a microwave heating process using cotton fabric and inorganic salt as precursors, which have shown better anti-corrosive performance and demonstrated great potential as novel electrochemical pseudocapacitor electrode.
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Magnetic carbon nanostructures from microwave assisted- and conventional-pyrolysis processes are compared. Unlike graphitized carbon shells from conventional heating, different carbon shell morphologies including nanotubes, nanoflakes and amorphous carbon were observed. Crystalline iron and cementite were observed in the magnetic core, different from a single cementite phase from the conventional process.
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Carbono/química , Magnetismo , Microondas , Nanoestructuras/química , Nanopartículas de Magnetita/química , Nanotubos/química , TemperaturaRESUMEN
Artemisinins are proposed to act in the malaria parasite cytosol by oxidizing dihydroflavin cofactors of redox-active flavoenzymes, and under aerobic conditions by inducing their autoxidation. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) ensues. Ascorbic acid-methylene blue (MB), N-benzyl-1,4-dihydronicotinamide (BNAH)-MB, BNAH-lumiflavine, BNAH-riboflavin (RF), and NADPH-FAD-E. coli flavin reductase (Fre) systems at pH 7.4 generate leucomethylene blue (LMB) and reduced flavins that are rapidly oxidized in situ by artemisinins. These oxidations are inhibited by the 4-aminoquinolines piperaquine (PPQ), chloroquine (CQ), and others. In contrast, the arylmethanols lumefantrine, mefloquine (MFQ), and quinine (QN) have little or no effect. Inhibition correlates with the antagonism exerted by 4-aminoquinolines on the antimalarial activities of MB, RF, and artemisinins. Lack of inhibition correlates with the additivity/synergism between the arylmethanols and artemisinins. We propose association via π complex formation between the 4-aminoquinolines and LMB or the dihydroflavins; this hinders hydride transfer from the reduced conjugates to the artemisinins. The arylmethanols have a decreased tendency to form π complexes, and so exert no effect. The parallel between chemical reactivity and antagonism or additivity/synergism draws attention to the mechanism of action of all drugs described herein. CQ and QN inhibit the formation of hemozoin in the parasite digestive vacuole (DV). The buildup of heme-Fe(III) results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme-Fe(III) complexes of CQ and QN that form in the DV are proposed to diffuse across the DV membrane. At the higher pH of the cytosol, the complexes decompose to liberate heme-Fe(III) . The quinoline or arylmethanol reenters the DV, and so transfers more heme-Fe(III) out of the DV. In this way, the 4-aminoquinolines and arylmethanols exert antimalarial activities by enhancing heme-Fe(III) and thence free Fe(III) concentrations in the cytosol. The iron species enter into redox cycles through reduction of Fe(III) to Fe(II) largely mediated by reduced flavin cofactors and likely also by NAD(P)H-Fre. Generation of ROS through oxidation of Fe(II) by oxygen will also result. The cytotoxicities of artemisinins are thereby reinforced by the iron. Other aspects of drug action are emphasized. In the cytosol or DV, association by π complex formation between pairs of lipophilic drugs must adversely influence the pharmacokinetics of each drug. This explains the antagonism between PPQ and MFQ, for example. The basis for the antimalarial activity of RF mirrors that of MB, wherein it participates in redox cycling that involves flavoenzymes or Fre, resulting in attrition of NAD(P)H. The generation of ROS by artemisinins and ensuing Fenton chemistry accommodate the ability of artemisinins to induce membrane damage and to affect the parasite SERCA PfATP6 Ca(2+) transporter. Thus, the effect exerted by artemisinins is more likely a downstream event involving ROS that will also be modulated by mutations in PfATP6. Such mutations attenuate, but cannot abrogate, antimalarial activities of artemisinins. Overall, parasite resistance to artemisinins arises through enhancement of antioxidant defense mechanisms.
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Antimaláricos/farmacología , Artemisininas/farmacología , Interacciones Farmacológicas , Cloroquina/farmacología , Compuestos Férricos/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Malaria/tratamiento farmacológico , Azul de Metileno/farmacología , NAD/análogos & derivados , NAD/metabolismo , NADP/metabolismo , Estrés Oxidativo/efectos de los fármacos , Quinolinas/metabolismo , Riboflavina/metabolismoRESUMEN
Artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB); they also oxidize dihydroflavins such as the reduced conjugates RFH2 of riboflavin (RF), and FADH2 of the cofactor flavin adenine dinucleotide (FAD), to the corresponding flavins. Like the artemisinins, MB oxidizes FADH2, but unlike artemisinins, it also oxidizes NAD(P)H. Like MB, artemisinins are implicated in the perturbation of redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases. The oxidation of LMB by artemisinin is inhibited by chloroquine (CQ), an inhibition that is abruptly reversed by verapamil (VP). CQ also inhibits artemisinin-mediated oxidation of RFH2 generated from N-benzyl-1,4-dihydronicotinamide (BNAH)-RF, or FADH2 generated from NADPH or NADPH-Fre, an effect that is also modulated by verapamil. The inhibition likely proceeds by the association of LMB or dihydroflavin with CQ, possibly involving donor-acceptor or πâ complexes that hinder oxidation by artemisinin. VP competitively associates with CQ, liberating LMB or dihydroflavin from their respective CQ complexes. The observations explain the antagonism between CQ-MB and CQ-artemisinins inâ vitro, and are reconcilable with CQ perturbing intraparasitic redox homeostasis. They further suggest that a VP-CQ complex is a means by which VP reverses CQ resistance, wherein such a complex is not accessible to the putative CQ-resistance transporter (PfCRT).
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Antimaláricos/farmacología , Artemisininas/farmacología , Cloroquina/antagonistas & inhibidores , Malaria/tratamiento farmacológico , Azul de Metileno/farmacología , Verapamilo/farmacología , Animales , Antimaláricos/química , Artemisininas/química , Cloroquina/química , Cloroquina/farmacología , Resistencia a Medicamentos , Sinergismo Farmacológico , Flavina-Adenina Dinucleótido/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Malaria/metabolismo , Malaria/patología , Azul de Metileno/química , Oxidación-Reducción/efectos de los fármacos , Verapamilo/químicaRESUMEN
1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small molecule ligands suggests that the disordered loop is folded in interaction with the substrates or reaction intermediates, supporting an induced-fit catalytic mechanism for the enzyme.
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Ácido Aspártico , Secuencia Conservada , Oxo-Ácido-Liasas/química , Oxo-Ácido-Liasas/metabolismo , Oxígeno/metabolismo , Análisis Espectral , Vitamina K 2/metabolismo , Absorción , Bacterias/enzimología , Dominio Catalítico , Coenzima A/química , Coenzima A/metabolismo , Coenzima A/farmacología , Estabilidad de Enzimas , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxo-Ácido-Liasas/antagonistas & inhibidores , Oxo-Ácido-Liasas/genética , Oxígeno/química , Fenoles/química , Fenoles/metabolismo , ProtonesRESUMEN
Flavin adenine dinucleotide (FAD) is reduced by NADPH-E.â coli flavin reductase (Fre) to FADH(2) in aqueous buffer at pHâ 7.4 under argon. Under the same conditions, FADH(2) in turn cleanly reduces the antimalarial drug methylene blue (MB) to leucomethylene blue. The latter is rapidly re-oxidized by artemisinins, thus supporting the proposal that MB exerts its antimalarial activity, and synergizes the antimalarial action of artemisinins, by interfering with redox cycling involving NADPH reduction of flavin cofactors in parasite flavin disulfide reductases. Direct treatment of the FADH(2) generated from NADPH-Fre-FAD by artemisinins and antimalaria-active tetraoxane and trioxolane structural analogues under physiological conditions at pHâ 7.4 results in rapid reduction of the artemisinins, and efficient conversion of the peroxide structural analogues into ketone products. Comparison of the relative rates of FADH(2) oxidation indicate optimal activity for the trioxolane. Therefore, the rate of intraparastic redox perturbation will be greatest for the trioxolane, and this may be significant in relation to its enhanced inâ vitro antimalarial activities. (1)Hâ NMR spectroscopic studies using the BNAH-riboflavin (RF) model system indicate that the tetraoxane is capable of using both peroxide units in oxidizing the RFH(2) generated inâ situ. Use of the NADPH-Fre-FAD catalytic system in the presence of artemisinin or tetraoxane confirms that the latter, in contrast to artemisinin, consumes two reducing equivalents of NADPH. None of the processes described herein requires the presence of ferrous iron. Ferric iron, given its propensity to oxidize reduced flavin cofactors, may play a role in enhancing oxidative stress within the malaria parasite, without requiring interaction with artemisinins or peroxide analogues. The NADPH-Fre-FAD system serves as a convenient mimic of flavin disulfide reductases that maintain redox homeostasis in the malaria parasite.
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Antimaláricos/química , FMN Reductasa/metabolismo , Flavinas/química , Azul de Metileno/análogos & derivados , Modelos Teóricos , Peróxidos/química , Azul de Metileno/químicaRESUMEN
The first committed step in the classical biosynthetic route to menaquinone (vitamin K(2)) is a Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 A) crystal structure of Bacillus subtilis MenD with cofactor and Mn(2+) has been determined. Based on structure-sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.
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Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Piruvato Oxidasa/química , Piruvato Oxidasa/metabolismo , Vitamina K 2/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Catálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , ADN Bacteriano/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Cuaternaria de Proteína , Piruvato Oxidasa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Sesquiterpenos , Especificidad de la Especie , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.
Asunto(s)
Bicarbonatos/química , Coenzimas/química , Escherichia coli/enzimología , Oxo-Ácido-Liasas/química , Vitamina K 2/química , Bicarbonatos/metabolismo , Sitios de Unión , Catálisis , Coenzimas/metabolismo , Escherichia coli/genética , Evolución Molecular , Cinética , Mutagénesis Sitio-Dirigida , Oxo-Ácido-Liasas/clasificación , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Homología Estructural de Proteína , Vitamina K 2/metabolismoRESUMEN
(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase (MenH) is an alpha/beta fold enzyme containing a catalytically essential serine-histidine-aspartate triad typical of serine proteases but catalyzes a pyruvate elimination reaction initiated by alpha-proton abstraction in the menaquinone biosynthetic pathway of Escherichia coli. In this study, we identify the active site residues in the synthase through sequence analysis and structural modeling and study their mechanistic roles in MenH catalysis. Steady-state kinetic characterization of site-directed mutants of the active site residues shows that three conserved arginine residues (Arg-90, Arg-124, and Arg-168) likely form ionic salt bridges with three carboxylate groups of the substrate in the Michaelis complex and that the side-chain polar groups of the conserved tyrosine (Tyr-85) and tryptophan (Trp-147) residues likely donate hydrogen bonds to form an "oxyanion hole". In addition, the pH dependence of the MenH kinetic properties reveals a catalytic base with a pK(a) highly dependent on the hydroxyl group of the triad serine residue in the enzymatic reaction. Moreover, proton inventory experiments demonstrate that the SHCHC synthase adopts one-proton catalysis like many serine proteases. These results allow the proposal of a mechanism in which the histidine residue of the MenH triad serves as a general base catalyst to deprotonate the triad seryl hydroxyl group in the alpha-proton abstraction from the substrate. As such, the MenH triad performs a simple and fundamental proton transfer reaction occurring repeatedly in the reactions catalyzed by serine proteases and alpha/beta fold hydrolases, suggesting a common evolutionary origin for all serine-histidine-aspartate triads serving different catalytic functions.