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Improvement of photosynthesis requires a thorough understanding of electron partitioning under both natural and strong electron sink conditions. We applied a wide array of state-of-the-art biophysical and biochemical techniques to thoroughly investigate the fate of photosynthetic electrons in the engineered cyanobacterium Synechocystis sp. PCC 6803, a blueprint for photosynthetic biotechnology, expressing the heterologous gene for ene-reductase, YqjM. This recombinant enzyme catalyses the reduction of an exogenously added substrate into the desired product by utilising photosynthetically produced NAD(P)H, enabling whole-cell biotransformation. Through coupling the biotransformation reaction with biophysical measurements, we demonstrated that the strong artificial electron sink, outcompetes the natural electron valves, the flavodiiron protein-driven Mehler-like reaction and cyclic electron transport. These results show that ferredoxin-NAD(P)H-oxidoreductase is the preferred route for delivering photosynthetic electrons from reduced ferredoxin and the cellular NADPH/NADP+ ratio as a key factor in orchestrating photosynthetic electron flux. These insights are crucial for understanding molecular mechanisms of photosynthetic electron transport and harnessing photosynthesis for sustainable bioproduction by engineering the cellular source/sink balance. Furthermore, we conclude that identifying the bioenergetic bottleneck of a heterologous electron sink is a crucial prerequisite for targeted engineering of photosynthetic biotransformation platforms.
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BACKGROUND: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon. RESULTS: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm. CONCLUSIONS: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.
Assuntos
Escherichia coli , Sacarose , beta-Frutofuranosidase , Escherichia coli/genética , Escherichia coli/metabolismo , beta-Frutofuranosidase/metabolismo , beta-Frutofuranosidase/genética , Sacarose/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Cicloexanonas/metabolismo , Oxirredução , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Elétrons , Biotransformação , Caproatos , LactonasRESUMO
BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.
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6-Fitase , Cupriavidus necator , Cupriavidus necator/metabolismo , 6-Fitase/genética , 6-Fitase/metabolismo , Dióxido de Carbono/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow decarboxylation reactions to be followed with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.
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Carboxiliases , Ácidos Cumáricos , Ácidos Cumáricos/química , Carboxiliases/química , FluorometriaRESUMO
Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.
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Cianobactérias , Elétrons , Biocatálise , Cianobactérias/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredução , Água/metabolismoRESUMO
Light-dependent enzymes are a rare type of biocatalyst with high potential for research and biotechnology. A recently discovered fatty acid photo-decarboxylase from Chlorella variabilis NC64A (CvFAP) converts fatty acids to the corresponding hydrocarbons only when irradiated with blue light (400 to 520â nm). To expand the available catalytic diversity for fatty acid decarboxylation, we reconstructed possible ancestral decarboxylases from a set of 12 extant sequences that were classified under the fatty acid decarboxylases clade within the glucose-methanol choline (GMC) oxidoreductase family. One of the resurrected enzymes (ANC1) showed activity in the decarboxylation of fatty acids, showing that the clade indeed contains several photo-decarboxylases. ANC1 has a 15 °C higher melting temperature (Tm ) than the extant CvFAP. Its production yielded 12-fold more protein than this wild type decarboxylase, which offers practical advantages for the biochemical investigation of this photoenzyme. Homology modelling revealed amino acid substitutions to more hydrophilic residues at the surface and shorter flexible loops compared to the wild type. Using ancestral sequence reconstruction, we have expanded the existing pool of confirmed fatty acid photo-decarboxylases, providing access to a more robust catalyst for further development via directed evolution.
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Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Oxirredutases/metabolismo , Proteínas de Bactérias/classificação , Chlorella/enzimologia , Descarboxilação , Ácidos Graxos/química , Simulação de Dinâmica Molecular , Oxirredutases/classificação , Filogenia , Estabilidade Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Temperatura de TransiçãoRESUMO
Racemic camphor and isoborneol are readily available as industrial side products, whereas (1R)-camphor is available from natural sources. Optically pure (1S)-camphor, however, is much more difficult to obtain. The synthesis of racemic camphor from α-pinene proceeds via an intermediary racemic isobornyl ester, which is then hydrolyzed and oxidized to give camphor. We reasoned that enantioselective hydrolysis of isobornyl esters would give facile access to optically pure isoborneol and camphor isomers, respectively. While screening of a set of commercial lipases and esterases in the kinetic resolution of racemic monoterpenols did not lead to the identification of any enantioselective enzymes, the cephalosporin Esterase B from Burkholderia gladioli (EstB) and Esterase C (EstC) from Rhodococcus rhodochrous showed outstanding enantioselectivity (E>100) towards the butyryl esters of isoborneol, borneol and fenchol. The enantioselectivity was higher with increasing chain length of the acyl moiety of the substrate. The kinetic resolution of isobornyl butyrate can be easily integrated into the production of camphor from α-pinene and thus allows the facile synthesis of optically pure monoterpenols from a renewable side-product.
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Monoterpenos Bicíclicos/química , Cânfora/síntese química , Monoterpenos Bicíclicos/metabolismo , Burkholderia gladioli/enzimologia , Cânfora/química , Cânfora/metabolismo , Cefalosporinas/metabolismo , Estrutura Molecular , Rhodococcus/enzimologia , Serina Endopeptidases/metabolismo , EstereoisomerismoRESUMO
Several thousand different terpenoid structures are known so far, and many of them are interesting for applications as pharmaceuticals, flavors, fragrances, biofuels, insecticides, or fine chemical intermediates. One prominent example is camphor, which has been utilized since ancient times in medical applications. Especially (-)-camphor is gaining more and more interest for pharmaceutical applications. Hence, a commercial reliable source is needed. The natural sources for (-)-camphor are limited, and the oxidation of precious (-)-borneol would be too costly. Hence, synthesis of (-)-camphor from renewable alpha-pinene would be an inexpensive alternative. As the currently used route for the conversion of alpha-pinene to camphor produces a mixture of both enantiomers, preferably catalytic methods for the separation of this racemate are demanded to yield enantiopure camphor. Enzymatic kinetic resolution is a sustainable way to solve this challenge but requires suitable enzymes. In this study, the first borneol dehydrogenase from Pseudomonas sp. ATCC 17453, capable of catalyzing the stereoselective reduction of camphor, was examined. By using a targeted enzyme engineering approach, enantioselective enzyme variants were created with E-values > 100. The best variant was used for the enzymatic kinetic resolution of camphor racemate, yielding 79% of (-)-camphor with an ee of > 99%. KEY POINTS: ⢠Characterization of a novel borneol dehydrogenase (BDH) from P. putida. ⢠Development of enantioselective BDH variants for the reduction of camphor. ⢠Enzymatic kinetic resolution of camphor with borneol dehydrogenase.
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Oxirredutases do Álcool , Cânfora , Engenharia de Proteínas , Pseudomonas/enzimologia , OxirreduçãoRESUMO
Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol's enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.
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Metiltransferases/química , Metiltransferases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Resveratrol/metabolismo , Estilbenos/metabolismo , Vitis/enzimologia , Engenharia Metabólica , Metiltransferases/genética , Modelos Moleculares , Filogenia , Proteínas de Plantas/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Bacterial arylmalonate decarboxylase (AMDase) and evolved variants have become a valuable tool with which to access both enantiomers of a broad range of chiral arylaliphatic acids with high optical purity. Yet, the molecular principles responsible for the substrate scope, activity, and selectivity of this enzyme are only poorly understood to date, greatly hampering the predictability and design of improved enzyme variants for specific applications. In this work, empirical valence bond and metadynamics simulations were performed on wild-type AMDase and variants thereof to obtain a better understanding of the underlying molecular processes determining reaction outcome. Our results clearly reproduce the experimentally observed substrate scope and support a mechanism driven by ground-state destabilization of the carboxylate group being cleaved by the enzyme. In addition, our results indicate that, in the case of the nonconverted or poorly converted substrates studied in this work, increased solvent exposure of the active site upon binding of these substrates can disturb the vulnerable network of interactions responsible for facilitating the AMDase-catalyzed cleavage of CO2. Finally, our results indicate a switch from preferential cleavage of the pro-(R) to the pro-(S) carboxylate group in the CLG-IPL variant of AMDase for all substrates studied. This appears to be due to the emergence of a new hydrophobic pocket generated by the insertion of the six amino acid substitutions, into which the pro-(S) carboxylate binds. Our results allow insight into the tight interaction network determining AMDase selectivity, which in turn provides guidance for the identification of target residues for future enzyme engineering.
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Proteínas de Bactérias/química , Carboxiliases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Bordetella/enzimologia , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Domínio Catalítico , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Teoria Quântica , TermodinâmicaRESUMO
Protein design is limited by the diversity of functional groups provided by the canonical protein "building blocks". Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split-GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3-phenylbutyrate.
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Aminoácidos , Engenharia de Proteínas , Catálise , Esterases/química , Esterases/metabolismo , ProteínasRESUMO
In this study, we coupled a well-established whole-cell system based on E.â coli via light-harvesting complexes to Rieske oxygenase (RO)-catalyzed hydroxylations in vivo. Although these enzymes represent very promising biocatalysts, their practical applicability is hampered by their dependency on NAD(P)H as well as their multicomponent nature and intrinsic instability in cell-free systems. In order to explore the boundaries of E.â coli as chassis for artificial photosynthesis, and due to the reported instability of ROs, we used these challenging enzymes as a model system. The light-driven approach relies on light-harvesting complexes such as eosinâ Y, 5(6)-carboxyeosin, and rose bengal and sacrificial electron donors (EDTA, MOPS, and MES) that were easily taken up by the cells. The obtained product formations of up to 1.3â g L-1 and rates of up to 1.6â mm h-1 demonstrate that this is a comparable approach to typical whole-cell transformations in E.â coli. The applicability of this photocatalytic synthesis has been demonstrated and represents the first example of a photoinduced RO system.
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Escherichia coli/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Oxigenases/metabolismo , Biocatálise , Escherichia coli/citologia , HidroxilaçãoRESUMO
Arylmalonate decarboxylase (AMDase) catalyzes the cofactor-free asymmetric decarboxylation of prochiral arylmalonic acids and produces the corresponding monoacids with rigorous R selectivity. Alteration of catalytic cysteine residues and of the hydrophobic environment in the active site by protein engineering has previously resulted in the generation of variants with opposite enantioselectivity and improved catalytic performance. The substrate spectrum of AMDase allows it to catalyze the asymmetric decarboxylation of small methylvinylmalonic acid derivatives, implying the possibility to produce short-chain 2-methylalkanoic acids with high optical purity after reduction of the nonactivated C=C double bond. Use of diimide as the reductant proved to be a simple strategy to avoid racemization of the stereocenter during reduction. The developed chemoenzymatic sequential cascade with use of R- and S-selective AMDase variants produced optically pure short-chain 2-methylalkanoic acids in moderate to full conversion and gave both enantiomers in excellent enantiopurity (up to 83 % isolated yield and 98 % ee).
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Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.
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Alquil e Aril Transferases/química , Modelos Moleculares , Análise de Sequência de Proteína/métodos , Motivos de Aminoácidos , Catálise , Domínio CatalíticoRESUMO
Multi-catalytic cascade reactions bear a great potential to minimize downstream and purification steps, leading to a drastic reduction of the produced waste. In many examples, the compatibility of chemo- and biocatalytic steps could be easily achieved. Problems associated with the incompatibility of the catalysts and their reactions, however, are very frequent. Cascade-like reactions can hardly occur in this way. One possible solution to combine, in principle, incompatible chemo- and biocatalytic reactions is the defined control of the microenvironment by compartmentalization or scaffolding. Current methods for the control of the microenvironment of biocatalysts go far beyond classical enzyme immobilization and are thus believed to be very promising tools to overcome incompatibility issues and to facilitate the synthetic application of cascade reactions. In this Minireview, we will summarize recent synthetic examples of (chemo)enzymatic cascade reactions and outline promising methods for their spatial control either by using bio-derived or synthetic systems.
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Enzimas/metabolismo , Biocatálise , Butanóis/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Clostridium acetobutylicum/metabolismo , Clostridium tyrobutyricum/metabolismo , Enzimas/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Metais/química , Água/química , Água/metabolismoRESUMO
Immobilizing enzymes for biocatalysis offers many advantages, including easy separation of the enzyme from the product and repeated and continuous use. ATR-FTIR spectroscopy is a versatile tool to monitor immobilized enzymes and has been applied to many proteins. However, while the common and convenient immobilization via oligohistidine on mono-NTA layers is adequate for the measurement of difference spectra induced by ligand binding or photochemistry, it lacks the long term stability that is necessary for monitoring biocatalysis. Here, we report a new immobilization methodology based on maleimido-thiol chemistry. A 12-mercaptododecanoic acid NHS ester monolayer is reacted with 1-(2-aminoethyl)-maleimide to build a thiol reactive surface. Subsequently, NTA-C16-thiol is covalently attached and finally oligohistidine tagged enzymes were immobilized to this surface, which remained bound with a five times higher EC50-value compared to typical mono-NTA layers. To demonstrate the high potential of the surface we analysed decarboxylation reactions catalyzed by arylmalonate decarboxylase. With ATR-FTIR both the enzyme and its substrate conversion can be monitored label free. Correct folding of the enzyme can be evaluated based on the amide band of the immobilized enzyme. In addition, the infrared absorption spectra of educt and product are monitored in real time. We show that hybrid hard-soft multivariate curve resolution improves separation of the product and educt spectra from other effects during the experiments, leading to clean kinetic traces and reaction rates for the catalytic process. Our approach can in principle be extended to any enzyme and is ideally suited for the development of biocatalysts.
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Enzimas Imobilizadas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Compostos de Sulfidrila/química , Indicadores e Reagentes , Propriedades de SuperfícieRESUMO
Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.
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Lisossomos/metabolismo , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Quinolinas/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Receptor ErbB-2/metabolismo , Análise Espectral RamanRESUMO
Bacterial arylmalonate decarboxylase (AMDase) shows high enantioselectivity and a broad substrate spectrum in the asymmetric synthesis of optically pure arylaliphatic carboxylic acids. The determination of the structure of AMDase has greatly extended the understanding of the catalytic mechanism of this unique cofactor-free decarboxylase and allowed the generation of tailor-made enzyme variants with improved catalytic properties. Despite this increase in knowledge and applicability, the natural role of the enzyme remains unknown. This mini-review summarizes the recent findings on the molecular mechanism and the synthetic application of the enzyme.
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Bordetella bronchiseptica/enzimologia , Carboxiliases/metabolismo , Ácidos Graxos/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
The combination of enzymes with traditional chemical catalysts unifies the high selectivity of the former with the versatility of the latter. A major challenge of this approach is the difference in the optimal reaction conditions for each catalyst type. In this work, we combined a cofactor-free decarboxylase with a ruthenium metathesis catalyst to produce high-value antioxidants from bio-based precursors. As suitable ruthenium catalysts did not show satisfactory activity under aqueous conditions, the reaction required the use of an organic solvent, which in turn significantly reduced enzyme activity. Upon encapsulation of the decarboxylase in a cryogel, the decarboxylation could be conducted in an organic solvent, and the recovery of the enzyme after the reaction was facilitated. After an intermediate drying step, the subsequent metathesis in pure organic solvent proved to be straightforward. The synthetic utility of the cascade was demonstrated by the synthesis of the antioxidant 4,4'-dihydroxystilbene in an overall yield of 90 %.
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A recombinant enoate reductase was expressed in cyanobacteria and used for the light-catalyzed, enantioselective reduction of C=C bonds. The coupling of oxidoreductases to natural photosynthesis allows asymmetric syntheses fueled by the oxidation of water. Bypassing the addition of sacrificial cosubstrates as electron donors significantly improves the atom efficiency and avoids the formation of undesired side products. Crucial factors for product formation are the availability of NADPH and the amount of active enzyme in the cells. The efficiency of the reaction is comparable to typical whole-cell biotransformations in E.â coli. Under optimized conditions, a solution of 100â mg prochiral 2-methylmaleimide was reduced to optically pure 2-methylsuccinimide (99 %â ee, 80 % yield of isolated product). High product yields and excellent optical purities demonstrate the synthetic usefulness of light-catalyzed whole-cell biotransformations using recombinant cyanobacteria.