RESUMEN
An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.
Asunto(s)
Biocatálisis , Nitrilos , Oxidorreductasas , Pirazoles , Pirimidinas , Nitrilos/química , Nitrilos/metabolismo , Pirimidinas/química , Pirimidinas/biosíntesis , Pirimidinas/metabolismo , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Pirazoles/química , Pirazoles/metabolismo , Iminas/química , Iminas/metabolismo , Estructura Molecular , Hidrazinas/química , Ingeniería de ProteínasRESUMEN
The mechanisms of insecticide resistance are complex. Recent studies have revealed a novel mechanism involving the chemosensory system in insecticide resistance. However, the specific binding mechanism between olfactory-related genes and insecticides needs to be clarified. In this study, the binding mechanism between pyrethroid insecticide deltamethrin and RpCSP6 from Rhopalosiphum padi was investigated by using computational and multiple experimental methods. RpCSP6 was expressed in different tissues and developmental stages of R. padi and can be induced by deltamethrin. Knockdown of RpCSP6 significantly increased the susceptibility of R. padi to deltamethrin. The binding affinity of RpCSP6 to 24 commonly used insecticides was measured. Seven key residues were found to steadily interact with deltamethrin, indicating their significance in the binding affinity to the insecticide. Our research provided insights for effectively analyzing the binding mechanism of insect CSPs with insecticides, facilitating the development of new and effective insecticides that target insect CSPs.
Asunto(s)
Proteínas de Insectos , Resistencia a los Insecticidas , Insecticidas , Nitrilos , Piretrinas , Piretrinas/metabolismo , Piretrinas/farmacología , Nitrilos/metabolismo , Nitrilos/farmacología , Nitrilos/química , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Insecticidas/farmacología , Insecticidas/metabolismo , Insecticidas/química , Resistencia a los Insecticidas/genética , Animales , Unión ProteicaRESUMEN
The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y456 differ from that of AgCPR-His456. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.
Asunto(s)
Anopheles , Resistencia a los Insecticidas , Insecticidas , NADPH-Ferrihemoproteína Reductasa , Piretrinas , Anopheles/genética , Anopheles/efectos de los fármacos , Anopheles/enzimología , Anopheles/metabolismo , Animales , Resistencia a los Insecticidas/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Insecticidas/farmacología , Insecticidas/metabolismo , Piretrinas/farmacología , Piretrinas/metabolismo , Oxidación-Reducción , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Especificidad por Sustrato , Nitrilos/metabolismo , Nitrilos/farmacología , Permetrina/farmacologíaRESUMEN
Etravirine (ETV) is an antiretroviral agent that belongs to the class of non-nucleoside reverse transcriptase inhibitors. This study explores the uptake and distribution of ETV in human aortic endothelial cells (HAECs) using Raman spectroscopy combined with chemometrics. The distinctive chemical structure of ETV facilitates tracking of its uptake by observing the Raman band at 2225 cm-1 in the Raman-silent region. The perinuclear distribution pattern in HAECs depends on drug concentration and incubation time. The uptake of ETV is observed within 5 minutes at a concentration of 10 µM, as evidenced by Raman images. Lower ETV concentrations, reflective of those found in human plasma, are detectable in HAECs by applying chemometric methods to Raman spectra from the perinuclear region. The ETV accumulation process is crucial in advancing our understanding of the drug's impact on biochemical alterations within endothelial cells. Additionally, ETV emerges as a promising Raman reporter for marking subcellular compartments, leveraging the 2225 cm-1 band in the cellular Raman silent region. This research contributes valuable insights into the behavior of ETV at the subcellular level, shedding light on its potential applications and impact on subcellular dynamics.
Asunto(s)
Aorta , Células Endoteliales , Nitrilos , Piridazinas , Pirimidinas , Espectrometría Raman , Espectrometría Raman/métodos , Humanos , Nitrilos/química , Nitrilos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/química , Pirimidinas/química , Pirimidinas/metabolismo , Aorta/metabolismo , Aorta/citología , Piridazinas/química , Piridazinas/metabolismo , Análisis de la Célula Individual/métodos , Células CultivadasRESUMEN
The objective of this investigation is to learn more about the structural, electrical, spectroscopic, and physiochemical characteristics of biologically active cyano-4'-hydroxybiphenyl (CHBP). The title molecule's optimized conformational analysis was computed using the DFT/B3LYP/6-311++G (d, p) level of theory. The observed wavenumbers were compared with theoretical FT-IR and FT-Raman spectra. 1H and 13C NMR experimental spectra in CDCl3 solution (solvent phase) were recorded and the chemical shift was calculated. NBO analysis was used to examine the transfer of charge as well as the intermolecular and intramolecular bonding of orbitals. The TD-DFT (time-dependent DFT) approach was used to estimate theoretical values for both the gas and solvent (ethanol) in the corresponding transitional research, which was conducted using UV-Vis's spectra. Energy gap (Eg = 0.26764 eV) implies that the strong potential for charge transfer, and the stability of the CHBP compound. CHBP compound's has bioactive nature, its drug-likeness and biological properties were evaluated. The predicted topological polar surface area of 44.02 \AA2 for the molecule falls within the permissible range of < 140 \AA2. Based on the docking results, the most stable docking score value is -6.84 kcal/mol. In that interaction, MET 165 affects both phenyl rings in a pi-sulphur fashion and a single bond hydrogen with protein moieties GLN 192. This suggests that the pi-alkyl in PRO 168 is a hydroxyl substitutional ring. Our findings demonstrate the CHBP compound is a good inhibitor against the SAR COVID-19 viral protein.
Asunto(s)
Simulación del Acoplamiento Molecular , Unión Proteica , SARS-CoV-2 , Espectrometría Raman , SARS-CoV-2/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de Fourier , Humanos , Antivirales/química , Antivirales/farmacología , Compuestos de Bifenilo/química , COVID-19/virología , Teoría Funcional de la Densidad , Conformación Molecular , Nitrilos/química , Nitrilos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Leaf mustard (Brassica juncea L.) is explored for its biofumigant properties, derived from its secondary metabolites, particularly allyl isothiocyanate (AITC), produced during the enzymatic breakdown of glucosinolates like sinigrin. The research examines eight leaf mustard cultivars developed in Yeosu city, South Korea, focusing on their genetic characteristics, AITC concentration and nitriles formation rates from glucosinolates. Results indicate that the allelopathic effects, largely dependent on AITC concentration and enzymatic activity, vary across cultivar. Sinigrin and AITC constitute 79% and 36%, respectively, of glucosinolate and its hydrolysis products. The cultivar 'Nuttongii' demonstrates significant potential for inhibiting weeds, exhibiting the highest AITC concentration at 27.47 ± 6.46 µmole g-1 These outcomes highlight the importance of selecting mustard cultivars for biofumigation based on their glucosinolate profiles and hydrolysis product yields. The study also identifies a significant genetic influence on AITC and nitrile formation, suggesting that epithiospecifier protein modulation could enhance both allelopathic and other beneficial effects. Collectively, the research underscores the promise of mustard as a sustainable, environmentally friendly alternative to traditional herbicides.
Asunto(s)
Glucosinolatos , Isotiocianatos , Planta de la Mostaza , Nitrilos , Glucosinolatos/metabolismo , Glucosinolatos/química , Isotiocianatos/farmacología , Isotiocianatos/metabolismo , Isotiocianatos/química , Nitrilos/metabolismo , Nitrilos/farmacología , Nitrilos/química , Planta de la Mostaza/metabolismo , Planta de la Mostaza/genética , República de Corea , AlelopatíaRESUMEN
Aerocyanidin and amycomicin are two antibiotics derived from long-chain acids with a rare epoxy isonitrile moiety, the complexity of which renders the total synthesis of these two natural products rather challenging. How this functionality is biosynthesized has also remained obscure. While the biosynthetic gene clusters for these compounds have been identified, both appear to be deficient in genes encoding enzymes seemingly necessary for the oxidative modifications observed in these antibiotics. Herein, the biosynthetic pathways of aerocyanidin and amycomicin are fully elucidated. They share a conserved pathway to isonitrile intermediates that involves a bifunctional thioesterase and a nonheme iron α-ketoglutarate-dependent enzyme. In both cases, the isonitrile intermediates are then loaded onto an acyl carrier protein (ACP) catalyzed by a ligase. The isonitrile-tethered ACP is subsequently processed by polyketide synthase(s) to undergo chain extension, thereby assembling a long-chain γ-hydroxy isonitrile acid skeleton. The epoxide is installed by the cupin domain-containing protein AecF to conclude the biosynthesis of aerocyanidin. In contrast, three P450 enzymes AmcB, AmcC, and AmcQ are involved in epoxidation and keto formation to finalize the biosynthesis of amycomicin. These results thus explain the sequence of oxidation events that result in the final structures of aerocyanidin and amycomicin as well as the biosynthesis of the key γ-hydroxy epoxy isonitrile functional group.
Asunto(s)
Antibacterianos , Nitrilos , Antibacterianos/química , Antibacterianos/biosíntesis , Nitrilos/química , Nitrilos/metabolismo , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Estructura MolecularRESUMEN
Some feed source plants will produce secondary metabolites such as cyanogenic glycosides during metabolism, which will produce some poisonous nitrile compounds after hydrolysis and remain in plant tissues. The consumption of feed-source plants without proper treatment affect the health of the animals' bodies. Nitrilases can convert nitriles and have been used in industry as green biocatalysts. However, due to their bottleneck problems, their application in agriculture is still facing challenges. Acid-resistant nitrilase preparations, high-temperature resistance, antiprotease activity, strong activity, and strict reaction specificity urgently need to be developed. In this paper, the application potential of nitrilase in agriculture, especially in feed processing industry was explored, the source properties and catalytic mechanism of nitrilase were reviewed, and modification strategies for nitrilase application in agriculture were proposed to provide references for future research and application of nitrilase in agricultural and especially in the biological feed scene.
Asunto(s)
Aminohidrolasas , Nitrilos , Aminohidrolasas/metabolismo , Aminohidrolasas/genética , Aminohidrolasas/química , Nitrilos/metabolismo , Nitrilos/química , Agricultura , Alimentación Animal/análisis , Biocatálisis , AnimalesRESUMEN
Occupancy of prostate cancer (PCa) cell androgen receptors (AR) signals proliferation, therefore testosterone biosynthesis inhibitors and AR antagonists are important PCa treatments. Conversely, androgen mimics (e.g., prednisone) used in management of PCa might cause proliferation. The balance between PCa proliferation and inhibition predicts treatment success. We used in silico molecular modelling to explore interactions between ARs, androgens (testosterone, dihydrotestosterone (DHT)) and drugs used to treat (bicalutamide) and manage (dexamethasone, prednisone, hydrocortisone) PCa. We found that hydrogen (H-) bonds between testosterone, DHT and Arg752, Asn705 and Thr877 followed by ligand binding cleft hydrophobic interactions signal proliferation, whereas bicalutamide antagonism is via Phe764 interactions. Hydrocortisone, dexamethasone and prednisone H-bond Asn705 and Thr877, but not Arg752 in the absence of a water molecule. Studies with a bicalutamide agonist AR mutation showed different amino acid interactions, indicating testosterone and DHT would not promote proliferation as effectively as via the native receptor. However, hydrocortisone and bicalutamide form Arg752 and Asn705 H-bonds indicating agonism. Our results suggest that as PCa progresses the resulting mutations will change the proliferative response to androgens and their drug mimics, which have implications for the treatment of prostate cancer.
Asunto(s)
Neoplasias de la Próstata , Receptores Androgénicos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Masculino , Receptores Androgénicos/metabolismo , Humanos , Anilidas/farmacología , Anilidas/química , Compuestos de Tosilo/farmacología , Compuestos de Tosilo/química , Compuestos de Tosilo/metabolismo , Simulación por Computador , Simulación del Acoplamiento Molecular , Modelos Moleculares , Nitrilos/química , Nitrilos/farmacología , Nitrilos/metabolismo , Esteroides/metabolismo , Esteroides/química , Testosterona/metabolismo , Testosterona/farmacología , Unión Proteica , Dihidrotestosterona/metabolismoRESUMEN
In this study, g-C3N4/PANI was prepared by in situ oxidative polymerization. Graphite-phase carbon nitride (g-C3N4) with surface defects was deposited onto the surface of conductive polyaniline (PANI) to form a p-n heterojunction. This construction aimed to create an efficient heterogeneous catalyst, increasing the surface defect level and active sites of the composite, and augmenting its capability to capture and transfer extracellular electrons under anaerobic conditions. This addresses the challenge of low efficiency in direct interspecies electron transfer between bacteria and archaea during anaerobic digestion for methane production. The results showed that the prepared g-C3N4/PANI increased the CH4 yield and CH4 production rate by 82% and 96%, respectively. Notably, the conductivity and XPS test results showed that the ratio of g-C3N4 to PANI was 0.15, and the composite exhibited favorable conductivity, with a uniform distribution of pyrrolic nitrogen, pyridinic nitrogen, and graphitic nitrogen, each accounting for approximately 30%. Furthermore, g-C3N4/PANI effectively enhanced the metabolic efficiency of intermediate products such as acetate and butyrate. Analysis of the microbial community structure revealed that g-C3N4/PANI led to a significant increase in the abundance of hydrogenotrophic methanogen Methanolinea (from 48% to 64%) and enriched Clostridium (a rise of 1%) with direct interspecies electron transfer capability. Microbial community function analysis demonstrated that the addition of g-C3N4/PANI boosted the activities of key enzymes involved in anaerobic digestion, including phosphate transacetylase (PTA), phospho-butyryl transferase (PTB), and NAD-independent lactate dehydrogenase (NNLD), by 47%, 135%, and 153%, respectively. This acceleration in enzymatic activity promoted the metabolism of acetyl-CoA, butyryl-CoA, and pyruvate. Additionally, the function of ABC transporters was enhanced, thereby improving the efficiency of material and energy exchange among microorganisms.
Asunto(s)
Compuestos de Anilina , Metano , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Anaerobiosis , Metano/metabolismo , Eliminación de Residuos Líquidos/métodos , Aguas Residuales/química , Grafito/química , Nitrilos/metabolismo , Nitrilos/químicaRESUMEN
Isonitrile lipopeptides discovered from Actinobacteria have attracted wide attention due to their fascinating biosynthetic pathways and relevance to the virulence of many human pathogens including Mycobacterium tuberculosis. Specifically, the identification of the new class of isonitrile-forming enzymes that belong to non-heme iron (II) and α-ketoglutarate dependent dioxygenases has intrigued several research groups to investigate their catalytic mechanism. Here we summarize the recent studies on the biosynthesis of isonitrile lipopeptides from Streptomyces and Mycobacterium. The latest research on the core and tailoring enzymes involved in the pathway as well as the isonitrile metabolic enzymes are discussed in this review.
Asunto(s)
Lipopéptidos , Nitrilos , Lipopéptidos/biosíntesis , Lipopéptidos/química , Lipopéptidos/metabolismo , Nitrilos/metabolismo , Nitrilos/química , Streptomyces/metabolismo , Humanos , Mycobacterium/metabolismo , Mycobacterium/enzimología , Vías BiosintéticasRESUMEN
Benzyl nitrile from tea plants attacked by various pests displays a diurnal pattern, which may be closely regulated by the endogenous circadian clock. However, the molecular mechanism by the circadian clock of tea plants that regulates the biosynthesis and release of volatiles remains unclear. In this study, the circadian clock gene CsPCL1 can activate both the expression of the benzyl nitrile biosynthesis-related gene CsCYP79 and the jasmonic acid signaling-related transcription factor CsMYC2 involved in upregulating CsCYP79 gene, thereby resulting in the accumulation and release of benzyl nitrile. Therefore, the anti-insect function of benzyl nitrile was explored in the laboratory. The application of slow-release beads of benzyl nitrile in tea plantations significantly reduced the number of tea geometrids and had positive effects on the yield of fresh tea leaves. These findings reveal the potential utility of herbivore-induced plant volatiles for the green control of pests in tea plantations.
Asunto(s)
Camellia sinensis , Relojes Circadianos , Nitrilos , Proteínas de Plantas , Compuestos Orgánicos Volátiles , Camellia sinensis/genética , Camellia sinensis/química , Camellia sinensis/metabolismo , Camellia sinensis/parasitología , Animales , Compuestos Orgánicos Volátiles/química , Compuestos Orgánicos Volátiles/metabolismo , Compuestos Orgánicos Volátiles/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Relojes Circadianos/genética , Nitrilos/farmacología , Nitrilos/química , Nitrilos/metabolismo , Regulación de la Expresión Génica de las Plantas , Mariposas Nocturnas/genética , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/metabolismo , Insecticidas/farmacología , Insecticidas/químicaRESUMEN
The appropriate use of human biomonitoring data to model population chemical exposures is challenging, especially for rapidly metabolized chemicals, such as agricultural chemicals. The objective of this study is to demonstrate a novel approach integrating model predicted dietary exposures and biomonitoring data to potentially inform regulatory risk assessments. We use lambda-cyhalothrin as a case study, and for the same representative U.S. population in the National Health and Nutrition Examination Survey (NHANES), an integrated exposure and pharmacokinetic model predicted exposures are calibrated to measurements of the urinary metabolite 3-phenoxybenzoic acid (3PBA), using an approximate Bayesian computing (ABC) methodology. We demonstrate that the correlation between modeled urinary 3PBA and the NHANES 3PBA measurements more than doubled as ABC thresholding narrowed the acceptable tolerance range for predicted versus observed urinary measurements. The median predicted urinary concentrations were closer to the median measured value using ABC than using current regulatory Monte Carlo methods.
Asunto(s)
Monitoreo Biológico , Exposición Dietética , Nitrilos , Piretrinas , Humanos , Piretrinas/orina , Piretrinas/metabolismo , Nitrilos/orina , Nitrilos/metabolismo , Exposición Dietética/análisis , Monitoreo Biológico/métodos , Adulto , Teorema de Bayes , Masculino , Femenino , Persona de Mediana Edad , Insecticidas/orina , Insecticidas/metabolismo , Adulto Joven , Adolescente , Encuestas Nutricionales , BenzoatosRESUMEN
Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.
Asunto(s)
Histidina , Nitrilos , Pseudomonas , Pseudomonas/enzimología , Pseudomonas/metabolismo , Nitrilos/metabolismo , Nitrilos/química , Histidina/química , Histidina/metabolismo , Hidrólisis , Biocatálisis , Dominio Catalítico , Fungicidas Industriales/química , Fungicidas Industriales/metabolismo , Halogenación , Hidrolasas/metabolismo , Hidrolasas/químicaRESUMEN
Barley produces several specialized metabolites, including five α-, ß-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production.
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Glucósidos , Hordeum , Hordeum/genética , Hordeum/metabolismo , Hordeum/microbiología , Glucósidos/metabolismo , Nitrilos/metabolismo , Sitios de Carácter Cuantitativo , Uretano/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estudio de Asociación del Genoma CompletoRESUMEN
Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.
Asunto(s)
Aminohidrolasas , Microscopía por Crioelectrón , Nitrilos , Multimerización de Proteína , Rhodococcus , Aminohidrolasas/química , Aminohidrolasas/metabolismo , Aminohidrolasas/ultraestructura , Microscopía por Crioelectrón/métodos , Rhodococcus/enzimología , Nitrilos/química , Nitrilos/metabolismo , Especificidad por Sustrato , Modelos Moleculares , Dominio Catalítico , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , CatálisisRESUMEN
Until now, bacteria able to degrade, 3,3'-iminodipropionitrile (IDPN), a neurotoxin that destroys vestibular hair cells, causing ototoxicity, culminating in irreversible movement disorders, had never been isolated. The aim of this study was to isolate a novel IDPN-biodegrading microorganism and characterize its metabolic pathway. Enrichment was performed by inoculating activated sludge from a wastewater treatment bioreactor that treated IDPN-contaminated wastewater in M9 salt medium, with IDPN as the sole carbon source. A bacterial strain with a spherical morphology that could grow at high concentrations was isolated on a solid medium. Growth of the isolated strain followed the Monod kinetic model. Based on the 16S rRNA gene, the isolate was Paracoccus communis. Whole-genome sequencing revealed that the isolated P. communis possessed the expected full metabolic pathway for IDPN biodegradation. Transcriptome analyses confirmed the overexpression of the gene encoding hydantoinase/oxoprolinase during the exponential growth phase under IDPN-fed conditions, suggesting that the enzyme involved in cleaving the imine bond of IDPN may promote IDPN biodegradation. Additionally, the newly discovered P. communis isolate seems to metabolize IDPN through cleavage of the imine bond in IDPN via nitrilase, nitrile hydratase, and amidase reactions. Overall, this study lays the foundation for the application of IDPN-metabolizing bacteria in the remediation of IDPN-contaminated environments.
Asunto(s)
Biodegradación Ambiental , Reactores Biológicos , Nitrilos , Paracoccus , Eliminación de Residuos Líquidos , Aguas Residuales , Nitrilos/metabolismo , Paracoccus/metabolismo , Paracoccus/genética , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/metabolismo , ARN Ribosómico 16SRESUMEN
Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0â Å resolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.
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Aldehído-Liasas , Mutagénesis Sitio-Dirigida , Aldehído-Liasas/metabolismo , Aldehído-Liasas/química , Aldehído-Liasas/genética , Animales , Benzaldehídos/metabolismo , Benzaldehídos/química , Acetonitrilos/química , Acetonitrilos/metabolismo , Modelos Moleculares , Cristalografía por Rayos X , Nitrilos/metabolismo , Nitrilos/química , EstereoisomerismoAsunto(s)
Piretrinas , Rutina , Masculino , Ratas , Animales , Rutina/farmacología , Testículo , Piretrinas/toxicidad , Nitrilos/metabolismo , Estrés Oxidativo , Antioxidantes/farmacologíaRESUMEN
Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15â¯mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5â¯g/L/h and 48.3â¯g/g of immobilized protein, respectively.