RESUMEN
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
OBJECTIVES: Cancer cells undergo metabolic reprogramming to adapt to high oxidative stress, but little is known about how metabolic remodeling enables gastric cancer cells to survive stress associated with aberrant reactive oxygen species (ROS) production. Here, we aimed to identify the key metabolic enzymes that protect gastric cancer (GC) cells from oxidative stress. METHODS: ROS level was detected by DCFH-DA probes. Multiple cell biological studies were performed to identify the underlying mechanisms. Furthermore, cell-based xenograft and patient-derived xenograft (PDX) model were performed to evaluate the role of MTHFD2 in vivo. RESULTS: We found that overexpression of MTHFD2, but not MTHFD1, is associated with reduced overall and disease-free survival in gastric cancer. In addition, MTHFD2 knockdown reduces the cellular NADPH/NADP+ ratio, colony formation and mitochondrial function, increases cellular ROS and cleaved PARP levels and induces in cell death under hypoxia, a hallmark of solid cancers and a common inducer of oxidative stress. Moreover, genetic or pharmacological inhibition of MTHFD2 reduces tumor burden in both tumor cell lines and patient-derived xenograft-based models. DISCUSSION: our study highlights the crucial role of MTHFD2 in redox regulation and tumor progression, demonstrating the therapeutic potential of targeting MTHFD2.
Asunto(s)
Progresión de la Enfermedad , Homeostasis , Metilenotetrahidrofolato Deshidrogenasa (NADP) , Estrés Oxidativo , Neoplasias Gástricas , Animales , Humanos , Ratones , Aminohidrolasas/metabolismo , Aminohidrolasas/genética , Línea Celular Tumoral , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/genética , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nitrilases represent a distinct class of enzymes that play a pivotal role in catalyzing the hydrolysis of nitrile compounds, leading to the formation of corresponding carboxylic acids. These enzymatic entities have garnered significant attention across a spectrum of industries, encompassing pharmaceuticals, agrochemicals, and fine chemicals. Moreover, their significance has been accentuated by mounting environmental pressures, propelling them into the forefront of biodegradation and bioremediation endeavors. Nevertheless, the natural nitrilases exhibit intrinsic limitations such as low thermal stability, narrow substrate selectivity, and inadaptability to varying environmental conditions. In the past decade, substantial efforts have been made in elucidating the structural underpinnings and catalytic mechanisms of nitrilase, providing basis for engineering of nitrilases. Significant breakthroughs have been made in the regulation of nitrilases with ideal catalytic properties and application of the enzymes for industrial productions. This review endeavors to provide a comprehensive discourse and summary of recent research advancements related to nitrilases, with a particular emphasis on the elucidation of the structural attributes, catalytic mechanisms, catalytic characteristics, and strategies for improving catalytic performance of nitrilases. Moreover, the exploration extends to the domain of process engineering and the multifarious applications of nitrilases. Furthermore, the future development trend of nitrilases is prospected, providing important guidance for research and application in the related fields.
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Aminohidrolasas , Nitrilos , Aminohidrolasas/genética , Aminohidrolasas/química , Catálisis , Biodegradación AmbientalRESUMEN
Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In some Pseudomonas strains, the assimilation of cyanide has been linked to NitC, such as the cyanotrophic model strain Pseudomonas pseudoalcaligenes CECT 5344, which has been recently reclassified as Pseudomonas oleovorans CECT 5344. In this work, a phylogenomic approach established a more precise taxonomic position of the strain CECT 5344 within the species P. oleovorans. Furthermore, a pan-genomic analysis of P. oleovorans and other species with cyanotrophic strains, such as P. fluorescens and P. monteilii, allowed for the comparison and identification of the cioAB and mqoAB genes involved in cyanide resistance, and the nitC and cynS genes required for the assimilation of cyanide or cyanate, respectively. While cyanide resistance genes presented a high frequency among the analyzed genomes, genes responsible for cyanide or cyanate assimilation were identified in a considerably lower proportion. According to the results obtained in this work, an in silico approach based on a comparative genomic approach can be considered as an agile strategy for the bioprospection of putative cyanotrophic bacteria and for the identification of new genes putatively involved in cyanide biodegradation.
Asunto(s)
Biodegradación Ambiental , Cianuros , Genoma Bacteriano , Filogenia , Pseudomonas , Cianuros/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Genómica/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Pseudomonas pseudoalcaligenes/metabolismo , Pseudomonas pseudoalcaligenes/genéticaRESUMEN
Ovarian cancer (OC) is a deadliest gynecological cancer with the highest mortality rate. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a crucial tumor-promoting factor, is over-expressed in several malignancies including OC. The present study aimed to explore the role and mechanisms of MTHFD2 in OC malignant progression. Thus, cell proliferation, cycling, apoptosis, migration, and invasion were evaluated by CCK-8 assay, EdU assay, flow cytometry, wound healing, transwell assay and western blotting. Additionally, glycolysis was assessed by measuring the level of glucose and lactate production, as well as the expressions of GLUT1, HK2 and PKM2. Then the expression of ferroptosis-related proteins and ERK signaling was detected using western blotting. Ferroptosis was detected through the measurement of iron level, GSH, MDA and ROS activities. The results revealed that MTHFD2 was highly expressed in OC cells. Besides, interference with MTHFD2 induced ferroptosis, promoted ROS accumulation, destroyed mitochondrial function, reduced ATP content and inhibited glycolysis in OC cells. Subsequently, we further found that interference with MTHFD2 affected mitochondrial function and glycolysis in OC cells through ERK signaling. Moreover, interference with MTHFD2 affected ferroptosis to inhibit the malignant progression of OC cells. Collectively, our present study disclosed that interference with MTHFD2 induced ferroptosis in OC to inhibit tumor malignant progression through regulating ERK signaling.
Asunto(s)
Ferroptosis , Sistema de Señalización de MAP Quinasas , Metilenotetrahidrofolato Deshidrogenasa (NADP) , Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Ferroptosis/fisiología , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Enzimas Multifuncionales/metabolismo , Línea Celular Tumoral , Aminohidrolasas/metabolismo , Aminohidrolasas/genética , Progresión de la Enfermedad , RatonesRESUMEN
Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.
Asunto(s)
Citidina Desaminasa , Mapas de Interacción de Proteínas , Humanos , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Desaminación , Desaminasas APOBEC/metabolismo , Aminohidrolasas/metabolismo , Aminohidrolasas/genética , Células HEK293 , Citosina Desaminasa/metabolismo , Desaminasa APOBEC-3G/metabolismo , Desaminasa APOBEC-3G/genética , Empalmosomas/metabolismo , Unión Proteica , Espectrometría de Masas , ARN/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genéticaRESUMEN
PURPOSE: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1). METHODS: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees. RESULTS: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries. CONCLUSION: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage.
Asunto(s)
Hemorragia Cerebral , Enfermedades de los Pequeños Vasos Cerebrales , Trastornos del Movimiento , Linaje , Humanos , Femenino , Masculino , Enfermedades de los Pequeños Vasos Cerebrales/genética , Enfermedades de los Pequeños Vasos Cerebrales/patología , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Persona de Mediana Edad , Hemorragia Cerebral/genética , Hemorragia Cerebral/patología , Hemorragia Cerebral/diagnóstico por imagen , Trastornos del Movimiento/genética , Trastornos del Movimiento/patología , Trastornos del Movimiento/diagnóstico por imagen , Imagen por Resonancia Magnética , Alelos , Adulto , Anciano , Sistema Glinfático/patología , Sistema Glinfático/diagnóstico por imagen , Secuenciación del Exoma , Encéfalo/patología , Encéfalo/diagnóstico por imagen , Aminohidrolasas/genéticaRESUMEN
4-cyanobenzoic acid serves as a crucial intermediate for the synthesis of various high-value organic compounds. The enzymatic hydrolysis of terephthalonitrile to produce 4-cyanobenzoic acid using nitrilase offers the advantages of a simple reaction pathway, environmental friendliness, and easy product separation. In order to efficiently develop nitrilases that meet industrial production requirements, the virtual screening method used in the study is established and mature. From a total of 371 amino acids in the nitrilase AfNIT, which exhibits activity in terephthalonitrile hydrolysis, three candidate sites (F168, S192, and T201) were identified, and a "small and accurate" mutant library was constructed. The triple mutant F168V/T201N/S192F was screened from this small mutant library with a specific activity of 227.3 U mg-1 , which was 3.8 times higher than that of the wild-type AfNIT. Using the whole-cell biocatalyst containing the mutant F168V/T201N/S192F, terephthalonitrile was successfully hydrolyzed at a concentration of 150 g L-1 to produce 4-cyanobenzoic acid with a final yield of 170.3 g L-1 and a conversion rate of 98.7%. The obtained nitrilase mutant F168V/T201N/S192F in this study can be effectively applied in the biomanufacturing of 4-cyanobenzoic acid using terephthalonitrile as a substrate. Furthermore, the results also demonstrate the significant improvement in predictive accuracy achieved through the latest AI-assisted computer simulation methods. This approach represents a promising and feasible new technological pathway for assisting enzyme engineering research, laying a theoretical foundation for other related studies.
Asunto(s)
Aminohidrolasas , Benzoatos , Simulación por Computador , Aminohidrolasas/genética , Aminohidrolasas/químicaRESUMEN
Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.
Asunto(s)
Aminohidrolasas , Cytophagaceae , Nitrilos , Pirimidinas , Triazoles , Nitrilos/química , Aminohidrolasas/genética , Aminohidrolasas/química , Aminohidrolasas/metabolismo , Amidas , Niacinamida , Especificidad por SustratoRESUMEN
Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.
Asunto(s)
Aminohidrolasas , Nitrilos , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Catálisis , Ingeniería de Proteínas , Especificidad por SustratoRESUMEN
The seven human APOBEC3 enzymes (APOBEC3A through H, excluding E) are host restriction factors. Most of the APOBEC3 enzymes can restrict HIV-1 replication with different efficiencies. The HIV-1 Vif protein combats APOBEC3-mediated restriction by inducing ubiquitination and degradation in the proteasome. APOBEC3F and APOBEC3G can hetero-oligomerize, which increases their restriction capacity and resistance to Vif. Here we determined if APOBEC3C, APOBEC3F, or APOBEC3G could hetero-oligomerize with APOBEC3H haplotype I. APOBEC3H haplotype I has a short half-life in cells due to ubiquitination and degradation by host proteins, but is also resistant to Vif. We hypothesized that hetero-oligomerization with APOBEC3H haplotype I may result in less Vif-mediated degradation of the interacting APOBEC3 and stabilize APOBEC3H haplotype I, resulting in more efficient HIV-1 restriction. Although we found that all three APOBEC3s could interact with APOBEC3H haplotype I, only APOBEC3F affected APOBEC3H haplotype I by surprisingly accelerating its proteasomal degradation. However, this increased APOBEC3F levels in cells and virions in the absence or presence of Vif and enabled APOBEC3F-mediated restriction of HIV-1 in the presence of Vif. Altogether, the data suggest that APOBEC3 enzymes can co-regulate each other at the protein level and that they cooperate to ensure HIV-1 inactivation rather than evolution.
Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , Haplotipos , Citidina Desaminasa , Citoplasma , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Citosina Desaminasa , Desaminasas APOBEC , Aminohidrolasas/genéticaRESUMEN
The coagulation factor 9 gene (FIX) point mutation contributes to most hemophilia B cases, providing ideal gene correction models. Here we identified the frequent mutation G20519A (R226Q) in FIX, which resulted in many severe and moderate hemophilia B patients. This study aimed to investigate the effect of HDR and base editing in correcting FIX mutant. We first constructed HEK293 and liver-derived cell lines Huh7 cells stabling carrying mutated FIX containing G20519A (HEK293-FIXmut and Huh7-FIXmut). Then, CRISPR/Cas9-based homology-directed repair (HDR) and base editing were used for the correction of this mutated point. We used Cas9 nickase (nCas9) mediated HDR and the advanced base editor ABE8e to correct G20519A and then measured the concentration and activity of FIX. Furthermore, we used the star-shaped poly(lysine) gene nanocarriers to deliver the ABE8e correction systems into HEK293-FIXmut and Huh7-FIXmut stem cells to correct mutated FIX. As a result, we found that gRNAs directed inefficient HDR in correcting G20519A. The ABE8e corrected the mutation efficiently in both HEK293-FIXmut and Huh7-FIXmut stem cells. In addition, the star-shaped poly(lysine) carriers delivered non-viral vectors into stem cells efficiently. The nanocarriers-delivered ABE8e system corrected mutated FIX in stem cells, and the stem cells secreted active FIX in high concentration. In conclusion, our study provides a potential alternative for correcting mutated FIX in hemophilia B patients.
Asunto(s)
Edición Génica , Hemofilia A , Hemofilia B , Aminohidrolasas/genética , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/metabolismo , Sistemas CRISPR-Cas/genética , Desoxirribonucleasa I/metabolismo , Edición Génica/métodos , Células HEK293 , Hemofilia A/genética , Hemofilia A/metabolismo , Hemofilia B/genética , Hemofilia B/terapia , Humanos , Mutación , Mutación Missense , Polilisina/química , Células Madre/metabolismoRESUMEN
Purpose: Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has been reported to be overexpressed in non-small-cell lung cancer (NSCLC) and to correlate with malignant proliferation. However, the mechanism of high MTHFD2 expression in NSCLC has not been clarified. Methods: qPCR, western blot, and immunofluorescence experiments were used to measure the expression of related mRNAs and proteins. Cell apoptosis was measured by flow cytometry and TUNEL assays. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to analyze the cell cycle. ROS, H2O2, MDA, SOD, and NADPH/NADP+ were evaluated by relevant assay kits. Transfection of siRNA or vectors was used to downregulate or upregulate gene expression. Dual-luciferase reporter gene assays were used to evaluate the regulated relationship between MTHFD2 and ATF4 or MYC. Results: MTHFD2 was highly expressed in NSCLC cells. Knockdown of MTHFD2 inhibited proliferation and increased apoptosis. Furthermore, oxidative factors significantly increased, while antioxidant factors significantly decreased in NSCLC cells with MTHFD2 knockdown, indicating that MTHFD2 was involved in NSCLC progression through the redox pathway. Although MTHFD2 was downregulated with ATF4 silencing, the dual-luciferase reporter assay suggested that ATF4 did not directly mediate MTHFD2 transcription. Further studies revealed that MYC had a transcriptional effect on MTHFD2 and was also regulated by ATF4. PCR, and western blotting experiments with ATF4 knockdown and MYC overexpression as well as ATF4 overexpression and MYC knockdown proved that ATF4 stimulated MTHFD2 through MYC mediation. Conclusions: ATF4 promoted high expression of MTHFD2 in NSCLC dependent on MYC.
Asunto(s)
Factor de Transcripción Activador 4 , Aminohidrolasas , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Metilenotetrahidrofolato Deshidrogenasa (NADP) , Enzimas Multifuncionales , Proteínas Proto-Oncogénicas c-myc , Factor de Transcripción Activador 4/genética , Aminohidrolasas/genética , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Homeostasis , Humanos , Peróxido de Hidrógeno/metabolismo , Luciferasas/genética , Neoplasias Pulmonares/patología , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Enzimas Multifuncionales/genética , Oxidación-Reducción , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Recurrent loss-of-function deletions cause frequent inactivation of tumour suppressor genes but often also involve the collateral deletion of essential genes in chromosomal proximity, engendering dependence on paralogues that maintain similar function. Although these paralogues are attractive anticancer targets, no methodology exists to uncover such collateral lethal genes. Here we report a framework for collateral lethal gene identification via metabolic fluxes, CLIM, and use it to reveal MTHFD2 as a collateral lethal gene in UQCR11-deleted ovarian tumours. We show that MTHFD2 has a non-canonical oxidative function to provide mitochondrial NAD+, and demonstrate the regulation of systemic metabolic activity by the paralogue metabolic pathway maintaining metabolic flux compensation. This UQCR11-MTHFD2 collateral lethality is confirmed in vivo, with MTHFD2 inhibition leading to complete remission of UQCR11-deleted ovarian tumours. Using CLIM's machine learning and genome-scale metabolic flux analysis, we elucidate the broad efficacy of targeting MTHFD2 despite distinct cancer genetic profiles co-occurring with UQCR11 deletion and irrespective of stromal compositions of tumours.
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Aminohidrolasas , Metilenotetrahidrofolato Deshidrogenasa (NADP) , Enzimas Multifuncionales , Neoplasias Ováricas , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Femenino , Humanos , Hidrolasas , Redes y Vías Metabólicas , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Mitocondrias/metabolismo , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , NAD/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismoRESUMEN
Nitrilases are promising biocatalysts to produce high-value-added carboxylic acids through hydrolysis of nitriles. However, since the enzymes always show low activity and sometimes with poor reaction specificity toward 2-chloronicotinonitrile (2-CN), very few robust nitrilases have been reported for efficient production of 2-chloronicotinic acid (2-CA) from 2-CN. Herein, a nitrilase from Paraburkholderia graminis (PgNIT) was engineered to improve its catalytic properties. We identified the beneficial residues via computational analysis and constructed the mutant library. The positive mutants were obtained and the activity of the "best" mutant F164G/I130L/N167Y/A55S/Q260C/T133I/R199Q toward 2-CN was increased from 0.14 × 10-3 to 4.22 U/mg. Its reaction specificity was improved with elimination of hydration activity. Molecular docking and molecular dynamics simulation revealed that the conformational flexibility, the nucleophilic attack distance, as well as the interaction forces between the enzyme and substrate were the main reason alternating the catalytic properties of PgNIT. With the best mutant as biocatalyst, 150 g/L 2-CN was completely converted, resulting in 2-CA accumulated to 169.7 g/L. When the substrate concentration was increased to 200 g/L, 203.1 g/L 2-CA was obtained with yield of 85.7%. The results laid the foundation for industrial production of 2-CA with the nitrilase-catalyzed route.
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Aminohidrolasas , Burkholderiaceae , Ácidos Nicotínicos , Aminohidrolasas/química , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Burkholderiaceae/genética , Burkholderiaceae/metabolismo , Simulación del Acoplamiento Molecular , Especificidad por Sustrato , Ácidos Nicotínicos/biosíntesis , Ácidos Nicotínicos/metabolismo , CatálisisRESUMEN
OBJECTIVES: To study the association of maternal methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) gene polymorphisms with congenital heart disease (CHD) in offspring. METHODS: A hospital-based case-control study was conducted. The mothers of 683 children with CHD alone who attended Hunan Children's Hospital, from November 2017 to March 2020 were enrolled as the case group, and the mothers of 740 healthy children who attended the same hospital during the same period and did not have any deformity were enrolled as the control group. A questionnaire survey was performed to collect related exposure data, and then venous blood samples (5 mL) were collected from the mothers to detect MTHFD1 and MTHFD2 gene polymorphisms. A multivariate logistic regression analysis was used to evaluate the association of MTHFD1 and MTHFD2 gene polymorphisms with CHD. The four-gamete test in Haploview 4.2 software was used to construct haplotypes and evaluate the association between haplotypes and CHD. The generalized multifactor dimensionality reduction method and logistic regression analysis were used to examine gene-gene interaction and its association with CHD. RESULTS: The multivariate logistic regression analysis showed that maternal MTHFD1 gene polymorphisms at rs11849530 (GA vs AA: OR=1.49; GG vs AA: OR=2.04) andat rs1256142 (GA vs GG: OR=2.34; AA vs GG: OR=3.25) significantly increased the risk of CHD in offspring (P<0.05), while maternal MTHFD1 gene polymorphisms at rs1950902 (AA vs GG: OR=0.57) and MTHFD2 gene polymorphisms at rs1095966 (CA vs CC: OR=0.68) significantly reduced the risk of CHD in offspring (P<0.05). The haplotypes of G-G-G (OR=1.86) and G-A-G (OR=1.35) in mothers significantly increased the risk of CHD in offspring (P<0.05). The gene-gene interaction analyses showed that the first-order interaction between MTHFD1 rs1950902 and MTHFD1 rs2236222 and the second-order interaction involving MTHFD1 rs1950902, MTHFD1 rs1256142, and MTHFD2 rs1095966 might be associated with risk of CHD (P<0.05). CONCLUSIONS: Maternal MTHFD1 and MTHFD2 gene polymorphisms and their haplotypes, as well as the interaction between MTHFD1 rs1950902 and MTHFD1 rs2236222 and between MTHFD1 rs1950902, MTHFD1 rs1256142, and MTHFD2 rs1095966, are associated with the risk of CHD in offspring.
Asunto(s)
Aminohidrolasas , Cardiopatías Congénitas , Metilenotetrahidrofolato Deshidrogenasa (NADP) , Enzimas Multifuncionales , Aminohidrolasas/genética , Estudios de Casos y Controles , Niño , Femenino , Predisposición Genética a la Enfermedad , Cardiopatías Congénitas/genética , Humanos , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Antígenos de Histocompatibilidad Menor/genética , Madres , Enzimas Multifuncionales/genética , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNITM0 ) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA). However, BaNITM0 also catalyzed the hydration of IBSN to produce by-product (S)-3-cyano-5-methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of BaNITM0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S (BaNITM2 ) displayed higher enantioselectivity (E = 515), increased enzyme activity (5.4-fold) and reduced amide formation (from 15.8% to 1.9%) compared with BaNITM0 . Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio- and regio-selective hydrolysis of 150 g/L IBSN using 1.5 g/L Escherichia coli cells harboring BaNITM2 as biocatalyst afforded (S)-CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of BaNITM2 for efficient manufacturing of pregabalin.
Asunto(s)
Aminohidrolasas , Escherichia coli , Amidas , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pregabalina/química , Especificidad por SustratoRESUMEN
The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (Kd). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the Kd at four different temperatures. These Kd values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the Kd determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the Kd caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the Kd, can be carried out using common laboratory equipment and is scalable.
Asunto(s)
Aminohidrolasas , Thermotoga maritima , Aminohidrolasas/genética , Histidina/metabolismoRESUMEN
AIMS: To characterize the functions of nitrilases of Variovorax boronicumulans CGMCC 4969 and evaluate flonicamid (FLO) degradation and ß-cyano-L-alanine (Ala(CN)) detoxification by this bacterium. METHODS AND RESULTS: Variovorax boronicumulans CGMCC 4969 nitrilases (NitA and NitB) were purified, and substrate specificity assay indicated that both of them degraded insecticide FLO to N-(4-trifluoromethylnicotinoyl)glycinamide (TFNG-AM) and 4-(trifluoromethyl)nicotinol glycine (TFNG). Ala(CN), a plant detoxification intermediate, was hydrolysed by NitB. Escherichia coli overexpressing NitA and NitB degraded 41.2 and 93.8% of FLO (0.87 mmol·L-1 ) within 1 h, with half-lives of 1.30 and 0.25 h, respectively. NitB exhibited the highest nitrilase activity towards FLO. FLO was used as a substrate to compare their enzymatic properties. NitB was more tolerant to acidic conditions and organic solvents than NitA. Conversely, NitA was more tolerant to metal ions than NitB. CGMCC 4969 facilitated FLO degradation in soil and surface water and utilized Ala(CN) as a sole nitrogen source for growth. CONCLUSIONS: CGMCC 4969 efficiently degraded FLO mediated by NitA and NitB; NitB was involved in Ala(CN) detoxification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study promotes our understanding of versatile functions of nitrilases from CGMCC 4969 that is promising for environmental remediation.
Asunto(s)
Insecticidas , Alanina/análogos & derivados , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Comamonadaceae , Escherichia coli/genética , Escherichia coli/metabolismo , Insecticidas/metabolismo , Niacinamida/análogos & derivadosRESUMEN
Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond time scale interconversion between these functional states hamper their experimental and computational characterization. Here, we combine extensive molecular dynamics simulations, enhanced sampling techniques, and dynamical networks to describe the allosteric activation of imidazole glycerol phosphate synthase (IGPS) from the substrate-free form to the active ternary complex. IGPS is a heterodimeric bienzyme complex whose HisH subunit is responsible for hydrolyzing glutamine and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation of IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that the combined effector and substrate binding dramatically decreases the conformational barrier associated with oxyanion hole formation, in line with the experimentally observed 4500-fold activity increase in glutamine hydrolysis. The allosteric activation is controlled by correlated time-evolving dynamic networks connecting the effector and substrate binding sites. This computational strategy tailored to describe millisecond events can be used to rationalize the effect of mutations on the allosteric regulation and guide IGPS engineering efforts.