RESUMEN
Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alcaloides Indólicos/metabolismo , Catharanthus/enzimología , Catharanthus/genética , Catharanthus/metabolismo , Catálisis , Alcaloides de Triptamina Secologanina/metabolismo , Evolución Molecular , Simulación del Acoplamiento Molecular , Secuencia de Aminoácidos , Hidroxilación , Simulación de Dinámica Molecular , Monoterpenos/metabolismo , FilogeniaRESUMEN
The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carota L.) and described the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. At the same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.
Asunto(s)
Aldehído-Liasas , Sistema Enzimático del Citocromo P-450 , Daucus carota , Proteínas de Plantas , Daucus carota/enzimología , Daucus carota/genética , Daucus carota/metabolismo , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Peróxidos Lipídicos/metabolismo , Especificidad por Sustrato , Secuencia de Aminoácidos , Ácidos LinoleicosRESUMEN
BACKGROUND: Cystic fibrosis (CF), an inherited autosomal recessive disorder, is linked with high morbidity and mortality rates due to bacteria, filamentous, yeast and black yeast-like fungi colonisation in the upper respiratory tract. Although Candida species are the most common fungi isolated from CF patients, azole-resistant Aspergillus fumigatus (ARAf) is a big concern for invasive aspergillosis. Notably, the exact prevalences of Aspergillus species and the prevalence of ARAf isolates among Iranian CF patients have yet to be previously reported and are unknown. We aimed to investigate the prevalence of ARAf isolates in CF patients among Iranian populations by focusing on molecular mechanisms of the mutations in the target gene. METHODS: The 1 year prospective study recovered 120 sputum samples from 103 CF patients. Of these, 55.1% (86/156) yielded Aspergillus species, screened for ARAf using plates containing itraconazole (4 mg/L) and voriconazole (1 mg/L). According to the CLSI-M38 guidelines, antifungal susceptibility testing was performed using the broth microdilution method. In all phenotypically resistant isolates, the target of azole agents, the cyp51A gene, was sequenced to detect any possible single nucleotide polymorphisms (SNP) mediating resistance. RESULTS: Of 120 samples, 101 (84.2%) were positive for filamentous fungi and yeast-like relatives, with 156 fungal isolates. The most common colonising fungi were Aspergillus species (55.1%, 86/156), followed by Candida species (39.8%, 62/156), Exophiala species (3.8%, 6/156) and Scedosporium species (1.3%, 2/156). Forty out of 86 (46.5%) were identified for section Fumigati, 36 (41.9%) for section Flavi, 6 (7%) for section Nigri and 4 (4.6%) for section Terrei. Fourteen out of 40 A. fumigatus isolates were phenotypically resistant. The overall proportion of ARAf in total fungal isolates was 9% (14/156). cyp51A gene analysis in resistant isolates revealed that 13 isolates harboured G448S, G432C, T289F, D255E, M220I, M172V, G138C, G54E and F46Y mutations and one isolate carried G448S, G432C, T289F, D255E, M220I, G138C, G54E and F46Y mutations. Additionally, this study detects two novel cyp51A single-nucleotide polymorphisms (I242V and D490E). CONCLUSIONS: This study first investigated ARAf isolates in Iranian CF patients. Due to a resistance rate of up to 9%, it is recommended that susceptibility testing of Aspergillus isolates from CF patients receiving antifungal treatment be a part of the routine diagnostic workup. However, extensive multicentre studies with a high volume of CF patients are highly warranted to determine the impact of ARAf on CF patients.
Asunto(s)
Antifúngicos , Aspergillus fumigatus , Azoles , Fibrosis Quística , Sistema Enzimático del Citocromo P-450 , Farmacorresistencia Fúngica , Proteínas Fúngicas , Pruebas de Sensibilidad Microbiana , Humanos , Fibrosis Quística/microbiología , Fibrosis Quística/complicaciones , Irán/epidemiología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Farmacorresistencia Fúngica/genética , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Estudios Prospectivos , Prevalencia , Sistema Enzimático del Citocromo P-450/genética , Azoles/farmacología , Azoles/uso terapéutico , Proteínas Fúngicas/genética , Masculino , Femenino , Aspergilosis/microbiología , Aspergilosis/epidemiología , Aspergilosis/tratamiento farmacológico , Adulto , Niño , Adolescente , Polimorfismo de Nucleótido Simple , Adulto Joven , Esputo/microbiología , Itraconazol/farmacología , Voriconazol/farmacología , Voriconazol/uso terapéutico , Preescolar , MutaciónRESUMEN
The implementation of biocatalytic steroid hydroxylation processes plays a crucial role in the pharmaceutical industry due to a plethora of medicative effects of hydroxylated steroid derivatives and their crucial role in drug approval processes. Cytochrome P450 monooxygenases (CYP450s) typically constitute the key enzymes catalyzing these reactions, but commonly entail drawbacks such as poor catalytic rates and the dependency on additional redox proteins for electron transfer from NAD(P)H to the active site. Recently, these bottlenecks were overcome by equipping Escherichia coli cells with highly active variants of the self-sufficient single-component CYP450 BM3 together with hydrophobic outer membrane proteins facilitating cellular steroid uptake. The combination of the BM3 variant KSA14m and the outer membrane pore AlkL enabled exceptionally high testosterone hydroxylation rates of up to 45 U gCDW-1 for resting (i.e., living but non-growing) cells. However, a rapid loss of specific activity heavily compromised final product titers and overall space-time yields. In this study, several stabilization strategies were evaluated on enzyme-, cell-, and reaction level. However, neither changes in biocatalyst configuration nor variation of cultivation media, expression systems, or inducer concentrations led to considerable improvement. This qualified the so-far used genetic construct pETM11-ksa14m-alkL, M9 medium, and the resting-cell state as the best options enabling comparatively efficient activity along with fast growth prior to biotransformation. In summary, we report several approaches not enabling a stabilization of the high testosterone hydroxylation rates, providing vital guidance for researchers tackling similar CYP450 stability issues. A comparison with more stable natively steroid-hydroxylating CYP106A2 and CYP154C5 in equivalent setups further highlighted the high potential of the investigated CYP450 BM3-based whole-cell biocatalysts. The immense and continuously developing repertoire of enzyme engineering strategies provides promising options to stabilize the highly active biocatalysts.
Asunto(s)
Biocatálisis , Sistema Enzimático del Citocromo P-450 , Escherichia coli , Hidroxilación , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Testosterona/metabolismo , Esteroides/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Estabilidad de EnzimasRESUMEN
The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.
Asunto(s)
Pruebas de Mutagenicidad , Mutágenos , Salmonella typhimurium , Animales , Humanos , Pruebas de Mutagenicidad/métodos , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Mutágenos/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Activación Metabólica , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Mutación , Daño del ADN/efectos de los fármacos , Fallopia multiflora/química , MasculinoRESUMEN
While much attention has been devoted to understanding the transcriptional changes underlying resistance to insecticides, comparatively little is known about the transcriptional response of naive insects to agrochemicals. In this study, we analyze the transcriptomic response of an insecticide susceptible strain of Drosophila melanogaster to nine agrochemicals using a robust method that goes beyond classical replication standards. Our findings demonstrate that exposure to piperonyl butoxide (PBO), but not to eight other compounds, elicits a robust transcriptional response in a wild-type strain of Drosophila melanogaster. PBO exposure leads to the upregulation of a subset of Cyps, GSTs, UGTs and EcKls. This response is both time and concentration-dependent, suggesting that the degree of inhibition of P450 activity correlates with the magnitude of the transcriptional response. Furthermore, the upregulation of these enzymes is excluded from reproductive organs. Additionally, different sets of genes are regulated in the digestive/secretory tract and the carcass. Our results suggest that P450s play a role in metabolizing yet unidentified endogenous compounds and are involved in an as-yet-unknown physiological regulatory feedback loop.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Drosophila melanogaster , Butóxido de Piperonilo , Animales , Butóxido de Piperonilo/farmacología , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Insecticidas/toxicidad , Insecticidas/farmacología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The spider mite Tetranychus urticae is a major agricultural pest with a global distribution, extremely diverse host range and a remarkable ability to develop resistance to a wide variety of acaricides. P450 mono-oxygenases have been frequently associated with resistance development in this species. In particular enzymes of the CYP392A-subfamily were shown to metabolize a number of key acaricides, including abamectin, amitraz, fenpyroximate and the active metabolite of pyflubumide. However, transcriptomic studies comparing highly resistant and susceptible populations have often revealed high expression of members of the CYP392D-subfamily, but these have been only poorly studied. Here, we conducted a meta-analysis of gene expression data of 20 populations and identified two key enzymes of this family, CYP392D2 and CYP392D8, whose expression is associated with resistance. We subsequently functionally expressed these enzymes, together with CYP392A11 and CYP392A16 as known metabolizers, and compared their potential to accept a wide diversity of acaricides as substrate. This study overall confirms previous discovered substrates for CYP392A11 and CYP392A16, but also reveals unreported metabolic activity towards new acaricides. These include carbaryl, chlorpyrifos and etoxazole for CYP392A16 and carbaryl, chlorpyrifos and NNI-0711-NH pyflubumide for CYP392A11. For the newly studied CYP392D-family, we show that CYP392D2 metabolizes pyridaben, fenpyroximate, etoxazole and chlorpyrifos, while CYP392D8 metabolizes carbaryl, fenazaquin and tebufenpyrad. Last, we observed that both CYP392A- and CYP392D-subfamily enzymes activate chlorpyrifos to its corresponding oxon. Our study indicates that there is both overlap and specificity in the activity of A- and D-subfamily enzymes against acaricides and model substrates. With the recent advent of highly efficient CRISPR/Cas9 gene editing protocols in T. urticae, the way is now paved to conduct further genetic experiments revealing and quantifying the role of these enzymes in the resistance phenotype in field populations.
Asunto(s)
Acaricidas , Sistema Enzimático del Citocromo P-450 , Tetranychidae , Animales , Acaricidas/metabolismo , Acaricidas/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Tetranychidae/genética , Tetranychidae/efectos de los fármacos , Tetranychidae/metabolismo , Tetranychidae/enzimologíaRESUMEN
Weed resistance to a range of herbicides has rapidly evolved, often with different mechanisms of action. The resulting uninhibited growth of weeds poses demonstrable threats to crop production and sustainable agriculture. Digitaria sanguinalis (L.) Scop., a troublesome weed in corn and other agricultural fields, has developed resistance to herbicides that inhibiting ALS (Acetolactate Synthase), such as nicosulfuron. Understanding the weed's resistance patterns and mechanisms is crucial. However, little is known of the non-target site resistance (NTSR) mechanisms of D. sanguinalis owing to a lack of relevant genome sequences and other materials. Therefore, in this study, a population of D.sanguinalis presenting multiple resistance was tested and found that its high level of resistance to ALS-inhibiting herbicides was not associated with target-related alterations.Administration of P450 inhibitors reversed the resistance to ALS-inhibiting herbicides. Following the application of ALS-inhibiting herbicides, the activities of NADPH-P450 reductase and p-nitroanisole O-demethylase (PNOD) were notably greater in the resistant population of D. sanguinalis than those in the susceptible population. The results suggested P450 enzyme familyplays a major role in the metabolic resistance mechanism, that increased P450 enzyme activity promote cross-resistance in D. sanguinalis to ALS-inhibiting herbicides. RNA-seq analysis showed that five genes from the P450 family (CYP709B2, CYP714C2, CYP71A1, CYP76C2, and CYP81E8) were upregulated in resistant D. sanguinalis. In conclusion, the upregulation of several P450 genes is responsible for establishing resistance to ALS-inhibiting herbicides in D. sanguinalis.
Asunto(s)
Acetolactato Sintasa , Sistema Enzimático del Citocromo P-450 , Digitaria , Resistencia a los Herbicidas , Herbicidas , Herbicidas/farmacología , Herbicidas/toxicidad , Acetolactato Sintasa/metabolismo , Acetolactato Sintasa/genética , Acetolactato Sintasa/antagonistas & inhibidores , Resistencia a los Herbicidas/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Digitaria/efectos de los fármacos , Compuestos de Sulfonilurea/farmacología , Malezas/efectos de los fármacos , Malezas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , PiridinasRESUMEN
Xenobiotic response element (XRE) to flavone was the cis- regulatory elements that mediates the induction of the allelochemical-metabolizing CYP321A1 gene from Helicoverpa zea. However, it was unknown whether the XRE-Fla element existed in other species. Recently we have identified and cloned the CYP321A1 gene with promoter region in a related species, Helicoverpa armigera. Sequence similarity of two orthologous CYP321A1 genes was 97.27%, but the promoter sequence similarity was only 56.32%. Sequence alignment showed the XRE-Fla like element owns three mutations in H. armigera compared with H. zea. Progressive 5' deletions and internal mutation indicated that H. armigera XRE-Fla was the essential element of CYP321A1 gene in response to flavone. XRE-Fla mutations and EMSA analysis confirmed that the H. armigera XRE-Fla element binding factor was stronger than H. zea. The findings indicate the XRE element mutations mainly contribute to the differences between the flavone-induced expressions of two CYP321A1 genes, which improve the flexibility and adaptability for allelochemical response of H. armigera.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Flavonas , Mariposas Nocturnas , Animales , Mariposas Nocturnas/genética , Mariposas Nocturnas/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Flavonas/farmacología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Regiones Promotoras Genéticas , Secuencia de Bases , Elementos de Respuesta , Helicoverpa armigeraRESUMEN
Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.
Asunto(s)
Oxidación-Reducción , Oxigenasas , Oxigenasas/metabolismo , Oxigenasas/química , Oxigenasas/genética , Oxigenasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Rhodococcus/enzimología , Rhodococcus/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/químicaRESUMEN
Nitenpyram, taking the place of imidacloprid, is a widely used neonicotinoid insecticide to control Nilaparvata lugens in Asia. Two P450s, CYP4CE1 and CYP6ER1, are key factors in the metabolic resistance against nitenpyram and imidacloprid. In this study, we found that CYP4CE1 expression was strongly associated with nitenpyram resistance in 8 field-collected populations, whereas CYP6ER1 expression correlated with imidacloprid resistance. Hence, we focused on nitenpyram metabolism by CYP4CE1, due to that imidacloprid metabolism by CYP6ER1 has intensively investigated. Mass spectrometry analysis revealed that recombinant CYP4CE1 metabolized nitenpyram into three products, N-desmethyl nitenpyram, hydroxy-nitenpyram, and N-desmethyl hydroxy-nitenpyram, with a preference for hydroxylation. In contrast, CYP6ER1 metabolized nitenpyram into a single product, N-desmethyl nitenpyram. These results provide new insights into the specific catalytic mechanisms of P450 enzymes in neonicotinoid metabolism and underscore the importance of different catalytic reactions in neonicotinoid insecticide resistance.
Asunto(s)
Proteínas de Insectos , Insecticidas , Neonicotinoides , Oxidación-Reducción , Neonicotinoides/metabolismo , Neonicotinoides/química , Insecticidas/metabolismo , Insecticidas/química , Hidroxilación , Animales , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/química , Desmetilación , Hemípteros/metabolismo , Hemípteros/genética , Hemípteros/enzimología , Nitrocompuestos/metabolismo , Nitrocompuestos/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Resistencia a los Insecticidas/genéticaRESUMEN
BACKGROUND: The Flavonoid 3'-hydroxylase gene(F3'H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear. RESULTS: In order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3'H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3'H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein. CONCLUSIONS: The results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke.
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Antocianinas , Sistema Enzimático del Citocromo P-450 , Regulación de la Expresión Génica de las Plantas , Antocianinas/biosíntesis , Antocianinas/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilación de la Expresión Génica , Transcriptoma , Redes Reguladoras de Genes , Pigmentación/genéticaRESUMEN
Cytochromes P450 monooxygenases (CYP450s) constitute the largest enzymic protein family that is widely present in plants, animals, and microorganisms, participate in numerous metabolic pathways, and play diverse roles in development, metabolism, and defense. Rapeseed (Brassica napus) is an important oil crop worldwide and have many versions of reference genome. However, there is no systemically comparative genome-wide analysis of CYP450 family genes in rapeseed and its parental species B. rapa and B. oleracea. In this study, we identified 765, 293 and 437 CYP450 genes in B. napus, B. rapa and B. oleracea, respectively, which were unevenly located in A01-A10 and/or C01-C09 chromosomes in corresponding species. Phylogenetic relationship analysis indicated that 1745 CYP450 proteins from three Brassica species and Arabidopsis were divided into 4 groups. Whole genome duplication (WGD) or segmental duplication resulted in gene expansion of CYP450 family in three Brassica species. There were 33-83 SSR loci in CYP450 genes of three Brassica species, and numerous transcription factor binding sites were identified in their promoters. A total of 459-777 miRNAs were predicted to target 174-426 CYP450 genes in three Brassica species. Based on transcriptome data, BnCYP450s, BrCYP450s and BoCYP450s were differentially expressed in various tissues. There existed numerous BnCYP450 DEGs in response to pathogens and abiotic stresses. Besides, many BnCYP450 DEGs were involved in the regulation of important traits, such as seed germination, seed ALA content, and yellow-seed. The qRT-PCR experiment confirmed the transcriptome analysis results by validating two representative Sclerotinia-responsive BnCYP450 DEGs as an example. Three BnCYP450s genes (CYP707A1, CYP81F1, CYP81H1) might be regulated by seed-specific transcription factors BnTT1 and BnbZIP67 to participate in the development and metabolism of seed coat and embryo by undertaking related metabolic reactions.
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Brassica napus , Sistema Enzimático del Citocromo P-450 , Filogenia , Semillas , Estrés Fisiológico , Brassica napus/genética , Brassica napus/enzimología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Semillas/genética , Estrés Fisiológico/genética , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Zealexin A1 is a nonvolatile sesquiterpene phytoalexin, which not only exhibits extensive antifungal and insecticidal activities but also has the ability to enhance the drought resistance of plants, and thus has potential applications in agricultural and food fields. In this study, the biosynthetic pathway of zealexin A1 was constructed in Saccharomyces cerevisiae for the first time, and the highest production of zealexin A1 reported to date was achieved. First, through screening of sesquiterpene synthases from various plants, BdMAS11 had a stronger (S)-ß-macrocarpene synthesis ability was obtained, and the heterologous synthesis of zealexin A1 was achieved by coexpressing BdMAS11 with cytochrome P450 oxygenase ZmCYP71Z18. Subsequently, after the site-directed mutagenesis of BdMAS11, fusion expression of farnesyl diphosphate synthase ERG20 and BdMAS11, and tailored truncation of BdMAS11 and ZmCYP71Z18, the strain coexpressing the manipulated BdMAS11 and original ZmCYP71Z18 produced 119.31 mg/L of zealexin A1 in shake-flask fermentation. Finally, the production of zealexin A1 reached 1.17 g/L through fed-batch fermentation in a 5 L bioreactor, which was 261.7-fold that of the original strain. This study lays the foundation for the industrial production of zealexin A1 and other terpenoids.
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Ingeniería Metabólica , Saccharomyces cerevisiae , Sesquiterpenos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fermentación , Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , FitoalexinasRESUMEN
The liver, a pivotal organ in human metabolism, serves as a primary site for heme biosynthesis, alongside bone marrow. Maintaining precise control over heme production is paramount in healthy livers to meet high metabolic demands while averting potential toxicity from intermediate metabolites, notably protoporphyrin IX. Intriguingly, our recent research uncovers a disrupted heme biosynthesis process termed 'porphyrin overdrive' in cancers that fosters the accumulation of heme intermediates, potentially bolstering tumor survival. Here, we investigate heme and porphyrin metabolism in both healthy and oncogenic human livers, utilizing primary human liver transcriptomics and single-cell RNA sequencing (scRNAseq). Our investigations unveil robust gene expression patterns in heme biosynthesis in healthy livers, supporting electron transport chain (ETC) and cytochrome P450 function without intermediate accumulation. Conversely, liver cancers exhibit rewired heme biosynthesis and a massive downregulation of cytochrome P450 gene expression. Notably, despite diminished drug metabolism, gene expression analysis shows that heme supply to the ETC remains largely unaltered or even elevated with patient cancer progression, suggesting a metabolic priority shift. Liver cancers selectively accumulate intermediates, which are absent in normal tissues, implicating their role in disease advancement as inferred by expression analysis. Furthermore, our findings in genomics establish a link between the aberrant gene expression of porphyrin metabolism and inferior overall survival in aggressive cancers, indicating potential targets for clinical therapy development. We provide in vitro proof-of-concept data on targeting porphyrin overdrive with a drug synergy strategy.
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Hemo , Neoplasias Hepáticas , Porfirinas , Humanos , Porfirinas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Hemo/metabolismo , Genómica , Hígado/metabolismo , Hígado/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
A genome of Rhodococcus rhodochrous IEGM 1362 was sequenced and annotated. This strain can transform monoterpene alcohol (-)-isopulegol with the formation of two novel pharmacologically promising metabolites. Nine genes encoding cytochrome P450, presumably involved in (-)-isopulegol transformation, were found in the genome of R. rhodochrous IEGM 1362. Primers and PCR conditions for their detection were selected. The obtained data can be used for the further investigation of genes encoding enzymes involved in monoterpene biotransformation.
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Biotransformación , Biología Computacional , Genoma Bacteriano , Rhodococcus , Rhodococcus/genética , Rhodococcus/metabolismo , Biología Computacional/métodos , Biotransformación/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Monoterpenos/metabolismoRESUMEN
Orthotospovirus tomatomaculae (tomato spotted wilt virus, TSWV) is transmitted by the western flower thrips, Frankliniella occidentalis. Epoxyoctadecamonoenoic acids (EpOMEs) function as immune-suppressive factors, particularly in insects infected by viral pathogens. These oxylipins are produced by cytochrome P450 monooxygenases (CYPs) and are degraded by soluble epoxide hydrolase (sEH). In this study, we tested the hypothesis that TSWV modulates the EpOME level in the thrips to suppress antiviral responses and enhance its replication. TSWV infection significantly elevated both 9,10-EpOME and 12,13-EpOME levels. Following TSWV infection, the larvae displayed apoptosis in the midgut along with the upregulated expression of four caspase genes. However, the addition of EpOME to the viral treatment notably reduced apoptosis and downregulated caspase gene expressions, which led to a marked increase in TSWV titers. The CYP and sEH genes of F. occidentalis were identified, and their expression manipulation using RNA interference (RNAi) treatments led to significant alternations in the insect's immune responses and TSWV viral titers. To ascertain which viral factor influences the host EpOME levels, specialized RNAi treatments targeting genes encoded by TSWV were administered to larvae infected with TSWV. These treatments demonstrated that NSS expression is pivotal in manipulating the genes involved in EpOME metabolism. These results indicate that NSs of TSWV are crucially linked with the elevation of host insect EpOME levels and play a key role in suppressing the antiviral responses of F. occidentalis.
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Oxilipinas , Thysanoptera , Tospovirus , Animales , Tospovirus/fisiología , Oxilipinas/metabolismo , Thysanoptera/virología , Insectos Vectores/virología , Insectos Vectores/inmunología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Larva/virología , Larva/inmunología , Apoptosis/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Epóxido Hidrolasas/metabolismo , Epóxido Hidrolasas/genéticaRESUMEN
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.
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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
BACKGROUND: Fatty acid metabolism constitutes a significant and intricate biochemical process within microorganisms. Cytochrome P450 (CYP450) enzymes are found in most organisms and occupy a pivotal position in the metabolism of fatty acids. However, the role of CYP450 enzyme mediated fatty acid metabolism in the pathogenicity of pathogenic fungi remains unclear. METHODS: In this study, a CYP450 enzyme-encoding gene, SsCYP86, was identified in the sugarcane smut fungus Sporisorium scitamineum and its functions were characterized using a target gene homologous recombination strategy and metabonomics. RESULTS: We found that the expression of SsCYP86 was induced by or sugarcane wax or under the condition of mating/filamentation. Sexual reproduction assay demonstrated that the SsCYP86 deletion mutant was defective in mating/filamentation and significantly reduced its pathogenicity. Further fatty acid metabolomic analysis unravelled the levels of fatty acid metabolites were reduced in the SsCYP86 deletion mutant. Exogenous addition of fatty acid metabolites cis-11-eicosenoic acid (C20:1N9), pentadecanoic acid (C15:0), and linolenic acid (C18:3N3) partially restored the mating/filamentation ability of the SsCYP86 deletion mutant and restored the transcriptional level of the SsPRF1, a pheromone response transcription factor that is typically down-regulated in the absence of SsCYP86. Moreover, the constitutive expression of SsPRF1 in the SsCYP86 deletion mutant restored its mating/filamentation. CONCLUSION: Our results indicated that SsCyp86 modulates the SsPRF1 transcription by fatty acid metabolism, and thereby regulate the sexual reproduction of S. scitamineum. These findings provide insights into how CYPs regulate sexual reproduction in S. scitamineum.
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Sistema Enzimático del Citocromo P-450 , Ácidos Grasos , Proteínas Fúngicas , Enfermedades de las Plantas , Ácidos Grasos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Enfermedades de las Plantas/microbiología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharum/microbiología , Virulencia , ReproducciónRESUMEN
Cytochromes P450 (P450s) are one of the largest enzymatic protein families and play critical roles in the synthesis and metabolism of plant secondary metabolites. Astragaloside IV (AS-IV) is one of the primary active components in Astragalus herbs, exhibiting diverse biological activities and pharmacological effects. However, P450s involved in the astragaloside biosynthesis have not been systematically analyzed in Astragalus mongholicus (A. mongholicus). In this study, we identified 209 P450 genes from the genome of A. mongholicus (AmP450s), which were classified into nine clans and 47 families and performed a systematic overview of their physical and chemical properties, phylogeny, gene structures and conserved motifs. Weighted gene co-expression network analysis (WGCNA) revealed that AmP450s are critical in the astragaloside biosynthesis pathway. The expression levels of these AmP450s were verified by quantitative real-time PCR (qRT-PCR) analysis in the root, stem and leaf, showing that most AmP450s are abundant in the root. Additionally, the correlation analysis between gene expressions and AS-IV content showed that twelve AmP450s, especially CYP71A28, CYP71D16 and CYP72A69, may have significant potential in the biosynthesis of astragaloside. This study systematically investigates the P450s of A. mongholicus and offers valuable insights into further exploring the functions of CYP450s in the astragaloside biosynthesis pathway.