RESUMEN
Cytochrome P450 enzymes are known to catalyse bimodal oxidation of aliphatic acids via radical intermediates, which partition between pathways of hydroxylation and desaturation1,2. Developing analogous catalytic systems for remote C-H functionalization remains a significant challenge3-5. Here, we report the development of Cu(I)-catalysed bimodal dehydrogenation/lactonization reactions of synthetically common N-methoxyamides through radical abstractions of the γ-aliphatic C-H bonds. The feasibility of switching from dehydrogenation to lactonization is also demonstrated by altering reaction conditions. The use of a readily available amide as both radical precursor and internal oxidant allows for the development of redox-neutral C-H functionalization reactions with methanol as the sole side product. These C-H functionalization reactions using a Cu(I) catalyst with loading as low as 0.5 mol.% is applied to the diversification of a wide range of aliphatic acids including drug molecules and natural products. The exceptional compatibility of this catalytic system with a wide range of oxidatively sensitive functionality demonstrates the unique advantage of using a simple amide substrate as a mild internal oxidant.
Asunto(s)
Carbono , Cobre , Hidrógeno , Lactonas , Amidas/química , Amidas/metabolismo , Carbono/química , Catálisis , Cobre/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/química , Hidrógeno/química , Hidrogenación , Lactonas/química , Metanol/química , Oxidantes/química , Oxidantes/metabolismo , Oxidación-ReducciónRESUMEN
Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms.
Asunto(s)
Citocromos b5 , Lignina , Proteínas de Plantas , Lignina/biosíntesis , Lignina/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Filogenia , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Evolución Molecular , Magnoliopsida/genética , Magnoliopsida/metabolismo , Embryophyta/genética , Carofíceas/genética , Carofíceas/metabolismoRESUMEN
Lateral branches are important components of shoot architecture and directly affect crop yield and production cost. Although sporadic studies have implicated abscisic acid (ABA) biosynthesis in axillary bud outgrowth, the function of ABA catabolism and its upstream regulators in shoot branching remain elusive. Here, we showed that the MADS-box transcription factor AGAMOUS-LIKE 16 (CsAGL16) is a positive regulator of axillary bud outgrowth in cucumber (Cucumis sativus). Functional disruption of CsAGL16 led to reduced bud outgrowth, whereas overexpression of CsAGL16 resulted in enhanced branching. CsAGL16 directly binds to the promoter of the ABA 8'-hydroxylase gene CsCYP707A4 and promotes its expression. Loss of CsCYP707A4 function inhibited axillary bud outgrowth and increased ABA levels. Elevated expression of CsCYP707A4 or treatment with an ABA biosynthesis inhibitor largely rescued the Csagl16 mutant phenotype. Moreover, cucumber General Regulatory Factor 1 (CsGRF1) interacts with CsAGL16 and antagonizes CsAGL16-mediated CsCYP707A4 activation. Disruption of CsGRF1 resulted in elongated branches and decreased ABA levels in the axillary buds. The Csagl16 Csgrf1 double mutant exhibited a branching phenotype resembling that of the Csagl16 single mutant. Therefore, our data suggest that the CsAGL16-CsGRF1 module regulates axillary bud outgrowth via CsCYP707A4-mediated ABA catabolism in cucumber. Our findings provide a strategy to manipulate ABA levels in axillary buds during crop breeding to produce desirable branching phenotypes.
Asunto(s)
Ácido Abscísico , Cucumis sativus , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Cucumis sativus/crecimiento & desarrollo , Cucumis sativus/genética , Cucumis sativus/metabolismo , Ácido Abscísico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Brotes de la Planta/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Plantas Modificadas Genéticamente , Sistema Enzimático del Citocromo P-450RESUMEN
A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.
Asunto(s)
Enfermedades Transmisibles , Descubrimiento de Drogas , Ratones , Animales , Interacciones Farmacológicas , Modelos Animales de Enfermedad , Sistema Enzimático del Citocromo P-450/metabolismo , AceleraciónRESUMEN
Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.
Asunto(s)
Hemípteros , Proteínas de Insectos , Resistencia a los Insecticidas , Sistema de Señalización de MAP Quinasas , Receptores Acoplados a Proteínas G , Animales , Hemípteros/genética , Hemípteros/metabolismo , Resistencia a los Insecticidas/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Femenino , Insecticidas/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genéticaRESUMEN
Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.
Asunto(s)
Anopheles , Sistema Enzimático del Citocromo P-450 , Elementos Transponibles de ADN , Resistencia a los Insecticidas , Insecticidas , Malaria , Mosquitos Vectores , Piretrinas , Animales , Anopheles/genética , Anopheles/parasitología , Anopheles/efectos de los fármacos , Piretrinas/farmacología , Resistencia a los Insecticidas/genética , Mosquitos Vectores/genética , Mosquitos Vectores/parasitología , Mosquitos Vectores/efectos de los fármacos , Malaria/transmisión , Malaria/parasitología , Malaria/genética , Elementos Transponibles de ADN/genética , Sistema Enzimático del Citocromo P-450/genética , Insecticidas/farmacología , Uganda , Humanos , CamerúnRESUMEN
This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have never lost the enthusiasm for research that I learned in the summer of 1968 with Harry Broquist, and I have tried to instill this in the many trainees I have worked with. A sentence I use on closing slides is "It's not just a laboratory-it's a fraternity."
Asunto(s)
Bioquímica , Sistema Enzimático del Citocromo P-450 , Humanos , Docentes , Mentores , Universidades , EnseñanzaRESUMEN
The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cß. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Hordeum , Alcaloides Indólicos , Proteínas de Plantas , Triptófano , Hordeum/genética , Hordeum/enzimología , Hordeum/metabolismo , Triptófano/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alcaloides Indólicos/metabolismo , Plantas Modificadas Genéticamente , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Oryza/genética , Oryza/enzimología , Oryza/metabolismo , Regulación de la Expresión Génica de las Plantas , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ciclopentanos , Sistema Enzimático del Citocromo P-450 , Regulación de la Expresión Génica de las Plantas , Lípidos de la Membrana , Oxilipinas , Hojas de la Planta , Ceras , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Ceras/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , Lípidos de la Membrana/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Transducción de Señal , Epidermis de la Planta/metabolismo , Epidermis de la Planta/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Liasas de Carbono-Carbono , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-HéliceRESUMEN
Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.
Asunto(s)
Alcaloides , Aporfinas , Aristolochia , Sistema Enzimático del Citocromo P-450 , Filogenia , Proteínas de Plantas , Aporfinas/metabolismo , Aristolochia/enzimología , Aristolochia/metabolismo , Aristolochia/genética , Aristolochia/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Alcaloides/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/enzimología , Raíces de Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Flores/enzimología , Flores/genética , Flores/metabolismo , Tallos de la Planta/metabolismo , Tallos de la Planta/enzimología , Tallos de la Planta/genéticaRESUMEN
Cytochrome P450 proteins (CYPs) play critical roles in plant development and adaptation to fluctuating environments. Previous reports have shown that CYP86A proteins are involved in the biosynthesis of suberin and cutin in Arabidopsis. However, the functions of these proteins in rice remain obscure. In this study, a rice mutant with incomplete male sterility was identified. Cytological analyses revealed that this mutant was defective in anther development. Cloning of the mutant gene indicated that the responsible mutation was on OsCYP86A9. OsMYB80 is a core transcription factor in the regulation of rice anther development. The expression of OsCYP86A9 was abolished in the anther of osmyb80 mutant. In vivo and in vitro experiments showed that OsMYB80 binds to the MYB-binding motifs in OsCYP86A9 promoter region and regulates its expression. Furthermore, the oscyp86a9 mutant exhibited an impaired suberin deposition in the root, and was more susceptible to drought stress. Interestingly, genetic and biochemical analyses revealed that OsCYP86A9 expression was regulated in the root by certain MYB transcription factors other than OsMYB80. Moreover, mutations in the MYB genes that regulate OsCYP86A9 expression in the root did not impair the male fertility of the plant. Taken together, these findings revealed the critical roles of OsCYP86A9 in plant development and proposed that OsCYP86A9 functions in anther development and root suberin formation via two distinct tissue-specific regulatory pathways.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Lípidos , Oryza , Proteínas de Plantas , Factores de Transcripción , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Lípidos/biosíntesis , Mutación , Oryza/genética , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Insect crop pests threaten global food security. This threat is amplified through the spread of nonnative species and through adaptation of native pests to control measures. Adaptations such as pesticide resistance can result from selection on variation within a population, or through gene flow from another population. We investigate these processes in an economically important noctuid crop pest, Helicoverpa zea, which has evolved resistance to a wide range of pesticides. Its sister species Helicoverpa armigera, first detected as an invasive species in Brazil in 2013, introduced the pyrethroid-resistance gene CYP337B3 to South American H. zea via adaptive introgression. To understand whether this could contribute to pesticide resistance in North America, we sequenced 237 H. zea genomes across 10 sample sites. We report H. armigera introgression into the North American H. zea population. Two individuals sampled in Texas in 2019 carry H. armigera haplotypes in a 4 Mbp region containing CYP337B3. Next, we identify signatures of selection in the panmictic population of nonadmixed H. zea, identifying a selective sweep at a second cytochrome P450 gene: CYP333B3. We estimate that its derived allele conferred a â¼5% fitness advantage and show that this estimate explains independently observed rare nonsynonymous CYP333B3 mutations approaching fixation over a â¼20-year period. We also detect putative signatures of selection at a kinesin gene associated with Bt resistance. Overall, we document two mechanisms of rapid adaptation: the introduction of fitness-enhancing alleles through interspecific introgression, and selection on intraspecific variation.
Asunto(s)
Introgresión Genética , Resistencia a los Insecticidas , Mariposas Nocturnas , Animales , Mariposas Nocturnas/genética , Resistencia a los Insecticidas/genética , Sistema Enzimático del Citocromo P-450/genética , América del Norte , Adaptación Biológica/genética , Adaptación Fisiológica/genética , Selección Genética , Especies IntroducidasRESUMEN
BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Drosophila melanogaster , Hormonas Juveniles , Animales , Corpora Allata/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Hormonas Juveniles/biosíntesis , Hormonas Juveniles/metabolismo , Larva/crecimiento & desarrollo , Larva/genética , Metamorfosis Biológica/genética , Oxidorreductasas , Pupa/crecimiento & desarrollo , Pupa/genética , Pupa/metabolismoRESUMEN
Mammalian cytochrome P450 1A (CYP1A) are key phase I xenobiotic-metabolizing enzymes that play a distinctive role in metabolic activation or metabolic clearance of a variety of procarcinogens, drugs, and endogenous substances. Human CYP1A subfamily contains two members (hCYP1A1 and hCYP1A2), which are known to catalyze the oxidative activation of some environmental procarcinogens into carcinogenic species. Increasing evidence has demonstrated that CYP1A inhibitor therapies are promising strategies for cancer chemoprevention or overcoming CYP1A-associated drug toxicity and resistance. Herein, we reviewed recent advances in the discovery and characterization of hCYP1A inhibitors, from the discovery approaches to structural features and biomedical applications of hCYP1A inhibitors. The inhibition potentials, inhibition modes, and inhibition constants of all reported hCYP1A inhibitors are comprehensively summarized. Meanwhile, the structural features and structure-activity relationships of different classes of hCYP1A1 and hCYP1A2 inhibitors are analyzed and discussed in depth. Furthermore, the major challenges and future directions for this field are presented and highlighted. Collectively, the information and knowledge presented here will strongly facilitate the researchers to discover and develop more efficacious CYP1A inhibitors for specific purposes, such as chemo-preventive agents or as tool molecules in hCYP1A-related fundamental studies.
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Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1A2 , Animales , Humanos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Mamíferos/metabolismoRESUMEN
The cytochrome P450 enzyme CYP121A1 endogenously catalyzes the formation of a carbon-carbon bond between the two phenol groups of dicyclotyrosine (cYY) in Mycobacterium tuberculosis (Mtb). One of 20 CYP enzymes in Mtb, CYP121A1 continues to garner significant interest as a potential drug target. The accompanying reports the use of 19F NMR spectroscopy, reconstituted activity assays, and molecular dynamics simulations to investigate the significance of hydrogen bonding interactions that were theorized to stabilize a static active site water network. The active site residue Asn-85, whose hydrogen bonds with the diketopiperazine ring of cYY contributes to a contiguous active site water network in the absence of cYY, was mutated to a serine (N85S) and to a glutamine (N85Q). These conservative changes in the hydrogen bond donor side chain result in inactivation of the enzyme. Moreover, the N85S mutation induces reverse type-I binding as measured by absorbance difference spectra. NMR spectra monitoring the ligand-adaptive FG-loop and the active site Trp-182 side chain confirm that disruption of the active site water network also significantly alters the structure of the active site. These data were consistent with dynamics simulations of N85S and N85Q that demonstrate that a compromised water network is responsible for remodeling of the active site B-helix and a repositioning of cYY toward the heme. These findings implicate a slowly exchanging water network as a critical factor in CYP121A1 function and a likely contributor to the unusual rigidity of the structure.
Asunto(s)
Mycobacterium tuberculosis , Dominio Catalítico , Asparagina , Agua , Sistema Enzimático del Citocromo P-450/metabolismo , Carbono , Enlace de HidrógenoRESUMEN
The mitochondrial enzyme cytochrome P450 11B2 (aldosterone synthase) catalyzes the 3 terminal transformations in the biosynthesis of aldosterone from 11-deoxycorticosterone (DOC): 11ß-hydroxylation to corticosterone, 18-hydroxylation, and 18-oxidation. Prior studies have shown that P450 11B2 produces more aldosterone from DOC than from the intermediate corticosterone and that the reaction sequence is processive, with intermediates remaining bound to the active site between oxygenation reactions. In contrast, P450 11B1 (11ß-hydroxylase), which catalyzes the terminal step in cortisol biosynthesis, shares a 93% amino acid sequence identity with P450 11B2, converts DOC to corticosterone, but cannot synthesize aldosterone from DOC. The biochemical and biophysical properties of P450 11B2, which enable its unique 18-oxygenation activity and processivity, yet are not also represented in P450 11B1, remain unknown. To understand the mechanism of aldosterone biosynthesis, we introduced point mutations at residue 320, which partially exchange the activities of P450 11B1 and P450 11B2 (V320A and A320V, respectively). We then investigated NADPH coupling efficiencies, binding kinetics and affinities, and product formation of purified P450 11B1 and P450 11B2, wild-type, and residue 320 mutations in phospholipid vesicles and nanodiscs. Coupling efficiencies for the 18-hydroxylase reaction with corticosterone as the substrate failed to correlate with aldosterone synthesis, ruling out uncoupling as a relevant mechanism. Conversely, corticosterone dissociation rates correlated inversely with aldosterone production. We conclude that intermediate dissociation kinetics, not coupling efficiency, enable P450 11B2 to synthesize aldosterone via a processive mechanism. Our kinetic data also suggest that the binding of DOC to P450 11B enzymes occurs in at least two distinct steps, favoring an induced-fit mechanism.
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Aldosterona , Esteroide 11-beta-Hidroxilasa , Esteroide 11-beta-Hidroxilasa/química , Esteroide 11-beta-Hidroxilasa/genética , Esteroide 11-beta-Hidroxilasa/metabolismo , Corticosterona/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/química , Citocromo P-450 CYP11B2/metabolismo , Catálisis , CinéticaRESUMEN
BACKGROUND: Insect Cytochrome P450 monooxygenase (CYPs or P450s) plays an important role in detoxifying insecticides, causing insect populations to develop resistance. However, the molecular functions of P450 gene family in Cyrtotrachelus buqueti genome are still lacking. RESULTS: In this study, 71 CbuP450 genes have been identified. The amino acids length of CbuP450 proteins was between 183 aa ~ 1041 aa. They are proteins with transmembrane domains. The main component of their secondary structure is α-helix and random coils. Phylogenetic analysis showed that C. buqueti and Rhynchophorus ferrugineus were the most closely related. This gene family has 29 high-frequency codons, which tend to use A/T bases and A/T ending codons. Gene expression analysis showed that CbuP450_23 in the female adult may play an important role on high temperature resistance, and CbuP450_17 in the larval may play an important role on low temperature tolerance. CbuP450_10, CbuP450_17, CbuP450_23, CbuP450_10, CbuP450_16, CbuP450_20, CbuP450_23 and CbuP450_ 29 may be related to the regulation of bamboo fiber degradation genes in C. buqueti. Protein interaction analysis indicates that most CbuP450 proteins are mainly divided into three aspects: encoding the biosynthesis of ecdysteroids, participating in the decomposition of synthetic insecticides, metabolizing insect hormones, and participating in the detoxification of compounds. CONCLUSIONS: We systematically analyzed the gene and protein characteristics, gene expression, and protein interactions of CbuP450 gene family, revealing the key genes involved in the stress response of CbuP450 gene family in the resistance of C. buqueti to high or low temperature stress, and identified the key CbuP450 proteins involved in important life activity metabolism. These results provided a reference for further research on the function of P450 gene family in C. buqueti.
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Sistema Enzimático del Citocromo P-450 , Evolución Molecular , Filogenia , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Familia de Multigenes , Genoma de los Insectos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Femenino , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: Hylurgus ligniperda (Coleoptera: Curculionidae) is a worldwide forest quarantine pest. It is widely distributed, has many host tree species, and possesses strong adaptability. To explore its environmental adaptability and the related molecular mechanisms, we conducted chromosome-level genome sequencing and analyzed the transcriptome under different environmental factors, identifying key expressed genes. RESULTS: We employed PacBio, Illumina, and Hi-C sequencing techniques to assemble a 520 Mb chromosomal-level genome of H. ligniperda, obtaining an N50 of 39.97 Mb across 138 scaffolds. A total of 10,765 protein-coding genes were annotated after repeat masking. Fourteen chromosomes were identified, among which Hyli14 was determined to be the sex chromosome. Survival statistics were tested over various growth periods under high temperature and low humidity conditions. The maximum survival period of adults reached 292 days at 25 °C, 65% relative humidity. In comparison, the maximum survival period was 14 days under 35 °C, 65% relative humidity, and 106 days under 25°C, 40% relative humidity. This indicated that environmental stress conditions significantly reduced adults' survival period. We further conducted transcriptome analysis to screen for potentially influential differentially expressed genes, such as CYP450 and Histone. Subsequently, we performed gene family analysis to gain insights into their functions and interactions, such as CYP450 and Histone. CYP450 genes affected the detoxification metabolism of enzymes in the Cytochrome P450 pathway to adapt to different environments. Histone genes are involved in insect hormone biosynthesis and longevity-regulating pathways in H. ligniperda to adapt to environmental stress. CONCLUSIONS: The genome at the chromosome level of H. ligniperda was assembled for the first time. The mortality of H. ligniperda increased significantly at 35 â, 65% RH, and 25 â, 40% RH. CYP450 and Histone genes played an important role in response to environmental stress. This genome offers a substantial genetic resource for investigating the molecular mechanisms behind beetle invasion and spread.
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Adaptación Fisiológica , Animales , Adaptación Fisiológica/genética , Genoma de los Insectos , Gorgojos/genética , Transcriptoma , Cromosomas de Insectos/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Perfilación de la Expresión Génica , Estrés Fisiológico/genéticaRESUMEN
BACKGROUND: Cytochrome P450 monooxygenases (CYP450s) play a crucial role in various biochemical reactions involved in the synthesis of antioxidants, pigments, structural polymers, and defense-related compounds in plants. As sweet potato (Ipomoea batatas L.) holds significant economic importance, a comprehensive analysis of CYP450 genes in this plant species can offer valuable insights into the evolutionary relationships and functional characteristics of these genes. RESULTS: In this study, we successfully identified and categorized 95 CYP450 genes from the sweet potato genome into 5 families and 31 subfamilies. The predicted subcellular localization results indicate that CYP450s are distributed in the cell membrane system. The promoter region of the IbCYP450 genes contains various cis-acting elements related to plant hormones and stress responses. In addition, ten conserved motifs (Motif1-Motif10) have been identified in the IbCYP450 family proteins, with 5 genes lacking introns and only one exon. We observed extensive duplication events within the CYP450 gene family, which may account for its expansion. The gene duplication analysis results showed the presence of 15 pairs of genes with tandem repeats. Interaction network analysis reveals that IbCYP450 families can interact with multiple target genes and there are protein-protein interactions within the family. Transcription factor interaction analysis suggests that IbCYP450 families interact with multiple transcription factors. Furthermore, gene expression analysis revealed tissue-specific expression patterns of CYP450 genes in sweet potatoes, as well as their response to abiotic stress and plant hormones. Notably, quantitative real-time polymerase chain reaction (qRTâPCR) analysis indicated the involvement of CYP450 genes in the defense response against nonbiological stresses in sweet potatoes. CONCLUSIONS: These findings provide a foundation for further investigations aiming to elucidate the biological functions of CYP450 genes in sweet potatoes.
Asunto(s)
Ipomoea batatas , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación de la Expresión Génica de las Plantas , FilogeniaRESUMEN
The genomes of species belonging to the genus Colletotrichum harbor a substantial number of cytochrome P450 monooxygenases (CYPs) encoded by a broad diversity of gene families. However, the biological role of their CYP complement (CYPome) has not been elucidated. Here, we investigated the putative evolutionary scenarios that occurred during the evolution of the CYPome belonging to the Colletotrichum Graminicola species complex (s.c.) and their biological implications. The study revealed that most of the CYPome gene families belonging to the Graminicola s.c. experienced gene contractions. The reductive evolution resulted in species restricted CYPs are predominant in each CYPome of members from the Graminicola s.c., whereas only 18 families are absolutely conserved among these species. However, members of CYP families displayed a notably different phylogenetic relationship at the tertiary structure level, suggesting a putative convergent evolution scenario. Most of the CYP enzymes of the Graminicola s.c. share redundant functions in secondary metabolite biosynthesis and xenobiotic metabolism. Hence, this current work suggests that the presence of a broad CYPome in the genus Colletotrichum plays a critical role in the optimization of the colonization capability and virulence.