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1.
Nat Commun ; 11(1): 4914, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004788

RESUMO

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with D-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the D-Phe residue back to L-form, which is essential for the final methylation by OpaF.


Assuntos
Amidas/metabolismo , Aspergillus/enzimologia , Proteínas Fúngicas/metabolismo , Oxepinas/metabolismo , Amidas/química , Amidas/isolamento & purificação , Aspergillus/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/genética , Hidroxilação , Isomerismo , Metilação , Oxepinas/química , Oxepinas/isolamento & purificação , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Proteína O-Metiltransferase/genética , Proteína O-Metiltransferase/metabolismo , Quinazolinonas/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo
2.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
3.
Chem Biol Interact ; 330: 109247, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866466

RESUMO

This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.


Assuntos
Fígado/metabolismo , Oxazinas/metabolismo , Frações Subcelulares/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Feminino , Herbicidas/química , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Ratos , Fatores Sexuais , Estereoisomerismo
4.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1346-1355, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748592

RESUMO

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.


Assuntos
Comamonadaceae , Sistema Enzimático do Citocromo P-450 , Biologia Sintética , Catálise , Comamonadaceae/enzimologia , Comamonadaceae/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Expressão Gênica
5.
Sci Total Environ ; 745: 140639, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-32758758

RESUMO

The Deepwater Horizon (DWH) oil spill marked the largest environmental oil spill in human history, where it was estimated a large amount of the polycyclic aromatic hydrocarbons (PAHs) were released with crude oil into the environment. In this study, common PAH compounds were quantitatively determined in crude oil from the DWH spill by gas chromatography-mass spectroscopy (GC-MS). Twelve PAH compounds were identified and quantified from a 100× dilution of DWH crude oil: naphthalene (7800 ng/mL), acenaphthylene (590 ng/mL), acenaphtehen (540 ng/mL), fluorene (2550 ng/mL), phenanthrene (2910 ng/mL), anthracene (840 ng/mL), fluoranthene (490 ng/mL), pyrene (290 ng/mL), benzo(k) fluoranthene (1050 ng/mL), benzo(b)fluoranthene (1360 ng/mL), dibenz(a,h)anthracene (2560 ng/mL), and benzo(g, h, i) perylene (630 ng/mL). Toxicity assays using the nematode, Caenorhabditis elegans (C. elegans), indicated a single PAH compound naphthalene, exposure increased C. elegans germ cell apoptosis which may adversely affect progeny reproduction. The number of apoptotic germ cells significantly increased from 1.4 to 2.5 when worms were treated with 10 µg/mL of naphthalene and from 1.3 to 2.5 and 3.5 cells in presence of 1 µg/mL and 5 µg/mL of benzo(a)pyrene, respectively. Five CYP450 genes (CYP14A3, CYP35A1, CYP35A2, CYP35A5, and CYP35C1) were significantly upregulated following 500× dilution of dispersed crude oil exposure (p < 0.05). These results suggest that CYP450s may play a role in bioactivation of PAHs in crude oil, resulting in DNA damage related germ cell apoptosis.


Assuntos
Petróleo/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Animais , Apoptose , Caenorhabditis elegans , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Células Germinativas , Humanos , Regulação para Cima
6.
Plant Mol Biol ; 104(3): 327-337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761540

RESUMO

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.


Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Transcriptoma
7.
Chem Biol Interact ; 329: 109147, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32738202

RESUMO

Acacetin is a natural flavonoid that is widely distributed in plants and possesses numerous pharmacological activities. The aim of the present study was to investigate the effects of acacetin on the activities of the cytochrome P450 family members CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1, and CYP3A2 in rat liver microsomes in vitro and rats in vivo to evaluate potential herb-drug interactions by using a cocktail approach. Phenacetin, bupropion, tolbutamide, dextromethorphan, chlorzoxazone, and midazolam were chosen as the probe substrates. An ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the probe substrates and their metabolites. In vitro, the mode of acacetin inhibition of CYP2B1, CYP2C11, and CYP2E1 was competitive, while mixed inhibition was observed for CYP1A2 and CYP3A2. The Ki values in this study were less than 8.32 µM. In vivo, the mixed probe substrates were administered by gavage after daily intraperitoneal injection with 50 mg/kg acacetin or saline for 2 weeks. The main pharmacokinetic parameters, area under the plasma concentration-time curve (AUC), plasma clearance (CL), and maximum plasma concentration (Cmax) of the probe substrates were significantly different in the experimental group than in the control group. Overall, the in vitro and in vivo results indicated that acacetin would be at high risk to cause toxicity and drug interactions via cytochrome P450 inhibition.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Flavonas/metabolismo , Animais , Área Sob a Curva , Sistema Enzimático do Citocromo P-450/química , Flavonas/química , Flavonas/farmacocinética , Meia-Vida , Concentração Inibidora 50 , Cinética , Masculino , Microssomos Hepáticos/metabolismo , Curva ROC , Ratos , Ratos Sprague-Dawley
8.
Mol Cell ; 79(5): 768-781.e7, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32738194

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Dobramento de Proteína , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo
9.
Aquat Toxicol ; 226: 105565, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32682195

RESUMO

Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 µM) and FLU (0.6 and 1.2 µM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 µM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 µM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.


Assuntos
Crassostrea/efeitos dos fármacos , Fluorenos/toxicidade , Pirenos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Biotransformação/efeitos dos fármacos , Crassostrea/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fluorenos/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Glutationa Transferase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pirenos/metabolismo , Poluentes Químicos da Água/metabolismo
10.
Eur J Cancer ; 136: 1-3, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32610172
11.
Nature ; 584(7819): 148-153, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32699417

RESUMO

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.


Assuntos
Vias Biossintéticas , Colchicina/biossíntese , Engenharia Metabólica , Vias Biossintéticas/genética , Colchicaceae/enzimologia , Colchicaceae/genética , Colchicaceae/metabolismo , Colchicina/química , Colchicina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolômica , Fenilalanina/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transcriptoma , Tirosina/metabolismo
12.
PLoS One ; 15(7): e0235746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678853

RESUMO

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.


Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Esterol 14-Desmetilase/genética
13.
Food Chem ; 331: 127365, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32619909

RESUMO

Phenolic acids from Salvia miltiorrhiza have been widely used in nutritious, health-promoting products with an increasing demand. In the current study, two biosynthetic genes RAS (rosmarinic acid synthase) and CYP98A14 (a cytochrome P450-dependent monooxygenase) were successfully introduced into S. miltiorrhiza hairy roots. Overexpression of RAS and CYP98A14 resulted in higher content of phenolic acids (up to over 3-fold) in transgenic lines compared to the control. Meanwhile, DPPH results revealed that engineered S. miltiorrhiza hairy roots had stronger antioxidant activities than the control. In addition, phenolic acid crude extracts of the engineered hairy root lines overexpressing RAS or CYP98A14 showed improved antibacterial activities compared to the control lines. Our work exhibits a useful strategy for enhancement of phenolic acid production and bioactivities of S. miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14, and also provides a new resource material to obtain active phenolic acids for food and healthy products.


Assuntos
Aciltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxibenzoatos/metabolismo , Raízes de Plantas/química , Salvia miltiorrhiza/genética , Aciltransferases/genética , Antibacterianos/química , Antibacterianos/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Salmonella/efeitos dos fármacos , Salvia miltiorrhiza/química , Salvia miltiorrhiza/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(26): 15262-15269, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541022

RESUMO

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina, thyroid hormone receptor ß (thrb) is required for expression of long-wavelength-sensitive opsin (lws) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme, cyp27c1, that converts vitamin A1 into vitamin A2 to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors (thraa, thrab, and thrb). We found that no single TH nuclear receptor is required for TH-mediated induction of cyp27c1 but that deletion of all three (thraa -/- ;thrab -/- ;thrb -/- ) completely abrogates its induction and the resulting conversion of A1- to A2-based retinoids. In the retina, loss of thrb resulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adult thrb -/- retina, of which three (lws1, lws2, and miR-726) occur in a single syntenic cluster. In the thrb -/- retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation of cyp27c1 by TH receptors and a requirement for thrb in red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.


Assuntos
Visão de Cores/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Opsinas/metabolismo , Receptores dos Hormônios Tireóideos/fisiologia , Percepção Visual/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Visão de Cores/genética , Sistema Enzimático do Citocromo P-450/genética , Deleção de Genes , Regulação da Expressão Gênica , Opsinas/genética , Células Fotorreceptoras Retinianas Cones , Raios Ultravioleta , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
15.
16.
Nat Commun ; 11(1): 2676, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32472090

RESUMO

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Elétrons/fisiologia , Hydrogenophilaceae/enzimologia , Cristalografia por Raios X , Conformação Proteica
17.
BMC Bioinformatics ; 21(1): 160, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349673

RESUMO

BACKGROUND: Cytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner. RESULTS: CYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences. CONCLUSIONS: CYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.


Assuntos
Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Análise de Dados , Bases de Dados Genéticas , Genoma , Filogenia , Automação , Análise por Conglomerados , Fungos/genética , Redes Reguladoras de Genes , Software , Interface Usuário-Computador
18.
PLoS One ; 15(5): e0231980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357188

RESUMO

Triterpenoids are high-value plant metabolites with numerous applications in medicine, agriculture, food, and home and personal care products. However, plants produce triterpenoids in low abundance, and their complex structures make their chemical synthesis prohibitively expensive and often impossible. As such, the yeast Saccharomyces cerevisiae has been explored as an alternative means of production. An important triterpenoid is oleanolic acid because it is the precursor to many bioactive triterpenoids of commercial interest, such as QS-21 which is being evaluated as a vaccine adjuvant in clinical trials against HIV and malaria. Oleanolic acid is derived from 2,3-oxidosqualene (natively produced by yeast) via a cyclisation and a multi-step oxidation reaction, catalysed by a ß-amyrin synthase and a cytochrome P450 of the CYP716A subfamily, respectively. Although many homologues have been characterised, previous studies have used arbitrarily chosen ß-amyrin synthases and CYP716As to produce oleanolic acid and its derivatives in yeast. This study presents the first comprehensive comparison of ß-amyrin synthase and CYP716A enzyme activities in yeast. Strains expressing different homologues are compared for production, revealing 6.3- and 4.5-fold differences in ß-amyrin and oleanolic acid productivities and varying CYP716A product profiles, which are important to consider when engineering strains for the production of bioactive oleanolic acid derivatives.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/biossíntese , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de Sequência
19.
Toxicol Appl Pharmacol ; 398: 115034, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387183

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor (AhR) agonist that elicits a broad spectrum of dose-dependent hepatic effects including lipid accumulation, inflammation, and fibrosis. To determine the role of inflammatory lipid mediators in TCDD-mediated hepatotoxicity, eicosanoid metabolism was investigated. Female Sprague-Dawley (SD) rats were orally gavaged with sesame oil vehicle or 0.01-10 µg/kg TCDD every 4 days for 28 days. Hepatic RNA-Seq data was integrated with untargeted metabolomics of liver, serum, and urine, revealing dose-dependent changes in linoleic acid (LA) and arachidonic acid (AA) metabolism. TCDD also elicited dose-dependent differential gene expression associated with the cyclooxygenase, lipoxygenase, and cytochrome P450 epoxidation/hydroxylation pathways with corresponding changes in ω-6 (e.g. AA and LA) and ω-3 polyunsaturated fatty acids (PUFAs), as well as associated eicosanoid metabolites. Overall, TCDD increased the ratio of ω-6 to ω-3 PUFAs. Phospholipase A2 (Pla2g12a) was induced consistent with increased AA metabolism, while AA utilization by induced lipoxygenases Alox5 and Alox15 increased leukotrienes (LTs). More specifically, TCDD increased pro-inflammatory eicosanoids including leukotriene LTB4, and LTB3, known to recruit neutrophils to damaged tissue. Dose-response modeling suggests the cytochrome P450 hydroxylase/epoxygenase and lipoxygenase pathways are more sensitive to TCDD than the cyclooxygenase pathway. Hepatic AhR ChIP-Seq analysis found little enrichment within the regulatory regions of differentially expressed genes (DEGs) involved in eicosanoid biosynthesis, suggesting TCDD-elicited dysregulation of eicosanoid metabolism is a downstream effect of AhR activation. Overall, these results suggest alterations in eicosanoid metabolism may play a key role in TCDD-elicited hepatotoxicity associated with the progression of steatosis to steatohepatitis.


Assuntos
Eicosanoides/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fígado/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Fígado Gorduroso/metabolismo , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/metabolismo
20.
Virology ; 546: 133-140, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452413

RESUMO

Auxin has profound effects on plant growth and development. In addition to participating in plant growth and development, the auxin signaling pathway is involved in plant defense against pathogens. In this study, we investigated the molecular mechanism by which helper-component protease (HCPro) encoded by the Tobacco vein banding mosaic virus (TVBMV) activates auxin biosynthesis genes (YUCs) and interferes with the auxin signaling pathway. Our results demonstrated that the viral suppressor HCPro decreased the DNA methylation of dispersed repeats (DRs) within the promoters of YUC1, YUC5 and YUC10 and transcriptional activated these YUC genes targeted by RNA-directed DNA methylation (RdDM), leading to an increase in auxin accumulation in plants. Furthermore, we found that the induction of these YUCs by HCPro was attenuated in ros1 mutant plants, suggesting that HCPro-mediated transcriptional activation of the genes was partly dependent on ROS1-mediated DNA demethylation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Ácidos Indolacéticos/metabolismo , Oxigenases/genética , Doenças das Plantas/virologia , Potyvirus/enzimologia , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Oxigenases/metabolismo , Doenças das Plantas/genética , Potyvirus/genética , Regiões Promotoras Genéticas
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