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1.
J Agric Food Chem ; 69(1): 55-66, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33356240

RESUMO

Hyphantria cunea (Drury) is a destructive invasive pest species in China that uses type II sex pheromone components. To date, however, the binding mechanisms of its sex pheromone components to their respective pheromone-binding proteins (HcunPBPs 1/2/3) have not been explored. In the current study, all three HcunPBPs were expressed in the antennae of both sexes. The prokaryotic expression and ligand binding assays were employed to study the binding of the moth's four sex pheromone components, including two aldehydes and two epoxides, and 24 plant volatiles to the HcunPBPs. Our results showed that the abilities of these HcunPBPs to bind to the aldehydes were significantly different from binding to the epoxides. These three HcunPBPs also selectively bind to some of the plant volatiles tested. Our molecular docking results indicated that some crucial hydrophobic residues might play a role in the binding of HcunPBPs to their sex pheromone components. Three HcunPBPs have different selectivities for pheromone components with both major and minor structural differences. Our study provides a fundamental insight into the olfactory mechanism of moths at the molecular level, especially for moth species that use various type II pheromone components.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Insetos/metabolismo , Atrativos Sexuais/metabolismo , Aldeídos/química , Aldeídos/metabolismo , Animais , Proteínas de Transporte/química , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Feminino , Proteínas de Insetos/química , Masculino , Simulação de Acoplamento Molecular , Mariposas/química , Mariposas/metabolismo , Ligação Proteica , Atrativos Sexuais/química , Olfato
2.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33374956

RESUMO

Epoxide hydrolases (EHs) are key enzymes involved in the detoxification of xenobiotics and biotransformation of endogenous epoxides. They catalyze the hydrolysis of highly reactive epoxides to less reactive diols. EHs thereby orchestrate crucial signaling pathways for cell homeostasis. The EH family comprises 5 proteins and 2 candidate members, for which the corresponding genes are not yet identified. Although the first EHs were identified more than 30 years ago, the full spectrum of their substrates and associated biological functions remain partly unknown. The two best-known EHs are EPHX1 and EPHX2. Their wide expression pattern and multiple functions led to the development of specific inhibitors. This review summarizes the most important points regarding the current knowledge on this protein family and highlights the particularities of each EH. These different enzymes can be distinguished by their expression pattern, spectrum of associated substrates, sub-cellular localization, and enzymatic characteristics. We also reevaluated the pathogenicity of previously reported variants in genes that encode EHs and are involved in multiple disorders, in light of large datasets that were made available due to the broad development of next generation sequencing. Although association studies underline the pleiotropic and crucial role of EHs, no data on high-effect variants are confirmed to date.


Assuntos
Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Transdução de Sinais , Xenobióticos/metabolismo , Biocatálise , Biotransformação , Epóxido Hidrolases/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Especificidade por Substrato
3.
Toxicol Lett ; 332: 82-87, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32569803

RESUMO

BACKGROUND: Glycidol, a probable human carcinogen, is a reactive chemical released in the gastrointestinal tract from glycidyl fatty acid esters, which are heat-induced dietary contaminants. OBJECTIVES: To investigate the prenatal transfer of glycidol, a specific hemoglobin adduct was measured as a biomarker for internal glycidol exposure in paired cord and maternal blood samples. METHODS: In 100 mother-newborn pairs from the Belgian ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort, we studied the correlation between levels of the glycidol-derived hemoglobin adduct N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val) in paired cord and maternal blood samples. The adduct levels were determined after cleavage with a modified Edman degradation by using ultra-high performance liquid chromatography-tandem mass spectrometry and an isotope-labeled reference standard. RESULTS: 2,3-DiHOPr-Val was detectable in all 100 maternal blood samples and in 96 cord blood samples (LOD =0.5 pmol 2,3-diHOPr-Val/g hemoglobin), with medians of 5.4 (range: 2.3-29.2) and 1.6 (range: LOD - 8.9) pmol/g hemoglobin), respectively. In blood samples of mothers who smoked during pregnancy and in the cord blood samples of their newborns (n = 6), the median 2,3-diHOPr-Val levels were 16.7 (range: 6.4-29.2) and 6.2 (range: LOD - 8.6) pmol/g hemoglobin, respectively. The median ratio of 2,3-diHOPr-Val levels of cord to maternal blood was 0.35 (range: 0.19-1.14) (n = 49). The Spearman correlation coefficient between 2,3-diHOPr-Val levels in cord and maternal blood samples was 0.63 (p < 0.001) among all mother-newborn pairs and 0.59 (p < 0.001) among mother-newborn pairs of non-smoking mothers. DISCUSSION: Maternal data confirm widespread exposure to glycidol, also in non-smokers. Neonatal levels indicate prenatal exposure to glycidol, due to an obviously relatively unhindered passive transfer through the placental barrier. Possible health effects of fetal (and postnatal) glycidol exposure in children may be addressed in epidemiological studies.


Assuntos
Compostos de Epóxi/metabolismo , Sangue Fetal/química , Hemoglobinas/metabolismo , Troca Materno-Fetal , Propanóis/metabolismo , Valina/análogos & derivados , Adulto , Biomarcadores/sangue , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Eritrócitos/química , Feminino , Hemoglobinas/análise , Humanos , Recém-Nascido , Gravidez , Fumar/sangue , Espectrometria de Massas em Tandem , Valina/sangue
4.
Toxicology ; 440: 152478, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32437779

RESUMO

Thiazoles are biologically active aromatic heterocyclic rings occurring frequently in natural products and drugs. These molecules undergo typically harmless elimination; however, a hepatotoxic response can occur due to multistep bioactivation of the thiazole to generate a reactive thioamide. A basis for those differences in outcomes remains unknown. A textbook example is the high hepatotoxicity observed for sudoxicam in contrast to the relative safe use and marketability of meloxicam, which differs in structure from sudoxicam by the addition of a single methyl group. Both drugs undergo bioactivation, but meloxicam exhibits an additional detoxification pathway due to hydroxylation of the methyl group. We hypothesized that thiazole bioactivation efficiency is similar between sudoxicam and meloxicam due to the methyl group being a weak electron donator, and thus, the relevance of bioactivation depends on the competing detoxification pathway. For a rapid analysis, we modeled epoxidation of sudoxicam derivatives to investigate the impact of substituents on thiazole bioactivation. As expected, electron donating groups increased the likelihood for epoxidation with a minimal effect for the methyl group, but model predictions did not extrapolate well among all types of substituents. Through analytical methods, we measured steady-state kinetics for metabolic bioactivation of sudoxicam and meloxicam by human liver microsomes. Sudoxicam bioactivation was 6-fold more efficient than that for meloxicam, yet meloxicam showed a 6-fold higher efficiency of detoxification than bioactivation. Overall, sudoxicam bioactivation was 15-fold more likely than meloxicam considering all metabolic clearance pathways. Kinetic differences likely arise from different enzymes catalyzing respective metabolic pathways based on phenotyping studies. Rather than simply providing an alternative detoxification pathway, the meloxicam methyl group suppressed the bioactivation reaction. These findings indicate the impact of thiazole substituents on bioactivation is more complex than previously thought and likely contributes to the unpredictability of their toxic potential.


Assuntos
Meloxicam/metabolismo , Tiazinas/metabolismo , Ativação Metabólica , Biotransformação , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Elétrons , Compostos de Epóxi/metabolismo , Humanos , Hidroxilação , Técnicas In Vitro , Cinética , Redes e Vias Metabólicas/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Tiazóis/metabolismo
5.
Chem Commun (Camb) ; 56(18): 2799-2802, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32030396

RESUMO

Both the activity and regioselectivity of Phaseolus vulgaris epoxide hydrolase were remarkably improved via reshaping two substrate tunnels based on rational design. The elegant one-step enantioconvergent hydrolysis of seven rac-epoxides was achieved by single mutants, allowing green and efficient access to valuable (R)-1,2 diols with high eep (90.1-98.3%) and yields.


Assuntos
Álcoois/metabolismo , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Phaseolus/enzimologia , Álcoois/química , Epóxido Hidrolases/química , Compostos de Epóxi/química , Modelos Moleculares , Conformação Molecular , Estereoisomerismo
6.
Sci Rep ; 10(1): 1680, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015448

RESUMO

The investigation of substrate spectrum towards five racemic (rac-) aryl glycidyl ethers (1a-5a) indicated that E. coli/pveh3, an E. coli BL21(DE3) transformant harboring a PvEH3-encoding gene pveh3, showed the highest EH activity and enantiomeric ratio (E) towards rac-3a. For efficiently catalyzing the kinetic resolution of rac-3a, the activity and E value of PvEH3 were further improved by site-directed mutagenesis of selected residues. Based on the semi-rational design of an NC-loop in PvEH3, four single-site variants of pveh3 were amplified by PCR, and intracellularly expressed in E. coli BL21(DE3), respectively. E. coli/pveh3E134K and /pveh3T137P had the enhanced EH activities of 15.3 ± 0.4 and 16.1 ± 0.5 U/g wet cell as well as E values of 21.7 ± 1.0 and 21.2 ± 1.1 towards rac-3a. Subsequently, E. coli/pveh3E134K/T137P harboring a double-site variant gene was also constructed, having the highest EH activity of 22.4 ± 0.6 U/g wet cell and E value of 24.1 ± 1.2. The specific activity of the purified PvEH3E134K/T137P (14.5 ± 0.5 U/mg protein) towards rac-3a and its catalytic efficiency (kcat/Km of 5.67 mM-1 s-1) for (S)-3a were 1.7- and 3.54-fold those (8.4 ± 0.3 U/mg and 1.60 mM-1 s-1) of PvEH3. The gram-scale kinetic resolution of rac-3a using whole wet cells of E. coli/pveh3E134K/T137P was performed at 20 °C for 7.0 h, producing (R)-3a with 99.4% ees and 38.5 ± 1.2% yield. Additionally, the mechanism of PvEH3E134K/T137P with remarkably improved E value was analyzed by molecular docking simulation.


Assuntos
Cresóis/metabolismo , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Catálise , Epóxido Hidrolases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Simulação de Acoplamento Molecular/métodos , Mutagênese Sítio-Dirigida/métodos , Phaseolus/genética , Estereoisomerismo , Especificidade por Substrato/fisiologia
7.
Molecules ; 25(3)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019202

RESUMO

The importance of the gut microbiota in drug metabolism, especially in that of nonabsorbable drugs, has become known. The aim of this study was to explore the metabolites of triptolide by the gut microbiota. With high-performance liquid chromatography coupled with tandem mass spectrometry and ion trap time-of-flight multistage mass spectrometry (LC-MS/MS and LC/MSn-IT-TOF), four metabolites of triptolide (M1, M2, M3, and M4) were found in the intestinal contents of rats. M1 and M2, were isomeric monocarbonyl-hydroxyl-substituted metabolites with molecular weights of 390. M3 and M4 were isomeric dehydrogenated metabolites with molecular weights of 356. Among the four metabolites, the dehydrogenated metabolites (M3 and M4) were reported in the gut microbiota for the first time. The metabolic behaviors of triptolide in the gut microbiota and liver microsomes of rats were further compared. The monocarbonyl-hydroxyl-substituted metabolites (M1 and M2) were generated in both systems, and another monohydroxylated metabolite (M5) was found only in the liver microsomes. The combined results suggested that the metabolism of triptolide in the gut microbiota was specific, with two characteristic, dehydrogenated metabolites. This investigation might provide a theoretical basis for the elucidation of the metabolism mechanism of triptolide and guide its proper application in clinical administration.


Assuntos
Diterpenos/metabolismo , Microbioma Gastrointestinal , Imunossupressores/metabolismo , Microssomos Hepáticos/metabolismo , Fenantrenos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Diterpenos/química , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Imunossupressores/química , Masculino , Fenantrenos/química , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem
8.
J Biotechnol ; 311: 19-24, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32067982

RESUMO

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts involved in the synthesis of ß-substituted alcohols via their nucleophile-mediated ring-opening activity. To use directed evolution to unleash the latent potential of HHDHs for the synthesis of ß-substituted alcohols, we report a high-throughput assay for screening HHDHs mutagenesis libraries. The assay is performed in a 96-well microtiter plate format using a cell-free extract or whole-cells in the presence of the desired nucleophile. The developed method relies upon the color change of bromothymol blue, due to the pH change caused by HHDH-catalyzed ring-opening of the epoxide substrate. The assay was validated using gas chromatography and subsequently applied to high-throughput screening of halohydrin dehalogenase HheC mutagenesis library. Active mutants were found for the tested substrates. Due to its simplicity and flexibility towards the use of nucleophiles and epoxides, the method is an attractive alternative to the existing assays for HHDH epoxide ring-opening reaction and could be helpful in the rapid discovery of industrial biocatalysts.


Assuntos
Bioensaio/métodos , Compostos de Epóxi/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Mutação/genética
9.
Nat Commun ; 11(1): 971, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080175

RESUMO

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tripterygium/genética , Tripterygium/metabolismo , Abietanos/metabolismo , Antineoplásicos Fitogênicos/biossíntese , Vias Biossintéticas/genética , Medicamentos de Ervas Chinesas/metabolismo , Compostos de Epóxi/metabolismo , Perfilação da Expressão Gênica , Genoma de Planta , Humanos , Engenharia Metabólica , Metaboloma , Oxirredução , Filogenia , Plantas Medicinais/genética , Plantas Medicinais/metabolismo
10.
Appl Microbiol Biotechnol ; 104(5): 2067-2077, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31932896

RESUMO

Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and ß-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2-7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.


Assuntos
Proteínas de Bactérias/metabolismo , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Hidrolases/metabolismo , Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Hidrolases/química , Hidrolases/genética , Hidrolases/isolamento & purificação , Cinética , Modelos Moleculares , Mutação , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato
11.
Enzyme Microb Technol ; 132: 109391, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731956

RESUMO

Styrene monooxygenases (SMOs) are two-component enzymes known to catalyze the epoxidation of styrene to (S)-styrene oxide. In this work, we identified a new oxygenase component, named StStyA, from the genome of Streptomyces sp. NRRL S-31. StStyA displayed complementary stereoselectivity to all of the known SMOs when coupled with a known reductase component (PsStyB), which made it the first natural SMO that produces (R)-styrene oxide. Accordingly, a plasmid co-expressing StStyA and PsStyB was constructed, which led to an artificial two-component SMO, named StStyA/B. When applied in the bio-epoxidation of nine aromatic alkenes, the enzyme showed activity toward five alkenes, and consistently displayed (R)-selectivity. Excellent stereoselectivity was achieved for all five substrates with enantiomeric excesses ranging from 91% to >99%ee.


Assuntos
Proteínas de Bactérias/metabolismo , Oxigenases/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Compostos de Epóxi/metabolismo , Cinética , Oxigenases/genética , Streptomyces/genética
12.
Plant Sci ; 290: 110293, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31779893

RESUMO

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Tripterygium/genética , Alcaloides/biossíntese , Sequência de Aminoácidos , Catharanthus/metabolismo , Diterpenos/metabolismo , Compostos de Epóxi/metabolismo , Fenantrenos/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Metabolismo Secundário , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tripterygium/metabolismo
13.
J Agric Food Chem ; 67(47): 13061-13072, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31738551

RESUMO

In this study, we purified three new sesquiterpenyl epoxy-cyclohexenoid (SEC) analogues, arthrobotrisin D (11) and its two derivatives, from nematode-trapping fungus Arthrobotrys oligospora. Our results revealed that arthrobotrisin type SEC metabolites could be detected in all the test fungal strains from geographically distinct regions grown on different nutrient media, indicative of unique diagnostic character as chemical indicators for A. oligospora. The time course designs over short-term intervals of the fungus under direct contact and indirect contact with living or dead nematodes revealed that arthrobotrisin B and D (6 and 11) displayed significant relationships (positive or negative correlation) with fungal saprophytic and pathogenic stages during a nematode predation event. Interestingly, fungus on nutrient-limiting medium conducive to fungal trap formation could rapidly drop the concentration levels of arthrobotrisins B and D within 6 h when dead nematodes were around, in great contrast to that for living nematodes. Moreover, only in the fungal strain under direct contact with living dominant soil bacteria, arthrobotrisins B and D exhibited significant increase in amounts. Among them, the new SEC, arthrobotrisin D (11) was found to be a key unique metabolic signal for fungal colony growth and fungal interaction with prey and bacteria. Our study suggested that chemical analysis of SEC metabolites in A. oligospora provides a window into the fungal growth status and much valuable information about ecological environments associated with the nematode infections.


Assuntos
Ascomicetos/química , Compostos de Epóxi/química , Nematoides/microbiologia , Sesquiterpenos/química , Animais , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Compostos de Epóxi/metabolismo , Estrutura Molecular , Sesquiterpenos/metabolismo
14.
J Am Chem Soc ; 141(51): 20397-20406, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31769979

RESUMO

(S)-2-Hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis-epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves ∼95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE·FeII·1 and HppE·ZnII·2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues-Leu120, Leu144, Phe182, and Leu193-close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans ∼99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (∼82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (∼13:87), trifluorination of C3 (∼23:77), or both (∼1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (∼56:44) and Leu144Phe/Leu193Phe (∼90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical-as proposed in a recent computational study-to direct formation of the more potently antibacterial cis-epoxide by radicaloid C-O coupling.


Assuntos
Compostos de Epóxi/metabolismo , Fosfomicina/biossíntese , Oxirredutases/metabolismo , Compostos de Epóxi/química , Fosfomicina/química , Radicais Livres/química , Radicais Livres/metabolismo , Conformação Molecular , Oxirredutases/química , Oxirredutases/genética , Estereoisomerismo
15.
Physiol Rep ; 7(22): e14275, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31782268

RESUMO

Fatty acid (FA)-derived lipid products generated by cytochrome P450 (CYP), lipoxygenase (LOX), and cyclo-oxygenase (COX) influence cardiovascular function. However, plasma measurements invariably ignore 40% of the blood specimen, namely the erythrocytes. These red blood cells (RBCs) represent a cell mass of about 3 kg. RBCs are a potential reservoir for epoxy fatty acids, which on release could regulate vascular capacity. We tested the hypothesis that maximal physical activity would influence the epoxy fatty acid status in RBCs. We used a standardized maximal treadmill exercise according to Bruce to ensure a robust hemodynamic and metabolic response. Central hemodynamic monitoring was performed using blood pressure and heart rate measurements and maximal workload was assessed in metabolic equivalents (METs). We used tandem mass spectrometry (LC-MS/MS) to measure epoxides derived from CYP monooxygenase, as well as metabolites derived from LOX, COX, and CYP hydroxylase pathways. Venous blood was obtained for RBC lipidomics. With the incremental exercise test, increases in the levels of various CYP epoxy-mediators in RBCs, including epoxyoctadecenoic acids (9,10-EpOME, 12,13-EpOME), epoxyeicosatrienoic acids (5,6-EET, 11,12-EET, 14,15-EET), and epoxydocosapentaenoic acids (16,17-EDP, 19,20-EDP) occurred, as heart rate, systolic blood pressure, and plasma lactate concentrations increased. Maximal (13.5 METs) exercise intensity had no effect on diols and various LOX, COX, and hydroxylase mediators. Our findings suggest that CYP epoxy-metabolites could contribute to the cardiovascular response to maximal exercise.


Assuntos
Compostos de Epóxi/metabolismo , Eritrócitos/metabolismo , Exercício Físico/fisiologia , Ácidos Graxos/metabolismo , Lipidômica/métodos , Adulto , Teste de Esforço/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
16.
Chem Res Toxicol ; 32(11): 2268-2280, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31613099

RESUMO

Metabolism of bardoxolone methyl (BARD-Me), an oleanolic acid derivative, and its epoxide metabolite was studied in different in vitro systems. BARD-Me also undergoes glutathione (GSH)-adduct formation via direct nucleophilic attack at the ß-carbon of the α,ß-unsaturated ketone substituent on the A-ring. The presence of an electron-withdrawing nitrile residue on the α-carbon increases the α,ß-unsaturated ketone's susceptibility to nucleophilic attack by thiols. This allows BARD-Me to generate reversible adducts with the thiol groups of cysteine residues in target proteins without the potential toxic liabilities of irreversible covalent adduct formation. However, BARD-Me epoxide can also react with thiols irreversibly. Therefore, the epoxide was synthesized and its metabolic fate studied in vitro. BARD-Me epoxide was found to undergo two novel metabolic biotransformations: epoxide reduction and oxidative elimination of nitrile moiety. Both metabolic pathways proceed via nucleophilic attack of the thiol group of GSH at each of the two carbon atoms of the epoxide as evidenced by the formation of two ß-hydroxy sulfide regioisomers. Oxidative elimination of nitrile moiety proceeds via nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is ß to the cyano group to give a cyanohydrin metabolite, which spontaneously decomposes to release cyanide and the corresponding ketone. Nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is α to the cyano group results in the formation of the GSH monoadduct that undergoes intermolecular reduction with another GSH molecule, followed by elimination of oxidized GSH (GS-SG) and the formation of an enolate intermediate. Upon protonation, the enolate intermediate gives rise to hydroxylated BARD-Me, which is readily converted back to BARD-Me through the elimination of water. The chemical reactivity of the epoxide metabolite and the liberation of cyanide are of significant toxicological interest for the potential utility of BARD-Me as a therapeutic agent.


Assuntos
Compostos de Epóxi/metabolismo , Glutationa/metabolismo , Ácido Oleanólico/análogos & derivados , Biotransformação , Citosol/metabolismo , Hepatócitos/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Fator 2 Relacionado a NF-E2 , Nitrilos/metabolismo , Ácido Oleanólico/metabolismo , Oxirredução
17.
Anal Chem ; 91(23): 14865-14872, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31660733

RESUMO

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme's physiological substrates. The methods described herein will enable further studies on EpFAs' metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.


Assuntos
Calorimetria/métodos , Ensaios Enzimáticos , Epóxido Hidrolases/química , Compostos de Epóxi/química , Ácidos Graxos/química , Adamantano/análogos & derivados , Adamantano/química , Biocatálise , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Ácidos Graxos/metabolismo , Análise de Injeção de Fluxo/métodos , Humanos , Hidrólise , Cinética , Ácidos Láuricos/química , Metabolismo dos Lipídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluções , Especificidade por Substrato
18.
Commun Biol ; 2: 378, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31633069

RESUMO

Metabolism is a major defense mechanism of the body against xenobiotic threats. Here we unravel a critical role of Bmal1 for circadian clock-controlled Cyp3a11 expression and xenobiotic metabolism. Bmal1 deficiency decreases the mRNA, protein and microsomal activity of Cyp3a11, and blunts their circadian rhythms in mice. A screen for Cyp3a11 regulators identifies two circadian genes Dbp and Hnf4α as potential regulatory mediators. Cell-based experiments confirm that Dbp and Hnf4α activate Cyp3a11 transcription by their binding to a D-box and a DR1 element in the Cyp3a11 promoter, respectively. Bmal1 binds to the P1 distal promoter to regulate Hnf4α transcriptionally. Cellular regulation of Cyp3a11 by Bmal1 is Dbp- and Hnf4α-dependent. Bmal1 deficiency sensitizes mice to toxicities of drugs such as aconitine and triptolide (and blunts circadian toxicity rhythmicities) due to elevated drug exposure. In summary, Bmal1 connects circadian clock and Cyp3a11 metabolism, thereby impacting drug detoxification as a function of daily time.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Ritmo Circadiano/genética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Preparações Farmacêuticas/metabolismo , Fatores de Transcrição ARNTL/deficiência , Fatores de Transcrição ARNTL/genética , Aconitina/metabolismo , Aconitina/toxicidade , Animais , Linhagem Celular , Relógios Circadianos/genética , Ritmo Circadiano/fisiologia , Proteínas de Ligação a DNA/genética , Diterpenos/metabolismo , Diterpenos/toxicidade , Compostos de Epóxi/metabolismo , Compostos de Epóxi/toxicidade , Regulação Enzimológica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Humanos , Inativação Metabólica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenantrenos/metabolismo , Fenantrenos/toxicidade , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Xenobióticos/metabolismo , Xenobióticos/toxicidade
19.
Chin J Nat Med ; 17(8): 575-584, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472894

RESUMO

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.


Assuntos
Diterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Domínio Catalítico , Compostos de Epóxi/metabolismo , Hidroximetilglutaril-CoA Sintase/química , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Interferência de RNA , Terpenos/metabolismo , Tripterygium/enzimologia , Tripterygium/genética , Triterpenos/metabolismo
20.
Adv Exp Med Biol ; 1161: 219-232, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31562632

RESUMO

Accumulating evidence suggests that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) offer protection against vascular inflammation, neuroinflammation, hypertension, and thrombosis. Recently, biochemical studies have demonstrated that these benefits are partially mediated by their conversion to ω-3 endocannabinoid epoxide metabolites. These lipid metabolites originate from the epoxidation of ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by cytochrome P450 (CYP) epoxygenases to form epoxydocosapentaenoic acid-ethanolamides (EDP-EAs) and epoxyeicosatetraenoic acid-ethanolamides (EEQ-EAs), respectively. The EDP-EAs and EEQ-EAs are endogenously produced in rat brain and peripheral organs. Additionally, EDP-EAs and EEQ-EAs dose-dependently decrease pro-inflammatory IL-6 cytokine and increased anti-inflammatory IL-10 cytokine. Furthermore, the EEQ-EAs and EDP-EAs attenuate angiogenesis and cell migration in cancer cells, induce vasodilation in bovine coronary arteries, and reciprocally regulate platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides represent a new class of dual acting molecules that display unique pharmacological properties.


Assuntos
Endocanabinoides , Compostos de Epóxi , Ácidos Graxos Ômega-3 , Animais , Anti-Inflamatórios/metabolismo , Endocanabinoides/metabolismo , Compostos de Epóxi/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Humanos , Vasodilatação , Vasodilatadores/metabolismo
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