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1.
Nat Commun ; 12(1): 1095, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597523

RESUMO

Active-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.


Assuntos
Proteínas de Bactérias/metabolismo , Domínio Catalítico , Dioxigenases/metabolismo , Engenharia de Proteínas/métodos , Pseudomonas fluorescens/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Dioxigenases/química , Dioxigenases/genética , Modelos Moleculares , Conformação Proteica , Pseudomonas fluorescens/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
2.
Nat Commun ; 12(1): 1301, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637718

RESUMO

Biodegradation of aromatic and heterocyclic compounds requires an oxidative ring cleavage enzymatic step. Extensive biochemical research has yielded mechanistic insights about catabolism of aromatic substrates; yet much less is known about the reaction mechanisms underlying the cleavage of heterocyclic compounds such as pyridine-ring-containing ones like 2,5-hydroxy-pyridine (DHP). 2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 uses a mononuclear nonheme Fe(II) to catalyze the oxidative pyridine ring cleavage reaction by transforming DHP into N-formylmaleamic acid (NFM). Herein, we report a crystal structure for the resting form of NicX, as well as a complex structure wherein DHP and NFM are trapped in different subunits. The resting state structure displays an octahedral coordination for Fe(II) with two histidine residues (His265 and His318), a serine residue (Ser302), a carboxylate ligand (Asp320), and two water molecules. DHP does not bind as a ligand to Fe(II), yet its interactions with Leu104 and His105 function to guide and stabilize the substrate to the appropriate position to initiate the reaction. Additionally, combined structural and computational analyses lend support to an apical dioxygen catalytic mechanism. Our study thus deepens understanding of non-heme Fe(II) dioxygenases.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dioxigenases/química , Dioxigenases/metabolismo , Compostos Heterocíclicos/metabolismo , Pseudomonas putida/enzimologia , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Dioxigenases/classificação , Dioxigenases/genética , Ferro , Ligantes , Modelos Moleculares , Oxigênio/metabolismo , Filogenia , Conformação Proteica
3.
Nat Commun ; 12(1): 1300, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637735

RESUMO

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.


Assuntos
Vias Biossintéticas , Dioxigenases/biossíntese , Dioxigenases/genética , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Sequência de Aminoácidos , Vias Biossintéticas/genética , Colesterol/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Hidroxilação , Ácidos Cetoglutáricos/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/genética , Filogenia , Plantas Geneticamente Modificadas , Metabolismo Secundário/genética , Metabolismo Secundário/fisiologia , Solanina/análogos & derivados , Solanum melongena/enzimologia , Solanum melongena/genética , Tomatina/análogos & derivados , Tomatina/metabolismo
4.
Gene ; 764: 145078, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32858175

RESUMO

In maize, eat rot and stalk rot caused by Fusarium verticillioides and Fusarium graminearum lead to contamination of moldy grains to produce mycotoxins. Identification of resistance genes against these pathogens for maize breeding is an effective way for disease control. Several 2-oxoglutarate-dependent dioxygenase (2OGD) proteins have been found to confer resistance to different pathogens in diverse plant species. However, little is known about the 2OGD superfamily in maize. Here, we identified 103 putative 2OGD genes in maize from a genome-wide analysis, and divided them into three classes - DOXA, DOXB, and DOXC. We further comprehensively investigated their gene structure, chromosome distribution, phylogenetic tree, gene-function enrichment, and expression profiles among different tissues. The genes encoding three 2OGD proteins, ACO, F3H, and NCS involved in ethylene biosynthesis, flavonoids biosynthesis, and alkaloids biosynthesis pathways, respectively, were identified to be induced by F. verticillioides and F. graminearum. The promoters of the three genes contain the binding sites for the transcription factor ZmDOF and ZmHSF, which are also induced by the two pathogens. The results imply that the three 2OGDs and the two transcription factors might be involved in the resistance to the two pathogens. This study provided a comprehensive understanding of the 2OGD superfamily in maize and laid the foundation for the further functional analysis of their roles in maize resistance to eat rot and stalk rot.


Assuntos
Dioxigenases/genética , Fusarium/imunologia , Proteínas de Plantas/genética , Zea mays/fisiologia , Sequência de Bases/genética , Sítios de Ligação/genética , Cromossomos de Plantas/genética , Coenzimas/metabolismo , Sequência Conservada/genética , Dioxigenases/imunologia , Dioxigenases/metabolismo , Resistência à Doença/genética , Evolução Molecular , Fusarium/patogenicidade , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas/fisiologia , Estudo de Associação Genômica Ampla , Ácidos Cetoglutáricos/metabolismo , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Caules de Planta/enzimologia , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/microbiologia , Regiões Promotoras Genéticas/genética , RNA-Seq , Fatores de Transcrição/metabolismo , Zea mays/microbiologia
5.
Methods Mol Biol ; 2198: 321-331, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822042

RESUMO

DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.


Assuntos
5-Metilcitosina/análogos & derivados , Epigenômica/métodos , Análise de Sequência de DNA/métodos , 5-Metilcitosina/análise , 5-Metilcitosina/química , Animais , Citosina/análise , Citosina/metabolismo , DNA/genética , Metilação de DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Epigênese Genética/genética , Genoma , Humanos , Oxirredução , Sulfitos/química
6.
Methods Mol Biol ; 2198: 333-348, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32822043

RESUMO

Bisulfite sequencing (BS-seq) remains the gold standard technique to quantitively map DNA methylation at a single-base resolution. However, BS-seq cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Oxidative bisulfite sequencing (oxBS-seq) was one of the first techniques that enabled absolute quantification of 5mC and 5hmC at single-base resolution. OxBS-seq uses chemical oxidation of 5hmC prior to bisulfite treatment to provide a direct readout of 5mC; comparison with BS-seq data can then be used to infer 5hmC levels. Here we describe in detail an updated version of our laboratory's oxBS-seq protocol, which uses potassium perruthenate (KRuO4) as an oxidant. We also describe a bioinformatics pipeline designed to handle Illumina short read sequencing data from whole-genome oxBS-seq.


Assuntos
5-Metilcitosina/análogos & derivados , Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , 5-Metilcitosina/análise , Animais , Citosina/análise , Citosina/metabolismo , DNA/genética , Metilação de DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oxirredução , Estresse Oxidativo , Sulfitos/química
7.
Plant Physiol Biochem ; 154: 439-450, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32912484

RESUMO

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7', 8' double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.


Assuntos
Carotenoides/análise , Cicloexenos/análise , Dioxigenases , Iridaceae , Terpenos/análise , Clonagem Molecular , Dioxigenases/genética , Iridaceae/enzimologia , Iridaceae/genética , Filogenia , Proteínas de Plantas/genética
8.
PLoS One ; 15(9): e0238179, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881902

RESUMO

Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 ('ZS11') revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.


Assuntos
Brassica napus/enzimologia , Dioxigenases/genética , Genoma de Planta , Proteínas de Plantas/genética , Arabidopsis/enzimologia , Brassica napus/genética , Carotenoides/metabolismo , Mapeamento Cromossômico , Dioxigenases/classificação , Dioxigenases/metabolismo , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas
9.
Proc Natl Acad Sci U S A ; 117(33): 19914-19925, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747548

RESUMO

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward ß-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce ß-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/química , Carotenoides/metabolismo , Dioxigenases/química , Archaea/química , Archaea/classificação , Archaea/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carotenoides/química , Catálise , Domínio Catalítico , Dioxigenases/genética , Dioxigenases/metabolismo , Especificidade por Substrato , Synechocystis/química , Synechocystis/enzimologia , Synechocystis/genética
10.
Chemosphere ; 261: 127779, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32736249

RESUMO

Rhizo-box experiments were conducted to analyze the phyto-microbial remediation potential of a grass (Lolium multiflorum L.) and a crop (Glycine max L.) combined with exogenous strain (Pseudomonas sp.) for polycyclic aromatic hydrocarbons (PAHs) contaminated soils. The dynamics of bacterial community composition, abundances of 16 S rDNA and ring hydroxylating dioxygenases (RHDα) genes, and removal of PAHs were evaluated and compared on four culture stages (days 0, 10, 20, and 30). The results showed that 8.65%-47.42% of Σ12 PAHs were removed after 30 days of cultivation. Quantitative polymerase chain reaction (qPCR) analysis indicated that treatments with soybean and ryegrass rhizosphere markedly increased the abundances of total bacteria and PAH-degraders, especially facilitated the growth of gram-negative degrading bacteria. Flavobacterium sp. and Pseudomonas sp. were the main and active strains in the control soil. However, the presence of plants and/or exogenous Pseudomonas sp. changed the soil bacterial community structure and modified the bacterial diversity of PAH-degraders. On the whole, this study showed that the high molecular weight PAHs removal efficiency of phyto-microbial remediation with ryegrass was better than those of remediation with soybean. Furthermore, the removals of PAHs strongly coincided with the abundance of PAH-degraders and bacterial community structure.


Assuntos
Biodegradação Ambiental , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Microbiologia do Solo , Poluentes do Solo/toxicidade , Bactérias/metabolismo , Dioxigenases/genética , Lolium , Hidrocarbonetos Policíclicos Aromáticos/análise , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Pseudomonas , Rizosfera , Solo/química , Poluentes do Solo/metabolismo
11.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709719

RESUMO

Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO), catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of a broader substrate range, the BphAES283M, BphAEp4-S283M, and BphAERR41-S283M variants were created from the parent enzymes BphAELB400, BphAEp4, and BphAERR41, respectively, by a substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the k cat/Km values of BphAES283M, BphAEp4-S283M, and BphAERR41-S283M were significantly increased compared to those of their parent enzymes. Meanwhile, we determined the steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested that the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3',4,4'-tetrachlorobiphenyl (2,3',4,4'-CB), 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-CB), and 2,3',4,4',5-pentachlorobiphenyl (2,3',4,4',5-CB). We compared the catalytic reactions of BphAELB400 and its variants toward 2,2'-dichlorobiphenyl (2,2'-CB), 2,5-dichlorobiphenyl (2,5-CB), and 2,6-dichlorobiphenyl (2,6-CB). The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site and, thereby, its site of hydroxylation, and this was confirmed by docking experiments. We also assessed the substrate ranges of BphAELB400 and its variants with degradation activity. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the 3-6-chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. Collectively, the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO. These will certainly be meaningful elements for understanding the effect of the residue corresponding to position 283 in other Rieske oxygenase enzymes.IMPORTANCE The segment from positions 280 to 283 in BphAEs is located at the entrance of the catalytic pocket, and it shows variation in conformation. In previous works, results have suggested but never proved that residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found that the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3',4,4'-CB, 2,2',6,6'-CB, and 2,3',4,4',5-CB. Meanwhile, the Ser283Met substitution altered the regiospecificity of BphAE toward 2,2'-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3 to 6 chlorines), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, the results of this study provide better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.


Assuntos
Proteínas de Bactérias/genética , Burkholderiaceae/genética , Mutagênese Sítio-Dirigida , Bifenilos Policlorados/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderiaceae/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Engenharia Genética
12.
Science ; 368(6496): 1270-1274, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527835

RESUMO

Sexual dichromatism, a difference in coloration between males and females, may be due to sexual selection for ornamentation and mate choice. Here, we show that carotenoid-based dichromatism in mosaic canaries, a hybrid phenotype that arises in offspring of the sexually dichromatic red siskin and monochromatic canaries, is controlled by the gene that encodes the carotenoid-cleaving enzyme ß-carotene oxygenase 2 (BCO2). Dichromatism in mosaic canaries is explained by differential carotenoid degradation in the integument, rather than sex-specific variation in physiological functions such as pigment uptake or transport. Transcriptome analyses suggest that carotenoid degradation in the integument might be a common mechanism contributing to sexual dichromatism across finches. These results suggest that differences in ornamental coloration between sexes can evolve through simple molecular mechanisms controlled by genes of major effect.


Assuntos
Canários/fisiologia , Carotenoides/metabolismo , Dioxigenases/genética , Tentilhões/fisiologia , Pigmentação/genética , Caracteres Sexuais , Animais , Evolução Biológica , Canários/anatomia & histologia , Canários/genética , Feminino , Tentilhões/anatomia & histologia , Tentilhões/genética , Masculino , Fatores Sexuais , Transcriptoma
13.
J Nutr ; 150(8): 2023-2030, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32433733

RESUMO

BACKGROUND: Plasma cholesterol is one of the strongest risk factors associated with the development of atherosclerotic cardiovascular disease (ASCVD) and myocardial infarction. Human studies suggest that elevated plasma ß-carotene is associated with reductions in circulating cholesterol and the risk of myocardial infarction. The molecular mechanisms underlying these observations are unknown. OBJECTIVE: The objective of this study was to determine the impact of dietary ß-carotene and the activity of ß-carotene oxygenase 1 (BCO1), which is the enzyme responsible for the conversion of ß-carotene to vitamin A, on circulating cholesterol concentration. METHODS: In our preclinical study, we compared the effects of a 10-d intervention with a diet containing 50 mg/kg of ß-carotene on plasma cholesterol in 5-wk-old male and female C57 Black 6 wild-type and congenic BCO1-deficient mice. In our clinical study, we aimed to determine whether 5 common small nucleotide polymorphisms located in the BCO1 locus affected serum cholesterol concentrations in a population of young Mexican adults from the Universities of San Luis Potosí and Illinois: A Multidisciplinary Investigation on Genetics, Obesity, and Social-Environment (UP AMIGOS) cohort. RESULTS: Upon ß-carotene feeding, Bco1-/- mice accumulated >20-fold greater plasma ß-carotene and had ∼30 mg/dL increased circulating total cholesterol (P < 0.01) and non-HDL cholesterol (P < 0.01) than wild-type congenic mice. Our results in the UP AMIGOS cohort show that the rs6564851 allele of BCO1, which has been linked to BCO1 enzymatic activity, was associated with a reduction in 10 mg/dL total cholesterol concentrations (P = 0.009) when adjusted for vitamin A and carotenoid intakes. Non-HDL-cholesterol concentration was also reduced by 10 mg/dL when the data were adjusted for vitamin A and total carotenoid intakes (P = 0.002), or vitamin A and ß-carotene intakes (P = 0.002). CONCLUSIONS: Overall, our results in mice and young adults show that BCO1 activity impacts circulating cholesterol concentration, linking vitamin A formation with the risk of developing ASCVD.


Assuntos
Colesterol/sangue , Dioxigenases/metabolismo , beta Caroteno/administração & dosagem , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , Adolescente , Animais , Colesterol/metabolismo , Dioxigenases/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , beta Caroteno/farmacologia , beta-Caroteno 15,15'-Mono-Oxigenase/genética
14.
Nat Commun ; 11(1): 2680, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471981

RESUMO

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Desmetilação do DNA , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Epigênese Genética/genética , Genoma/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/genética
15.
Cancer Res ; 80(12): 2599-2611, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32312832

RESUMO

Zinc finger protein 143 (ZNF143) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence-specific DNA. The exact biological role of ZNF143 in hepatocellular carcinoma (HCC) has not been investigated. Here we report that ZNF143 is overexpressed in HCC tissues and its overexpression correlates with poor prognosis. Gain- and loss-of-function experiments showed that ZNF143 promoted HCC cell proliferation, colony formation, and tumor growth in vitro and in vivo. ZNF143 accelerated HCC cell-cycle progression by activating cell division cycle 6 (CDC6). Mechanistically, ZNF143 promoted expression of CDC6 by directly activating transcription of histone demethylase mineral dust-induced gene (MDIG), which in turn reduced H3K9me3 enrichment in the CDC6 promoter region. Consistently, ZNF143 expression correlated significantly with MDIG and CDC6 expression in HCC. Collectively, we propose a model for a ZNF143-MDIG-CDC6 oncoprotein axis that provides novel insight into ZNF143, which may serve as a therapeutic target in HCC. SIGNIFICANCE: These findings describe the mechanism by which ZNF143 promotes HCC proliferation and provide important clues for exploring new targets and strategies for clinical treatment of human liver cancer.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Dioxigenases/genética , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Transativadores/metabolismo , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Estudos de Coortes , Metilação de DNA , Conjuntos de Dados como Assunto , Dioxigenases/metabolismo , Células HEK293 , Hepatectomia , Histona Desmetilases/metabolismo , Histonas/genética , Humanos , Fígado/patologia , Fígado/cirurgia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Análise Serial de Tecidos , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Zhonghua Gan Zang Bing Za Zhi ; 28(3): 259-265, 2020 Mar 20.
Artigo em Chinês | MEDLINE | ID: mdl-32306660

RESUMO

Objective: To detect the expression of Myc-induced nuclear antigen 53 (Mina53) and liver tissue >5 cm from the edge of the tumor (LTM5), and analyze its relationship with tumorigenesis, clinicopathological characteristics, and patient survival and prognosis in hepatocellular carcinoma (HCC). Methods: The expression levels of Mina53 mRNA and protein in 18 pairs of fresh HCC and LTM5 were assessed by qRT-PCR and Western blot, respectively. The expression of Mina53 in 284 pairs of HCC and LTM5 sample was determined by immunohistochemistry. Paired-sample t-test was used for the comparison of measurement data among groups, and heterogeneity of variance was tested using Wilcoxon rank-sum test. χ (2) test was used for the comparison of measurement data among groups. Kaplan-Meier method and log-rank test were used for survival analysis. Cox regression model was used for single factor and multi factor analysis. Results: The relative expression levels of Mina53 mRNA and protein in 18 fresh HCC tissues were significantly higher than those in LTM5 tissues (mRNA: -4.41 ± 1.48 and -5.93 ± 1.65, t = 3.100, P = 0.007; protein: 1.12 ± 0.29 and 0.46 ± 0.21, t = 10.616, P < 0.001). The relative expression level of Mina53 in 284 HCC tissues was higher than that of LTM5 (z = -18.739, P < 0.001). The expression level of Mina53 was associated with tumor size (χ (2) = 5.474, P = 0.019), vascular invasion (χ (2) = 8.965, P = 0.003), pathological grade (χ (2) = 12.006, P = 0.002), and TNM stage (χ (2) = 16.686, P < 0.001). The overall postoperative survival time and disease-free survival time of patients with high expression of Mina53 (28.5 months and 22.7 months, respectively) were shorter than those with low expression (33.0 months and 31.8 months, respectively) (P < 0.05) in HCC. Cox multivariate regression analysis showed that Mina53 and multiple tumors were independent prognostic factors affecting the overall postoperative survival time and disease-free survival time of HCC patients (P < 0.05). Conclusion: Mina53 may play an important role in the occurrence of HCC and participate in the process of tumor growth as well as invasion and metastasis. The high expression of Mina53 signifies that the patient has a poor prognosis and thus can be used as a potential marker for judging the prognosis of HCC patients.


Assuntos
Carcinoma Hepatocelular/genética , Dioxigenases/genética , Histona Desmetilases/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Antígenos Nucleares , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/diagnóstico , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico , Estadiamento de Neoplasias , Prognóstico
17.
RNA ; 26(8): 960-968, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32345632

RESUMO

Dozens of candidate orphan riboswitch classes have been discovered previously by using comparative sequence analysis algorithms to search bacterial genomic sequence databases. Each orphan is classified by the presence of distinct conserved nucleotide sequences and secondary structure features, and by its association with particular types of genes. One previously reported orphan riboswitch candidate is the "NMT1 motif," which forms a hairpin structure with an internal bulge that includes numerous highly conserved nucleotides. This motif associates with genes annotated to encode various dioxygenase enzymes, transporters, or proteins that have roles associated with thiamin or histidine metabolism. Biochemical evaluation of numerous ligand candidates revealed that NMT1 motif RNA constructs most tightly bind 8-azaxanthine, xanthine, and uric acid, whereas most other closely related compounds are strongly rejected. Genetic assays revealed that NMT1 motif RNAs function to turn off gene expression upon ligand binding, likely by regulating translation initiation. These results suggest that NMT1 motif RNAs function as aptamer domains for a riboswitch class that specifically responds to high concentrations of oxidized purines. Members of this "xanthine riboswitch" class appear to regulate genes predominantly related to purine transport and oxidation, thus avoiding the effects of overproduction of these common purine derivatives.


Assuntos
Purinas/metabolismo , RNA Bacteriano/genética , Riboswitch/genética , Ácido Úrico/metabolismo , Xantina/metabolismo , Aptâmeros de Nucleotídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Oxirredução , Xantinas/metabolismo
18.
Chemosphere ; 247: 125844, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32069708

RESUMO

Biphenyl 2,3-dioxygenase (BphA), a Rieske-type and first enzyme in the aerobic degradation process, plays a key role in the metabolizing process of biphenyl/polychlorinated biphenyl aromatic pollutants in the environment. To understand the catalytic mechanism of biphenyl 2,3-dioxygenase, the conversions leading to the cis-diols are investigated by means of quantum mechanics/molecular mechanics (QM/MM) method. A hydroperoxo-iron (III) species is involved in the enzyme-catalyzed reaction. Herein, we explored the direct reaction mechanism of hydroperoxo-iron (III) species with biphenyl and 4-4'-dichlorobiphenyl. The reaction process involves an epoxide intermediate, it could develop into a carbocation intermediate, and ultimately evolve into a cis-diol product. The important roles of several residues during the dioxygenation process were highlighted. This study may provide theoretical support for further directed mutations and enzymatic engineering of BphA, as well as promote the development of degrading environmentally persistent biphenyl/polychlorinated biphenyl aromatic contaminants.


Assuntos
Compostos de Bifenilo/química , Dioxigenases/metabolismo , Poluentes Ambientais/química , Bifenilos Policlorados/química , Catálise , Dioxigenases/genética , Hidroxilaminas , Hidroxilação , Oxigenases/metabolismo , Bifenilos Policlorados/metabolismo
19.
Appl Microbiol Biotechnol ; 104(7): 2987-2997, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32060694

RESUMO

Metarhizium species are the most abundant fungi that can be isolated from soil, with a well-known biopesticide capacity. Metarhizium recognizes their hosts when the conidium interacts with insects, where the fungi are in contact with the hydrocarbons of the outermost lipid layer cuticle. These cuticular hydrocarbons comprise a mixture of n-alkanes, n-alkenes, and methyl-branched chains. Metarhizium can degrade insect hydrocarbons and use these hydrocarbons for energy production and the biosynthesis of cellular components. The metabolism of nitroalkanes involves nitronate monooxygenase activity. In this work, we isolated a family of six genes with potential nitronate monooxygenase activity from Metarhizium brunneum. The six genes were expressed in Escherichia coli, and the nitronate monooxygenase activity was verified in the recombinant proteins. Additionally, when the conidia of M. brunneum were grown in medium with nitroalkanes, virulence against Plutella xylostella increased. Furthermore, we analyzed the expression of the six Npd genes during the infection to this insect, which showed differential expression of the six Npd genes during infection.


Assuntos
Agentes de Controle Biológico/metabolismo , Dioxigenases/metabolismo , Metarhizium/enzimologia , Mariposas/microbiologia , Alcanos/metabolismo , Animais , DNA Fúngico/genética , Dioxigenases/genética , Hidrocarbonetos/metabolismo , Proteínas de Insetos/metabolismo , Metarhizium/genética , Metarhizium/patogenicidade , Controle Biológico de Vetores , Virulência/genética
20.
Proc Natl Acad Sci U S A ; 117(7): 3621-3626, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32024762

RESUMO

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.


Assuntos
Reprogramação Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Dioxigenases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Domínio Catalítico , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/química , Dioxigenases/genética , Dioxigenases/metabolismo , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
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