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1.
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
2.
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
3.
J Mol Microbiol Biotechnol ; 28(4): 183-189, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30566957

RESUMO

Nowadays, contamination of soil and marine sediments by polycyclic aromatic hydrocarbons (PAHs) has become a serious problem all over the world. Rhodococcus sp. P14 was isolated from sediments with crude oil contaminate and showed degradation ability on various PAHs. The genome of Rhodococcus sp. P14 was sequenced. A gene cluster encoding a ring-hydroxylating dioxygenase Baa related to PAH degradation was identified by bioinformatics. The expression level of gene baaA was increased when P14 was cultured with anthracene, pyrene, phenanthrene, or benz[a]-anthracene as the single carbon source. The recombinant protein Baa was overexpressed in E. coli BL21 (DE3). Further investigations on the recombinant protein Baa in E. coli demonstrated that it was able to oxidize anthracene and benz [a]anthracene, resulting in 9,10-dihydroxyanthracene and 7, 12-dihydroxybenz[a]anthracene as metabolites, respectively. These results indicate that Baa plays an important role in PAH degradation in Rhodococcus sp. P14 and Baa has potential application in the bioremediation of PAHs in the contaminated environment.


Assuntos
Antracenos/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Sequência de Bases , Biodegradação Ambiental , Dioxigenases/classificação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Sedimentos Geológicos/microbiologia , Hidroxilação , Família Multigênica , Fenantrenos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Pirenos , Proteínas Recombinantes/genética , Alinhamento de Sequência
4.
BMC Biochem ; 19(1): 8, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30115012

RESUMO

BACKGROUND: Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,ß-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum. METHODS: In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations. RESULTS: In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed ß-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation. CONCLUSIONS: The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme's molecular environment on substrate specificities for future investigation.


Assuntos
Dioxigenases/química , Dioxigenases/metabolismo , Pseudomonas/enzimologia , Cristalografia por Raios X , Dioxigenases/classificação , Simulação de Acoplamento Molecular , Filogenia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Microbiologia do Solo , Especificidade por Substrato
5.
Aquat Toxicol ; 200: 62-72, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29727772

RESUMO

To better understand heavy metal tolerance in Chlamydomonas acidophila, an extremophilic green alga, we assembled its transcriptome and measured transcriptomic expression before and after Cd exposure in this and the neutrophilic model microalga Chlamydomonas reinhardtii. Genes possibly related to heavy metal tolerance and detoxification were identified and analyzed as potential key innovations that enable this species to live in an extremely acid habitat with high levels of heavy metals. In addition we provide a data set of single orthologous genes from eight green algal species as a valuable resource for comparative studies including eukaryotic extremophiles. Our results based on differential gene expression, detection of unique genes and analyses of codon usage all indicate that there are important genetic differences in C. acidophila compared to C. reinhardtii. Several efflux family proteins were identified as candidate key genes for adaptation to acid environments. This study suggests for the first time that exposure to cadmium strongly increases transposon expression in green algae, and that oil biosynthesis genes are induced in Chlamydomonas under heavy metal stress. Finally, the comparison of the transcriptomes of several acidophilic and non-acidophilic algae showed that the Chlamydomonas genus is polyphyletic and that acidophilic algae have distinctive aminoacid usage patterns.


Assuntos
Chlamydomonas/efeitos dos fármacos , Metais Pesados/toxicidade , Poluentes Químicos da Água/toxicidade , Actinas/genética , Actinas/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Cádmio/metabolismo , Cádmio/toxicidade , Hidrolases de Éster Carboxílico/classificação , Hidrolases de Éster Carboxílico/genética , Chlamydomonas/classificação , Chlamydomonas/metabolismo , Dioxigenases/classificação , Dioxigenases/genética , Tolerância a Medicamentos/genética , Metais Pesados/metabolismo , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , RNA de Plantas/química , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Análise de Sequência de RNA , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/química , Poluentes Químicos da Água/metabolismo
6.
Plant Physiol ; 173(3): 1583-1593, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28100450

RESUMO

Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. Natural selection has led to diversity in the GSL composition even within individual species. However, in radish (Raphanus sativus), glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for more than 90% of the total GSLs, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables.


Assuntos
Dioxigenases/metabolismo , Glucosinolatos/biossíntese , Ácidos Cetoglutáricos/metabolismo , Raphanus/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Transporte Biológico , Vias Biossintéticas/genética , Cromatografia Líquida de Alta Pressão , Dioxigenases/classificação , Dioxigenases/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosinolatos/análise , Engenharia Metabólica/métodos , Mutação , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Raphanus/enzimologia , Raphanus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Plântula/genética , Plântula/metabolismo , Plântula/fisiologia , Homologia de Sequência de Aminoácidos
7.
J Mol Cell Biol ; 7(6): 494-504, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26003568

RESUMO

The DNA repair enzyme AlkB was identified in E. coli more than three decades ago. Since then, nine mammalian homologs, all members of the superfamily of alpha-ketoglutarate and Fe(II)-dependent dioxygenases, have been identified (designated ALKBH1-8 and FTO). While E. coli AlkB serves as a DNA repair enzyme, only two mammalian homologs have been confirmed to repair DNA in vivo. The other mammalian homologs have remarkably diverse substrate specificities and biological functions. Substrates recognized by the different AlkB homologs comprise erroneous methyl- and etheno adducts in DNA, unique wobble uridine modifications in certain tRNAs, methylated adenines in mRNA, and methylated lysines on proteins. The phenotypes of organisms lacking or overexpressing individual AlkB homologs include obesity, severe sensitivity to inflammation, infertility, growth retardation, and multiple malformations. Here we review the present knowledge of the mammalian AlkB homologs and their implications for human disease and development.


Assuntos
Dioxigenases/química , Proteínas de Escherichia coli/química , Ácidos Cetoglutáricos/química , Oxigenases de Função Mista/química , RNA de Transferência/metabolismo , Animais , Biologia Computacional , Metilação de DNA , Reparo do DNA/fisiologia , Dioxigenases/classificação , Dioxigenases/metabolismo , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/metabolismo , Humanos , Camundongos , Oxigenases de Função Mista/classificação , Oxigenases de Função Mista/metabolismo , Filogenia , Processamento de Proteína Pós-Traducional , Processamento Pós-Transcricional do RNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
8.
J Plant Res ; 128(4): 519-34, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25929830

RESUMO

During different environmental stress conditions, plant growth is regulated by the hormone abscisic acid (an apocarotenoid). In the biosynthesis of abscisic acid, the oxidative cleavage of cis-epoxycarotenoid catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is the crucial step. The NCED genes were isolated in numerous plant species and those genes were phylogenetically investigated to understand the evolution of NCED genes in various plant lineages comprising lycophyte, gymnosperm, dicot and monocot. A total of 93 genes were obtained from 48 plant species to statistically estimate their sequence conservation and functional divergence. Selaginella moellendorffii appeared to be evolutionarily distinct from those of the angiosperms, insisting the substantial influence of natural selection pressure on NCED genes. Further, using exon-intron structure analysis, the gene structures of NCED were found to be conserved across some species. In addition, the substitution rate ratio of non-synonymous (Ka) versus synonymous (Ks) mutations using the Bayesian inference approach, depicted the critical amino acid residues for functional divergence. A significant functional divergence was found between some subgroups through the co-efficient of type-I functional divergence. Our results suggest that the evolution of NCED genes occurred by duplication, diversification and exon intron loss events. The site-specific profile and functional diverge analysis revealed NCED genes might facilitate the tissue-specific functional divergence in NCED sub-families, that could combat different environmental stress conditions aiding plant survival.


Assuntos
Dioxigenases/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Variação Genética , Filogenia , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Dioxigenases/classificação , Dioxigenases/genética , Genômica , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Plantas/classificação , Especificidade da Espécie
9.
Mol Genet Genomics ; 290(4): 1589-603, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25749981

RESUMO

In plants, the carotenoid cleavage dioxygenase 4 (CCD4) could target on plastoglobules and cleave specific carotenoids, producing apocarotenoids and volatile compounds. These compounds are important for color and aroma formation in fruits and flowers. In this study, five CCD4 gene members (CCD4a, b, c, d, and e) were investigated in different citrus species including mandarin, pummelo, and sweet orange. Sequence analysis showed that the CCD4 genes from all the species examined exhibited extensive allelic variability (including SNPs and frame-shift mutations). Furthermore, the distribution of the CCD4 allelic mutation sites supported our previous hypothesis that the sweet orange originated from the hybridization of mandarin and pummelo. A derived cleaved amplified polymorphic sequence (dCAPs) marker was then successfully developed based on the allelic polymorphism of CCD4c, providing an ideal molecular marker for studying the genetic relationship between citrus species. Quantitative RT-PCR analysis identified differential expression patterns for the CCD4 genes in tissues/organs, and CCD4b was shown to have a high-level expression in citrus fruit flavedos (especially those with a deep orange-reddish color). HPLC-based detection of a key component (i.e., ß-citraurin) for orange-reddish flavedo formation in different citrus revealed a positive correlation between CCD4b expression levels and the presence of ß-citraurin, suggesting that CCD4b may be responsible for ß-citraurin biosynthesis in flavedo. In summary, this study not only reinforced the anticipated roles of CCD4 genes in flavedo color formation in citrus, but also provided new information about gene expression patterns, allelic polymorphism characteristics, and sequence variability for this gene subfamily.


Assuntos
Carotenoides/metabolismo , Citrus/genética , Dioxigenases/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Citrus/classificação , Citrus/metabolismo , Dioxigenases/classificação , Dioxigenases/metabolismo , Mutação da Fase de Leitura , Frutas/genética , Frutas/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Variação Genética , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , beta Caroteno/análogos & derivados , beta Caroteno/biossíntese
10.
Plant Mol Biol ; 86(4-5): 555-69, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25204497

RESUMO

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC-DAD and the volatile products by GC/MS. ß-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9',10') bonds and generating ß-ionone, although ß-cyclocitral resulting from a 7,8 (7',8') cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.


Assuntos
Carotenoides/metabolismo , Crocus/metabolismo , Dioxigenases/metabolismo , Proteínas de Plantas/metabolismo , Aldeídos/metabolismo , Sequência de Aminoácidos , Crocus/enzimologia , Crocus/genética , Dioxigenases/classificação , Dioxigenases/genética , Diterpenos/metabolismo , Flores/enzimologia , Flores/genética , Flores/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Isoenzimas/genética , Isoenzimas/metabolismo , Luteína/metabolismo , Dados de Sequência Molecular , Família Multigênica , Norisoprenoides/metabolismo , Pressão Osmótica , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Homologia de Sequência de Aminoácidos , Estresse Mecânico , Especificidade por Substrato , Tabaco/enzimologia , Tabaco/genética , Tabaco/metabolismo , Xantofilas/metabolismo , beta Caroteno/metabolismo
11.
Nucleic Acids Res ; 41(16): 7635-55, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23814188

RESUMO

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.


Assuntos
DNA/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Bacteriófagos/enzimologia , Bacteriófagos/genética , Biologia Computacional , DNA/química , Dioxigenases/classificação , Dioxigenases/genética , Endodesoxirribonucleases/genética , Evolução Molecular , Genoma Bacteriano , Glicosilação , Glicosiltransferases/genética , Dados de Sequência Molecular , Oxirredução , Filogenia , Estrutura Terciária de Proteína , S-Adenosilmetionina/análogos & derivados , Alinhamento de Sequência , Timina/metabolismo
12.
Science ; 337(6098): 1104-7, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22936780

RESUMO

Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic data sets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox.


Assuntos
Organismos Aquáticos/metabolismo , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Metano/biossíntese , Compostos Organofosforados/metabolismo , Aerobiose , Organismos Aquáticos/genética , Archaea/genética , Proteínas Arqueais/classificação , Proteínas Arqueais/genética , Dioxigenases/classificação , Dioxigenases/genética , Dioxigenases/metabolismo , Ordem dos Genes , Metagenoma , Filogenia , Água do Mar/química , Água do Mar/microbiologia
14.
FEMS Microbiol Lett ; 331(2): 97-104, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22448874

RESUMO

L-isoleucine-4-hydroxylase (IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. L-methionine, L-leucine, L-isoleucine and L-threonine were found to be catalysed by the investigated enzymes, producing L-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free L-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed.


Assuntos
Aminoácidos/metabolismo , Bactérias/enzimologia , Dioxigenases/metabolismo , Escherichia coli/enzimologia , Bactérias/classificação , Bactérias/genética , Clonagem Molecular , Dioxigenases/classificação , Dioxigenases/genética , Escherichia coli/genética , Hidroxilação , Isoleucina/metabolismo , Cinética , Leucina/metabolismo , Metionina/metabolismo , Especificidade por Substrato , Treonina/metabolismo
15.
Curr Opin Chem Biol ; 16(1-2): 60-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22356841

RESUMO

Heme iron is often used in biology for activation of oxygen. The mechanisms of oxygen activation by heme-containing monooxygenases (the cytochrome P450s) are well known, and involve formation of a Compound I species, but information on the heme-containing dioxygenase enzymes involved in tryptophan oxidation lags far behind. In this review, we gather together information emerging recently from structural, mechanistic, spectroscopic, and computational approaches on the heme dioxygenase enzymes involved in tryptophan oxidation. We explore the subtleties that differentiate various heme enzymes from each other, and use this to piece together a developing picture for oxygen activation in this particular class of heme-containing dioxygenases.


Assuntos
Dioxigenases/metabolismo , Heme/metabolismo , Biocatálise , Dioxigenases/química , Dioxigenases/classificação , Heme/química , Humanos , Oxirredução , Especificidade por Substrato , Triptofano/química , Triptofano/metabolismo
17.
Nucleic Acids Res ; 37(21): 7124-36, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19786499

RESUMO

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m(1)A) and 3-methylcytosine (m(3)C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N(6)-ethenoadenine (epsilonA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.


Assuntos
Proteínas de Bactérias/classificação , Enzimas Reparadoras do DNA/classificação , Dioxigenases/classificação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biologia Computacional , DNA/metabolismo , Dano ao DNA , Metilação de DNA , Reparo do DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , DNA de Cadeia Simples/metabolismo , Dioxigenases/química , Dioxigenases/genética , Proteínas de Escherichia coli/química , Teste de Complementação Genética , Oxigenases de Função Mista/química , Dados de Sequência Molecular , Filogenia , RNA/metabolismo , Análise de Sequência de Proteína
18.
Cell Cycle ; 8(11): 1698-710, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19411852

RESUMO

Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a potential regulatory role. Through a wider analysis of other poorly characterized DNA-modifying enzymes we also show that the phage Mu Mom-like proteins, which catalyze the N6-carbamoylmethylation of adenines, are also linked to diverse families of bacterial transposases, suggesting that DNA modification by transposable elements might have a more general presence than previously appreciated. Among the other families of 2-oxoglutarate- and iron(II)-dependent dioxygenases identified in this study, one which is found in algae, is predicted to mainly comprise of RNA-modifying enzymes and shows a striking diversity in protein domain architectures suggesting the presence of RNA modifications with possibly unique adaptive roles. The results presented here are likely to provide the means for future investigation of unexpected epigenetic modifications, such as hydroxymethyl cytosine, that could profoundly impact our understanding of gene regulation and processes such as DNA demethylation.


Assuntos
Dioxigenases/genética , Nucleotídeos/química , Sequência de Aminoácidos , Enzimas Reparadoras do DNA/genética , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Dioxigenases/classificação , Humanos , Ferro/química , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Dados de Sequência Molecular , Nucleotídeos/biossíntese , Oxirredução , Valor Preditivo dos Testes , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transposases/genética
19.
Appl Environ Microbiol ; 75(9): 2969-72, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19251892

RESUMO

Separate quantification of three classes of tfdA genes was performed using TaqMan quantitative real-time PCR for 13 different soils subsequent to mineralization of three phenoxy acids. Class III tfdA genes were found to be involved in mineralization more often than class I and II tfdA genes.


Assuntos
Dioxigenases/classificação , Dioxigenases/genética , Herbicidas , Reação em Cadeia da Polimerase/métodos , Microbiologia do Solo
20.
Huan Jing Ke Xue ; 29(6): 1655-9, 2008 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-18763518

RESUMO

Pseudomonas nitroreducens J5-1 is able to use monochlorobenzene, 1,2-dichlorobenzene, 1,3-dichlorobenzene and 1,2,4-trichlorobenzene as sole carbon and energy sources, and it differs from those 1,2,4-trichlorobenzene degrading bacteria reported in substrate utilizing characters. PCR technique was used to amplify the genes of chlorobenzene dioxygenase and dehydrogenase of J5-1, and they were named as tcbA and tcbB, respectively. Homology analysis indicated that these genes and gene products were most closely related to those of Burkholderia sp. PS12. By alignment of the amino acid sequences of the a subunits of TcbAa (from J5-1) and TecA1 (from PS12), four amino acid residues from site 307 to site 310 were found to be different (I307L, M308T, I309V, Q310E), which probably retarded the preference for the substrate 1,2,4,5-tetrachlorobenzene. Furthermore, the phylogenetic analysis of the dioxygenase alpha subunits showed that TcbAa was belong to the toluene/diphenyl subfamily, and was most closely related to the poly-chlorinated benzene dioxygenase alpha subunit.


Assuntos
Proteínas de Bactérias/genética , Clorobenzenos/metabolismo , Dioxigenases/genética , Oxirredutases/genética , Pseudomonas/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Biodegradação Ambiental , Clonagem Molecular , Dioxigenases/classificação , Poluentes Ambientais/metabolismo , Dados de Sequência Molecular , Oxirredução , Filogenia , Subunidades Proteicas/classificação , Subunidades Proteicas/genética , Pseudomonas/genética , Pseudomonas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
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