RESUMO
Aminoacyl-tRNA synthetases (aaRSs) are fundamental components of the protein translation machinery. In light of their pivotal role in protein synthesis and structural divergence among species, they have always been considered potential targets for the development of antimicrobial compounds. Arginyl-tRNA synthetase from Trypanosoma cruzi (TcArgRS), the parasite responsible for causing Chagas Disease, contains a 100-amino acid insertion that was found to be completely absent in the human counterpart of similar length, as ascertained from multiple sequence alignment results. Thus, we were prompted to perform a preliminary characterization of TcArgRS using biophysical, biochemical, and bioinformatics tools. We expressed the protein in E. coli and validated its in-vitro enzymatic activity. Additionally, analysis of DTNB kinetics, Circular dichroism (CD) spectra, and ligand-binding studies using intrinsic tryptophan fluorescence measurements aided us to understand some structural features in the absence of available crystal structures. Our study indicates that TcArgRS can discriminate between L-arginine and its analogues. Among the many tested substrates, only L-canavanine and L-thioarginine, a synthetic arginine analogue exhibited notable activation. The binding of various substrates was also determined using in silico methods. This study may provide a viable foundation for studying small compounds that can be targeted against TcArgRS.
Assuntos
Aminoacil-tRNA Sintetases , Arginina-tRNA Ligase , Humanos , Arginina-tRNA Ligase/química , Arginina-tRNA Ligase/genética , Arginina-tRNA Ligase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Alinhamento de Sequência , Canavanina/química , Canavanina/genética , Canavanina/metabolismoRESUMO
Error-free protein synthesis relies on the precise recognition by the aminoacyl-tRNA synthetases of their cognate tRNAs in order to attach the corresponding amino acid. A concept of universal tRNA identity elements requires the aminoacyl-tRNA synthetases provided by the genome of an organism to match the identity elements found in the cognate tRNAs in an evolution-independent manner. Identity elements tend to cluster in the tRNA anticodon and acceptor stem regions. However, in the arginine system, in addition to the anticodon, the importance of nucleotide A20 in the tRNA D-loop for cognate enzyme recognition has been a sustained feature for arginyl-tRNA synthetase in archaea, bacteria and in the nuclear-encoded cytosolic form in mammals and plants. However, nuclear-encoded mitochondrial arginyl-tRNA synthetase, which can be distinguished from its cytosolic form by the presence or absence of signature motifs, dispenses with the A20 requirement. An examination of several hundred non-metazoan organisms and their corresponding tRNAArg substrates has confirmed this general concept to a large extent and over numerous phyla. However, some Stramenopiles, and in particular, Diatoms (Bacillariophyta) present a notable exception. Unusually for non-fungal organisms, the nuclear genome encodes tRNAArg isoacceptors with C or U at position 20. In this case one of two nuclear-encoded cytosolic arginyl-tRNA synthetases has evolved to become insensitive to the nature of the D-loop identity element. The other, with a binding pocket that is compatible with tRNAArg-A20 recognition, is targeted to organelles that encode solely such tRNAs.
Assuntos
Aminoacil-tRNA Sintetases , Diatomáceas , Aminoácidos , Aminoacil-tRNA Sintetases/genética , Animais , Anticódon/genética , Diatomáceas/genética , Mamíferos , RNA de Transferência/genéticaRESUMO
During the endosymbiotic evolution of mitochondria, the genes for aminoacyl-tRNA synthetases were transferred to the ancestral nucleus. A further reduction of mitochondrial function resulted in mitochondrion-related organisms (MRO) with a loss of the organelle genome. The fate of the now redundant ancestral mitochondrial aminoacyl-tRNA synthetase genes is uncertain. The derived protein sequence for arginyl-tRNA synthetase from thirty mitosomal organisms have been classified as originating from the ancestral nuclear or mitochondrial gene and compared to the identity element at position 20 of the cognate tRNA that distinguishes the two enzyme forms. The evolutionary choice between loss and retention of the ancestral mitochondrial gene for arginyl-tRNA synthetase reflects the coevolution of arginyl-tRNA synthetase and tRNA identity elements.
Assuntos
Aminoacil-tRNA Sintetases , Arginina-tRNA Ligase , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Arginina-tRNA Ligase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA de TransferênciaRESUMO
The evolutionary origin of the family of eukaryotic aminoacyl-tRNA synthetases that are essential to all living organisms is a matter of debate. In order to shed molecular light on the ancient source of arginyl-tRNA synthetase, a total of 1347 eukaryotic arginyl-tRNA synthetase sequences were mined from databases and analyzed. Their multiple sequence alignment reveals a signature sequence that is characteristic of the nuclear-encoded enzyme, which is imported into mitochondria. Using this molecular beacon, the origins of this gene can be traced to modern prokaryotes. In this way, a previous phylogenetic analysis linking Myxococcus to the emergence of the eukaryotic mitochondrial arginyl-tRNA synthetase is supported by the unique existence of the molecular signature within the suborder Cystobacterineae that includes Myxococcus.
Assuntos
Arginina-tRNA Ligase/genética , Eucariotos/enzimologia , Mitocôndrias/enzimologia , Myxococcales/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Mineração de Dados , Eucariotos/genética , Evolução Molecular , Mitocôndrias/genética , Myxococcales/genética , Filogenia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (ß-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232.
Assuntos
Aminoaciltransferases/metabolismo , Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Anticorpos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Massas , Proteínas de Plantas/química , Mapas de Interação de Proteínas , Proteômica/métodosRESUMO
Codon-anticodon recognition between triplets of an mRNA and a specific tRNA is the key element in the translation of the genetic code. In general, the precision of this process is dominated by a strict Watson-Crick base-pairing scheme. However, the degeneracy of the genetic code led Crick to propose the Wobble Hypothesis, permitting a less restraining interaction with the third base of the codon and involving the participation of inosine for decoding C-ending codons. The concept that the anticodon base A34 of tRNAACGArg in all eukaryotes, eubacteria, and plant chloroplasts is converted to I34 is firmly anchored in the literature despite conflicting evidence for its existence in higher eukaryote cytoplasmic tRNAACGArg. Here, we provide additional data and summarize the arguments favoring and contradicting post-transcriptional deamination of this position. A hypothesis that resolves the apparent conflict is proposed. © 2016 IUBMB Life, 68(6):419-422, 2016.
Assuntos
Anticódon , Códon , Inosina/genética , Edição de RNA , RNA de Transferência de Arginina/metabolismo , Adenosina/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Células Eucarióticas , Código Genético , Humanos , Inosina/metabolismo , RNA de Transferência de Arginina/genéticaRESUMO
The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.
Assuntos
Aminoaciltransferases/metabolismo , Padronização Corporal , Bryopsida/enzimologia , Bryopsida/crescimento & desenvolvimento , Células Germinativas Vegetais/crescimento & desenvolvimento , Arabidopsis/metabolismo , Padronização Corporal/genética , Bryopsida/genética , Bryopsida/ultraestrutura , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutação/genética , Especificidade de Órgãos , Fenótipo , Desenvolvimento Vegetal , Reação em Cadeia da Polimerase em Tempo Real , Amido/metabolismo , Frações Subcelulares/metabolismoRESUMO
Increasingly, the discovery and characterization of small regulatory RNAs from a variety of organisms have all required deep-sequencing methodologies. However, the crux to successful deep-sequencing analysis depends upon optimal construction of a cDNA library compatible for the high-throughput sequencing platform. Challenges to small RNA library constructions arise when dealing with minute tissue samples because certain structural RNA fragments can dominate and mask the desired characterization of regulatory small RNAs like microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs). Here, we describe methods that improve the chances of constructing a successful library from small RNAs isolated from minute tissues such as enriched follicle cells from the Drosophila ovarium. Because the ribosomal RNA (rRNA) fragments are frequently the major contaminants in small RNA preparations from minute amounts of tissue, we demonstrate the utility of antisense oligonucleotide depletion and an acryloylaminophenylboronic acid (APB) polyacrylamide gel system for separating the abundant 2S rRNA in Drosophila from endo-siRNAs and piRNAs. Finally, our methodology generates libraries amenable to multiplex sequencing on the Illumina Hi-Seq platform.
Assuntos
Biblioteca Gênica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/isolamento & purificação , Manejo de Espécimes , Animais , Ácidos Borônicos/química , Drosophila melanogaster/citologia , Eletroforese em Gel de Poliacrilamida , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Oligorribonucleotídeos Antissenso/genética , Especificidade de Órgãos , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Análise de Sequência de RNARESUMO
Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNA(Ser)UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distinct from those possessed by the cytoplasmic tRNAArg in which the major identity elements permit the arginylation of all 5 encoded isoacceptors. We have performed comparative in vitro aminoacylation using an insect mitochondrial arginyl-tRNA synthetase and tRNAArgUCG structural variants. The established identity elements are sufficient to maintain the fidelity of tRNASerUCU reassignment. tRNAs having a UCU anticodon cannot be arginylated but can be converted to arginine acceptance by identity element transplantation. We have examined the evolutionary distribution and functionality of these tRNA elements within metazoan taxa. We conclude that the identity elements that have evolved for the recognition of mitochondrial tRNAArgUCG by the nuclear encoded mitochondrial arginyl-tRNA synthetases of eumetazoans have been extensively, but not universally conserved, throughout this clade. They ensure that the AGR codon reassignment in eumetazoan mitochondria is not compromised by misaminoacylation. In contrast, in other metazoans, such as Porifera, whose mitochondrial translation is dictated by the universal genetic code, recognition of the 2 encoded tRNAArgUCG/UCU isoacceptors is achieved through structural features that resemble those employed by the yeast cytoplasmic system.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Anticódon/genética , Evolução Biológica , Mitocôndrias/genética , RNA de Transferência de Arginina/metabolismo , Aminoacilação de RNA de Transferência/fisiologia , Aminoacil-tRNA Sintetases/genética , Animais , Sequência de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Besouros , Código Genético , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência de Arginina/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
L-canavanine, the toxic guanidinooxy analogue of L-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the Lcanavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism. A discriminating arginyl-tRNA synthetase, detected in a crude C. brasiliensis larval extract, was proposed to be responsible for insect's ability to survive the diet of L-canavanine (Rosenthal, G. A., Dahlman, D. L., and Janzen, D. H. (1976) A novel means for dealing with L-canavanine, a toxic metabolite. Science 192, 256e258). Since the arginyl-tRNA synthetase of at least three genetic compartments (insect cytoplasmic, insect mitochondrial and insect gut microflora) may participate in conferring L-canavanine resistance, we investigated whether the nuclear-encoded C. brasiliensis mitochondrial arginyl-tRNA synthetase plays a role in this discrimination. Steady state kinetics of the cloned, recombinant enzyme have revealed and quantified an amino acid discriminating potential of the mitochondrial enzyme that is sufficient to account for the overall L-canavanine misincorporation rate observed in vivo. As in the cytoplasmic enzyme of the L-canavanine producer plant, the mitochondrial arginyl-tRNA synthetases from a specialist seed predator relies on a kinetic discrimination that prevents L-canavanine misincorporation into proteins.
Assuntos
Arginina-tRNA Ligase/metabolismo , Canavanina/toxicidade , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Aminoácidos/genética , Animais , Arginina-tRNA Ligase/química , Arginina-tRNA Ligase/genética , Canavanina/química , Núcleo Celular/genética , Besouros/efeitos dos fármacos , Besouros/enzimologia , Besouros/metabolismo , Costa Rica , Dioclea/química , Cinética , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Mitocôndrias/genéticaRESUMO
It is a prevalent concept that, in line with the Wobble Hypothesis, those tRNAs having an adenosine in the first position of the anticodon become modified to an inosine at this position. Sequencing the cDNA derived from the gene coding for cytoplasmic tRNA (Arg) ACG from several higher plants as well as mass spectrometric analysis of the isoacceptor has revealed that for this kingdom an unmodified A in the wobble position of the anticodon is the rule rather than the exception. In vitro translation shows that in the plant system the absence of inosine in the wobble position of tRNA (Arg) does not prevent decoding. This isoacceptor belongs to the class of tRNA that is imported from the cytoplasm into the mitochondria of higher plants. Previous studies on the mitochondrial tRNA pool have demonstrated the existence of tRNA (Arg) ICG in this organelle. In moss the mitochondrial encoded distinct tRNA (Arg) ACG isoacceptor possesses the I34 modification. The implication is that for mitochondrial protein biosynthesis A-to-I editing is necessary and occurs by a mitochondrion-specific deaminase after import of the unmodified nuclear encoded tRNA (Arg) ACG.
Assuntos
Adenosina/metabolismo , Anticódon/metabolismo , Glycine max/genética , Inosina/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Arginina/metabolismo , Triticum/genética , Adenosina/genética , Adenosina Desaminase/metabolismo , Anticódon/química , Anticódon/genética , Pareamento de Bases , Sequência de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Sistema Livre de Células , Citoplasma/genética , Citoplasma/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Código Genético , Inosina/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência de Arginina/química , RNA de Transferência de Arginina/genética , Glycine max/metabolismo , Sphagnopsida/genética , Sphagnopsida/metabolismo , Triticum/metabolismoRESUMO
Identity elements determine the accurate recognition between tRNAs and aminoacyl-tRNA synthetases. The arginine system from yeast and Escherichia coli has been studied extensively in the past. However, information about the enzymes from higher eukaryotes is limited and plant aminoacyl-tRNA synthetases have been largely ignored in this respect. We have designed in vitro tRNA transcripts, based on the soybean tRNA(Arg) primary structure, aiming to investigate its specific aminoacylation by two recombinant plant arginyl-tRNA synthetases and to compare this with the enzyme from E. coli. Identity elements at positions 20 and 35 in plants parallel those previously established for bacteria. Cryptic identity elements in the plant system that are not revealed within a tRNA(Arg) consensus sequence compiled from isodecoders corresponding to nine distinct cytoplasmic, mitochondrial and plastid isoaccepting sequences are located in the acceptor stem. Additionally, it has been shown that U20a and A38 are essential for a fully efficient cognate E. coli arginylation, whereas, for the plant arginyl-tRNA synthetases, these bases can be replaced by G20a and C38 with full retention of activity. G10, a constituent of the 10:25:45 tertiary interaction, is essential for both plant and E. coli activity. Amino acid recognition in terms of discriminating between arginine and canavanine by the arginyl-tRNA synthetase from both kingdoms may be manipulated by changes at different sites within the tRNA structure.
Assuntos
Arginina-tRNA Ligase/metabolismo , Arginina/metabolismo , Canavanina/metabolismo , Escherichia coli/enzimologia , Glycine max/enzimologia , Aminoacil-RNA de Transferência/metabolismo , Arginina-tRNA Ligase/genética , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Aminoacil-RNA de Transferência/genética , Especificidade por Substrato , Aminoacilação de RNA de TransferênciaRESUMO
L-canavanine occurs as a toxic non-protein amino acid in more than 1500 leguminous plants. One mechanism of its toxicity is its incorporation into proteins, replacing L-arginine and giving rise to functionally aberrant polypeptides. A comparison between the recombinant arginyl-tRNA synthetases from a canavanine producer (jack bean) and from a related non-producer (soybean) provided an opportunity to study the mechanism that has evolved to discriminate successfully between the proteinogenic amino acid and its non-protein analogue. In contrast to the enzyme from jack bean, the soybean enzyme effectively produced canavanyl-tRNA(Arg) when using RNA transcribed from the jack bean tRNA(ACG) gene. The corresponding k(cat)/K(M) values gave a discrimination factor of 485 for the jack bean enzyme. The arginyl-tRNA synthetase does not possess hydrolytic post-transfer editing activity. In a heterologous system containing either native Escherichia coli tRNA(Arg) or the modification-lacking E. coli transcript RNA, efficient discrimination between L-arginine and L-canavanine by both plant enzymes (but not by the E. coli arginyl-tRNA synthetase) occurred. Thus, interaction of structural features of the tRNA with the enzyme plays a significant role in determining the accuracy of tRNA arginylation. Of the potential amino acid substrates tested, apart from L-canavanine, only L-thioarginine was active in aminoacylation. As it is an equally good substrate for the arginyl-tRNA synthetase from both plants, it is concluded that the higher discriminatory power of the jack bean enzyme towards L-canavanine does not necessarily provide increased protection against analogues in general, but appears to have evolved specifically to avoid auto-toxicity.
Assuntos
Arginina-tRNA Ligase/química , Variação Genética , Sequência de Aminoácidos , Arginina-tRNA Ligase/metabolismo , Canavanina/metabolismo , DNA de Plantas/metabolismo , Cinética , Dados de Sequência Molecular , RNA de Plantas/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Especificidade por Substrato , Aminoacilação de RNA de TransferênciaRESUMO
The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.
Assuntos
Arginina-tRNA Ligase/biossíntese , Arginina-tRNA Ligase/química , Canavalia/enzimologia , Expressão Gênica , Arginina-tRNA Ligase/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
A virtually identical nicotine catabolic pathway including the heterotrimeric molybdenum enzyme nicotine and 6-hydroxy-pseudo-oxynicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, 2,6-dihydroxy-pseudo-oxynicotine hydrolase, and 2,6-dihydroxypyridine hydroxylase have been identified in A. nicotinovorans and Nocardioides sp. JS614. Enzymes catalyzing the same reactions and similar protein antigens were detected in the extracts of the two microorganisms. Nicotine blue and methylamine, two end products of nicotine catabolism were detected in the growth medium of both bacterial species. Nicotine catabolic genes are clustered on pAO1 in A. nicotinovorans, but located chromosomally in Nocardioides sp. JS614.
Assuntos
Actinomycetales/enzimologia , Arthrobacter/enzimologia , Oxigenases de Função Mista/metabolismo , Nicotina/metabolismo , Actinomycetales/genética , Sequência de Aminoácidos , Arthrobacter/genética , Western Blotting , Mapeamento Cromossômico , Cromossomos Bacterianos , Hidroxilação , Metilaminas/metabolismo , Família Multigênica , Fases de Leitura Aberta , Plasmídeos , Piridonas/metabolismoRESUMO
The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.
Assuntos
Arthrobacter/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nicotina/metabolismo , 2,4-Dinitrofenol/farmacologia , Sequência de Aminoácidos , Aminobutiratos/metabolismo , Arthrobacter/efeitos dos fármacos , Proteínas de Bactérias/genética , Radioisótopos de Carbono/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Deleção de Genes , Teste de Complementação Genética , Ionóforos/farmacologia , Marcação por Isótopo , Metilaminas/metabolismo , Dados de Sequência Molecular , Nigericina/farmacologia , Nitrilas/farmacologia , Alinhamento de Sequência , Desacopladores/farmacologiaRESUMO
New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrate (CH(3)-4-aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH(3)-4-aminobutyrate could be identified. CH(3)-4-aminobutyrate may be demethylated to gamma-N-aminobutyrate by the recently identified gamma-N-methylaminobutyrate oxidase. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH(3)-4-aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP(+)-dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, gamma-N-methylaminobutyrate oxidase and amine oxidase share the same substrate. The K(m) of 2.5 mM and k(cat) of 1230 s(-1) for amine oxidase vs. K(m) of 140 microM and k(cat) of 800 s(-1) for gamma-N-methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH(3)-4-aminobutyrate. However, bacteria grown on [(14)C]nicotine secreted [(14)C]methylamine into the medium, indicating that the pathway to succinate is active in vivo.
Assuntos
Arthrobacter/metabolismo , Proteínas de Bactérias/metabolismo , Nicotina/metabolismo , Oxirredutases/metabolismo , Aminobutiratos/metabolismo , Arthrobacter/genética , Proteínas de Bactérias/genética , Humanos , Metilaminas/metabolismo , Estrutura Molecular , Nicotina/química , Oxirredutases/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.
Assuntos
Arthrobacter/enzimologia , Hidrolases/química , Hidrolases/metabolismo , Nicotina/metabolismo , Alcaloides/metabolismo , Arthrobacter/metabolismo , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Ordem dos Genes , Compostos Heterocíclicos/metabolismo , Nicotina/análogos & derivados , Fases de Leitura Aberta , Plasmídeos/genética , Piridinas/metabolismoRESUMO
Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation gamma-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating gamma-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between -48 and -88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the -35 promoter region of the purU-mabO-folD operon.
Assuntos
Arthrobacter/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Plasmídeos , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Arthrobacter/fisiologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Mutagênese Insercional , Nicotina/metabolismo , Óperon/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica , RNA Bacteriano/análise , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência , Transativadores/isolamento & purificação , Sítio de Iniciação de Transcrição/fisiologia , Transcrição GênicaRESUMO
The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid L-nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids. Of the 165 ORFs, approximately 50% were related to metabolic functions. pAO1 conferred to A. nicotinovorans the ability to take up L-[(14)C]nicotine from the medium, with an K(m) of 5.6 +/- 2.2 micro M. ORFs of putative nicotine transporters formed a cluster with the gene of the D-nicotine-specific 6-hydroxy-D-nicotine oxidase. ORFs related to replication, chromosome partitioning, and natural transformation functions (dprA) were identified on pAO1. Few ORFs showed similarity to known conjugation-promoting proteins, but pAO1 could be transferred by conjugation to a pAO1-negative strain at a rate of 10(-2) to 10(-3) per donor. ORFs with no known function represented approximately 35% of the pAO1 sequence. The positions of insertion sequence elements and composite transposons, corroborated by the G+C content of the pAO1 sequence, suggest a modular composition of the plasmid.