Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Elife ; 102021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34665128

RESUMO

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

2.
Nat Commun ; 11(1): 5682, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173168

RESUMO

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λmax = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λmax = 690 nm) highly fluorescent state (QF = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.


Assuntos
Quitridiomicetos/genética , Rodopsina , Biologia Computacional , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/isolamento & purificação , Genes Fúngicos , Genoma Fúngico , Mutagênese Sítio-Dirigida , Processos Fotoquímicos , Células Fotorreceptoras/fisiologia , Rodopsina/biossíntese , Rodopsina/química , Rodopsina/genética
3.
Nat Commun ; 10(1): 3315, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346176

RESUMO

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.


Assuntos
Ânions/metabolismo , Bactérias/genética , Channelrhodopsins/genética , Família Multigênica , Vírus/genética , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Humanos , Cinética , Luz , Metagenoma , Neurônios/metabolismo , Optogenética , Filogenia , Água do Mar/microbiologia , Água do Mar/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírus/classificação , Vírus/isolamento & purificação , Vírus/metabolismo
4.
Plant Physiol ; 162(1): 63-73, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23569108

RESUMO

The NADPH-dependent thioredoxin reductase C (NTRC) is involved in redox-related regulatory processes in chloroplasts and nonphotosynthetic active plastids. Together with 2-cysteine peroxiredoxin, it forms a two-component peroxide-detoxifying system that acts as a reductant under stress conditions. NTRC stimulates in vitro activity of magnesium protoporphyrin IX monomethylester (MgPMME) cyclase, most likely by scavenging peroxides. Reexamination of tetrapyrrole intermediate levels of the Arabidopsis (Arabidopsis thaliana) knockout ntrc reveals lower magnesium protoporphyrin IX (MgP) and MgPMME steady-state levels, the substrate and the product of MgP methyltransferase (CHLM) preceding MgPMME cyclase, while MgP strongly accumulates in mutant leaves after 5-aminolevulinic acid feeding. The ntrc mutant has a reduced capacity to synthesize 5-aminolevulinic acid and reduced CHLM activity compared with the wild type. Although transcript levels of genes involved in chlorophyll biosynthesis are not significantly altered in 2-week-old ntrc seedlings, the contents of glutamyl-transfer RNA reductase1 (GluTR1) and CHLM are reduced. Bimolecular fluorescence complementation assay confirms a physical interaction of NTRC with GluTR1 and CHLM. While ntrc contains partly oxidized CHLM, the wild type has only reduced CHLM. As NTRC also stimulates CHLM activity in vitro, it is proposed that NTRC has a regulatory impact on the redox status of conserved cysteine residues of CHLM. It is hypothesized that a deficiency of NTRC leads to a lower capacity to reduce cysteine residues of GluTR1 and CHLM, affecting the stability and, thereby, altering the activity in the entire tetrapyrrole synthesis pathway.


Assuntos
Arabidopsis/enzimologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Metiltransferases , NADP/genética , NADP/metabolismo , Oxirredução , Peroxirredoxinas , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plastídeos/enzimologia , Plastídeos/genética , Plastídeos/metabolismo , Protoporfirinas/genética , Protoporfirinas/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/metabolismo , Tetrapirróis/genética , Tetrapirróis/metabolismo , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
5.
Methods Mol Biol ; 775: 357-85, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21863454

RESUMO

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.


Assuntos
Arabidopsis/metabolismo , Técnicas de Química Analítica/métodos , Fotossíntese , Pigmentos Biológicos/biossíntese , Tetrapirróis/biossíntese , Ácido Aminolevulínico/análise , Ácido Aminolevulínico/isolamento & purificação , Ácido Aminolevulínico/metabolismo , Apoenzimas/metabolismo , Calibragem , Carotenoides/análise , Carotenoides/biossíntese , Carotenoides/isolamento & purificação , Clorofilídeos/análise , Clorofilídeos/biossíntese , Clorofilídeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Heme/análise , Heme/isolamento & purificação , Peroxidase do Rábano Silvestre/metabolismo , Pigmentos Biológicos/análise , Pigmentos Biológicos/isolamento & purificação , Porfobilinogênio/análise , Porfobilinogênio/isolamento & purificação , Porfobilinogênio/metabolismo , Protoclorifilida/análise , Protoclorifilida/biossíntese , Protoclorifilida/isolamento & purificação , Espectrometria de Fluorescência
6.
Plant Cell Physiol ; 51(7): 1229-41, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20460500

RESUMO

Mg protoporphyrin monomethylester (MgProtoME) cyclase catalyzes isocyclic ring formation to form divinyl protochlorophyllide. The CHL27 protein is part of the cyclase complex. Deficiency of CHL27 has been previously reported to compromise photosynthesis and nuclear gene expression. In a comprehensive analysis of different CHL27 antisense tobacco lines grown under different light conditions, the physiological consequences of gradually reduced CHL27 expression on the tetrapyrrole biosynthetic pathway were explored. Excessive amounts of MgProtoME, the substrate of the cyclase reaction, accumulated in response to the reduced CHL27 content. Moreover, 5-aminolevulinic acid (ALA) synthesis, Mg chelatase and Mg protoporphyrin methyltransferase activities were reduced in transgenic plants. Compared with growth under continuous light exposure, the CHL27-deficient plants showed a stronger reduction in Chl content, cell death and leaf necrosis during diurnal light/dark cycles. This photooxidative phenotype correlated with a rapidly increasing MgProtoME steady-state level at the beginning of each light period. In contrast, the same transformants grown under continuous light exposure possessed a permanently elevated amount of MgProtoME. Its lower phototoxicity correlated with increased activities of ascorbate peroxidase and catalase, and a higher amount of reduced ascorbate. It is proposed that improved stress acclimation during continuous light in comparison with light-dark growth increases the capacity to prevent photooxidation by excess tetrapyrrole precursors and lowers the susceptibility to secondary photodynamic damage.


Assuntos
Luz , Estresse Oxidativo , Oxigenases/metabolismo , Tetrapirróis/biossíntese , Liases/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Porfirinas/análise , RNA Antissenso/genética , Tabaco/enzimologia , Tabaco/genética
7.
Plant Cell Physiol ; 51(5): 670-81, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20375109

RESUMO

In photosynthetic organisms chlorophyll and heme biosynthesis is tightly regulated at various levels in response to environmental adaptation and plant development. The formation of 5-aminolevulinic acid (ALA) is the key regulatory step and provides adequate amounts of the common precursor molecule for the Mg and Fe branches of tetrapyrrole biosynthesis. Pathway control prevents accumulation of metabolic intermediates and avoids photo-oxidative damage. In angiosperms reduction of protochlorophyllide (Pchlide) to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR). Although a correlation between down-regulated ALA synthesis and accumulation of Pchlide in the dark was proposed a long time ago, the time-resolved mutual dependency has never been analyzed. Taking advantage of the high metabolic activity of young barley (Hordeum vulgare L.) seedlings, in planta ALA synthesis could be determined with high time-resolution. ALA formation declined immediately after transition from light to dark and correlated with an immediate accumulation of POR-bound Pchlide within the first 60 min in darkness. The flu homologous barley mutant tigrina d(12) uncouples ALA synthesis from dark-suppression and continued to form ALA in darkness without a significant change in synthesis rate in this time interval. Similarly, inhibition of protoporphyrinogen IX oxidase by acifluorfen resulted in a delayed accumulation of Pchlide during the entire dark period and a weak repression of ALA synthesis in darkness. Moreover, it is demonstrated that dark repression of ALA formation relies rather on rapid post-translational regulation in response to accumulating Pchlide than on changes in nuclear gene expression.


Assuntos
Ácido Aminolevulínico/análogos & derivados , Escuridão , Hordeum/metabolismo , Protoclorifilida/biossíntese , Ácido Aminolevulínico/metabolismo , Heme/metabolismo , Hordeum/genética , Peroxidação de Lipídeos , Nitrobenzoatos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Protoporfirinogênio Oxidase/metabolismo , RNA de Plantas/análise
8.
Mol Plant ; 2(6): 1198-210, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19995725

RESUMO

In aerobic photosynthetic organisms, GUN4 binds the chlorophyll intermediates protoporphyrin and Mg protoporphyrin, stimulates Mg chelatase activity, and is implicated in plastidic retrograde signaling. GUN4 expression is most abundant in young and greening tissues and parallels the activity of 5-aminolevulinic acid (ALA) ALA and Mg porphyrin biosynthesis during photoperiodic growth. We explored function and mode of action of GUN4 using GUN4-deficient and overexpressing plants. GUN4 overexpression leads to a general activation of the enzymes of chlorophyll biosynthesis. During photoperiodic growth GUN4 deficiency prevents ALA synthesis and chlorophyll accumulation. All these metabolic changes do not correlate with altered gene expression or changes of protein abundance in tetrapyrrole biosynthesis. While ALA feeding fails to compensate GUN4 deficiency during light-dark growth, this approach results in chlorophyll accumulation under continuous dim light. A new model defines the involvement of GUN4 in posttranslational regulation of ALA and Mg porphyrin synthesis, to sustain chlorophyll synthesis, namely under varying environmental conditions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Plantas/metabolismo , Tetrapirróis/biossíntese , Ácido Aminolevulínico/metabolismo , Proteínas de Arabidopsis/genética , Clorofila/biossíntese , Clorofila/genética , Clorofila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Iluminação , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Processamento de Proteína Pós-Traducional , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , Tetrapirróis/metabolismo , Transcrição Genética
9.
Plant Mol Biol ; 71(4-5): 425-36, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19680747

RESUMO

At the last step of the chlorophyll biosynthetic pathway chlorophyll synthase (CHLG) esterifies chlorophyllide a and b with phytyl or geranyl-geranyl pyrophosphate in chloroplasts. Transgenic tobacco plants expressing CHLG RNA in sense and antisense orientation were examined for the effects of excessive and reduced ectopic CHLG expression, respectively, on the chlorophyll biosynthetic pathway and the expression of chlorophyll-binding proteins. Reduced chlorophyll synthase activity does not result in accumulation of chlorophyllide and caused reduced ALA formation and Mg and ferrochelatase activity, while CHLG overexpression correlated with enhanced ALA synthesizing capacity and more chelatase activities. The transcript levels of genes expressing proteins of chlorophyll biosynthesis and chlorophyll-binding proteins were down-regulated in response to reduced CHLG expression. Thus, reduced expression and activity of chlorophyll synthase caused a feedback-controlled inactivation of the initial and rate limiting step of the pathway leading to down regulation of the metabolic flow, while overexpression can mediate a stimulation of the pathway. Chlorophyll synthase is proposed to be important for the co-regulation of the entire pathway and the coordination of synthesis of chlorophyll and the chlorophyll-binding proteins.


Assuntos
Carbono-Oxigênio Ligases/fisiologia , Clorofila/biossíntese , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/metabolismo , Tabaco/enzimologia , Tabaco/metabolismo , Northern Blotting , Western Blotting , Carbono-Oxigênio Ligases/genética , Cromatografia Líquida de Alta Pressão , Heme/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , RNA Antissenso/genética , RNA Antissenso/fisiologia , Tetrapirróis/biossíntese , Tetrapirróis/genética , Tabaco/genética
10.
Biochim Biophys Acta ; 1787(12): 1458-67, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19540827

RESUMO

The two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI(-) mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI(-) mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity.


Assuntos
Oxigenases/fisiologia , Synechocystis/genética , Aerobiose , Mutação , Fases de Leitura Aberta , Oxigenases/genética , Protoporfirinas/biossíntese , Synechocystis/crescimento & desenvolvimento
11.
J Biol Chem ; 283(38): 25794-802, 2008 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-18625715

RESUMO

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.


Assuntos
Proteínas de Transporte/fisiologia , Clorofila/metabolismo , Cianobactérias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/fisiologia , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Clorofila/química , Cloroplastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Liases/química , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Mutação , Fenótipo , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II/metabolismo , Porfirinas/química , Espectrometria de Fluorescência/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...