Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
Mais filtros










Filtros aplicados
Base de dados
Intervalo de ano de publicação
1.
Environ Microbiol Rep ; 7(4): 623-33, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25990300

RESUMO

'Bacterial consortium' sensu lato applies to mutualism or syntrophy-based systems consisting of unrelated bacteria. Consortia of cyanobacteria have been preferentially studied on Anabaena epibioses; non-photosynthetic satellites of other filamentous or unicellular cyanobacteria were also considered although structure-functional data are few. At the same time, information about consortia of cyanobacteria which have light-harvesting antennae distinct from standard phycobilisome was missing. In this study, we characterized first, via a polyphasic approach, the cultivable consortium of Prochlorothrix hollandica CCAP 1490/1 (filamentous cyanobacterium which contains chlorophylls a, b/carotenoid/protein complex in the absence of phycobilisome) and non-photosynthetic heterotrophic bacteria. The strains of most abundant satellites were isolated and identified. Consortium metagenome reconstructed via 454-pyro and Illumina sequencing was shown to include, except for P. hollandica, several phylotypes of Proteobacteria and Bacteroidetes. The ratio of consortium members was essentially stable irrespective of culture age, and restored after artificially imposed imbalance. The consortium had a complex spatial arrangement as demonstrated by FISH and SEM images of the association, epibiosis, and biofilm type. Preliminary data of metagenome annotation agreed with the hypothesis that satellite bacteria contribute to P. hollandica protection from reactive oxygen species (ROS).


Assuntos
Bacteroidetes/classificação , Biota , Metagenoma , Consórcios Microbianos , Prochlorothrix/crescimento & desenvolvimento , Proteobactérias/classificação , Bacteroidetes/genética , Bacteroidetes/crescimento & desenvolvimento , Bacteroidetes/isolamento & purificação , Análise por Conglomerados , Hibridização in Situ Fluorescente , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Filogenia , Prochlorothrix/genética , Proteobactérias/genética , Proteobactérias/crescimento & desenvolvimento , Proteobactérias/isolamento & purificação , Análise de Sequência de DNA
2.
Biochim Biophys Acta ; 1817(11): 1992-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22728755

RESUMO

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different host species and cultured Prochlorothrix by means of spectroscopic measurements, including time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb, PS II-PS I connection, and pigment status, especially that of PS I Red Chls, were different among the four samples. We also discuss relationships between these observed characteristics and the light conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host colonies, presence or absence of tunic spicules, and microenvironments within the ascidians' habitat.


Assuntos
Prochloron/metabolismo , Prochlorothrix/metabolismo , Simbiose , Urocordados/microbiologia , Animais , Complexo de Proteína do Fotossistema I/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Espectrometria de Fluorescência
3.
Plant Cell Physiol ; 53(3): 518-27, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22302713

RESUMO

In oxygenic photosynthetic organisms, the properties of photosynthetic reaction systems primarily depend on the Chl species used. Acquisition of new Chl species with unique optical properties may have enabled photosynthetic organisms to adapt to various light environments. The artificial production of a new Chl species in an existing photosynthetic organism by metabolic engineering provides a model system to investigate how an organism responds to a newly acquired pigment. In the current study, we established a transformation system for a Chl d-dominated cyanobacterium, Acaryochloris marina, for the first time. The expression vector (constructed from a broad-host-range plasmid) was introduced into A. marina by conjugal gene transfer. The introduction of a gene for chlorophyllide a oxygenase, which is responsible for Chl b biosynthesis, into A. marina resulted in a transformant that synthesized a novel Chl species instead of Chl b. The content of the novel Chl in the transformant was approximately 10% of the total Chl, but the level of Chl a, another Chl in A. marina, did not change. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d(P) by mass spectrometry and nuclear magnetic resonance spectroscopy. [7-Formyl]-Chl d(P) is hypothesized to be produced by the combined action of chlorophyllide a oxygenase and enzyme(s) involved in Chl d biosynthesis. These results demonstrate the flexibility of the Chl biosynthetic pathway for the production of novel Chl species, indicating that a new organism with a novel Chl might be discovered in the future.


Assuntos
Clorofila/metabolismo , Cianobactérias/enzimologia , Cianobactérias/genética , Genes Bacterianos/genética , Engenharia Metabólica/métodos , Oxigenases/genética , Transformação Genética , Vias Biossintéticas/genética , Clorofila/química , Cromatografia Líquida de Alta Pressão , Conjugação Genética , Cianobactérias/citologia , Vetores Genéticos/genética , Especificidade de Hospedeiro/genética , Oxigenases/metabolismo , Plasmídeos/genética , Prochlorothrix/enzimologia , Reprodutibilidade dos Testes , Análise Espectral
4.
J Phys Chem B ; 114(28): 9275-82, 2010 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-20583762

RESUMO

Chlorophyll (Chl) a/b-binding proteins from Prochlorothrix hollandica known as Pcb antennae were studied by femtosecond transient absorption technique to identify energy transfer rates and pathways in Pcb and Pcb-PS I complexes. Carotenoids transfer energy to Chl with low efficiency of approximately 25% in Pcb complexes. Interestingly, analysis of transient absorption spectra identified a pathway from the hot S(1) state of zeaxanthin and/or beta-carotene as the major energy transfer channel between carotenoids and chlorophylls in Pcb whereas the S(2) state contributes only marginally to energy transfer. Due to energetic reasons, no energy transfer is possible via the relaxed S(1) state of carotenoids. The low overall energy transfer efficiency of carotenoids recognizes chlorophylls as the main light-harvesting pigments. Besides Chl a, presence of Chl b, which transfers energy to Chl a with nearly 100% efficiency, significantly broadens the spectral range accessible for light-harvesting and improves cross section of Pcb complexes. The major role of carotenoids in Pcb is photoprotection.


Assuntos
Carotenoides/química , Clorofila/química , Complexo de Proteína do Fotossistema I/química , Prochlorothrix/enzimologia , Clorofila A , Transferência de Energia , Complexo de Proteína do Fotossistema I/metabolismo , Espectrometria de Fluorescência
5.
Biochim Biophys Acta ; 1797(1): 89-97, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19761753

RESUMO

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment-protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (>200 mumol photons m(-2) s(-1)) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment-protein complexes function as low light-induced mobile antennae. Further, we have detected alpha-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.


Assuntos
Fotossíntese/fisiologia , Prochlorothrix/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Luz , Complexo de Proteína do Fotossistema I/isolamento & purificação , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/isolamento & purificação , Complexo de Proteína do Fotossistema II/metabolismo , Proclorófitas/metabolismo , Tilacoides/metabolismo , Tilacoides/ultraestrutura
6.
J Am Chem Soc ; 130(6): 1985-91, 2008 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-18201089

RESUMO

The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type Pc and mutant Pc, but the chemical shift perturbations are smaller for the complex with mutant Pc. Docking calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures. Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded that the double mutation shifts the complex further from the well-defined toward the encounter state.


Assuntos
Simulação por Computador , Citocromos f/química , Modelos Químicos , Plastocianina/química , Prochlorothrix/química , Prochlorothrix/enzimologia , Cádmio/química , Cobre/química , Citocromos f/biossíntese , Citocromos f/isolamento & purificação , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Conformação Molecular , Plastocianina/biossíntese , Plastocianina/isolamento & purificação , Padrões de Referência
7.
Microb Ecol ; 55(4): 685-96, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-17874260

RESUMO

Ecophysiological investigations on the salinity acclimation of the cyanobacterium Prochlorothrix hollandica SAG 10.89 led to significantly revised salinity tolerance limits. Besides potential effects of cultivation techniques, clear ion composition effects mainly explain formerly described hypersensitivity to NaCl-mediated salinity and lack of osmolyte detection. An extraordinarily broad plasticity of cellular chlorophyll a/b ratios occurred with variations of NaCl-induced salinity. Photosynthesis characteristics, pigment regulation, respiration, and biomass yield in growth medium with field-like ion composition indicated generally reduced acclimation pressure. A simultaneously significant increase in osmolyte (sucrose) accumulation indicated more efficient osmotic acclimation. Minor growth inhibition up to salinities of 10 practical salinity units enlarged the potential habitat of P. hollandica but at the most to about 300,000 km2 in the Baltic Sea. This supports probable observations of Prochlorothrix sp. in phytoplankton assemblages of open waters in Baltic Sea-monitoring studies. Brackish habitats differ from so far known habitats of Prochlorothrix spp. in turbidity, productivity, and plankton composition. Adjusted physiological features dispel fundamental doubts on the establishment of filamentous prochlorophytes in brackish waters.


Assuntos
Clorofila/biossíntese , Prochlorothrix/efeitos dos fármacos , Prochlorothrix/crescimento & desenvolvimento , Cloreto de Sódio/farmacologia , Biomassa , Meios de Cultura , Concentração Osmolar , Fotossíntese/efeitos dos fármacos , Prochlorothrix/metabolismo , Salinidade , Sacarose/metabolismo , Microbiologia da Água
8.
Biochim Biophys Acta ; 1767(6): 801-6, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17174934

RESUMO

Action spectra for photosystem II (PSII)-driven oxygen evolution and of photosystem I (PSI)-mediated H(2) photoproduction and photoinhibition of respiration were used to determine the participation of chlorophyll (Chl) a/b-binding Pcb proteins in the functions of pigment apparatus of Prochlorothrix hollandica. Comparison of the in situ action spectra with absorption spectra of PSII and PSI complexes isolated from the cyanobacterium Synechocystis 6803 revealed a shoulder at 650 nm that indicated presence of Chl b in the both photosystems of P. hollandica. Fitting of two action spectra to absorption spectrum of the cells showed a chlorophyll ratio of 4:1 in favor of PSI. Effective antenna sizes estimated from photochemical cross-sections of the relevant photoreactions were found to be 192+/-28 and 139+/-15 chlorophyll molecules for the competent PSI and PSII reaction centers, respectively. The value for PSI is in a quite good agreement with previous electron microscopy data for isolated Pcb-PSI supercomplexes from P. hollandica that show a trimeric PSI core surrounded by a ring of 18 Pcb subunits. The antenna size of PSII implies that the PSII core dimers are associated with approximately 14 Pcb light-harvesting proteins, and form the largest known Pcb-PSII supercomplexes.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Prochlorothrix/metabolismo , Clorofila/metabolismo , Clorofila A , Modelos Biológicos , Synechocystis/metabolismo
9.
J Biol Chem ; 281(22): 15385-93, 2006 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-16574646

RESUMO

The organization of pigment molecules in photosystems is strictly determined. The peripheral antennae have both chlorophyll a and b, but the core antennae consist of only chlorophyll a in green plants. Furthermore, according to the recent model obtained from the crystal structure of light-harvesting chlorophyll a/b-protein complexes II (LHCII), individual chlorophyll-binding sites are occupied by either chlorophyll a or chlorophyll b. In this study, we succeeded in altering these pigment organizations by introducing a prokaryotic chlorophyll b synthesis gene (chlorophyllide a oxygenase (CAO)) into Arabidopsis. In these transgenic plants (Prochlirothrix hollandica CAO plants), approximately 40% of chlorophyll a of the core antenna complexes was replaced by chlorophyll b in both photosystems. Chlorophyll a/b ratios of LHCII also decreased from 1.3 to 0.8 in PhCAO plants. Surprisingly, these transgenic plants were capable of photosynthetic growth similar to wild type under low light conditions. These results indicate that chlorophyll organizations are not solely determined by the binding affinities, but they are also controlled by CAO. These data also suggest that strict organizations of chlorophyll molecules are not essential for photosynthesis under low light conditions.


Assuntos
Arabidopsis/metabolismo , Oxigenases/metabolismo , Agrobacterium tumefaciens/genética , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clorofila/metabolismo , Clorofila A , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Genes Bacterianos , Genes de Plantas , Microscopia Eletrônica , Mutação , Oxigenases/genética , Fenótipo , Fotossíntese , Plantas Geneticamente Modificadas , Prochlorothrix/enzimologia , Prochlorothrix/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Genética
10.
Biochim Biophys Acta ; 1708(1): 1-5, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15949978

RESUMO

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called "prochlorophytes") that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.


Assuntos
Complexo de Proteína do Fotossistema I/ultraestrutura , Prochlorothrix/ultraestrutura , Centrifugação com Gradiente de Concentração , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/ultraestrutura , Microscopia Eletrônica , Estrutura Quaternária de Proteína
11.
Planta ; 218(6): 1019-25, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14716565

RESUMO

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b-protein complexes in land plants.


Assuntos
Oxigenases/genética , Oxigenases/metabolismo , Prochlorothrix/enzimologia , Prochlorothrix/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Dados de Sequência Molecular , Oryza/enzimologia , Oryza/genética , Oxigenases/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
12.
Rapid Commun Mass Spectrom ; 17(24): 2788-96, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14673828

RESUMO

Bacteriohopanepolyols (BHPs) fragment via characteristic pathways during atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry (APCI-LC/MS(n)). Comparison of the MS(2) spectra of bacteriohopane-32,33,34,35-tetrol (BHT) and 2 beta-methylbacteriohopane-32,33,34,35-tetrol has confirmed the previously proposed ring-C cleavage occurring between C-9 and 11 and C-8 and 14. This fragmentation, diagnostic of all hopanoids, also occurs in BHPs containing an amino group (-NH(2)) at C-35 although the higher relative stability of the ion limits this fragmentation to a minor process after protonation of the basic nitrogen function. Studies of a number of cell cultures including a prochlorophyte (Prochlorothrix hollandica) and a cyanobacterium (Chlorogloeopsis LA) demonstrate the power of this technique to detect composite BHPs with a complex biological functionality at C-35. We also report the first observation of intact pentafunctionalised bacteriohopanepolyols using this method.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cianobactérias/química , Cianobactérias/metabolismo , Prochlorothrix/química , Prochlorothrix/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Triterpenos/química , Triterpenos/metabolismo , Pressão Atmosférica , Sedimentos Geológicos/análise , Sedimentos Geológicos/química , Sedimentos Geológicos/microbiologia , Indicadores e Reagentes/química , Triterpenos/análise
13.
Appl Environ Microbiol ; 69(10): 6243-9, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14532086

RESUMO

Prochlorothrix hollandica is the only filamentous chlorophyll b (Chlb)-containing oxyphotobacterium that has been found in freshwater habitats to date. Chlb serves as a light-harvesting pigment which is bound to special binding proteins (Pcb). Even though Prochlorothrix was initially characterized as a highly salt-sensitive species, we detected it in a brackish water environment that is characterized by salinities of up to 12 practical salinity units. Using PCR and reverse transcription, we amplified pcb gene fragments of phytoplankton samples taken along a salinity gradient in the eutrophic Darss-Zingst estuary (southern Baltic Sea). After sequencing, high levels of homology to the pcbB and pcbC genes of P. hollandica were found. Furthermore, autofluorescence of Prochlorothrix-like filaments that indicated that Chlb was present was detected in enrichment cultures prepared from the estuarine phytoplankton. The detection of Chlb-containing filaments, as well as the pcb and 16S ribosomal DNA sequences, suggests that Prochlorothrix is an indigenous genus in the Darss-Zingst estuary and may also inhabit many other brackish water environments. The potential of using pcb gene detection to differentiate Prochlorothrix from morphologically indistinguishable species belonging to the genera Pseudanabaena and Planktothrix (Oscillatoria) in phytoplankton analyses is discussed.


Assuntos
Clorofila/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Prochlorothrix/classificação , Prochlorothrix/isolamento & purificação , Rios/microbiologia , Cloreto de Sódio/metabolismo , Animais , DNA Ribossômico/análise , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Filogenia , Fitoplâncton/microbiologia , Prochlorothrix/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
14.
J Biol Chem ; 278(10): 8179-83, 2003 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-12509429

RESUMO

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.


Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Plastocianina/metabolismo , Prochlorothrix/metabolismo , Sequência de Bases , Primers do DNA , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Plastocianina/química , Plastocianina/genética
15.
Biochemistry ; 41(52): 15698-705, 2002 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-12501198

RESUMO

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.


Assuntos
Citocromos/química , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Plastocianina/química , Sítios de Ligação/genética , Sequência Conservada/genética , Cianobactérias/enzimologia , Cianobactérias/genética , Citocromos/genética , Citocromos f , Glicina/genética , Histidina/química , Leucina/genética , Substâncias Macromoleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Plastocianina/genética , Prochlorothrix/química , Prochlorothrix/genética , Prolina/genética , Prótons , Tirosina/genética
16.
Biophys J ; 82(6): 3305-13, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12023253

RESUMO

We have used several docking algorithms (GRAMM, FTDOCK, DOT, AUTODOCK) to examine protein-protein interactions between plastocyanin (Pc)/photosystem I (PSI) in the electron transfer reaction. Because of the large size and complexity of this system, it is faster and easier to use computer simulations than conduct x-ray crystallography or nuclear magnetic resonance experiments. The main criterion for complex selection was the distance between the copper ion of Pc and the P700 chlorophyll special pair. Additionally, the unique tyrosine residue (Tyr(12)) of the hydrophobic docking surface of Prochlorothrix hollandica Pc yields a specific interaction with the lumenal surface of PSI, thus providing the second constraint for the complex. The structure that corresponded best to our criteria was obtained by the GRAMM algorithm. In this structure, the solvent-exposed histidine that coordinates copper in Pc is at the van der Waals distance from the pair of stacked tryptophans that separate the chlorophylls from the solvent, yielding the shortest possible metal-to-metal distance. The unique tyrosine on the surface of the Prochlorothrix Pc hydrophobic patch also participates in a hydrogen bond with the conserved Asn(633) of the PSI PsaB polypeptide (numbering from the Synechococcus elongatus crystal structure). Free energy calculations for complex formation with wild-type Pc, as well as the hydrophobic patch Tyr(12)Gly and Pro(14)Leu Pc mutants, were carried out using a molecular mechanics Poisson-Boltzman, surface area approach (MM/PBSA). The results are in reasonable agreement with our experimental studies, suggesting that the obtained structure can serve as an adequate model for P. hollandica Pc-PSI complex that can be extended for the study of other cyanobacterial Pc/PSI reaction pairs.


Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema I , Plastocianina/química , Algoritmos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Simulação por Computador , DNA Bacteriano/genética , Transporte de Elétrons , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Plastocianina/genética , Prochlorothrix/química , Prochlorothrix/genética , Homologia de Sequência de Aminoácidos , Termodinâmica
17.
Nature ; 413(6856): 590, 2001 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-11595938

RESUMO

The oceanic picoplankton Prochlorococcus - probably the most abundant photosynthetic organism on our planet - can grow at great depths where light intensity is very low. We have found that the chlorophyll-binding proteins in a deep-living strain of this oxyphotobacterium form a ring around a trimer of the photosystem I (PS I) photosynthetic reaction centre, a clever arrangement that maximizes the capture of light energy in such dim conditions.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Proteínas de Bactérias/fisiologia , Cianobactérias/fisiologia , Complexos de Proteínas Captadores de Luz , Fotossíntese , Complexo de Proteína do Fotossistema I , Prochloron/química , Prochloron/fisiologia , Prochlorothrix/química , Prochlorothrix/fisiologia
18.
J Biol Chem ; 276(40): 37501-5, 2001 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-11457853

RESUMO

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.


Assuntos
Proteínas de Bactérias/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I , Plastocianina/metabolismo , Prochlorothrix/metabolismo , Prolina/metabolismo , Proteínas de Bactérias/genética , Transporte de Elétrons , Cinética , Proteínas Motores Moleculares , Mutação , Plastocianina/genética , Prochlorothrix/genética
19.
Nature ; 400(6740): 159-62, 1999 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-10408441

RESUMO

Photosynthetic organisms have a variety of accessory pigments, on which their classification has been based. Despite this variation, it is generally accepted that all chloroplasts are derived from a single cyanobacterial ancestor. How the pigment diversity has arisen is the key to revealing their evolutionary history. Prochlorophytes are prokaryotes which perform oxygenic photosynthesis using chlorophyll b, like land plants and green algae (Chlorophyta), and were proposed to be the ancestors of chlorophyte chloroplasts. However, three known prochlorophytes (Prochloron didemni, Prochlorothrix hollandica and Prochlorococcus marinus) have been shown to be not the specific ancestors of chloroplasts, but only diverged members of the cyanobacteria, which contain phycobilins but lack chlorophyll b. Consequently it has been proposed that the ability to synthesize chlorophyll b developed independently several times in prochlorophytes and in the ancestor of chlorophytes. Here we have isolated the chlorophyll b synthesis genes (chlorophyll a oxygenase) from two prochlorophytes and from major groups of chlorophytes. Phylogenetic analyses show that these genes share a common evolutionary origin. This indicates that the progenitors of oxygenic photosynthetic bacteria, including the ancestor of chloroplasts, had both chlorophyll b and phycobilins.


Assuntos
Clorofila/análise , Cloroplastos/química , Cianobactérias/química , Evolução Molecular , Oxigenases/genética , Ficocianina/análise , Pirróis/análise , Sequência de Aminoácidos , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Cloroplastos/classificação , Cianobactérias/classificação , Cianobactérias/enzimologia , Cianobactérias/genética , Eucariotos/química , Eucariotos/enzimologia , Eucariotos/genética , Dados de Sequência Molecular , Ficobilinas , Filogenia , Prochloron/química , Prochloron/enzimologia , Prochloron/genética , Prochlorothrix/química , Prochlorothrix/enzimologia , Prochlorothrix/genética , Homologia de Sequência de Aminoácidos , Tetrapirróis
20.
Biochemistry ; 38(16): 4988-95, 1999 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-10213601

RESUMO

The solution structure of a divergent plastocyanin (PC) from the photosynthetic prokaryote Prochlorothrix hollandica was determined by homonuclear 1H NMR spectroscopy. Nineteen structures were calculated from 1222 distance restraints, yielding a family of structures having an average rmsd of 0.42 +/- 0.08 A for backbone atoms and 0.71 +/- 0.07 A for heavy atoms to the mean structure. No distance constraint was violated by more than 0.26 A in the structure family. Despite the low number of conserved residues shared with other PC homologues, the overall folding pattern of P. hollandica PC is similar to other PCs, in that the protein forms a two-sheet beta-barrel tertiary structure. The greatest variability among the backbone structures is seen in the loop region from residues 47-60. The differences seen in the P. hollandica PC homologue likely arise due to a small deletion of 2-4 residues compared to the PC consensus; this yields a less extended loop containing a short alpha-helix from residues Ala52-Leu55. Additionally, the protein has an altered hydrophobic patch thought to be important in binding reaction partners. Whereas the backbone structure is very similar within the loops of the hydrophobic region, the presence of two unique residues (Tyr12 and Pro14) yields a structurally different hydrophobic surface likely important in binding P. hollandica Photosystem I.


Assuntos
Plastocianina/química , Prochlorothrix/química , Sequência de Aminoácidos , Cristalografia por Raios X , Transporte de Elétrons , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fotossíntese , Prolina/química , Conformação Proteica , Estrutura Secundária de Proteína , Prótons , Homologia de Sequência de Aminoácidos , Soluções
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...