49 results on '"Avilan L"'
Search Results
2. Crystal structure of a cutinase enzyme from Saccharopolyspora flava (611)
- Author
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Zahn, M., primary, Avilan, L., additional, Beckham, G.T., additional, and McGeehan, J.E., additional
- Published
- 2022
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3. Crystal structure of cutinase 1 from Thermobifida fusca DSM44342 (703)
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Zahn, M., primary, Avilan, L., additional, Beckham, G.T., additional, and McGeehan, J.E., additional
- Published
- 2022
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4. Information Transfer in GAPdH-PRK Complex of Chlamydomonas Reinhardtii
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Gontero, B., Lebreton, S., Avilan, L., Ricard, J., and Garab, G., editor
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- 1998
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5. Enzymes and the supramolecular organization of the living cell. Information transfer within supramolecular edifices and imprinting effects
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Ricard, J., Gontero, B., Avilan, L., and Lebreton, S.
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- 1998
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6. Plasminogen interaction and activation on Streptococcus mutans surface
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Urdaneta, L., Vanegas, G., Premoli, G., and Avilan, L.
- Published
- 2004
7. Isolation of Different Forms of Phosphoribulokinase from Mutant Chlamydomonas Reinhardtii Cells
- Author
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Avilan, L., primary, Gontero, B., additional, and Ricard, J., additional
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- 1995
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8. Properties of a Multimeric Protein Complex From Chlamydomonas Reinhardtii Chloroplast Possessing Glyceraldehyde-3-Phosphate Dehydrogenase and Phosphoribulokinase Activities
- Author
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Gontero, B., primary, Lebreton, S., additional, Avilan, L., additional, and Ricard, J., additional
- Published
- 1995
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- View/download PDF
9. Golden magic: RSH enzymes for (p)ppGpp metabolism in the diatom Phaeodactylum tricornutum
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Avilan, L, primary, Puppo, C, additional, Villain, A, additional, Bouveret, E, additional, Menand, B, additional, Field, B, additional, and Gontero, B, additional
- Published
- 2018
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10. ESTADOS TIPO REPRODUCTIVOS DEL MANGO (MANGIFERA INDICA L.) EN ZONA CENTRAL DE VENEZUELAMODEL REPRODUCTIVE STAGES OF MANGO (MANGIFERA INDICA L.) IN THE CENTRAL ZONE OF VENEZUELA
- Author
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Pérez Macias, M., primary, Soto, E., additional, Puche, M., additional, Avilan, L., additional, and Gutiérrez, M., additional
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- 2015
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11. Exploring CP12 binding proteins revealed the aldolase as a new partner for PRK/GAPDH/CP12 complex. Purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii
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Erales, J., Avilan, L., Lebreton, S., Gontero, B., and Azzopardi, Laure
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ComputingMilieux_MISCELLANEOUS - Published
- 2008
12. Producción forzada del mango (Mangifera indica. L) en alta densidad (278 pl ha-1)durante el periodo de crecimiento
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Avilan, L., Marín R., C., Rodríguez, M., and Ruíz, J.
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poda ,regulador de crecimiento ,regulator ,pruning ,Mangifera indica ,yield ,rendimiento - Abstract
Resumen En árboles de mango dentro del período de crecimiento, de los cvs Haden, Tommy Atkins, Springfels y Edward, injertados sobre «criollo», distanciados a seis metros entre si (278 pl ha-1); se evaluó el efecto de la poda y el regulador de crecimiento Paclobutrazol (PBZ) aplicado al suelo (2,5 g ia planta-1) sobre el desarrollo vegetativo y la producción. Los tratamientos fueron: Testigo (T) en libre crecimiento, T + PBZ, Poda (P) a 2m de altura del suelo, P + PBZ, P + corte lateral de las ramas a un radio de 1,8 m del tronco (P+L) y P + entresaque de 1-2 ramas primarias desde su inserción en el tronco (P+E). En todos los tratamientos se aplicó el promotor de floración nitrato de potasio (KNO3) al 6% a los cinco meses de efectuada la poda. Las variables estudiadas fueron: incremento del volumen de copa (IVC) anual, rendimiento (kg de frutos planta-1) y eficiencia productiva (kg frutos m-3). Los resultados evidenciaron que a mayor intensidad de la poda, mayor el IVC y la reducción de la producción de frutos, pero la poda moderada (P) mejoró significativamente la eficiencia productiva. Los rendimientos (t ha-1) fueron 24,6 T+PBZ; 24,1 T; 19,7 P; 19,6 P + PBZ 18,0 P+E y 13,2 P+L, respectivamente, superiores en 200% a los obtenidos en huertos comerciales durante el mismo periodo empleando el sistema tradicional (69 plantas ha-1). Abstract A mango trial was established in 1996 using five-year-old trees. Cultivars Haden, Tommy Atkins, Springfels and Edward grafted on «Criollo» were planted at 6x6 m (278 trees ha-1) to evaluate the pruning effect at 2m height, the Paclobutrazol (PBZ) effect when applied to the soil (2.5 g ai per tree) and flowering promoter potassium nitrate (KNO3), at 6% on the vegetative growth and yield of the mango cultivars. The treatments were: Checker (T), T + PBZ, Pruning (P) and P + PBZ. P + lateral branches of a radius of 2 m (P+L) and P + pruning of internal primary branches (P+E). The studied variables were increase of canopy volume (ICV), yield (kg tree-1) and productive efficiency (kg tree m-3). It was found that the largest pruning increases the canopy volume, with a concomitant reduction of fruit yield, due to the moderate pruning increase significantly the productive efficiency. Yields (t ha-1) for the four cycles 1996-2000, for the different treatments were: 19.29 T; 24.6 T+PBZ; 19.7 P; 19.6 P+PBZ; 18.1 P+E and 13.2 P+L. These yields were higher (>200%) than the one reached by low tree density system (69 tree ha-1) in the same time. In general, the results showed that in the «Growth Period» of fruit trees, characterized by a large volume growth in the productive life cycle of the tree is the most critical to be controlled to reduce the free height. If we consider that 90% of the country fruit production came from small and medium size growers, the use of a high tree density system should provides an improvement of the income of these peoples.
- Published
- 2005
13. The function of glycosomes in the metabolism of trypanosomatid particles and the promise of glycososomal proteins as drug targets
- Author
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UCL - Autre, Gualdron-Lopez, M., Michels, Paulus, Quinones, W., Caceres, A.J., Avilan, L., Concepcion, J.L., UCL - Autre, Gualdron-Lopez, M., Michels, Paulus, Quinones, W., Caceres, A.J., Avilan, L., and Concepcion, J.L.
- Abstract
Trypanosomatids have the unique feature of compartmentalizing the major part of the glycolytic pathway inside peroxisome-related organelles called glycosomes. However, these organelles also contain enzymes of several other important pathways involved in both catabolic and anabolic processes. The enzyme content and the metabolic role of glycosomes differ between trypanosomatid species and between their life cycle stages. Several of the glycosomal pathways have been shown to be important for the viability, pathogenicity, and/or virulence of different trypanosomatid parasites. Additionally, the correct compartmentalization of glycosomal enzymes inside the organelles appeared to be vital for these pathogens. Therefore, many of these enzymes, as well as the proteins involved in the translocation of metabolites across the glycosomal membrane and peroxins (PEXs), proteins responsible for the biogenesis of glycosomes, are candidate drug targets. Glycosomal enzymes and PEX proteins of Trypanosoma brucei, T. cruzi, and Leishmania spp. are being studied, and compounds that interfere with their functioning are being developed for use as lead drugs against the diseases caused by these parasites. Potent, selective inhibitors of several enzymes have been obtained that exert trypanocidal activity on parasites cultured in vitro and have no or only little effect on growth of human cells. In addition, some compounds showed anti-parasite activity in experimentally infected animals.
- Published
- 2013
14. ID: 041 Plasminogen binding on the surface of Leishmania mexicana by enolase
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Avilan, L., primary, Vanegas, G., additional, Quiñones, W., additional, Peña, P., additional, Gonzalez, A., additional, Gualdron, M., additional, and Concepcion, J., additional
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- 2006
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15. Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts.
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Avilan, L, Lebreton, S, and Gontero, B
- Abstract
The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.
- Published
- 2000
16. The influence of preparation conditions on the surface area and phase formation of MoO3/ZrO2 catalysts
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Calafat, A., Avilan, L., and Aldana, J.
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- 2000
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17. Interaction of Leishmania mexicana promastigotes with the plasminogen-plasmin system
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Avilan, L., Calcagno, M., Figuera, M., Lemus, L., Puig, J., and Rodriguez, A. M.
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- 2000
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18. Naturally acquired immune responses to malaria vaccine candidate antigens MSP3 and GLURP in Guahibo and Piaroa indigenous communities of the Venezuelan Amazon
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Baumann Andreas, Magris Magda M, Urbaez Marie-Luz, Vivas-Martinez Sarai, Durán Rommy, Nieves Tahidid, Esen Meral, Mordmüller Benjamin G, Theisen Michael, Avilan Luisana, and Metzger Wolfram G
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Malaria transmission in most of Latin America can be considered as controlled. In such a scenario, parameters of baseline immunity to malaria antigens are of specific interest with respect to future malaria eradication efforts. Methods A cross-sectional study was carried out in two indigenous population groups in Amazonas/Venezuela. Data from the regional malaria documentation system were extracted and participants from the ethnic groups of the Guahibo (n = 180) and Piaroa (n = 295) were investigated for the presence of Plasmodium parasites and naturally acquired antibodies to Plasmodium falciparum antigens in serum. The GMZ2 vaccine candidate proteins MSP3 and GLURP were chosen as serological markers. Results The incidence of P. falciparum in both communities was found to be less than 2%, and none of the participants harboured P. falciparum at the time of the cross-sectional. Nearly a quarter of the participants (111/475; 23,4%) had positive antibody titres to at least one of the antigens. 53/475 participants (11.2%) were positive for MSP3, and 93/475 participants (19.6%) were positive for GLURP. High positive responses were detected in 36/475 participants (7.6%) and 61/475 participants (12.8%) for MSP3 and GLURP, respectively. Guahibo participants had significantly higher antibody titres than Piaroa participants. Conclusions Considering the low incidence of P. falciparum, submicroscopical infections may explain the comparatively high anti-P. falciparum antibody concentrations.
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- 2012
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19. Hysteresis of cytosolic NADP-malic enzyme II from Trypanosoma cruzi
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Avilan, L. and Garcia, P.
- Published
- 1994
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20. Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells.
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Launay H, Avilan L, Gérard C, Parsiegla G, Receveur-Brechot V, Gontero B, and Carriere F
- Subjects
- Carbon metabolism, Photosynthesis, Chloroplasts metabolism, Carbon Dioxide metabolism, Microalgae metabolism, Chlamydomonas reinhardtii
- Abstract
Carbon acquisition, assimilation and storage in eukaryotic microalgae and cyanobacteria occur in multiple compartments that have been characterised by the location of the enzymes involved in these functions. These compartments can be delimited by bilayer membranes, such as the chloroplast, the lumen, the peroxisome, the mitochondria or monolayer membranes, such as lipid droplets or plastoglobules. They can also originate from liquid-liquid phase separation such as the pyrenoid. Multiple exchanges exist between the intracellular microcompartments, and these are reviewed for the CO
2 concentration mechanism, the Calvin-Benson-Bassham cycle, the lipid metabolism and the cellular energetic balance. Progress in microscopy and spectroscopic methods opens new perspectives to characterise the molecular consequences of the location of the proteins involved, including intrinsically disordered proteins., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)- Published
- 2023
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21. Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.
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Avilan L, Lichtenstein BR, König G, Zahn M, Allen MD, Oliveira L, Clark M, Bemmer V, Graham R, Austin HP, Dominick G, Johnson CW, Beckham GT, McGeehan JE, and Pickford AR
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- Hydrolases, Ethylenes, Polyethylene Terephthalates chemistry, Phthalic Acids chemistry
- Abstract
Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability., (© 2023 The Authors. ChemSusChem published by Wiley-VCH GmbH.)
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- 2023
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22. Assembling Multiple Fragments: The Gibson Assembly.
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Avilan L
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- Cloning, Molecular, DNA Ligase ATP, DNA Primers, DNA Ligases, Nucleotidyltransferases
- Abstract
The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2023
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23. Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.
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Gérard C, Lebrun R, Lemesle E, Avilan L, Chang KS, Jin E, Carrière F, Gontero B, and Launay H
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- Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism, Photosynthesis genetics, Chlamydomonas reinhardtii genetics, Chlamydomonas reinhardtii metabolism
- Abstract
The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.
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- 2022
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24. ppGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.
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Avilan L, Lebrun R, Puppo C, Citerne S, Cuiné S, Li-Beisson Y, Menand B, Field B, and Gontero B
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- Chloroplasts metabolism, Guanosine Pentaphosphate metabolism, Photosynthesis, Diatoms genetics, Diatoms metabolism, Guanosine Tetraphosphate metabolism
- Abstract
Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta- and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression. We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent-like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments. Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)
- Published
- 2021
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25. A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana.
- Author
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Shao H, Huang W, Avilan L, Receveur-Bréchot V, Puppo C, Puppo R, Lebrun R, Gontero B, and Launay H
- Subjects
- Amino Acid Sequence, Chloroplast Proteins chemistry, Computer Simulation, Magnetic Resonance Spectroscopy, Protein Multimerization, Protein Structure, Secondary, Scattering, Small Angle, X-Ray Diffraction, Aquatic Organisms metabolism, Chloroplast Proteins metabolism, Diatoms metabolism
- Abstract
Background: CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background., Methods: A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12., Results: Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions., Conclusions: These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism. Video Abstract.
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- 2021
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26. Overproduction of the Flv3B flavodiiron, enhances the photobiological hydrogen production by the nitrogen-fixing cyanobacterium Nostoc PCC 7120.
- Author
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Roumezi B, Avilan L, Risoul V, Brugna M, Rabouille S, and Latifi A
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- Bacterial Proteins genetics, Genes, Homeobox, Hydrogenase genetics, Hydrogenase metabolism, Metabolic Engineering, Nitrogen metabolism, Nitrogen Fixation, Nostoc genetics, Photosynthesis, Bacterial Proteins metabolism, Hydrogen metabolism, Nostoc metabolism, Oxygen metabolism
- Abstract
Background: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H
2 ) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2 -consumption on H2 photoproduction yields in the heterocyte-forming, N2 -fixing cyanobacterium Nostoc PCC7120., Results: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses., Conclusion: We conclude that the overproduced Flv3B protein might have enhanced O2 -consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.- Published
- 2020
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27. Targeting TOR signaling for enhanced lipid productivity in algae.
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Prioretti L, Carriere F, Field B, Avilan L, Montané MH, Menand B, and Gontero B
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- Algal Proteins antagonists & inhibitors, Algal Proteins metabolism, Biofuels supply & distribution, Gene Expression Regulation, Homeostasis drug effects, Homeostasis genetics, Lipid Metabolism genetics, Microalgae enzymology, Microalgae genetics, Microalgae growth & development, Morpholines pharmacology, Signal Transduction, TOR Serine-Threonine Kinases antagonists & inhibitors, TOR Serine-Threonine Kinases metabolism, Algal Proteins genetics, Lipid Metabolism drug effects, Microalgae drug effects, Protein Kinase Inhibitors pharmacology, TOR Serine-Threonine Kinases genetics, Triglycerides biosynthesis
- Abstract
Microalgae can produce large quantities of triacylglycerols (TAGs) and other neutral lipids that are suitable for making biofuels and as feedstocks for green chemistry. However, TAGs accumulate under stress conditions that also stop growth, leading to a trade-off between biomass production and TAG yield. Recently, in the model marine diatom Phaeodactylum tricornutum it was shown that inhibition of the target of rapamycin (TOR) kinase boosts lipid productivity by promoting TAG production without stopping growth. We believe that basic knowledge in this emerging field is required to develop innovative strategies to improve neutral lipid accumulation in oleaginous microalgae. In this minireview, we discuss current research on the TOR signaling pathway with a focus on its control on lipid homeostasis. We first provide an overview of the well characterized roles of TOR in mammalian lipogenesis, adipogenesis and lipolysis. We then present evidence of a role for TOR in controlling TAG accumulation in microalgae, and draw parallels between the situation in animals, plants and microalgae to propose a model of TOR signaling for TAG accumulation in microalgae., (Copyright © 2019. Published by Elsevier B.V.)
- Published
- 2020
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28. RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms.
- Author
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Avilan L, Puppo C, Villain A, Bouveret E, Menand B, Field B, and Gontero B
- Subjects
- Acclimatization genetics, Algal Proteins metabolism, Amino Acid Sequence, Arabidopsis enzymology, Catalytic Domain, Chloroplasts metabolism, DNA, Algal genetics, Escherichia coli genetics, Evolution, Molecular, Gene Expression, Ligases metabolism, Photosynthesis, Phylogeny, Plant Proteins genetics, Plant Proteins metabolism, Algal Proteins genetics, Diatoms enzymology, Genetic Variation, Ligases genetics, Rhodophyta enzymology
- Abstract
The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.
- Published
- 2019
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29. Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives.
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Latifi A, Avilan L, and Brugna M
- Subjects
- Anaerobiosis, Fermentation, Industrial Waste, Clostridium enzymology, Clostridium metabolism, Hydrogen metabolism
- Abstract
Strictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects.
- Published
- 2019
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30. Hydrogenases and H 2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus.
- Author
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Baffert C, Kpebe A, Avilan L, and Brugna M
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- Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Biocatalysis, Desulfovibrio enzymology, Desulfovibrio genetics, Electrons, Gene Expression Regulation, Bacterial, Genetic Variation, Hydrogenase chemistry, Hydrogenase genetics, Hydrogenase metabolism, Models, Biological, Bacterial Proteins physiology, Desulfovibrio metabolism, Hydrogen metabolism, Hydrogenase physiology
- Abstract
Hydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H
2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H2 metabolism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
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31. Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.
- Author
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Avilan L, Roumezi B, Risoul V, Bernard CS, Kpebe A, Belhadjhassine M, Rousset M, Brugna M, and Latifi A
- Subjects
- Organisms, Genetically Modified genetics, Organisms, Genetically Modified metabolism, Clostridium acetobutylicum enzymology, Hydrogen metabolism, Hydrogenase genetics, Hydrogenase metabolism, Industrial Microbiology methods, Nostoc genetics
- Abstract
The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H
2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2 . As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2 . The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 μmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2 -sensitive hydrogenase.- Published
- 2018
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32. Fairy "tails": flexibility and function of intrinsically disordered extensions in the photosynthetic world.
- Author
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Thieulin-Pardo G, Avilan L, Kojadinovic M, and Gontero B
- Abstract
Intrinsically Disordered Proteins (IDPs), or protein fragments also called Intrinsically Disordered Regions (IDRs), display high flexibility as the result of their amino acid composition. They can adopt multiple roles. In globular proteins, IDRs are usually found as loops and linkers between secondary structure elements. However, not all disordered fragments are loops: some proteins bear an intrinsically disordered extension at their C- or N-terminus, and this flexibility can affect the protein as a whole. In this review, we focus on the disordered N- and C-terminal extensions of globular proteins from photosynthetic organisms. Using the examples of the A2B2-GAPDH and the α Rubisco activase isoform, we show that intrinsically disordered extensions can help regulate their "host" protein in response to changes in light, thereby participating in photosynthesis regulation. As IDPs are famous for their large number of protein partners, we used the examples of the NAC, bZIP, TCP, and GRAS transcription factor families to illustrate the fact that intrinsically disordered extremities can allow a protein to have an increased number of partners, which directly affects its regulation. Finally, for proteins from the cryptochrome light receptor family, we describe how a new role for the photolyase proteins may emerge by the addition of an intrinsically disordered extension, while still allowing the protein to absorb blue light. This review has highlighted the diverse repercussions of the disordered extension on the regulation and function of their host protein and outlined possible future research avenues.
- Published
- 2015
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33. Glyceraldehyde-3-phosphate dehydrogenase is regulated by ferredoxin-NADP reductase in the diatom Asterionella formosa.
- Author
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Mekhalfi M, Puppo C, Avilan L, Lebrun R, Mansuelle P, Maberly SC, and Gontero B
- Subjects
- Amino Acid Sequence, Darkness, Glyceraldehyde-3-Phosphate Dehydrogenases antagonists & inhibitors, Kinetics, Molecular Sequence Data, NADP metabolism, NADP pharmacology, Phylogeny, Surface Plasmon Resonance, Diatoms physiology, Ferredoxin-NADP Reductase metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism
- Abstract
Diatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions. The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition. GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts. Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)
- Published
- 2014
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34. CP12 residues involved in the formation and regulation of the glyceraldehyde-3-phosphate dehydrogenase-CP12-phosphoribulokinase complex in Chlamydomonas reinhardtii.
- Author
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Avilan L, Puppo C, Erales J, Woudstra M, Lebrun R, and Gontero B
- Subjects
- Chlamydomonas reinhardtii genetics, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases genetics, Phosphotransferases (Alcohol Group Acceptor) genetics, Plant Proteins genetics, Protein Binding, Chlamydomonas reinhardtii metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism, Plant Proteins metabolism
- Abstract
CP12, a member of the intrinsically disordered protein family, forms a stable complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). To understand the function of conserved residues of CP12 in the formation of the GAPDH-CP12-PRK complex and in the regulation of the enzymes within this complex, we have produced mutants of CP12 by site-directed mutagenesis. The GAPDH, CP12 and PRK recombinant proteins are able to reconstitute spontaneously the ternary complex that has been described in Chlamydomonas reinhardtii. Our analysis reveals that the central part ((35)WXXVEE(47)) of CP12 is required to form the GAPDH-CP12-PRK complex. Using the same series of single amino acid replacements, we have identified individual residues, which seem to represent also contact points for GAPDH. Most notably, substitution of glutamate 74 prevents the binding of GAPDH to CP12. This is similar to the mutant C66S, with which the GAPDH-CP12-PRK complex is not formed. In contrast, replacement of the three last residues ((78)YED(80)) of CP12 has no effect on the formation of the ternary supra-molecular complex. However, our findings strongly suggest that Y78 and D80 are involved in the regulation of the GAPDH activity within the supra-molecular complex, since the mutants, D80K and Y78S, do not down-regulate the activity of GAPDH. The replacement of the amino acid E79 weakens the interaction between GAPDH and CP12 as no GAPDH-CP12 sub-complex is formed. In this case, nevertheless, the supra-molecular complex is formed when PRK is present indicating that PRK strengthens the interaction between GAPDH and CP12 within the supra-molecular complex.
- Published
- 2012
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35. Consequences of the presence of 24-epibrassinolide, on cultures of a diatom, Asterionella formosa.
- Author
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Mekhalfi M, Avilan L, Lebrun R, Botebol H, and Gontero B
- Subjects
- Diatoms growth & development, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Photosynthesis drug effects, Protein Binding drug effects, Brassinosteroids pharmacology, Diatoms drug effects, Diatoms metabolism, Steroids, Heterocyclic pharmacology
- Abstract
Addition of the plant hormone 24-epibrassinolide to culture media stimulated the growth of a freshwater diatom, Asterionella formosa. The hormone stimulated activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme from Calvin cycle, by 6-fold. Other key metabolic enzymes, phosphofructokinase and malate dehydrogenase were also stimulated but to a lesser extent. The activity of glucose-6-phosphate dehydrogenase, involved in the oxidative pentose phosphate pathway, also increased in the presence of the hormone but only under non reducing conditions. In cells stimulated by epibrassinolide, activated enzymes were sensitive to oxidized-DTT. GAPDH purified from cells grown in the presence of the hormone was not associated with a small protein of 8.5 kDa shown to be similar to CP12. Consequently the activity of GAPDH was no longer regulated by either oxidizing or reducing conditions. Among enzymes that, like GAPDH, responded positively to reducing agent were fructose-1,6-bisphosphatase (FBPase) and glucose-6-phosphate dehydrogenase (G6PDH). These enzymes were also sensitive to, and were negatively regulated by, oxidized-DTT. The activities in extracts from illuminated cells differed from those from darkened cells: FBPase, G6PDH and GAPDH, that were activated by DTT in darkened cells were no more activated in illuminated cells, but were oxidized by oxidized-DTT. Thus, oxidizing or reducing conditions mimic the conditions in dark and light, respectively. Unlike the other enzymes, phosphofructokinase (PFK) was inhibited by DTT but oxidized-DTT reversed this effect. The enzymes shown to be redox regulated in vitro by reduction/oxidation are very likely candidates for regulation in vivo by thioredoxins.
- Published
- 2012
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36. Creating order out of disorder: structural imprint of GAPDH on CP12.
- Author
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Gontero B and Avilan L
- Abstract
The work presented by Matsumura et al. in this issue of Structure describes the structure of the ternary GAPDH-NAD-CP12 and the binary NAD-GAPDH complex in the cyanobacterium Synechococcus elongatus., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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37. Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein.
- Author
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Gómez-Arreaza A, Acosta H, Barros-Álvarez X, Concepción JL, Albericio F, and Avilan L
- Subjects
- Amino Acid Sequence, Animals, Antigens, Protozoan chemistry, Antigens, Protozoan genetics, Antigens, Protozoan immunology, Blotting, Western, Electrophoresis, Gel, Two-Dimensional, Fluorescent Antibody Technique, Humans, Immune Sera immunology, Leishmania mexicana genetics, Leishmania mexicana immunology, Molecular Structure, Mutagenesis, Site-Directed, Protozoan Proteins chemistry, Protozoan Proteins genetics, Protozoan Proteins immunology, Rabbits, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Antigens, Protozoan metabolism, Leishmania mexicana chemistry, Plasminogen metabolism, Protozoan Proteins metabolism
- Abstract
Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a K(d) value of 1.6±0.4 μM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif (260)VYDLESKAV(268), similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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38. Trypanosoma cruzi: A kinetoplast-associated protein of the photolyase/cryptochrome family.
- Author
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Carolaing Gabaldón M, Labrador L, Arraiz G, Concepción JL, and Avilan L
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Protozoan, Blotting, Western, Cloning, Molecular, Cryptochromes chemistry, Cryptochromes immunology, Deoxyribodipyrimidine Photo-Lyase chemistry, Deoxyribodipyrimidine Photo-Lyase immunology, Electrophoresis, Polyacrylamide Gel, Fluorescent Antibody Technique, Gene Expression Regulation, Enzymologic, Genome, Protozoan, Molecular Sequence Data, Protozoan Proteins chemistry, Protozoan Proteins immunology, Rabbits, Trypanosoma cruzi genetics, Cryptochromes genetics, DNA, Kinetoplast chemistry, Deoxyribodipyrimidine Photo-Lyase genetics, Protozoan Proteins genetics, Trypanosoma cruzi chemistry
- Abstract
A photolyase-like protein gene found in the Trypanosoma cruzi genome database was cloned and expressed in Escherichia coli resulting in the formation of inclusion bodies. Antibodies against this protein were used to determine expression of the protein in the different forms of the parasite. It was visualized in the epimastigote form but not in amastigote or trypomastigote forms obtained from culture in Vero cells. In epimastigotes, this protein is located at the level of the mitochondrion associated to both sides of the kinetoplast. Sequence analyses indicated that this protein, as well as other photolyases from Leishmania spp. and Trypanosoma brucei are related to single-stranded photolyases or cryptochromes DASH., (Copyright 2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
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39. Characteristics of plasminogen binding to Trypanosoma cruzi epimastigotes.
- Author
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Rojas M, Labrador I, Concepción JL, Aldana E, and Avilan L
- Subjects
- Animals, Binding Sites, Humans, Kinetics, Pancreatic Elastase metabolism, Peptide Fragments metabolism, Protein Binding, Rhodnius parasitology, Trypanosoma cruzi isolation & purification, Plasminogen metabolism, Trypanosoma cruzi physiology
- Abstract
The binding constants of the interaction between plasminogen and Trypanosoma cruzi epimastigotes were determined. An indirect method in which the bound plasminogen is detached from the cell by epsilon-aminocaproic acid and a direct method through biotinylated plasminogen were used. The analyses revealed a dissociation constant (Kd) from 0.4 to 1.2microM, these values being compatible with recognition in vivo. Moreover, epimastigotes from the gut of Rhodnius prolixus were able to bind plasminogen from the blood meal. Fragments derived from elastase digestion of plasminogen were tested for their ability to bind T. cruzi cells. The fragment with highest ability to interact with the parasite was miniplasminogen that bound in a concentration-dependent and saturable manner with a Kd similar to that for plasminogen. This binding was inhibited by epsilon-aminocaproic acid indicating that the lysine-binding site of kringle 5 may be responsible for the interaction of plasminogen with T. cruzi.
- Published
- 2008
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40. Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex--purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii.
- Author
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Erales J, Avilan L, Lebreton S, and Gontero B
- Subjects
- Algal Proteins chemistry, Animals, Fructose-Bisphosphate Aldolase chemistry, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, Immunoprecipitation, Kinetics, Phosphotransferases (Alcohol Group Acceptor) chemistry, Protein Binding, Algal Proteins metabolism, Chlamydomonas reinhardtii enzymology, Fructose-Bisphosphate Aldolase metabolism, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism
- Abstract
Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6-bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 +/- 0.05 mum and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3-phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55+/-5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis-Menten kinetics with fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, with a catalytic constant equal to 35 +/- 1 s(-1) and 4 +/- 0.1 s(-1), respectively. The K(m) value for fructose 1,6-bisphosphate was equal to 0.16 +/- 0.02 mm and 0.046 +/- 0.005 mm for sedoheptulose 1,7-bisphosphate. The catalytic efficiency of aldolase was thus 219 +/- 31 s(-1).mm(-1) with fructose 1,6-bisphosphate and 87 +/- 9 s(-1).mm(-1) with sedoheptulose 1,7-bisphosphate. In the presence of the complex, this parameter for fructose 1,6-bisphosphate increased to 310 +/- 23 s(-1).mm(-1), whereas no change was observed with sedoheptulose 1,7-bisphosphate. The condensation reaction of aldolase to form fructose 1,6-bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.
- Published
- 2008
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41. Leishmania mexicana: molecular cloning and characterization of enolase.
- Author
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Quiñones W, Peña P, Domingo-Sananes M, Cáceres A, Michels PA, Avilan L, and Concepción JL
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Protozoan immunology, Base Sequence, Blotting, Western, Cell Membrane Permeability drug effects, Cloning, Molecular, Digitonin pharmacology, Electrophoresis, Polyacrylamide Gel, Fluorescent Antibody Technique, Indicators and Reagents pharmacology, Kinetics, Leishmania mexicana genetics, Leishmania mexicana immunology, Mice, Molecular Sequence Data, Phosphopyruvate Hydratase biosynthesis, Phosphopyruvate Hydratase isolation & purification, Rabbits, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins genetics, Sequence Alignment, Leishmania mexicana enzymology, Phosphopyruvate Hydratase chemistry, Phosphopyruvate Hydratase genetics
- Abstract
The gene of Leishmania mexicana enolase was cloned and overexpressed in Escherichia coli as an active enzyme; the protein was biochemically analyzed. This enolase shares with enolases from other trypanosomatids the presence of three atypical residues, each with a reactive side group, near the active site, already described for the enzyme from Trypanosoma brucei. The natural enzyme was purified, using a three-step procedure, from a cytosolic fraction of L. mexicana promastigotes. The kinetic properties of the purified recombinant enzyme were similar to those of the natural enzyme. Both the recombinant and natural enzyme were inhibited by inorganic pyrophosphate. Subcellular localization analysis after differential centrifugation showed that the enzyme activity is only associated with the cytosolic fraction. However, an apparently inactive form of enolase was detected by Western blots in the microsomal fraction. Digitonin treatment of parasites and immunofluorescence studies with permeabilized and non-permeabilized parasites showed that enolase is also associated with membranes and it was found at the external face of the plasma membrane.
- Published
- 2007
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42. A study of cutaneous lesions caused by Leishmania mexicana in plasminogen-deficient mice.
- Author
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Maldonado J, Marina C, Puig J, Maizo Z, and Avilan L
- Subjects
- Animals, Female, Immunohistochemistry, Leishmania mexicana immunology, Leishmaniasis, Cutaneous immunology, Male, Mice, Sex Factors, Host-Parasite Interactions physiology, Leishmania mexicana pathogenicity, Leishmaniasis, Cutaneous pathology, Leishmaniasis, Cutaneous physiopathology, Plasminogen deficiency
- Abstract
The role of plasminogen, the zymogenic form of the serine protease plasmin, was investigated in the infection of Leishmania mexicana in plasminogen-deficient (plg(-/-)) and Plg wild-type (plg(+/+)) mice. Differences in the lesion size were observed between male plg(+/+) and plg(-/-) mice. However, these differences were not observed in female mice. In both genders, examination of the lesion tissues at 8 weeks post-infection showed differences in the immunoreactivity pattern with anti-Leishmania antibodies. The parasites were limited to isolated foci in the plg(-/-) mice lesion, in contrast to the scattered pattern observed in plg(+/+) mice. These results support the hypothesis that the interaction of the parasite with the host plasminogen-plasmin system might contribute to the virulence of L. mexicana.
- Published
- 2006
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43. Plasminogen interaction with Trypanosoma cruzi.
- Author
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Almeida L, Vanegas G, Calcagno M, Concepción JL, and Avilan L
- Subjects
- Animals, Enzyme Activation, Humans, Immunohistochemistry, Tissue Plasminogen Activator metabolism, Plasminogen metabolism, Trypanosoma cruzi metabolism
- Abstract
The ability of Trypanosoma cruzi to interact with plasminogen, the zimogenic form of the blood serin protease plasmin, was examined. Immunohistochemistry studies revealed that both forms, epimastigotes and metacyclic trypomastigotes, were able to fix plasminogen in a lysine dependant manner. This interaction was corroborated by plasminogen activation studies. Both forms of the parasite enhanced the plasminogen activation by tissue-type plasminogen activator. The maximal enhancements obtained were 15-fold and 3.4-fold with epimastigotes and metacyclic trypomastigotes, respectively, as compared to plasminogen activation in absence of cells. Ligand-blotting analysis of proteins extracted with Triton X-114 from a microsomal fraction of epimastigotes revealed at least five soluble proteins and one hydrophobic protein able to bind plasminogen.
- Published
- 2004
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44. Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi.
- Author
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Cáceres AJ, Portillo R, Acosta H, Rosales D, Quiñones W, Avilan L, Salazar L, Dubourdieu M, Michels PA, and Concepción JL
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA Primers, Escherichia coli enzymology, Escherichia coli genetics, Hexokinase chemistry, Humans, Isoenzymes chemistry, Isoenzymes genetics, Kinetics, Molecular Sequence Data, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Sequence Alignment, Sequence Homology, Amino Acid, Trypanosoma cruzi genetics, Hexokinase genetics, Hexokinase metabolism, Trypanosoma cruzi enzymology
- Abstract
The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli. Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human. It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins. The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV. The natural enzyme was purified to homogeneity from T. cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form. Its kinetic properties were compared with those of the purified recombinant protein. Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase. However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively). None of these enzymes presented activity with fructose. As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P). In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM)., (Copyright 2002 Elsevier Science B.V.)
- Published
- 2003
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45. Interaction of different Leishmania mexicana morphotypes with plasminogen.
- Author
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Calcagno M, Avilan L, Colasante C, Berrueta L, and Salmen S
- Subjects
- Animals, Cricetinae, Culture Media, Fibrinolysin metabolism, Fluorescent Antibody Technique, Hydrogen-Ion Concentration, Leishmania mexicana ultrastructure, Leishmaniasis, Cutaneous parasitology, Microscopy, Confocal, Temperature, Leishmania mexicana growth & development, Leishmania mexicana metabolism, Plasminogen metabolism
- Abstract
The interaction of plasminogen with Leishmania mexicana promastigotes was found, using immunoperoxidase assays, to occur with a specific morphotype. In in vitro cultured promastigotes, the morphotype that possessed the plasminogen binding capacity had round to ovoid cell bodies. In contrast, neither slender nor metacyclic promastigotes showed this property. In vivo plasminogen immunofluorescence assays showed deposits of plasminogen exclusively on the cell surface of promastigotes. This was observed as intense patches spread around the cell, with higher intensities towards the flagellar pocket. Plasminogen binding capacity detected by plasmin activity increased with the age of the promastigote culture, at pH 7.2 and pH 5.5, as well as after heat shock. The total amastigote population, freshly isolated from a hamster lesion, also bound plasminogen in a lysine-dependent manner as detected by immunoperoxidase studies.
- Published
- 2002
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46. Information transfer in multienzyme complexes--2. The role of Arg64 of Chlamydomonas reinhardtii phosphoribulokinase in the information transfer between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.
- Author
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Avilan L, Gontero B, Lebreton S, and Ricard J
- Subjects
- Animals, Arginine, Chloroplasts enzymology, Escherichia coli genetics, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Kinetics, Mutation, Phosphotransferases (Alcohol Group Acceptor) metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Chlamydomonas reinhardtii enzymology, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, Phosphotransferases (Alcohol Group Acceptor) chemistry
- Abstract
A mutant phosphoribulokinase has been isolated from the 12-2B mutant of Chlamydomonas reinhardtii. In this mutant, Arg64 has been replaced by Cys. The enzyme, which may exist in the dimeric and tetrameric states, is almost devoid of activity. Neither of these enzymes is able to form a complex with glyceraldehyde-3-phosphate dehydrogenase. The phosphoribulokinase gene has been expressed in Escherichia coli. The resulting recombinant protein, after isolation and purification, is apparently identical to the native enzyme extracted from the chloroplast. Three mutants have been generated by site directed mutagenesis. Arg64 has been replaced by Ala, Lys or Glu. With the exception of the latter, the two other mutants, [A64]phosphoribulokinase and [K64]phosphoribulokinase, are active when they are reduced, and nearly totally inactive in their oxidized state. Their activity, however, is decreased relative to that of the native, or to that of the wild-type recombinant phosphoribulokinase. Both the catalytic constant and the apparent affinity of ribulose 5-phosphate are decreased relative to the corresponding values obtained for the wild-type, the native or the recombinant enzyme. Whereas the [A64]phosphoribulokinase is unable to form a complex with glyceraldehyde-3-phosphate dehydrogenase, [K64]phosphoribulokinase does, but the stability of the resulting complex is much decreased relative to that of the wild-type complex. The oxidized mutant [K64]phosphoribulokinase becomes active in the presence of glyceraldehyde-3-phosphate dehydrogenase but this activity is smaller than that of the corresponding wild-type enzyme. Taken together, these results show that Arg64 plays a major role in the association of the two enzymes and in the information transfer that takes place between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. As this residue also appears to be important for catalytic activity, it may be tempting to consider that it stabilizes a conformation that is required for both the catalytic activity and the formation of the bienzyme complex.
- Published
- 1997
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47. Information transfer in multienzyme complexes--1. Thermodynamics of conformational constraints and memory effects in the bienzyme glyceraldehyde-3-phosphate-dehydrogenase-phosphoribulokinase complex of Chlamydomonas reinhardtii chloroplasts.
- Author
-
Lebreton S, Gontero B, Avilan L, and Ricard J
- Subjects
- Animals, Chloroplasts enzymology, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Kinetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Protein Conformation, Chlamydomonas reinhardtii enzymology, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, Phosphotransferases (Alcohol Group Acceptor) chemistry, Thermodynamics
- Abstract
Oxidized phosphoribulokinase is almost inactive in its isolated state but becomes active when associated with glyceraldehyde-3-phosphate dehydrogenase. There is therefore an information transfer that takes place between these two enzymes. However, when the complex dissociates, free oxidized phosphoribulokinase is even more active than when it is associated with glyceraldehyde-3-phosphate dehydrogenase. This means that glyceraldehyde-3-phosphate dehydrogenase exerts an imprinting effect upon phosphoribulokinase which persists for a while after the parting of the two proteins. Various methods derived from statistical thermodynamics can be used to estimate the fraction of energy transferred from glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase and which alters the kinetic parameters of the latter enzyme. In the complex, the decrease of the free energy associated with the binding of ribulose 5-phosphate is larger than that of ATP. This implies that the mutual association of the two enzymes facilitates the binding of the former substrate but is without effect on that of the latter. The main effect exerted by the association of the two enzymes is to decrease by about 10 kJ/mol the height of the energy barrier of the catalytic process. Phosphoribulokinase keeps an imprinting effect exerted by glyceraldehyde-3-phosphate dehydrogenase after the parting of the two enzymes. Part of the energy transferred from one protein to the other is used to decrease slightly the apparent binding free energy of the two substrates of phosphoribulokinase by about 1.5 kJ/mol. Whereas the previous association of the two enzymes does not significantly alter substrate binding to phosphoribulokinase, it greatly affects catalysis and decreases by about 16 kJ/mol the height of the energy barrier pertaining to this step. Therefore, within multienzyme complexes, information and energy can be transferred between proteins. Statistical thermodynamics offers the possibility of estimating how this energy is used to alter the various kinetic parameters of the reaction.
- Published
- 1997
- Full Text
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48. Memory and imprinting effects in multienzyme complexes--I. Isolation, dissociation, and reassociation of a phosphoribulokinase-glyceraldehyde-3-phosphate dehydrogenase complex from Chlamydomonas reinhardtii chloroplasts.
- Author
-
Avilan L, Gontero B, Lebreton S, and Ricard J
- Subjects
- Animals, Blotting, Western, Chloroplasts enzymology, Chromatography, DEAE-Cellulose, Chromatography, Gel, Dithiothreitol pharmacology, Durapatite, Glyceraldehyde-3-Phosphate Dehydrogenases isolation & purification, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Molecular Weight, Multienzyme Complexes isolation & purification, Multienzyme Complexes metabolism, NAD metabolism, NADP metabolism, Phosphotransferases (Alcohol Group Acceptor) isolation & purification, Phosphotransferases (Alcohol Group Acceptor) metabolism, Protein Conformation, Thioredoxins pharmacology, Chlamydomonas reinhardtii enzymology, Glyceraldehyde-3-Phosphate Dehydrogenases chemistry, Multienzyme Complexes chemistry, Phosphotransferases (Alcohol Group Acceptor) chemistry
- Abstract
A bienzyme complex made up of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight glyceraldehyde-3-phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and glyceraldehyde-3-phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two glyceraldehyde-3-phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two nonconsecutive reactions. The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase. The dissociation of the complex was reversible. If oxidized phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.
- Published
- 1997
- Full Text
- View/download PDF
49. Memory and imprinting effects in multienzyme complexes--II. Kinetics of the bienzyme complex from Chlamydomonas reinhardtii and hysteretic activation of chloroplast oxidized phosphoribulokinase.
- Author
-
Lebreton S, Gontero B, Avilan L, and Ricard J
- Subjects
- Adenosine Triphosphate metabolism, Animals, Chloroplasts enzymology, Enzyme Activation, Enzyme Stability, Fluorescence, Glutathione pharmacology, Kinetics, Models, Chemical, Oxidation-Reduction, Phosphotransferases (Alcohol Group Acceptor) chemistry, Protein Conformation, Ribulosephosphates metabolism, Tryptophan, Chlamydomonas reinhardtii enzymology, Glyceraldehyde-3-Phosphate Dehydrogenases metabolism, Multienzyme Complexes metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism
- Abstract
Oxidized, free, stable phosphoribulokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s(-1)/site). However, when it was bound to glyceraldehyde-3-phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s(-1)/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s(-1)/site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and k(cat)) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5-phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5-phosphate tended to stabilize this complex. Inactive, stable, oxidized phosphoribulokinase may form a complex with glyceraldehyde-3-phosphate dehydrogenase regaining its catalytic activity. In this case, glyceraldehyde-3-phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of glyceraldehyde-3-phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity. The potential biological significance of these effects remains to be discovered.
- Published
- 1997
- Full Text
- View/download PDF
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