ABSTRACT
BACKGROUND: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP+ to NADPH using the electrons provided by Ferredoxin (Fd), a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd. METHODS: The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5'RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking. RESULTS: The kinetic analysis shows KMNADPH of 12.5 M and a kcat of 86 s-1, data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS, sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS. CONCLUSION: The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.
Subject(s)
Gracilaria/enzymology , Ferredoxin-NADP Reductase/chemistry , Ferredoxins/metabolism , Oxidation-Reduction , Photosynthesis/physiology , Amino Acid Sequence , Gracilaria/chemistry , Electrophoresis, Polyacrylamide Gel , Ferredoxin-NADP Reductase/genetics , Ferredoxin-NADP Reductase/pharmacokineticsABSTRACT
En las últimas décadas se ha discutido ampliamente la necesidad de modernizar la docencia, por ejemplo generando espacios que promuevan que los estudiantes se involucren en el proceso enseñanza-aprendizaje. Metodologías consistentes con esta propuesta son las tecnologías de la información y de la comunicación, que presentan ventajas socioeconómicas y pedagógicas, destacando el estimular competencias genéricas. En este contexto, para motivar el autoaprendizaje y la responsabilidad social de difundir el conocimiento científico y avances tecnológicos a la comunidad, se utilizó el Wiki en la asignatura Bioquímica de carreras científicas. Los estudiantes redactaron artículos sobre temas de libre elección en la plataforma Arco, los cuales se evaluaron utilizando una rúbrica global de desempeño, mientras que para estimar la apreciación del estudiante se utilizó una lista de cotejo. Los estudiantes consideraron que la actividad los motivó a estudiar Bioquímica, que aprendieron sobre el tema seleccionado por su equipo y que comunicar a la sociedad sobre temas de salud y biotecnología es parte de su rol profesional. La estrategia fue exitosa tanto como metodología para el autoaprendizaje, como para fomentar la responsabilidad social del futuro científico.
Over the past decades, the need of modernizing teaching has been extensively discussed, for example by producing environments that promote the student engagement in the teaching and learning process. Methodologies that are consistent with this proposal are Information and Communication Technologies, which present socioeconomic and pedagogical advantages, especially the stimulation of generic competences. Within this context, for the purpose of motivating self-learning and the social responsibility of communicating scientific knowledge and technological advances to the community, we used Wiki in the biochemistry course of scientific undergraduate programs. Students wrote articles concerning freely chosen topics on the Arco platform, which were graded using a global perspective rubric, whereas, a checklist was used for student's assessment of the activity. The students felt motivated to study biochemistry, since they learnt about the topic selected by their team and that communicating health and biotechnological issues to the community was one of their professional roles. The strategy was successful as an approach to support self-learning, and promoting social responsibility of future scientists.
ABSTRACT
The structure of phycobiliproteins and their spatial organization in the phycobilisome provide the environment for high efficiency in light harvesting and conduction towards photosystem II. This article focuses on the analysis of R-phycoerythrin, a light harvesting hexameric phycobiliprotein that is part of the phycobilisomes. The interaction surfaces and the environment of the chromophores of R-phycoerythrin were studied in order to explain its structural stability and spectroscopic sensitivity, properties revealed by perturbation experiments. Three interaction surfaces are described (ab), (ab)3 and (ab)6. The analysis shows the importance of a subunits in the interaction between trimers, the homodimeric nature of the monomer (ab) and also the presence of anchor points in every interaction surface studied: a18Phe and b18Tyr for (ab), b76Asn for (ab)3 and a25Asn for (ab)6 . Side chains of arginine, lysine or glutamine residues are located in the proximity of the chromophores providing the correct stabilization of their carboxylates. Aspartic acids residues are associated through H-bonds to the N atom of the two central rings of the tetrapyrrolic chromophores. Changes in the spectroscopic properties are observed in perturbation experiments, confirming the spatial requirement for an efficient resonance energy transfer among chromophores and through the phycobilisome.