Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin

Abstract Backgroud 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 Ferredox...

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Autores principales:	Vorphal,María Alejandra, Bruna,Carola, Wandersleben,Traudy, Dagnino-Leone,Jorge, Lobos-González,Francisco, Uribe,Elena, Martínez-Oyanedel,José, Bunster,Marta
Lenguaje:	English
Publicado:	Sociedad de Biología de Chile 2017
Materias:	Ferredoxin NADP+ reductase Ferredoxin Sequence Kinetic parameters Structural features
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602017000100228
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spelling	oai:scielo:S0716-976020170001002282018-01-08Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxinVorphal,María AlejandraBruna,CarolaWandersleben,TraudyDagnino-Leone,JorgeLobos-González,FranciscoUribe,ElenaMartínez-Oyanedel,JoséBunster,Marta Ferredoxin NADP+ reductase Ferredoxin Sequence Kinetic parameters Structural features Abstract Backgroud 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.info:eu-repo/semantics/openAccessSociedad de Biología de ChileBiological Research v.50 20172017-01-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602017000100228en10.1186/s40659-017-0144-5
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Ferredoxin NADP+ reductase Ferredoxin Sequence Kinetic parameters Structural features
spellingShingle	Ferredoxin NADP+ reductase Ferredoxin Sequence Kinetic parameters Structural features Vorphal,María Alejandra Bruna,Carola Wandersleben,Traudy Dagnino-Leone,Jorge Lobos-González,Francisco Uribe,Elena Martínez-Oyanedel,José Bunster,Marta Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin
description	Abstract Backgroud 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.
author	Vorphal,María Alejandra Bruna,Carola Wandersleben,Traudy Dagnino-Leone,Jorge Lobos-González,Francisco Uribe,Elena Martínez-Oyanedel,José Bunster,Marta
author_facet	Vorphal,María Alejandra Bruna,Carola Wandersleben,Traudy Dagnino-Leone,Jorge Lobos-González,Francisco Uribe,Elena Martínez-Oyanedel,José Bunster,Marta
author_sort	Vorphal,María Alejandra
title	Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin
title_short	Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin
title_full	Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin
title_fullStr	Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin
title_full_unstemmed	Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin
title_sort	molecular and functional characterization of ferredoxin nadp(h) oxidoreductase from gracilaria chilensis and its complex with ferredoxin
publisher	Sociedad de Biología de Chile
publishDate	2017
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602017000100228
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Molecular and functional characterization of ferredoxin NADP(H) oxidoreductase from Gracilaria chilensis and its complex with ferredoxin

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