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

Abstract

BackgroudFerredoxin 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.MethodsThe 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.ResultsThe kinetic analysis shows KM NADPH of 12.5 M and a k cat 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s40659-017-0144-5) contains supplementary material, which is available to authorized users.

Iron Speciation and Iron Binding Proteins in Arthrospira platensis Grown in Media Containing Different Iron Concentrations

“…One other protein related to photosynthesis is ferredoxin NADP(H) oxidoreductase (EC 1.18.1.2) (FNR), identified in band 8 [ 28 ]. This enzyme receives electrons from ferredoxin and reduces NADP+ to NADPH [ 29 ].…”

What are the criteria for morphological cell death inDunaliella salina?

“…When serious damage is done to the reaction centers of photosynthetic organisms, the electron transfer systems and carbohydrate production will be stopped which will result in cell death. The electron transport chains operating in the light reaction phase of photosynthetic cells, are composed of oxidoreductase enzymes (20)(21)(22)(23)(24)(25) that also cause the reduction of tetrazolium salt which is a staining method used to study the survival, reproduction and the metabolic activity in mammalian cells. MTT assay is used to study mitochondrial activity (26)(27)(28).…”

Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli

“…The function of E. coli Fdx has yet to be fully elucidated, although the small size of the protein, its stability, and the highly negative reduction potential (2300 mV or less) of its [2Fe-2S] center suggest a role as a cellular electron transfer protein (30,31). Several studies have evaluated the physiological electron donor and acceptor partners for Fdx and its homologous proteins, which have contributed to an established model of a biological redox system involved in the biogenesis of iron-sulfur clusters (16,(31)(32)(33). Interestingly, however, holo-Fdx is a [2Fe-2S] cluster-containing protein in addition to an iron-sulfur cluster assembly protein.…”