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 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.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.
Highlights
Ferredoxin NADP (H) oxidoreductases (EC 1.18.1.2) (FNR) are enzymes of 34–45 kDa, involved in crucialVorphal et al Biol Res (2017) 50:39NADPH dehydrogenase [5], Tic62 [6, 7] and Fd in the stroma of chloroplasts [8]
We report here the sequence of one gene found in the genome of G. ch, the sequence analysis of the translated amino acid sequence and the molecular and kinetic characterization of the enzyme
In order to complete the molecular characterization of ferredoxin NADP+ reductase from G. ch, we report the sequence of the ferredoxins found in the transcriptome of G. ch and molecular models for FNR, its Fd partner and the corresponding FNR/Fd complex
Summary
Ferredoxin NADP (H) oxidoreductases (EC 1.18.1.2) (FNR) are enzymes of 34–45 kDa, involved in crucialVorphal et al Biol Res (2017) 50:39NADPH dehydrogenase [5], Tic62 [6, 7] and Fd in the stroma of chloroplasts [8]. Ferredoxin NADP (H) oxidoreductases (EC 1.18.1.2) (FNR) are enzymes of 34–45 kDa, involved in crucial. A general characteristic of FNR enzymes, is an optimum pH of 7.0 for the reduction of cytochrome c dependent of ferredoxin at 40–55 °C; under these conditions, for NADPH, Km is in the μM order with a turnover number or kcat of 80–100 s−1 [11]. In cyanobacteria FNR has been found associated to phycobilisomes (PBS) [14], which is an accessory light harvesting protein complex present in thylakoid membranes. It has been reported that PBS-associated FNR from Synechococcus presents the extra domain [15,16,17,18]. No molecular and functional information is available for FNR from this specie, nor if the protein presents the previously described extra domain. Considering the importance of FNR for mass production of this commercially important algae for polysaccharides and pigments production, this research was focused on the characterization of the enzyme
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