Part:BBa_K1884006
Ferredoxin-NADP(+)Reductase(FNR)
Ferredoxin-NADP+ reductase (FNR) is the key enzyme catalyzing the reaction of electron transfer from ferredoxin (Fd) to NADP+, leading to production of NADPH mainly used for carbon dioxide (CO2) fixation in photosynthesis. Under anaerobic condition, FNR has been proposed to be one of the components competing for photosynthetic electrons from Fd with hydrogenases in Chlamydomonas [1]. Recent data from an in vitro experiment indicated that, under anaerobic condition supporting H2 production, there is a significant loss of photosynthetic electrons toward NADPH formation [2]. Using a proteomic approach, we have previously identified FNR as one of the differentially expressed proteins in Chlamydomonas that undergo sulfur-deprived H2 photoproduction process [3].
Ferredoxin: NADP+ reductase is the last enzyme in the transfer of electrons during photosynthesis from photosystem I to NADPH[4]. The NADPH is then used as a reducing equivalent in the reactions of the Calvin cycle.[4] Electron cycling from ferredoxin to NADPH only occurs in the light in part because FNR activity is inhibited in the dark[5]. In nonphotosynthetic organisms, the FNR primarily works in reverse to provide reduced ferredoxin for various metabolic pathways. These pathways include nitrogen fixation, terpenoid biosynthesis, steroid metabolism, oxidative stress response, and iron–sulfur protein biogenesis[6].
FNR is a soluble protein that is found both free in the chloroplast stroma and bound to the thylakoid membrane. This binding occurs opposite to the active site of the enzyme and does not seem to affect the structure of the active site or have a significant impact on the enzyme’s activity. When bound to the thylakoid membrane, the enzyme exists as a dimer, but when it is free in the stroma, it is monomeric The binding of the FNR to the integral membrane proteins on the thylakoid membrane is enhanced under acidic conditions, so recruitment and binding of FNR to the thylakoid membrane may be a method of storing and stabilizing the enzyme in the dark when photosynthesis is not occurring[8]. The chloroplast stroma varies from being slightly acidic in the dark to more alkaline in the light[7]. Therefore, in the dark, more FNRs would be recruited and bound to the thylakoid membrane, and in the light, more FNRs would dissociate from the membrane and be free in the stroma.
Biology
Plant-type ferredoxin: NADP+ reductase has two structural domains. The first domain is an antiparallel beta barrel at the amino terminus of the protein that contains the binding domain for the FAD cofactor. The second domain is at the carboxyl terminus of the protein and contains an alpha helix-beta strand fold[6] This terminal domain is where the NADP+ binds[7]. The active site for the enzyme occurs at the interface between the two domains[8].
Binding of the enzyme to the thylakoid membrane involves a polyproline type II helix created between two FNR monomers and several proline rich integral membrane proteins[9](Fig 1)
Usage
FNR is 141bp in length. Fig 2 shows the DNA sequence of FNR is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of FNR PCR product is rather high compared with DNA Marker, which indicates that the PCR product of FNR is in a high concerntration.
For confirming the gene of our fusion protien gene has been transformate into green algae, we extracted genome DNA of green algae and designed a pair of primers in order to amplify FNR respectively. Fig 3 shows the DNA sequence of FNR is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of FNR PCR product is rather high compared with DNA Marker, which indicates that the PCR product of FNR is in a high concentration.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 20
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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