Molecular characterization of ferredoxin-NADP+ oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002 and studies on the gene product

Abstract

The petH gene encoding ferredoxin-NADP+ oxidoreductase (FNR) was cloned and sequenced from the cyanobacterium Synechococcus sp. PCC 7002. The deduced amino acid sequence of the FNR protein (402 amino acids) is approximately 110 amino acids longer at the N-terminus than FNR proteins which have been characterized from other cyanobacteria. N-Terminal amino acid sequence analysis of the protein confirms the assigned translational start codon and shows that the initiator methionine is not removed. Mapping of the petH transcript by primer extension demonstrates that transcription initiates 112-114 bp upstream from this translational initiation site. Analyses of the mature protein from whole-cell extracts by polyacrylamide gel electrophoresis and subsequent immunoblot analysis with anti-spinach FNR antibodies revealed two distinct forms of the mature protein; both had masses of approximately 45 kDa, corresponding to the predicted molecular mass deduced from the nucleotide sequence data. Analyses by Triton X-114 phase-partitioning indicate that one form of the protein is found exclusively in the cytosol and is hydrophilic when extracts are made at low ionic strength while the second form of the protein is hydrophobic and is tightly associated with the total membrane fraction. Hydroxylamine treatment converted a portion of the membrane-associated, hydrophobic form into a protein which then behaved like the hydrophilic form. These results suggest that a portion of the FNR pool may be acylated via an ester linkage to aid in attachment of the protein to the membranes. A computer database search revealed that the N-terminal extension of the FNR protein was 78% similar to the 9-kDa phycocyanin-associated linker protein CpcD, a structural component of the phycobilisomes. It is hypothesized that the N-terminal domain of FNR serves to localize the protein near the thylakoid membrane by docking FNR at the extremities of the peripheral rods of the phycobilisomes. Consistent with this notion, FNR is present in the phycobilisomes of Synechococcus sp. PCC 7002. Immunoblotting analyses of other cyanobacterial species showed that in all cases the major proteins recognized by the spinach FNR antibodies had masses of 42-55 kDa and were much larger than previously reported. Smaller cross-reactive species in the mass range 24-35 kDa appear to be proteolytic degradation products.

A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation

“…In CK and PAL cell extracts FNR L seemed to undergo proteolysis (Fig. 1CЈ) as has been observed (10). It should be noted that cell extracts from AB, PAL, and CK contain relatively high amounts of FNR S , a point that is addressed in Discussion.…”

“…9, which is published as supporting information on the PNAS web site), which indicates the action of similar proteases in both bacteria. The sizes of the FNR L proteolytic fragments also suggest that proteolysis occurs within the linker domain of the FNR and not only at a putative PEST site as has been proposed (6,10). Furthermore, heterologous expression of the mutant alleles MI6 and FS failed to produce FNR S and FNR L , respectively, indicating that also in E. coli translation initiates at both sites (Fig.…”

“…strains OS-A and OS-BЈ (9), encodes a Ϸ46-kDa FNR that contains an N-terminal domain, of Ϸ80 aa, whose sequence is similar to PBS rod-linker polypeptides (10). The PBS is a large and abundant bilin-protein complex that harvests light for photosynthesis.…”

“…Purified PBS from several cyanobacterial strains contain significant amounts of the 46-kDa FNR, one to two molecules per PBS (10,13,14). It was proposed that the FNR binds to the PBS rods via its linker domain to fulfill functions in both cyclic electron transport and respiration, in close proximity to the membrane (6,15).…”

“…strain PCC6803 (18). This result was attributed to proteolytic degradation of the N-terminal domain of the 46-kDa FNR (6,10).…”