ScholarWorks@UNO

The Role of the cpeF Deletion Mutant in the Biosynthesis of PE in Fremyella diplosiphon

Cyanobacteria have light-harvesting complexes known as phycobilisomes (PBS). PBS are composed of various phycobiliproteins (PBP) with a core and rod structures which transfer light energy to photosynthetic reaction centers. Fremyella diplosiphon contains the red-fluorescent protein phycoerythrin (PE) in the outer PBS rods. These fluorescent properties are attributed to the linear tetrapyrrole molecules known as bilins. Phycoerythrobilin (PEB) is attached to specific cysteine residues on the α (CpeA) and β (CpeB) subunits of PE through bilin lyase catalyzed reactions. In F. diplosiphon, CpeF belongs to the CpcEF family of bilin lyases according to its similarities in sequence and predicted 3D structure. Previous recombinant protein expression experiments in E. coli suggested that CpeF is a bilin lyase responsible for attaching PEB to CpeB on one of the three Cys sites (doublylinked PEB at Cys-48/59). To test the role of CpeF in native cyanobacteria, a cpeF deletion mutant (ΔcpeF) was generated in F. diplosiphon and compared to wild-type (WT). When compared to WT, the phenotype of the ΔcpeF mutant suggests low PE levels. PBS were purified from ΔcpeF mutant and WT cells using a sucrose gradient, and PE was also purified from cells using size-exclusion and ionexchange chromatography methods. Fractions from both the PE and PBS preps were analyzed using absorbance and fluorescence spectroscopy, SDS-PAGE, and Western blotting. The results indicate CpeF plays a major role is PE biosynthesis, possibly as a bilin lyase for CpeB. Samples are currently being processed by mass spectrometry to verify if CpeF attaches PEB to Cys-48/59 of CpeB, as suspected.