Event Title
Characterization of the Activities of CpeU Putative Phycoerythrin Lyase in Fremyella diplosiphon
Faculty Sponsor
Wendy Schluchter
Submission Type
Poster
Description
When grown in green light, the cyanobacterium Fremyella diplosiphon produces red-colored phycoerythrin (PE) in the outer rods of the light-harvesting complex, known as the phycobilisome (PBS). PE is composed of two subunits, CpeA and CpeB, which carry phycoerythrobilin (PEB) chromophores that are attached by enzymes called bilin lyases to specific cysteine residues. CpeU is a putative bilin lyase based on sequence similarity to the known lyase CpcS/CpcU from Synechococcus sp. PCC 7002, which is responsible for attaching phycocyanobilin (PCB) to β-subunit of phycocyanin (PC) and allophycocyanin (AP) α and β subunits at Cys-81 [1, 2]. Phycobilisomes (PBS) were isolated and purified from wild-type (WT) and ΔcpeU deletion mutant cells from F. diplosiphon that were grown under green light, optimal growth conditions for PE production. The PBS complex contains PE, PC and AP. The proteins in the PBS were separated based on their mass using a sucrose gradient. PE was also purified using size exclusion and ion-exchange chromatography. Purified PBS and PE fractions were analyzed using absorbance and fluorescence spectroscopy, SDS-PAGE and Western-blotting. The results thus far suggest that ΔcpeU mutant has a similar phenotype to WT including similar peak profiles with slight decreases in PE levels in the mutant. The samples will be further analyzed by mass spectrometry to study the function of CpeU in F. diplosiphon. 1. Saunée, N.A., et al., JBC, 2008. 283: p. 7513-7522. 2. Shen, G., W.M. Schluchter, and D.A. Bryant, JBC, 2008. 28: p. 7503-7512.
Characterization of the Activities of CpeU Putative Phycoerythrin Lyase in Fremyella diplosiphon
When grown in green light, the cyanobacterium Fremyella diplosiphon produces red-colored phycoerythrin (PE) in the outer rods of the light-harvesting complex, known as the phycobilisome (PBS). PE is composed of two subunits, CpeA and CpeB, which carry phycoerythrobilin (PEB) chromophores that are attached by enzymes called bilin lyases to specific cysteine residues. CpeU is a putative bilin lyase based on sequence similarity to the known lyase CpcS/CpcU from Synechococcus sp. PCC 7002, which is responsible for attaching phycocyanobilin (PCB) to β-subunit of phycocyanin (PC) and allophycocyanin (AP) α and β subunits at Cys-81 [1, 2]. Phycobilisomes (PBS) were isolated and purified from wild-type (WT) and ΔcpeU deletion mutant cells from F. diplosiphon that were grown under green light, optimal growth conditions for PE production. The PBS complex contains PE, PC and AP. The proteins in the PBS were separated based on their mass using a sucrose gradient. PE was also purified using size exclusion and ion-exchange chromatography. Purified PBS and PE fractions were analyzed using absorbance and fluorescence spectroscopy, SDS-PAGE and Western-blotting. The results thus far suggest that ΔcpeU mutant has a similar phenotype to WT including similar peak profiles with slight decreases in PE levels in the mutant. The samples will be further analyzed by mass spectrometry to study the function of CpeU in F. diplosiphon. 1. Saunée, N.A., et al., JBC, 2008. 283: p. 7513-7522. 2. Shen, G., W.M. Schluchter, and D.A. Bryant, JBC, 2008. 28: p. 7503-7512.
Comments
3rd place, Poster