Characterizing the function of lyases involved in the biosynthesis of Phycoerythrin I and II from marine Synechococcus cyanobacteria
Date of Award
Marine Synechococcus are the second most abundant oxygenic phototrophs in the open ocean. They are efficient at harvesting available light using complex antenna, or phycobilisome, composed of allophycocyanin proteins and extended rod structures of phycocyanin and one or two types of phycoerythrin (PEI and PEII). This dissertation focuses on the enzymes called bilin lyases required for the biogenesis of PEI and PEII in marine strains of Synechococcus which undergo type 4 chromatic acclimation (CA4). I used interruption mutant analysis, high performance liquid chromatography, a protein expression system in E. coli, and liquid chromatography tandem mass spectrometry to characterize the role of these bilin lyases.
A candidate lyase, CpeY, in the strain Synechococcus sp. RS9916 was characterized. Using molecular techniques and a cpeY- interruption mutant, I show that CpeY adds phycoerythrobilin to C82 of CpeA in the presence or absence of the chaperone-like protein CpeZ in a recombinant protein expression system. These studies demonstrate that CpeY attaches phycoerythrobilin to as much as 72% of CpeA, making it one of the most efficient phycoerythrin lyases characterized to date.
CpeU is similar to the CpcS/U family of lyases hypothesized to be a putative lyase on a phycoerythrin subunit. Analysis of purified phycobilisomes by HPLC and mass spectrometry shows that the cpeU- mutant was defective in bilin addition to CpeA and MpeA in green light and to CpeA in blue light, suggesting it plays a role in bilin changes during CA4. In the recombinant expression system, CpeU interacts with PEB, potentially sequestering it away from CpeY, thus decreasing the PEB attachment capabilities of CpeY.
I compared the activity of two homologs of the candidate lyase-isomerase MpeV from two closely related strains of Synechococcus. I show that Synechococcus sp. RS9916 MpeV is the lyase-isomerase responsible for doubly-ligating phycourobilin at C50, 61 positions on the CpeB and MpeB proteins from RS9916. However, the strain Synechococcus sp. WH8020 MpeV demonstrated lyase-isomerase activity on RS9916 CpeB and both WH8020 and RS9916 MpeB, yet it ligated phycoerythrobilin to WH8020 CpeB, suggesting the substrate protein CpeB may have an influence on the isomerization activity in addition to the bilin lyase.
Carrigee, Lyndsay A., "Characterizing the function of lyases involved in the biosynthesis of Phycoerythrin I and II from marine Synechococcus cyanobacteria" (2020). University of New Orleans Theses and Dissertations. 2734.
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