Event Title
Characterizing the Function of Bilin Lyase Isomerase MpeV in Marine Synechococcus sp. RS9916
Faculty Sponsor
Wendy Schluchter
College(s)
College of Sciences
Submission Type
Poster
Description
Cyanobacteria are prolific contributors to global primary production and are found ubiquitously around the world in both marine and freshwater environments. Synechococcus sp. RS9916 (hereafter, RS9916), a marine species of cyanobacteria, contain a membrane-bound protein complex with covalently bound bilin chromophores, called a phycobilisome (PBS), responsible for harvesting light energy and directing it into photosystem II. In RS9916, the PBS rods contain two types of phycoerythrin (PEI and PEII), the most distal phycobiliproteins (PBPs) of the complex. Enzymes known as bilin lyases catalyze attachment of bilin chromophores to these PBPs. MpeV is a putative lyase in RS9916 with homology to CpeF, a lyase from freshwater cyanobacteria Fremyella diplosiphon (Fd). MpeV is hypothesized to attach phycoerythrobilin (PEB) and simultaneously isomerize to phycourobilin (PUB) at the doubly ligated Cys-50, 61 residues on the beta subunit of PEI (CpeB). Using a heterologous expression system, recombinant MpeV was coexpressed with CpeB in E. coli, along with the genes required for PEB synthesis. Recombinant CpeB was then purified and analyzed by absorbance and fluorescence spectroscopy followed by SDS-PAGE. These samples were further analyzed by LC-MS-MS. MpeV was found to covalently attach PUB to Cys-50, 61 of CpeB. This activity increased when CpeB was partially chromophorylated by CpeS, a lyase that adds PEB to Cys-82. Additionally, there was an increase in MpeV activity when it was coexpressed with CpeZ, a homolog of a chaperone-like protein in Fd. A similar expression system was used to examine the activity of MpeV on the beta subunit of PEII (MpeB), with some PUB attachment detected at the Cys-50, 61 residues. Therefore MpeV is a lyase-isomerase for beta-phycoerythrin subunits and facilitates two thioether linkages to PUB at rings A and D to Cys-50 and Cys-61.
Characterizing the Function of Bilin Lyase Isomerase MpeV in Marine Synechococcus sp. RS9916
Cyanobacteria are prolific contributors to global primary production and are found ubiquitously around the world in both marine and freshwater environments. Synechococcus sp. RS9916 (hereafter, RS9916), a marine species of cyanobacteria, contain a membrane-bound protein complex with covalently bound bilin chromophores, called a phycobilisome (PBS), responsible for harvesting light energy and directing it into photosystem II. In RS9916, the PBS rods contain two types of phycoerythrin (PEI and PEII), the most distal phycobiliproteins (PBPs) of the complex. Enzymes known as bilin lyases catalyze attachment of bilin chromophores to these PBPs. MpeV is a putative lyase in RS9916 with homology to CpeF, a lyase from freshwater cyanobacteria Fremyella diplosiphon (Fd). MpeV is hypothesized to attach phycoerythrobilin (PEB) and simultaneously isomerize to phycourobilin (PUB) at the doubly ligated Cys-50, 61 residues on the beta subunit of PEI (CpeB). Using a heterologous expression system, recombinant MpeV was coexpressed with CpeB in E. coli, along with the genes required for PEB synthesis. Recombinant CpeB was then purified and analyzed by absorbance and fluorescence spectroscopy followed by SDS-PAGE. These samples were further analyzed by LC-MS-MS. MpeV was found to covalently attach PUB to Cys-50, 61 of CpeB. This activity increased when CpeB was partially chromophorylated by CpeS, a lyase that adds PEB to Cys-82. Additionally, there was an increase in MpeV activity when it was coexpressed with CpeZ, a homolog of a chaperone-like protein in Fd. A similar expression system was used to examine the activity of MpeV on the beta subunit of PEII (MpeB), with some PUB attachment detected at the Cys-50, 61 residues. Therefore MpeV is a lyase-isomerase for beta-phycoerythrin subunits and facilitates two thioether linkages to PUB at rings A and D to Cys-50 and Cys-61.
Comments
Winner, Tolmas Prize
1st place, Oral Presentation (Tie)