Cyanobacteria are extremely important contributors to primary productivity in the world's oceans. In order to perform photosynthesis and fix carbon, cyanobacteria must absorb light. They achieve efficient light absorption utilizing light-harvesting complexes called phycobilisomes that contain brilliantly colored phycobiliproteins, which absorb light in the visible region of the light spectrum. The actual light-absorbing molecules are bilin chromophores, which are attached to each phycobiliprotein by enzymes called bilin lyases. Additionally, some marine cyanobacteria in the genus Synechococcus can harvest the blue light that dominates these environments due to the presence of a yellow chromophore called phycourobilin. The goal of this research is to determine how the yellow phycourobilin chromophore is synthesized and how all of the chromophores are attached to each phycobiliprotein in three species of Synechococcus (PCC 7002, WH8020, and WH8102). Several genes that encode potential enzymes in these biosynthetic processes have been identified in the genome of Synechococcus WH8020 and WH8102 by sequence similarity to other known bilin lyases. The role of each candidate gene in chromophore attachment will be evaluated by recreating the biosynthetic pathway for each phycobiliprotein inside Escherichia coli cells. In addition, candidate recombinant bilin lyase enzymes will be assayed for phycourobilin synthesis and attachment in vitro. In collaboration with a structural biologist, one of the bilin lyases will be purified and crystallized in order to determine its three-dimensional structure.
Broader impacts: In addition to providing fundamental knowledge about photosynthetic light harvesting, this research will result in training two to four undergraduates per year along with two graduate students. Students will be recruited through mechanisms that encourage participation of underrepresented groups in science. The PI will continue to co-author publications with her high-school, undergraduate, MS, and PhD students. The PI has integrated her research with the curriculum of a Biochemistry and Molecular Biology lab course, reaching 20 students each year.
In order to perform photosynthesis, cyanobacteria utilize light-harvesting complexes called phycobilisomes. The brilliantly colored phycobiliproteins are the major components of these phycobilisome complexes and absorb light in the visible region of the spectrum due to the bilin chromophores that are attached to each protein by enzymes called bilin lyases. Cyanobacteria are extremely important contributors to primary productivity in the oceans, and some marine Synechococcus can harvest the blue light that dominates these environments due to the presence of the yellow chromophore called phycourobilin attached to its phycobiliproteins. The long-term goal of this research was to determine how the yellow phycourobilin chromophore is synthesized and how all of the chromophores are attached to each phycobiliprotein in cyanobacteria. The biosynthetic pathway for two of these proteins (phycocyanin and allophycocyanin) was completely determined and the role of three new enzymes was characterized. A structure of one of these bilin lyase enzymes was solved and modeled with its substrate, and six other bilin lyase enzymes involved in the biosynthesis of the phycobiliprotein phycoerythrin were also characterized. MpeZ, a major enzyme responsible for the attachment of phycourobilin to phycoerythrin in marine Synechococcus. Ten publications resulted from this work. In addition, one patent application was filed and a preliminary patent application for the use of MpeZ to attach phycourobilin was also filed. Both of these patents involve the synthesis of these fluorescent phycobiliproteins as biomarkers for any protein of interest in cells. Therefore, this work may have far-reaching applications to biotechnology. Broader impacts: Five graduate students were trained under this project (four PhD and 2 MS students). Fourteen undergraduates were trained on this project (6 from groups under-represented in science), and four undergraduates are listed as co-authors on publications during the grant period. Five of these undergraduates are in graduate programs in science, and four others plan to apply to graduate programs in the near future. In addition, a high school student and a high school teacher (from a group underrepresented in science) were mentored.
