Investigation of the amino acids responsible for isomerase activity through mutagenesis of bilin lyase MpeW from Synechococcus sp. A15-62
Date of Award
Synechococcus sp. A15-62 is a marine cyanobacterium that undergoes Type IV chromatic acclimation (CA4) by altering the bilin content of the phycobilisome (PBS) or light harvesting complex to match the light color of its environment. The PBS is a protein complex comprised of rods containing phycobiliproteins (PBP) and linker proteins. In this species, phycoerythrin I and phycoerythrin II (PEI and PEII) are the phycobiliproteins located on the distal ends of the rods and the alpha () subunits of these proteins are the ones with altered bilin content during CA4. Bilin chromophores such as phycoerythrobilin (PEB) and phycourobilin (PUB) are bound to these PBPs at specific Cysteine (Cys) residues by phycobilin lyases. In marine Synechococcus species, lyases and lyase-isomerases within the MpeQWYZ sub-family of lyases are specific for attaching bilin to Cys-83 site of the α –subunit (MpeA) of PEII. The similarities between the enzymes in this family were investigated by aligning them with Geneious 6.1.8. The four positions 104, 331, 335 and 364 were identical or similar between isomerases. The residues at these positions differ between lyases and lyase-isomerases, but are conserved among lyase-isomerases which suggests that they might be involved in the isomerase function. To investigate amino acids responsible for isomerase function, MpeW, the lyase responsible for attaching PEB to Cys-83 of MpeA, was mutated by overlapping PCR to create: a quintuple mutant, triple mutant, and double mutants. Point mutations were introduced in which the residue present in MpeW was mutated to the corresponding residue in MpeQ, a lyase isomerase. After co-expressions with HT-MpeA using a heterologous co-expression system in E. coli, purified HT-MpeA was analyzed by zinc-enhanced fluorescence and fluorescence spectroscopy for bilin attachment. It was observed that MpeW (T110A,G319V,V320T,Q323Y,A352T), MpeW(G319V,V320T,Q323Y), and MpeW(G319V,V320T) attached both PEB and some PUB. These results indicate that positions 319 and 320 are important for isomerase function.
Weaver, Kourtney, "Investigation of the amino acids responsible for isomerase activity through mutagenesis of bilin lyase MpeW from Synechococcus sp. A15-62" (2019). Senior Honors Theses. 134.
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