The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.

The goal of this research is to investigate the role of the slr1098 gene in the cyanobacterium Synechocystis sp. PCC 6803, a gene with similarity to cpcE which encodes a subunit of an enzyme involved in bilin attachment to phycocyanin. This protein is hypothesized to be involved in oligomerization of phycocyanin due to previous results showing the mutant made shorter phycocyanin rods. The recombinant Slr1098 protein was produced and purified from E. coli cells. Binding assays showed interaction between Slr1098 and both apo- and holo-phycocyanin, but not to apo-allophycocyanin. Slr1098 blocked bilin addition at Cys-82 on CpcB by the CpcS/CpcU bilin lyase. Size exclusion chromatography and sucrose density gradient analysis of complexes formed suggest that Slr1098 strongly interacts with all intermediate forms of phycocyanin and may be an important checkpoint in the biosynthesis and oligomerization of this protein, but that by itself, Slr1098 does not increase oligomerization of phycocyanin.

Cyanobacteria are photosynthetic prokaryotes that able to produce oxygen. They have light harvesting complexes called phycobilisomes (PBS). PBS are generally composed of an allophycocyanin core with phycocyanin and phycoerythrin rods connected to the core. PBS are able to efficiently harvest light energy from different wavelengths of visible light due to the evolution of PBP. Phycoerythrin has five chromophores that are attached to six cysteine residues and is essential for efficient green light capture and transfer of energy for use in photosynthesis. The attachment of these chromophores to PBP is facilitated by enzymes known as bilin lyases. In this study, we characterize and explore the role of enzymes that are involved in the biosynthesis of phycoerythrin in cyanobacteria. Biochemical and molecular techniques were used in the characterization of these proteins to gain a better understanding of their roles in the post-translational modification of phycobiliprotein. In F. diplosipohon, the lyase activity of CpeT was characterized and studied using a heterologous, co-expression system in E. coli. It was determined that CpeT was able to ligate PEB to Cys-165 of CpeB in the presence of CpeZ, a chaperone-like protein. Next, the roles of three proteins, MpeY from RS9916 and MpeQ and MpeW from A15-62, were analyzed using a combination of gene-interruption mutants and recombinant protein expression techniques. The absence of mpeY resulted in the reduction of PEB chromophorylation of MpeA in green light conditions, and recombinant protein coexpression confirmed that MpeY was responsible for PEB attachment to Cys-83 of MpeA. The interruption of mpeQ in A15-62 resulted in a reduced PUB phenotype in MpeA in blue light. Recombinant protein expressions revealed that MpeQ was a lyase-isomerase responsible for the attachment of PUB to Cys-83 of MpeA. Two regulatory proteins located in two conserved configurations of a genomic island present in species that are able to change their phycobilin content in response to different light environments, known as Type-IV chromatic acclimation (CA4), were investigated. FciA and FciB from RS9916 were studied using gene interruption mutants from RS9916 and they were found to be responsible for the CA4 response in CA4-A containing species of Synechococcus.

Cyanobacteria are photosynthetic organisms that efficiently capture light by utilizing the light-harvesting complexes called phycobilisomes. In many cyanobacteria, phycobilisomes are composed of an allophycocyanin core with phycocyanin and phycoerythrin (PE) rods radiating from the core. These phycobiliproteins have multiple bilin chromophores, such as phycoerythrobilin (PEB), covalently attached to specific cysteine (Cys) residues for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions. This study mainly focuses on characterizing the roles of enzymes that are involved in the biosynthetic pathway of the phycobiliproteins within two cyanobacteria Thermosynechococcus elongatus and Fremyella diplosiphon. A combination of molecular and biochemical techniques were used to better understand the roles of these proteins in the post-translational modification and/or stability of phycobiliproteins. Using a heterologous plasmid coexpression system in E. coli, recombinant CpcS-III from T. elongatus was shown to ligate three different bilins to both subunits of allophycocyanin and to the beta subunit of phycocyanin, thus, acting as a bilin lyase. The crystal structure of CpcS-III was also solved, the first bilin lyase structure. Next, the roles of three proteins from F. diplosiphon CpeY, CpeZ, and CpeF were analyzed using a combination of gene knock-out mutants and recombinant protein expression techniques. In the absence of cpeY, chromophorylation to the alpha subunit of PE at Cys-82 was reduced, coinciding with the recombinant data that CpeY is the lyase that attaches PEB to this site. Removing cpeZ from the genome resulted in the destabilization and reduced accumulation of PE, especially the beta subunit CpeB. Recombinant CpeZ was shown to act like a chaperone-like protein and increased the solubility and fluorescence of both recombinant and native CpeB by increasing the stability of the phycobiliprotein and/or by increasing the activities of other lyases. The deletion of cpeF resulted in a reduced-PE phenotype with the doubly attached PEB missing from CpeB at Cys-48/Cys-59. Recombinant CpeF was shown to ligate PEB to CpeB-Cys-48/Cys-59 in the presence of recombinant CpeS (lyase attaches PEB to CpeB-Cys-80) and CpeZ. CpeF also showed a chaperone-like function by stabilizing CpeB, but its main role appears to be as a bilin lyase.

Reported in this dissertation are the results of investigations performed at the New Orleans Center for Mass Spectrometry at the University of New Orleans. The projects that are detailed in the coming pages take on a variety of subjects, but a common thread is that each employs matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to solve a problem. Fundamental aspects of MALDI in-plume ionization are implicated in the introduction of a newly developed electron-transfer secondary ionization matrix. The remaining projects are related to the ever expanding field of proteomics. Mass spectrometry was used to investigate the site specificity of a newly developed bilin-lyase enzyme, a new approach was developed to distinguish between A-ring and D-ring attachment of bilins, and F. grandis protein expression pattern was investigated in several tissues. All obtained results were acquired using a MALDI TOF/TOF mass spectrometer. The sensitivity, mass accuracy, mass resolution and the ability to perform collision induced decomposition (CID) experiments were all valuable features that served to raise the quality of data, and thereby improved the detail of inferences to be drawn for the different projects.