Phycoerythrin (PE) present on the outer phycobilisome (PBS) rods in Fremyella diplosiphon contains covalently attached phycoerythrobilin (PEB) chromophores for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions. The cpeY and cpeZ genes in F. diplosiphon were shown to attach PEB on alph-82 of PE. To better understand the individual functions of cpeY and cpeZ in native cyanobacteria, we characterized PBS and PE purified from cpeY and cpeZ deletion mutants and compared them with wild type (WT). Both cpeY and cpeZ mutants generated much less PE than WT as well as assembling much less PE into the PBS. PE purified from cpeY mutant had phycocyanobilin on alpha-PE in place of PEB. The mutation of cpeZ affected the biosynthesis and accumulation of beta-PE with a red-shifted absorbance compared to WT PE. CpeY was shown to function as a bilin lyase, and CpeZ possibly functions as a chaperone.

Synechococcus, a genus of photosynthetic cyanobacteria, is the second most abundant oxygenic microorganism in the marine environment that contributes significantly to the ocean’s primary productivity (Humily et al. 2013; Shukla et al. 2012). They are capable of utilizing available light of different wavelengths in the visible spectrum to perform photosynthesis and fix carbon dioxide and thus inhabit a wide range of light niches in the ocean along horizontal (coast vs offshore) and vertical gradients (depth) (Humily et al. 2013). A gene encoding a putative lyase isomerase, mpeQ, is present in phycoerythrin-II encoding operon that is expressed constitutively and a gene encoding putative lyase, mpeW, is present in CA-4 genomic island whose expression is regulated by ambient light color were identified and characterized in Synechococcus sp. A15- 62, a strain having a blue light specialist phenotype in its basal state. The amino acid sequence of the proteins encoded by mpeW and mpeQ are similar to other characterized lyases and these genes are conserved in cyanobacteria strains containing the CA4-B genomic island, which controls CA4 (Humily et al. 2013). The MpeW and MpeQ proteins were produced in E. coli and co-expressed with recombinant HT-MpeA and phycoerythrobilin (PEB) synthesis machinery. Site directed mutants of the HT-MpeA protein (Cys75Ala, Cys83Ala, Cys140Ala) were used to investigate the site for bilin attachment. The recombinant protein co-expression experiments of MpeQ and MpeW demonstrated that MpeQ attaches phycoerythrobilin (PEB) to cysteine-83 site on a-phycoerythrin II and isomerizes it to phycourobilin (PUB) and MpeW attaches phycoerythrobilin (PEB) to the same site.

A multi-plasmid, co-expression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. PCC 7002 in E. coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB), -phycocyanin, and -phycocyanin. This system was used to demonstrate that CpcS-I and CpcU proteins are both required attaching PCB to allophycocyanin subunits ApcD (AP-B) and ApcF (18). The N-terminal, AP-like domain of ApcE (LCM99) was produced in soluble form and shown to have intrinsic bilin lyase activity. In addition, this system was used to chromophorylated CpcA from Synechococystis sp. PCC 6803 with a non-cognate bilin; PEB with the aid of CpcEF type bilin lyase. However, the CpcSU type lyase displays much higher specificity for PCB (the native bilin in these species) than PEB. Next, using a heterologous, co-expression system in E. coli, the PEB ligation activity of putative lyase subunits CpeY, CpeZ, and CpeS was tested on the CpeA and CpeB subunits from F. diplosiphon. CpeY/CpeZ was found to ligate PEB on CpeA, although CpeY alone had only 60% chromophorylation activity compared to CpeYZ together. Studies with site-directed variants of CpeA (C82S and C139S), revealed that CpeY/CpeZ attached PEB at Cys-82 on HT-CpeA. The CpeS bilin lyase ligated PEB at both Cys-82 and Cys-139 of CpeA, but the yield of attached PEB at Cys 82 was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys-82 of CpeB. Purified PE from cpeY deletion mutants in F. diplosiphon was found to have PCB added on α-PE instead of PEB, which was likely performed by CpcEF in vivo. However, a cpeZ knock-out mutant is affected in chromophorylation of both  and  subunits of PE with a red-shifted absorbance compared to wild type PE probably due to missing PEB on PE subunits. Next a new type of bilin lyase isomerase for PEII ( subunit) named MpeZ from Synechococcus sp. RS 9916, was analyzed using the E. coli heterologous coexpression system. MpeZ acted as bilin lyase/isomerase chromophorylating α-PEII (MpeA) with PUB on Cys 83.

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.