Date of Award

Summer 8-4-2011

Degree Type

Dissertation-Restricted

Degree Name

Ph.D.

Degree Program

Chemistry

Department

Chemistry

Major Professor

Schluchter, Wendy

Second Advisor

Liu, Zhengchang

Third Advisor

Stevens, Edwin D.

Fourth Advisor

Rick, Steven W.

Abstract

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.

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The University of New Orleans and its agents retain the non-exclusive license to archive and make accessible this dissertation or thesis in whole or part in all forms of media, now or hereafter known. The author retains all other ownership rights to the copyright of the thesis or dissertation.

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