Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria

<p> <p>A multi-plasmid, co-expression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. PCC 7002 in <em><em>E. coli</em></em>. 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 <em><em>Synechococystis sp. </em></em>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. </p> </p> <p> <p>Next, using a heterologous, co-expression system in <em><em>E. coli</em></em>, the PEB ligation activity of putative lyase subunits CpeY, CpeZ, and CpeS was tested on the CpeA and CpeB subunits from <em><em>F. diplosiphon</em></em>. 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 <em><em>cpeY </em></em>deletion mutants in <em><em>F. diplosiphon </em></em>was found to have PCB added on α-PE instead of PEB, which was likely performed by CpcEF <em><em>in vivo</em></em>. However, a <em><em>cpeZ </em></em>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. </p> </p> <p> <p>Next a new type of bilin lyase isomerase for PEII ( subunit) named MpeZ from <em><em>Synechococcus sp. </em></em>RS 9916, was analyzed using the <em><em>E. coli </em></em>heterologous coexpression system. MpeZ acted as bilin lyase/isomerase chromophorylating α-PEII (MpeA) with PUB on Cys 83. </p> </p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation-Restricted

Discipline thesis:degree_discipline

Chemistry

Year

2011

Author dc:creator

• Biswas, Avijit

Contributors dc:contributor

• Schluchter, Wendy
• Liu, Zhengchang
• Stevens, Edwin D.

• Phycobiliprotein, Allophycocyanin, Phycocyanin, Phycoerythrin, Phycocyanobilin, Phycoerythrobilin, Phycourobilin
• Biochemistry, Biophysics, and Structural Biology

Repository record dc:identifier

https://scholarworks.uno.edu/td/446

OAI identifier oai:identifier

oai:scholarworks.uno.edu:td-1244