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Biochemistry. 2011 Jun 7;50(22):4890-902. doi: 10.1021/bi200307s. Epub 2011 May 16.

Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli.

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1
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Abstract

Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin α-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin α-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin α-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.

PMID:
21553904
DOI:
10.1021/bi200307s
[Indexed for MEDLINE]

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