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Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host.

ABSTRACT: The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp. PCC6803) was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter. Genes for the apoprotein (C-phycocyanin alpha subunit; cpcA) and the heterodimeric lyase (cpcE and cpcF) that catalyzes chromophore attachment were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used the cellular pool of heme to produce holo-CpcA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria. About a third of the apo-CpcA was converted to holo-CpcA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks cpcE and cpcF. This approach should permit incisive analysis of many remaining questions in phycobiliprotein biosynthesis. These studies also demonstrate the feasibility of generating constructs of these proteins in situ for use as fluorescent protein probes in living cells.

SUBMITTER: Tooley AJ

PROVIDER: S-EPMC58505 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host.

Tooley A J AJ Cai Y A YA Glazer A N AN

Proceedings of the National Academy of Sciences of the United States of America 20010901 19

The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp. PCC6803) was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter. Genes for the apoprotein (C-phycocy ...[more]

PMID: 11553806

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Project description:Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin. Insertional inactivation of the phycocyanin α-subunit lyase (ΔcpcF mutant) prevents the ligation of phycocyanobilin to α-phycocyanin (CpcA), while disruption of the cpcB/A/C2/C1 operon in the CK mutant prevents synthesis of both apo-α-phycocyanin (apo-CpcA) and apo-β-phycocyanin (apo-CpcB). Both mutants exhibited similar light saturation curves under white actinic light illumination conditions, indicating the phycobilisomes in the ΔcpcF mutant are not fully functional in excitation energy transfer. Under red actinic light illumination, wild type and both phycocyanin mutant strains exhibited similar light saturation characteristics. This indicates that all three strains contain functional allophycocyanin cores associated with their phycobilisomes. Analysis of the phycobilisome content of these strains indicated that, as expected, wild type exhibited normal phycobilisome assembly and the CK mutant assembled only the allophycocyanin core. However, the ΔcpcF mutant assembled phycobilisomes which, while much larger than the allophycocyanin core observed in the CK mutant, were significantly smaller than phycobilisomes observed in wild type. Interestingly, the phycobilisomes from the ΔcpcF mutant contained holo-CpcB and apo-CpcA. Additionally, we found that the large form of FNR (FNR(L)) accumulated to normal levels in wild type and the ΔcpcF mutant. In the CK mutant, however, significantly less FNR(L) accumulated. FNRL has been reported to associate with the phycocyanin rods in phycobilisomes via its N-terminal domain, which shares sequence homology with a phycocyanin linker polypeptide. We suggest that the assembly of apo-CpcA in the phycobilisomes of ΔcpcF can stabilize FNR(L) and modulate its function. These phycobilisomes, however, inefficiently transfer excitation energy to Photosystem II.

Dataset Information

Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host.

Publications

Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host.

Similar Datasets

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