Biosynthesis of the cyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-alpha subunit in a heterologous host.
The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-alpha subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin alpha subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Tooley
Aaron J
AJ
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
Glazer
Alexander N
AN
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D052980:Phycobilins; D010798:Phycocyanin / Q000096:biosynthesis* / Q000737:chemistry; D021122:Protein Subunits
D052980:Phycobilins; D021122:Protein Subunits; C038397:phycoerythrocyanin; D010798:Phycocyanin