Molecular cloning of the cpeT gene encoding a bilin lyase responsible for attachment of phycoerythrobilin to Cys-158 on the β-subunit of phycoerythrin in Gracilariopsis lemaneiformis
Phycobilin lyase plays an important role in the binding of phycobilin chromophores to phycobiliprotein. The bilin lyase cpeT gene was cloned from Gracilariopsis lemaneiformis to study the synthesis of fluorescent phycobiliprotein. Two recombinant plasmids, one containing the genes (ho1 and pebA, pebB) that produce phycoerythrobilin and the other containing the two subunit genes (rpeB, rpeA) of phycoerythrin (R-PE), as well as the lyase gene cpeT, were constructed and transformed into Escherichia coli. The recombinant strain with lyase CpeT and the β-subunit of phycoerythrin (R-PeB) enhanced the optical activity of R-PE, indicating that lyase CpeT catalyzes the synthesis of the R-PeB with optical activity. Another two recombinant plasmids, one containing the genes (ho(314), pcyA(314)) that produce phycocyanobilin and the other containing the two subunit genes (rpcB, rpcA) of phycocyanin (R-PC), as well as the lyase gene cpeT, were constructed and transformed into Escherichia coli. However, there was no enhancement in fluorescence in this recombinant strain, revealing that lyase CpeT cannot catalyze the synthesis of optically active R-PC. Thereafter, Cys-82 and Cys-158 of R-PeB were individually mutated to identify the possible binding sites of apoproteins with phycobilins catalyzed by CpeT. We found that there was a significant decrease in the fluorescence of the mutated Cys-158 recombinant strain, suggesting that Cys-158 is the active site for the binding of the β-subunit with CpeT-catalyzed phycoerythrobilin in G. lemaneiformis. The results of this study lay the foundation for understanding the synthesis of fluorescent phycobiliprotein.
KeywordsGracilariopsis lemaneiformis Rhodophyta CpeT Site-directed mutation Fluorescence intensity
This research was supported by China Agriculture Research System (CRAS-50) and the National Natural Science Foundation of China (grant no. 31472255).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and institutional guidelines for the care and use of animals were followed.
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