Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481

Avijit Biswas, M. Nazim Boutaghou, Richard M. Alvey, Christina M. Kronfel, Richard B. Cole, Donald A. Bryant, Wendy M. Schluchter

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

When grown in green light, Fremyella diplosiphon strain UTEX481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His 6-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys 82 of CpeA. The CpeS bilin lyase ligated PEB at both Cys 82 and Cys 139 of CpeA but very inefficiently; the yield of PEB ligated at Cys 82 was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys 80 of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.

Original languageEnglish (US)
Pages (from-to)35509-35521
Number of pages13
JournalJournal of Biological Chemistry
Volume286
Issue number41
DOIs
StatePublished - Oct 14 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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