title: Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481.
abstract: When grown in green light, Fremyella diplosiphon strain UTEX 481 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.
authors.affiliation: Department of Biological Science, University of New Orleans, New Orleans, Louisiana 70148.
authors.forename: Avijit
authors.initials: A
authors.lastname: Biswas
authors.affiliation: Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148.
authors.forename: M Nazim
authors.initials: MN
authors.lastname: Boutaghou
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Alvey
authors.affiliation: Department of Biological Science, University of New Orleans, New Orleans, Louisiana 70148.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Kronfel
authors.affiliation: Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Cole
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802; Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
authors.affiliation: Department of Biological Science, University of New Orleans, New Orleans, Louisiana 70148. Electronic address: wschluch@uno.edu.
authors.forename: Wendy M
authors.initials: WM
authors.lastname: Schluchter
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010799:Phycoerythrin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C104232:CpeY protein, Fremyella diplosiphon; C104233:CpeZ protein, Fremyella diplosiphon; D010799:Phycoerythrin; D008190:Lyases