Identification and Characterization of a New Class of Bilin Lyase

Abstract

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin ␤-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin ␤-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin ␤-subunit. Cyanobacteria and red algae contain peripheral, light-harvesting complexes called phycobilisomes (PBS).2 These macromolecular complexes are composed of two types of proteins: colored phycobiliproteins (PBPs), which absorb and transmit light energy to the photosynthetic reaction centers, and non-pigmented linker proteins, which direct the assembly of PBS (1). In many cyanobacteria, including Synechococcus sp. PCC 7002, PBS are composed of two blue-colored PBPs: phycocyanin (PC) and allophycocyanin (AP). These PBPs are composed of two subunits, ␣ and ␤ (2), which are members of the globin superfamily (3). The ␣-and ␤-subunits of PC and AP carry at least one linear tetrapyrrole chromophore (bilin) called phycocyanobilin (PCB), which is covalently attached to specific cysteine residues via thioether linkages (see supplemental Fig. 1) (4). The bilin chromophores of PBPs are derived from heme and are related to the phytochromobilin (5), the photoisomerizable chromophore of the plant protein, phytochrome (6). X-ray crystallographic analyses of PC have shown that the chiral C3 1 carbons of the PCB chromophores attached at cysteines ␣84 and ␤82 are in the R configuration, whereas the C3

Biogenesis of Phycobiliproteins

“…Recombinant Synechococcus sp. PCC 7002 apo-PC (CpcB/CpcA) was purified from E. coli as described (24). The calculated molecular masses for apo-CpcB and apo-CpcA were 18,335 and 17,621 Da, respectively.…”

Characterization of the Activities of the CpeY, CpeZ, and CpeS Bilin Lyases in Phycoerythrin Biosynthesis in Fremyella diplosiphon Strain UTEX 481

“…They can typically recognize many different PBP substrates and attach bilins at their Cys 82 equivalent positions (30). The third family of lyases is called the T-type and is typified by CpcT, an enzyme that attaches PCB at Cys 153 of CpcB (8,34). Last, there is a family of PBPs that autocatalytically ligate bilin chromophores.…”

“…coli cells containing recombinant proteins were thawed and resuspended in buffer O (20 mM Tris-HCl, 100 mM NaCl, pH 8.0) at 2.5 ml g Ϫ1 (wet weight) along with protease inhibitor mixture tablets ("Complete Mini" from Roche Applied Science). The cells were lysed, and the His 6 -tagged recombinant proteins were purified as previously described (8). The recombinant protein(s) were exhaustively dialyzed with buffer O containing 10 mM 2-mercaptoethanol overnight at 4°C to remove the imidazole introduced during elution.…”

“…Protein and Bilin Analysis-Polypeptides were resolved by PAGE (15%, w/v) in the presence of SDS, and visualized by staining with Coomassie Blue as described (8). To detect PEB linked to proteins, gels were soaked in 100 mM ZnSO 4 for ϳ5 min (50,51) and the zinc-enhanced fluorescence, indicative of bilin attachment, was visualized using an FX imaging system (Bio-Rad) with excitation at 532 nm.…”

“…The reaction was quenched by adding 30% (v/v) glacial acetic acid, followed by passage through a C-18 Sep-Pak (Waters Corp., Milford, MA) cartridge as described (4). The eluted sample was vacuum dried and stored at Ϫ20°C for HPLC analysis as described (8).…”