BIOGENESIS OF PHYCOBILIPROTEINS. II. CpcS-I AND CpcU COMPRISE THE HETERODIMERIC BILIN LYASE THAT ATTACHES PHYCOCYANOBILIN TO CYS-82 OF β-PHYCOCYANIN AND CYS-81 OF ALLOPHYCOCYANIN SUBUNITS IN SYNECHOCOCCUS SP. PCC 7002*

The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcSI copurified with His6-tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apo-phycocyanin (PC), the reaction product had an absorbance maximum of 622 nm and was highly fluorescent (λmax = 643 nm). In control reactions with PCB and apo-PC, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero et al., 1988; J. Biol. Chem. 263, 18343-18349). Tryptic peptide mapping showed that the CpcSU-dependent reaction product had one major PCB-containing peptide that contained the PCB binding site Cys-82. The CpcSU lyase was also tested with recombinant, apo-HT-allophycocyanin (aporHT-AP) and PCB in vitro. Apo-rHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of ~27 kDa. When apo-rHTAP was incubated with PCB and CpcSU, the product had an absorbance maximum of 614 nm and a fluorescence emission maximum at 636 nm, the expected maxima for monomeric holo-AP. When no enzyme or CpcS-I or CpcU was added alone, the products had absorbance maxima between 645-647 nm and were not fluorescent. When these reaction products were analyzed by gel electrophoresis and zinc-enhanced fluorescence emission, only the reaction products from CpcSU had PCB attached to both AP subunits. Therefore, CpcSU is bilin lyase responsible for attachment of PCB to Cys-82 of CpcB and Cys-81 of ApcA and ApcB. Cyanobacteria are a morphologically and developmentally diverse group of prokaryotes. Their light-harvesting complexes, phycobilisomes (PBS), are very similar to those found in red algal chloroplasts but are quite distinct from the chlorophyll-based, lightharvesting protein complexes of higher plants (1-3). The PBS of the genetically amenable cyanobacterium, Synechococcus sp. PCC 7002 are ideal objects for detailed characterization, because they are among the simplest known PBS in structure and composition. These PBS contain only 12 polypeptides and are principally composed of only two phycobiliproteins (PBP): allophycocyanin (AP) and phycocyanin (PC) (312). Each of these major PBP are composed of two subunits, α and β, and each of these subunits carries at least one covalently attached phycocyanobilin (PCB) chromophore (1-3). The attachment of PCB to the polypeptide subunits occurs through thioether bonds to specific cysteine residues (1-3). For some PBP it has been demonstrated that lyase enzymes are required for the attachment, isomerization, and detachment of the bilin chromophores from the cysteine residues of the PBP (13-21). For example, enzymes appear to be involved in the attachment of PCB to each of the three Cys attachment sites of PC. The products of two genes, cpcE and cpcF, which occur downstream of the cpcBA structural genes that encode the β and α subunits of PC, respectively, comprise a heterodimeric lyase that specifically attaches PCB to Cys-84 of α-PC (CpcA) (13-17). The PecEF and CpeYZ lyases are similar in sequence to CpcEF but are active on different substrate proteins (18-21). A completely different family of genes, first http://www.jbc.org/cgi/doi/10.1074/jbc.M708165200 The latest version is at JBC Papers in Press. Published on January 16, 2008 as Manuscript M708165200