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• W1968729463 abstract "The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcS-I copurified with His6-tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apophycocyanin, the reaction product had an absorbance maximum of 622 nm and was highly fluorescent (λmax = 643 nm). In control reactions with PCB and apophycocyanin, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (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 apoHT-allophycocyanin (aporHT-AP) and PCB in vitro. AporHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of ∼27 kDa. When aporHT-AP 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 and 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 the bilin lyase-responsible for attachment of PCB to Cys-82 of CpcB and Cys-81 of ApcA and ApcB. The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcS-I copurified with His6-tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apophycocyanin, the reaction product had an absorbance maximum of 622 nm and was highly fluorescent (λmax = 643 nm). In control reactions with PCB and apophycocyanin, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (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 apoHT-allophycocyanin (aporHT-AP) and PCB in vitro. AporHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of ∼27 kDa. When aporHT-AP 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 and 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 the 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), 4The abbreviations used are: PBSphycobilisomeAPallophycocyaninHPLChigh performance liquid chromatographyHTHis6-taggedMBVmesobiliverdinNi-NTAnickel-nitrilotriacetic acidPBPphycobiliproteinPCphycocyaninPCBphycocyanobilinrHT-APrecombinant His6-tagged-ApcA/ApcBrCpcBArecombinant apoCpcB/CpcACpcSUrecombinant CpcS-I/HT-CpcU. 4The abbreviations used are: PBSphycobilisomeAPallophycocyaninHPLChigh performance liquid chromatographyHTHis6-taggedMBVmesobiliverdinNi-NTAnickel-nitrilotriacetic acidPBPphycobiliproteinPCphycocyaninPCBphycocyanobilinrHT-APrecombinant His6-tagged-ApcA/ApcBrCpcBArecombinant apoCpcB/CpcACpcSUrecombinant CpcS-I/HT-CpcU. are very similar to those found in red algal chloroplasts but are quite distinct from the chlorophyll-based, light-harvesting protein complexes of higher plants (1Glazer A.N. J. Biol. Chem. 1989; 264: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Press, Dordrecht, The Netherlands1994: 139-216Google Scholar, 3Bryant D.A. Bogarad L. Vasil I.K. The Photosynthetic Apparatus: Molecular Biology and Operation. Academic Press, Inc., New York1991: 255-298Google Scholar). 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) (3Bryant D.A. Bogarad L. Vasil I.K. The Photosynthetic Apparatus: Molecular Biology and Operation. Academic Press, Inc., New York1991: 255-298Google Scholar, 4de Lorimier R. Bryant D.A. Porter R.D. Liu W-Y. Jay E. Stevens Jr., S.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7946-7950Crossref PubMed Scopus (85) Google Scholar, 5Bryant D.A. Stevens Jr., S.E. Bryant D.A. Light-Energy Transduction in Photosynthesis: Higher Plant and Bacterial Models. American Society of Plant Physiologists, Rockville, MD1988: 62-90Google Scholar, 6Bryant D.A. de Lorimier R. Guglielmi G. Stevens Jr., S.E. Arch. Microbiol. 1990; 153: 550-560Crossref PubMed Scopus (39) Google Scholar, 7de Lorimier R. Guglielmi G. Bryant D.A. Stevens Jr., S.E. Arch. Microbiol. 1990; 153: 541-549Crossref PubMed Scopus (34) Google Scholar, 8de Lorimier R. Bryant D.A. Stevens Jr., S.E. Biochim. Biophys. Acta. 1990; 1019: 29-41Crossref PubMed Scopus (58) Google Scholar, 9Maxson P. Sauer K. Bryant D.A. Glazer A.N. Biochim. Biophys. Acta. 1989; 974: 40-51Crossref Scopus (42) Google Scholar, 10Gindt Y.M. Zhou J. Bryant D.A. Sauer K. J. Photochem. Photobiol. B. 1992; 15: 75-89Crossref PubMed Scopus (37) Google Scholar, 11Gindt Y.M. Zhou J. Bryant D.A. Sauer K. Biochim. Biophys. Acta. 1994; 1186: 153-162Crossref PubMed Scopus (54) Google Scholar, 12Gómez-Lojero C. Pérez-Gómez B. Shen G. Schluchter W.M. Bryant D.A. Biochemistry. 2003; 42: 13800-13811Crossref PubMed Scopus (45) Google Scholar). Each of these major PBP is composed of two subunits, α and β, and each of these subunits carries at least one covalently attached phycocyanobilin (PCB) chromophore (1Glazer A.N. J. Biol. Chem. 1989; 264: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Press, Dordrecht, The Netherlands1994: 139-216Google Scholar, 3Bryant D.A. Bogarad L. Vasil I.K. The Photosynthetic Apparatus: Molecular Biology and Operation. Academic Press, Inc., New York1991: 255-298Google Scholar). The attachment of PCB to the polypeptide subunits occurs through thioether bonds to specific cysteine residues (1Glazer A.N. J. Biol. Chem. 1989; 264: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Press, Dordrecht, The Netherlands1994: 139-216Google Scholar, 3Bryant D.A. Bogarad L. Vasil I.K. The Photosynthetic Apparatus: Molecular Biology and Operation. Academic Press, Inc., New York1991: 255-298Google Scholar). phycobilisome allophycocyanin high performance liquid chromatography His6-tagged mesobiliverdin nickel-nitrilotriacetic acid phycobiliprotein phycocyanin phycocyanobilin recombinant His6-tagged-ApcA/ApcB recombinant apoCpcB/CpcA recombinant CpcS-I/HT-CpcU. phycobilisome allophycocyanin high performance liquid chromatography His6-tagged mesobiliverdin nickel-nitrilotriacetic acid phycobiliprotein phycocyanin phycocyanobilin recombinant His6-tagged-ApcA/ApcB recombinant apoCpcB/CpcA recombinant CpcS-I/HT-CpcU. 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 (13Zhou J. Gasparich G.E. Stirewalt V.L. de Lorimier R. Bryant D.A. J. Biol. Chem. 1992; 267: 16138-16145Abstract Full Text PDF PubMed Google Scholar, 14Swanson R.V. Zhou J. Leary J.A. Williams T. de Lorimier R. Bryant D.A. Glazer A.N. J. Biol. Chem. 1992; 267: 16146-16154Abstract Full Text PDF PubMed Google Scholar, 15Fairchild C.D. Zhao J. Zhou J. Colson S.E. Bryant D.A. Glazer A.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7017-7021Crossref PubMed Scopus (113) Google Scholar, 16Zhou, J. (1992) Mutational Analysis of the Genes Encoding Phycobilisome Components in the Cyanobacterium Synechococcus sp. PCC 7002. Ph.D. thesis, The Pennsylvania State University, University Park, PAGoogle Scholar, 17Fairchild C.D. Glazer A.N. J. Biol. Chem. 1994; 269: 8686-8694Abstract Full Text PDF PubMed Google Scholar, 18Jung L.J. Chan C.F. Glazer A.N. J. Biol. Chem. 1995; 270: 12877-12884Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 19Zhao K.H. Deng M.G. Zheng M. Zhou M. Parbel A. Storf M. Meyer M. Strohmann B. Scheer H. FEBS Lett. 2000; 469: 9-13Crossref PubMed Scopus (68) Google Scholar, 20Zhao K.H. Wu D. Zhou M. Zhang L. Bohm S. Bubenzer C. Scheer H. Biochemistry. 2005; 44: 8126-8137Crossref PubMed Scopus (28) Google Scholar, 21Kahn K. Mazel D. Houmard J. Tandeau de Marsac N. Schaefer M.R. J. Bacteriol. 1997; 179: 998-1006Crossref PubMed Google Scholar). 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) (13Zhou J. Gasparich G.E. Stirewalt V.L. de Lorimier R. Bryant D.A. J. Biol. Chem. 1992; 267: 16138-16145Abstract Full Text PDF PubMed Google Scholar, 14Swanson R.V. Zhou J. Leary J.A. Williams T. de Lorimier R. Bryant D.A. Glazer A.N. J. Biol. Chem. 1992; 267: 16146-16154Abstract Full Text PDF PubMed Google Scholar, 15Fairchild C.D. Zhao J. Zhou J. Colson S.E. Bryant D.A. Glazer A.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7017-7021Crossref PubMed Scopus (113) Google Scholar, 16Zhou, J. (1992) Mutational Analysis of the Genes Encoding Phycobilisome Components in the Cyanobacterium Synechococcus sp. PCC 7002. Ph.D. thesis, The Pennsylvania State University, University Park, PAGoogle Scholar, 17Fairchild C.D. Glazer A.N. J. Biol. Chem. 1994; 269: 8686-8694Abstract Full Text PDF PubMed Google Scholar). The PecEF and CpeYZ lyases are similar in sequence to CpcEF but are active on different substrate proteins (18Jung L.J. Chan C.F. Glazer A.N. J. Biol. Chem. 1995; 270: 12877-12884Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 19Zhao K.H. Deng M.G. Zheng M. Zhou M. Parbel A. Storf M. Meyer M. Strohmann B. Scheer H. FEBS Lett. 2000; 469: 9-13Crossref PubMed Scopus (68) Google Scholar, 20Zhao K.H. Wu D. Zhou M. Zhang L. Bohm S. Bubenzer C. Scheer H. Biochemistry. 2005; 44: 8126-8137Crossref PubMed Scopus (28) Google Scholar, 21Kahn K. Mazel D. Houmard J. Tandeau de Marsac N. Schaefer M.R. J. Bacteriol. 1997; 179: 998-1006Crossref PubMed Google Scholar). A completely different family of genes, first sequenced as part of an operon encoding PBS linker proteins for phycoerythrin in Fremyella diplosiphon (22Cobley J.G. Clark A.C. Weerasurya S. Queseda F.A. Xiao J.Y. Bandrapali N. D'Silva I. Thounaojam M. Oda J.F. Sumiyoshi T. Chu M.H. Mol. Microbiol. 2002; 44: 1517-1531Crossref PubMed Scopus (71) Google Scholar), is involved in PCB attachment to the Cys-82 attachment site of β-PC as well as to Cys-153 of the same polypeptide (23Shen G. Saunee N.A. Gallo E. Begovic Z. Schluchter W.M. Bryant D.A. Niederman R.A. Blankenship R.E. Frank H. Robert B. van Grondelle R. Photosynthesis 2004 Light-Harvesting Systems Workshop, August 26–29, 2004. Saint Adele, Quebec, Canada2004: 14-15Google Scholar). The Synechococcus sp. PCC 7002 genome encodes four members of this second lyase family (23Shen G. Saunee N.A. Gallo E. Begovic Z. Schluchter W.M. Bryant D.A. Niederman R.A. Blankenship R.E. Frank H. Robert B. van Grondelle R. Photosynthesis 2004 Light-Harvesting Systems Workshop, August 26–29, 2004. Saint Adele, Quebec, Canada2004: 14-15Google Scholar, 24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 25Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Three genes show highest sequence similarity to cpeS and have been named cpcS-I, cpcU, and cpcV. One gene is most similar to cpeT of F. diplosiphon and has been named cpcT (23Shen G. Saunee N.A. Gallo E. Begovic Z. Schluchter W.M. Bryant D.A. Niederman R.A. Blankenship R.E. Frank H. Robert B. van Grondelle R. Photosynthesis 2004 Light-Harvesting Systems Workshop, August 26–29, 2004. Saint Adele, Quebec, Canada2004: 14-15Google Scholar, 24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 25Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). CpcT specifically attaches PCB to Cys-153 of β-PC in Synechococcus sp. PCC 7002 (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In Nostoc sp. PCC 7120 it was recently shown that the product of open reading frame alr0617, which we shall refer to as CpcS-III based on phylogenetic analyses (25Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), is another member of this lyase family (26Zhao K.H. Su P. Li J. Tu J.M. Zhou M. Bubenzer C. Scheer H. J. Biol. Chem. 2006; 281: 8573-8581Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Zhao et al. (26Zhao K.H. Su P. Li J. Tu J.M. Zhou M. Bubenzer C. Scheer H. J. Biol. Chem. 2006; 281: 8573-8581Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Zhao K-H. Su P. Tu M.-T. Wang X. Liu H. Ploscher M. Eichacker L. Yang B. Zhou M. Scheer H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14300-14305Crossref PubMed Scopus (85) Google Scholar) have shown that CpcS-III attaches PCB to Cys-82 on β-PC and β-phycoerythrocyanin as well as to several AP subunits in Nostoc sp. PCC 7120. The PBS core linker, also known as LCM or ApcE, contains a PBP-like domain at its N terminus that is related in sequence to all other PBP but has the Cys residue for PCB attachment at a structurally distinct position (5Bryant D.A. Stevens Jr., S.E. Bryant D.A. Light-Energy Transduction in Photosynthesis: Higher Plant and Bacterial Models. American Society of Plant Physiologists, Rockville, MD1988: 62-90Google Scholar, 10Gindt Y.M. Zhou J. Bryant D.A. Sauer K. J. Photochem. Photobiol. B. 1992; 15: 75-89Crossref PubMed Scopus (37) Google Scholar, 28Capuano V. Braux A.S. Tandeau de Marsac N. Houmard J. J. Biol. Chem. 1991; 266: 7239-7247Abstract Full Text PDF PubMed Google Scholar, 29Zhao K.H. Su P. Bohm S. Song B. Zhou M. Bubenzer C. Scheer H. Biochim. Biophys. Acta. 2005; 1706: 81-87Crossref PubMed Scopus (60) Google Scholar). ApcE may not require a lyase enzyme for bilin attachment. Zhao et al. (29Zhao K.H. Su P. Bohm S. Song B. Zhou M. Bubenzer C. Scheer H. Biochim. Biophys. Acta. 2005; 1706: 81-87Crossref PubMed Scopus (60) Google Scholar) have shown that PCB addition to ApcE can occur in the absence of lyases in vitro, although it is currently not known if this also occurs in vivo. Moreover, PCB addition to ApcA (α-AP) may not require a lyase when the protein is synthesized in Escherichia coli cells that co-express apcA and the genes required to synthesize PCB from heme (30Hu I.C. Lee T.R. Lin H.F. Chiueh C.C. Lyu P.C. Biochemistry. 2006; 45: 7092-7099Crossref PubMed Scopus (22) Google Scholar). When purified HT-ApcA and PCB were combined in vitro, PCB addition occurred, and the resulting product was reported to have an absorption spectrum that matched that of native holo-AP subunits (30Hu I.C. Lee T.R. Lin H.F. Chiueh C.C. Lyu P.C. Biochemistry. 2006; 45: 7092-7099Crossref PubMed Scopus (22) Google Scholar). In some studies in which no requirement for lyases was reported, detergents or urea were added to modify the conformation of the PBP, the bilin, or both (29Zhao K.H. Su P. Bohm S. Song B. Zhou M. Bubenzer C. Scheer H. Biochim. Biophys. Acta. 2005; 1706: 81-87Crossref PubMed Scopus (60) Google Scholar, 31Zhao K.H. Zhu J.P. Song B. Zhou M. Storf M. Bohm S. Bubenzer C. Scheer H. Biochim. Biophys. Acta. 2004; 1657: 131-145Crossref PubMed Scopus (26) Google Scholar). We have used a combination of reverse genetics and biochemical methods to identify and characterize the PCB lyases of Synechococcus sp. PCC 7002. In an accompanying manuscript (25Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we have presented strong evidence that null mutants for cpcS-I and cpcU define a heterodimeric PCB lyase that attaches PCB to Cys-82 of β-PC. The characterization of these mutants also suggested that CpcS-I and CpcU are required for PCB attachment to Cys-81 of the α and β subunits of AP (23Shen G. Saunee N.A. Gallo E. Begovic Z. Schluchter W.M. Bryant D.A. Niederman R.A. Blankenship R.E. Frank H. Robert B. van Grondelle R. Photosynthesis 2004 Light-Harvesting Systems Workshop, August 26–29, 2004. Saint Adele, Quebec, Canada2004: 14-15Google Scholar, 25Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In the studies presented here, the conclusions from these reverse genetics analyses are validated in vitro, and we show that CpcS-I and CpcU form a 1:1 heterodimer that attaches PCB to Cys-82 of β-PC. Neither CpcS-I nor CpcU alone is able to perform this lyase reaction. Additionally, we show that these two proteins are also required for correct attachment of PCB to the α and β subunits of AP in vitro. Construction of Recombinant Expression Plasmids—The cpcS-I gene was amplified by PCR from Synechococcus sp. PCC 7002 chromosomal DNA using primers 7002cpcS5 (5′-AATTTTTCCATATGCAAAGCTTTGCGGATGCC-3′) and 7002cpcS3 (5′-TTGACTCGAGCAACACGGATATCTCTGTGGG-3′). The PCR product was cloned into pAED4 T7 expression vector using the restriction enzymes NdeI and XhoI (underlined in the primer sequences). The cpcU gene was amplified by PCR from Synechococcus sp. PCC 7002 chromosomal DNA using two primers (7002cpcU5, 5′-GTAACTGTTCATATGGATATCAATGCCTTTATCC-3′; 7002cpcU3, 5′-CTAAAAGCTTTCGTTAGTTACTGGCTTCAGCGG-3′). The PCR product was cloned into pBS150v vector using NdeI and HindIII (underlined in the primer sequences). The pBS150v vector includes a His6 tag for easy purification of the protein. The cpcV gene was amplified by PCR using primers 7002cpcV5 (5′-GCTCTTCGCATATGAATTTACTTGCGAC-3′) and 7002cpcV3 (5′-TTTAAGCTTACTAAAGACGCGTTTCTAAATACTGCGC-3′). After PCR amplification, the cpcV gene was cloned into vectors pAED4 and pBS150v using restriction enzymes NdeI and HindIII (underlined in the primer sequences). The cpcB and cpcA genes were cloned into pAED4 as described (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The apcA and apcB genes were cloned in pET100 (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The expression constructs were sequenced at the W. M. Keck Conservation and Molecular Genetics laboratory (University of New Orleans) to confirm that no unwanted mutations had been introduced. Protein Overexpression and Purification—Expression plasmids were transformed into E. coli BL21 DE3 cells, and colonies were selected on Luria Bertani plates in the presence of ampicillin (100 μg ml–1) or spectinomycin (100 μg ml–1). For expression of cpcS-I, cpcU, and cpcV, cells from a 50-ml starter culture were added to 1 liter of Luria-Bertani medium with the appropriate antibiotic and grown for 4 h. Cells harboring plasmids encoding cpcS-I or cpcV were grown at 30 °C, whereas those encoding cpcU were grown at 37 °C. Production of proteins was induced by the addition of 0.5 mm isopropyl β-d-thiogalactoside. Cells were incubated with shaking for another 4 h before cells were harvested by centrifugation, and pellets were frozen at –20 °C until required. Purification of rCpcBA and aporHT-AP was performed as described (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). To purify CpcS-I, cells were resuspended in 50 mm Tris-HCl, pH 8.0, and lysed by three passages through a chilled French pressure cell at 138 megapascals. The lysed cell suspension was centrifuged for 25 min at 17,000 × g to remove unbroken cells and inclusion bodies. The supernatant was brought to 40% saturation with ammonium sulfate and left at 4 °C overnight. After centrifugation at 17,000 × g for 20 min, the pellet containing the CpcS-I protein was resuspended in a small amount and dialyzed exhaustively against the same buffer to remove the ammonium sulfate. Aliquots (10 ml) of the CpcS-I (pI 4.79) solution were loaded onto a DEAE column (Whatman DE-52: 2.5 × 12.5 cm) that had been equilibrated with 50 mm Tris-HCl, pH 8.0, 1 mm NaN3 (buffer 1; sodium azide was added to prevent microbial growth during storage of the column/buffer) by using the BioLogic LP system at room temperature (Bio-Rad). The column was developed using the same procedure described for CpcT (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Fractions containing CpcS-I were pooled and dialyzed against 50 mm Tris-HCl, pH 8.0. CpcS-I was concentrated using an Amicon YM-10 concentrator and stored in 2-ml aliquots at ×20 °C until required. The CpcS-I protein has a calculated molecular mass of 22,526.3 Da. Antibodies were raised against the CpcS-I protein present in inclusion bodies. Inclusion bodies were washed as described in Fairchild et al. (15Fairchild C.D. Zhao J. Zhou J. Colson S.E. Bryant D.A. Glazer A.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7017-7021Crossref PubMed Scopus (113) Google Scholar), and CpcS-I was purified by preparative SDS-PAGE on a 15% (w/v) acrylamide gel. The band corresponding to CpcS-I was excised and minced, and protein was electroeluted from the gel slices using a procedure recommended by Sigma Genosys, who generated rabbit polyclonal antibodies to the protein. Because CpcU is produced with an N-terminal His6 tag, it was purified by metal affinity chromatography using Ni-NTA resin (Qiagen, Valencia, CA). E. coli cells containing HTCpcU were resuspended in 20 mm Tris-HCl, pH 8.0, 50 mm NaCl, 50 mm KCl (buffer 0) and lysed by three passages through a chilled French pressure cell at 138 megapascals. After clarification of the extract by centrifugation at 17,000 × g for 25 min, the supernatant was added to 10 ml of Ni-NTA resin that had been washed with buffer 0. The resin and the extracts were incubated together for 15 min and then loaded into a column. The column was washed as described in Shen et al. (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and HT-CpcU was eluted from the Ni-NTA resin by the addition of 20 ml buffer C (20 mm Tris-HCl, pH 8.0, 50 mm NaCl, 50 mm KCl, 200 mm imidazole), dialyzed against Buffer 0, concentrated, and stored in 2-ml aliquots at 20 °C until required. The HT-CpcU protein has a calculated molecular mass of 23,471.71 Da. The HT-CpcV protein was purified in the same manner and had a molecular mass of 22,728.2 Da. CpcV (non-tagged; calculated molecular mass of 19,851.3 Da) was not purified; whole cell extracts containing CpcV were used in some enzyme assays. Interaction Assays—A pulldown interaction assay was performed to determine whether HT-CpcU and CpcS-I interacts in vitro. An aliquot (50 μl) of E. coli whole-cell extract containing HT-CpcU was mixed with a whole-cell extract (50 μl) containing CpcS-I, and the solution was incubated on ice for 15 min. The Ni-NTA resin (150 μl) was pelleted by centrifugation at 3000 × g for 4 min. The supernatant was discarded, and 500 μl of buffer 0 was added. The resin was pelleted again under the same conditions, and the supernatant was discarded. The protein solution was then added to the washed resin and incubated for 30 min with gentle agitation. The mixture was then centrifuged for 5 min at 3000 × g. The supernatant was discarded, and 500 μl of buffer A1 was added, and the resin was pelleted by centrifugation for 5 min at 3000 × g. The same procedure was followed for buffer B and buffer A2 (as described in Shen et al. (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar)). The bound proteins were eluted by the addition of buffer C (40 μl). An equal amount of 2× SDS-loading buffer was added to the sample, which was boiled and loaded on a 15% (w/v) acrylamide gel as described previously (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). For purification of large amounts of the CpcSU complex, whole cell extracts containing each subunit were incubated together on ice for 1 h, and then the complexes were purified using metal affinity chromatography in the same way as described above for HT-CpcU. In Vitro PcyA Reactions—A plasmid for the expression of pcyA was kindly provided by Dr. J. C. Lagarias (32Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This enzyme was overproduced as a glutathione S-transferase fusion and purified as described previously (32Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 33Frankenberg N. Mukougawa K.K. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (190) Google Scholar, 34Kohchi T. Mukougawa K. Frankenberg N. Masuda M. Yakota A. Lagarias J.C. Plant Cell. 2001; 13: 425-436Crossref PubMed Scopus (196) Google Scholar). PcyA reduces biliverdin in two sequential 2-electron reductions using reduced ferredoxin to produce PCB. rCpcBA (1 mg ml–1) reactions (total volume, 4 ml) were set up with one of the following additions; 200 μl of whole-cell extract from E. coli cells harboring pAED4 as the negative control (24Shen G. Saunée N.A. Williams S.R. Gallo E.F. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2006; 281: 17768-17778Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), 200 μl of CpcS-I (1 mg ml–1), 600 μl of HT-CpcU (0.33 mg ml–1), or 400 μl of co-purified CpcSU (1 mg ml–1). The following were added to the reaction mixture: 50 mm HEPES buffer, pH 7.3, 1 mm MgCl2 6.5 mm glucose-6-phosphate, 1.6 mm NADP+, 1.1 units of glucose-6-phosphate dehydrogenase ml–1, 4.6 μm recombinant ferredoxin from Synechococcus sp. PCC 7002 (35Schluchter, W. M. (1994) The Characterization of Photosystem I and Ferredoxin-NADP+ Oxidoreductase in the Cyanobacterium Synechococcus sp. PCC 7002. Ph.D. thesis, The Pennsylvania State University, University Park, PAGoogle Scholar) or spinach ferredoxin (Sigma), 0.025 units ml–1 of spinach (Sigma) or recombinant Synechococcus sp. PCC 7002 ferredoxin:NADP+ oxidoreductase (12Gómez-Lojero C. Pérez-Gómez B. Shen G. Schluchter W.M. Bryant D.A. Biochemistry. 2003; 42: 13800-13811Crossref PubMed Scopus (45) Google Scholar, 35Schluchter, W. M. (1994) The Characterization of Photosystem I and Ferredoxin-NADP+ Oxidoreductase in the Cyanobacterium Synechococcus sp. PCC 7002. Ph.D. thesis, The Pennsylvania State University, University Park, PAGoogle Scholar, 36Schluchter W.M. Bryant D.A. Biochemistry. 1992; 31: 3092-3102Crossref PubMed Scopus (116) Google Scholar), 10 μm bovine serum albumin, 5 μm biliverdin (Porphyrin Products, Logan, UT), and 10 μm PcyA (32Frankenberg N. Lagarias J.C. J. Biol. Chem. 2003; 278: 9219-9226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 33Frankenberg N. Mukougawa K.K. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (190) Google Scholar, 34Kohchi T. Mukougawa K. Frankenberg N. Masuda M. Yakota A. Lagarias J.C. Plant Cell. 2001; 13: 425-436Crossref PubMed Scopus (196) Google Scholar). All reactions were incubated in a 30 °C water bath for 1 h in the dark. Another aliquot of biliverdin was added (for a final concentration of 10 μm), and the reactions were allowed to continue for 1 h at 30 °C. The reaction solutions were clarified by centrifugation at 14,000 × g for 10 min. For reactions containing aporHT-AP (in a 1.5 ml volume), aporHT-AP (600 μl of 2.0 mg ml–1 solution)" @default.
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