Chromophore Attachment to Phycobiliprotein β-Subunits
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m513796200
ISSN1083-351X
AutoresKai‐Hong Zhao, Ping Su, Jian Li, Jun‐Ming Tu, Ming Zhou, Claudia Bubenzer, Hugo Scheer,
Tópico(s)Biocrusts and Microbial Ecology
ResumoThe gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-β84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-β84 of the apo-β-subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 °C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3μm, and a kcat = 1.7 × 10-5 and 1.1 × 10-5 s-1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS. The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-β84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-β84 of the apo-β-subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 °C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3μm, and a kcat = 1.7 × 10-5 and 1.1 × 10-5 s-1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, are supramolecular complexes of phycobiliproteins and linkers (1Gantt B. Grabowski B. Cunningham F.X. Green B. Parson W. 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Phycobilin chromophores are generally bound to the polypeptide, via thioether bonds, to conserved cysteine residues: phycocyanobilin (PCB) 4The abbreviations used are: PCB, phycocyanobilin; Cpes, PCB:Cys-β84 phycobiliprotein lyase for cysteine-β84 of phycobiliproteins; CPC, cyanobacterial phycocyanin; CpcA, apoprotein of α-CPC; CpcB, apoprotein of β-CPC; CpcE and CpcF, subunits of PCB:α-CPC lyase; HPLC, high performance liquid chromatography; KPP, potassium phosphate buffer; ME, mercaptoethanol; IPEB, phycoerythrobilin; PEC, phycoerythrocyanin; PecA, apoprotein of α-PEC; PecB, apoprotein of β-PEC; PecE and PecF, subunits of PVB:α-PEC isomerase-lyase; PVB, phycoviolobilin; TX100, Triton X-100; CPE, C-phycoerythrin. and phycoviolobilin (PVB) are singly bound to C-3 at ring A of the tetrapyrroles, whereas phycoerythrobilin (PEB) and phycourobilin often have an additional bond to C-18 at ring D (7Lagarias J.C. Klotz A.V. Dallas J.L. Glazer A.N. Bishop J.E. Oconnell J.F. Rapoport H. J. 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Biochemistry. 2004; 43: 3659-3669Crossref PubMed Scopus (113) Google Scholar, 13Terauchi K. Montgomery B.L. Grossman A.R. Lagarias J.C. Kehoe D.M. Mol. Microbiol. 2004; 51: 567-577Crossref PubMed Scopus (110) Google Scholar, 14Wu S.-H. Lagarias J.C. Biochemistry. 2000; 39: 13487-13495Crossref PubMed Scopus (160) Google Scholar, 15Zhao K.-H. Ran Y. Li M. Sun Y.-N. Zhou M. Storf M. Kupka M. Böhm S. Bubenzer C. Scheer H. Biochemistry. 2004; 43: 11576-11588Crossref PubMed Scopus (35) Google Scholar). In contrast, chromophore binding to other phycobiliproteins is more complex. Spontaneous addition of PCB has been observed to all binding sites (Cys-α84, Cys-β84, and Cys-β155) of CPC, but it is slow and generally leads to a mixture of products (16Arciero D.M. Dallas J.L. Glazer A.N. J. Biol. Chem. 1988; 263: 18350-18357Abstract Full Text PDF PubMed Google Scholar, 17Arciero D.M. Bryant D.A. Glazer A.N. J. Biol. Chem. 1988; 263: 18343-18349Abstract Full Text PDF PubMed Google Scholar). Lyases, specific for attaching PCB to Cys-α84 of CpcA, and PecA, have been identified in several cyanobacteria (9Schluchter W.M. Glazer A.N. Peschek G.A. Löffelhardt W. Schmetterer G. The Phototrophic Prokaryotes. Kluwer/Plenum Press, New York1999: 83-95Crossref Google Scholar). The PCB:α-CPC lyase reversibly binds PCB to cysteine-84 of the α-subunit of cyanobacterial phycocyanin (CpcA); it is composed of two subunits, CpcE and CpcF, which, in several species, are coded by genes on the respective biliprotein operon (18Bhalerao R.P. Gustafsson P. Physiol. Plant. 1994; 90: 187-197Crossref Scopus (10) Google Scholar, 19Zhou 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). Similar lyases probably catalyze the attachment of PEB to cysteine-α84 (20Fairchild 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 (116) Google Scholar, 21Fairchild C.D. Glazer A.N. J. Biol. Chem. 1994; 269: 8686-8694Abstract Full Text PDF PubMed Google Scholar). The phylogenetically related PVB:α-PEC lyases (EC 4.4.1.17), PecE/F (22Kaneko T. Nakamura Y. Wolk C.P. Kuritz T. Sasamoto S. Watanabe A. Iriguchi M. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kohara M. Matsumoto M. Matsuno A. Muraki A. Nakazaki N. Siumpo S. Sugimoto M. Takazawa M. Yamada M. Yasuda M. Tabata S. DNA Res. 2001; 8: 205-213Crossref PubMed Scopus (583) Google Scholar, 24Swanson R.V. Lorimier de R. Glazer A.N. J. Bacteriol. 1992; 174: 2640-2647Crossref PubMed Scopus (28) Google Scholar) 5W. Kufer, A. Högner, M. Eberlein, K. Mayer, A. Buchner, and L. Gottschalk (1991) GenBank™ accession number M75599. catalyze not only the addition of PCB to cysteine-84 of the α-subunit of phycoerythrocyanin, PecA, but also an isomerization that generates the photoactive PVB chromophore that is characteristic for α-PEC (25Storf M. Parbel A. Meyer M. Strohmann B. Scheer H. Deng M. Zheng M. Zhou M. Zhao K. Biochemistry. 2001; 40: 12444-12456Crossref PubMed Scopus (75) Google Scholar, 26Zhao 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, 27Zhao K.-H. Wu D. Wang L. Zhou M. Storf M. Bubenzer C. Strohmann B. Scheer H. Eur. J. Biochem. 2002; 269: 4542-4550Crossref PubMed Scopus (28) Google Scholar, 28Zhao K.H. Wu D. Zhou M. Zhang L. Böhm S. Bubenzer C. Scheer H. Biochemistry. 2005; 44: 8126-8137Crossref PubMed Scopus (28) Google Scholar). A similar reaction sequence would lead from PEB to the protein-bound phycourobilin, which is present in many marine cyanobacteria and red algae; a first example for such an enzyme may be the RpeE/F fusion protein from Synechococcus sp. WH8102 (29Six C. Thomas J.-C. Thion L. Lemoine Y. Zal F. Partensky F. J. Bacteriol. 2005; 187: 1685-1694Crossref PubMed Scopus (49) Google Scholar). Two chromophores, PCB and PEB, are biosynthesized from heme by ring opening at C-5 of the tetrapyrrole, followed by reduction and isomerization steps (30Beale S.I. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 519-558Crossref Google Scholar, 31Frankenberg N. Mukougawa K. Kohchi T. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (195) Google Scholar). Heterologous in vivo synthesis of PCB, e.g. in Escherichia coli, is possible by the expression of two genes encoding heme oxygenase 1 (ho1) and PCB:ferredoxin oxidoreductase (pcyA). When these were co-expressed in E. coli, together with the apophytochrome gene, cph1, by a dual plasmid system, photoreversibly active holophytochrome was produced (32Gambetta G.A. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10566-10571Crossref PubMed Scopus (197) Google Scholar, 33Landgraf F.T. Forreiter C. Hurtado Pico A. Lamparter T. Hughes J. FEBS Lett. 2001; 508: 459-462Crossref PubMed Scopus (49) Google Scholar). Similarly, the entire pathway for the synthesis of α-CPC or α-PEC could be reconstituted in E. coli, when ho1 and pcyA were co-expressed with cpcA, cpcE, and cpcF or with pecA, pecE, and pecF, respectively (34Tooley A.J. Cai Y.A. Glazer A.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10560-10565Crossref PubMed Scopus (111) Google Scholar, 35Tooley A.J. Glazer A.N. J. Bacteriol. 2002; 184: 4666-4671Crossref PubMed Scopus (46) Google Scholar). Recently, a group of four genes (cpcS, cpcT, cpcU, and cpcV) has been identified in Synechococcus sp. PCC7002 that code for lyases that attach PCB to cysteine-84 (consensus numbering) of the β-subunits of CPC and possibly allophycocyanin; homologous genes are also found in other cyanobacteria (36Shen 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. Saint Adele, Québec, Canada2004: 14-15Google Scholar). Open reading frame alr0617 in Anabaena sp. PCC7120 (22Kaneko T. Nakamura Y. Wolk C.P. Kuritz T. Sasamoto S. Watanabe A. Iriguchi M. Ishikawa A. Kawashima K. Kimura T. Kishida Y. Kohara M. Matsumoto M. Matsuno A. Muraki A. Nakazaki N. Siumpo S. Sugimoto M. Takazawa M. Yamada M. Yasuda M. Tabata S. DNA Res. 2001; 8: 205-213Crossref PubMed Scopus (583) Google Scholar) is homologous to this new type of lyase, in particular, to cpeS of Fremyella diplosiphon (Calothrix PCC7601) (37Cobley 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 (76) Google Scholar), Gloeobacter violaceus PCC 7421 (38Nakamura Y. Kaneko T. Sato S. Mimuro M. Miyashita H. Tsuchiya T. Sasamoto S. Watanabe A. Kawashima K. Kishida Y. Kiyokawa C. Kohara M. Matsumoto M. Matsuno A. Nakazaki N. Shimpo S. Takeuchi C. Yamada M. Tabata S. DNA Res. 2003; 10: 137-145Crossref PubMed Scopus (226) Google Scholar), and Synechococcus sp. WH8102 (39Palenik B. Brahamsha B. Larimer F.W. Land M. Hauser L. Chain P. Lamerdin J. Regala W. Allen E.E. McCarren J. Paulsen I. Dufresne A. Partensky F. Webb E.A. Waterbury J. Nature. 2003; 424: 1037-1042Crossref PubMed Scopus (534) Google Scholar). Using biochemical, enzymatic, and molecular biological methods, we now show that alr0617 codes for a PCB:phycobiliprotein lyase that catalyzes the covalent attachment of PCB to cysteine-84 of the β-subunits of CPC (CpcB) and PEC (PecB). In analogy to cpeS from Calothrix PCC7601 (37Cobley 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 (76) Google Scholar), we therefore annotate alr0617 as cpeS. 6Anabaena sp. PCC7120 does not produce CPE. The name cpeS was nonetheless retained for alr0617, at least for the time being, in view of the comparably broad substrate specificity of the lyase. The enzymatic function of CpeS from Anabaena sp. PCC7120 was further demonstrated by co-expression in E. coli of dual plasmids containing cpcB(C155I) or pecB(C155I), with cpeS (i.e. alr0617), ho1 and pcyA, resulting in biosynthesis of the respective β-subunits singly chromophorylated at cysteine-β84. Proteins—Cloning and expression generally followed the standard procedures of Sambrook et al. (40Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The integral genes, cpcE and cpcF, were PCR-amplified as described previously from Anabaena sp., pecA, pecE, and pecF from Anabaena sp. PCC 7120 and Mastigocladus laminosus (Fischerella PCC7603) (15Zhao K.-H. Ran Y. Li M. Sun Y.-N. Zhou M. Storf M. Kupka M. Böhm S. Bubenzer C. Scheer H. Biochemistry. 2004; 43: 11576-11588Crossref PubMed Scopus (35) Google Scholar, 25Storf M. Parbel A. Meyer M. Strohmann B. Scheer H. Deng M. Zheng M. Zhou M. Zhao K. Biochemistry. 2001; 40: 12444-12456Crossref PubMed Scopus (75) Google Scholar). Mutants cpcB(C155I), cpcB(C84S), pecB(C155I), and pecB(C84S) were generated from cpcB and pecB of M. laminosus (41Zhao K.H. Zhu J.P. Song B. Zhou M. Storf M. Böhm S. Bubenzer C. Scheer H. Biochim. Biophys. Acta. 2004; 1657: 131-145Crossref PubMed Scopus (26) Google Scholar). The plasmids containing cpeS (= alr0617), cpcB, pecB, and nblB (coding for a degrading lyase (42Dolganov N. Grossman A.R. J. Bacteriol. 1999; 181: 610-617Crossref PubMed Google Scholar) from Anabaena sp. PCC 7120) were constructed in this work using the primers P1-P8 shown below. They were cloned first into pBluescript (Stratagene), and then subcloned into pET30a (Novagen). CpeS, without an His tag, was obtained by expressing pGEMEX (Promega) containing alr0617 (26Zhao 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). For the construction of dual plasmids, the following genes from Anabaena sp. PCC 7120 were PCR-amplified via primers P8-P13: cpeS, ho1 (= all1897, annotated according to its high homology with ho1 from Synechococcus PCC 6803 (43Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2122) Google Scholar) (encoding heme oxygenase 1) and pcyA (= alr3707), annotated according to its homology with pcyA from Synechococcus PCC 6803 (43Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2122) Google Scholar) (encoding PCB:ferredoxin oxidoreductase). The PCR-amplified ho1 and pcyA were cloned together in pACYCDuet-1 (Novagen) to produce pHO-PcyA, and alr0617 was cloned in pCDFDuet-1 (Novagen) to produce pCDF-CpeS. All molecular constructions were verified by sequencing as follows. P1: 5′-GGACCCGGGATGACATTAGACGTATTTAC-3′, upstream; P2: 5′-ATTCTCGAGTTAACCTACAGCAGCAGCAG-3′, downstream; P3: 5′-ATACCCGGGATGCTCGATGCTTTTTC-3′, upstream; P4: 5′-GTGCTCGAGTTAAACAACCGCAGAAGC-3′, downstream; P5: 5′-GTACCCGGGATGAGTATTACACCTGAG-3′, upstream; P6: 5′-CGCCTCGAGCTAAACTGATTGTAAAGACT-3′, downstream; P7: 5′-GCGCCCGGGATGAATATCGAAGAGTTTTTT-3′, upstream; P8: 5′-GGGCTCGAGGTTTTAACTTGACGCAGAATT-3′, downstream; P9: 5′-GGCGATATCCGGGATGAATATTGAAGAGTTT-3′, upstream; P10: 5′-CTACCATGGCGATGAGCAGCAATTTAGCA-3′, upstream; P11: 5′-ATCCTGCAGTTACTCAGCCGTGGCAAGTT-3′, downstream; P12: 5′-CGCGATATCGATGTCACTTACTTCCATTC-3′, upstream; and P13: 5′-TACCTCGAGCCGTTATTCTGGGAGATC-3′, downstream. For P1-P8, all upstream primers have a SmaI site (CCCGGG) and the downstream primers have a XhoI site (CTCGAG) (both marked in bold), which ensure correct ligation of the fragments to pBluescript and then to pET30. P1 and P2 were used for cpcB, P3 and P4 for pecB, P5 and P6 for nblB, and P7 and P8 for alr0617. For P8-P13, the upstream primers for pcyA and alr0617 have an EcoRV site (GATATC), and the downstream primers have an XhoI site (CTCGAG); the upstream primer for ho1 has an NcoI site (CCATTG), and the downstream primer has a PstI site (CTGCAG). P8 and P9 were used for alr0617, P10 and P11 for ho1, and P12 and P13 for pcyA. Expressions—The pET-based plasmids were used to transform E. coli BL21(DE3). Cells were grown in Luria-Bertani (LB) medium containing kanamycin (30 μg·ml-1) at 37 °C for genes from M. laminosus or at 20 °C for genes from Anabaena sp. PCC 7120. When the cell density reached A600 = 0.5-0.7, isopropyl 1-thio-β-d-galactopyranoside (1 mm) was added. Cells were collected by centrifugation 5 h after induction (for genes from M. laminosus) or after 6 h (for genes from Anabaena sp.), then washed twice with doubly distilled water and stored at -20 °C until use (15Zhao K.-H. Ran Y. Li M. Sun Y.-N. Zhou M. Storf M. Kupka M. Böhm S. Bubenzer C. Scheer H. Biochemistry. 2004; 43: 11576-11588Crossref PubMed Scopus (35) Google Scholar, 27Zhao K.-H. Wu D. Wang L. Zhou M. Storf M. Bubenzer C. Strohmann B. Scheer H. Eur. J. Biochem. 2002; 269: 4542-4550Crossref PubMed Scopus (28) Google Scholar). The dual plasmids were transformed together into BL21(DE3) cells under the respective antibiotic selections (chloromycetin for pHO-PcyA and streptomycin for pCDF-cpeS). One of the four plasmids pET-CpcB(C155I), pET-CpcB, pET-PecB(C155I), or pET-PecB, was used together with pCDF-CpeS and pHO-PcyA to produce PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), or PCB-PecB, respectively. In the control experiment, pCDF-CpeS was omitted from the transformations. Cells were grown at 20 °C in LB-medium containing kanamycin (20 μg·ml-1), chloromycetin (17 μg·ml-1), and streptomycin (25 μg·ml-1). When the cell density reached A600 = 0.5-0.7, isopropyl 1-thio-β-d-galactopyranoside (1 mm) was added. Cells were collected by centrifugation 12 h after induction, washed twice with doubly distilled water, and stored at -20 °C until use. Cell pellets were resuspended in ice-cold potassium phosphate buffer (KPP, 20 mm, pH 7.2) containing NaCl (0.5 m) and disrupted by sonication (5 min, 45 W, Branson model 450W). The suspension was centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant containing the crude proteins was purified via Ni2+-affinity chromatography on chelating Sepharose (Amersham Biosciences). The initial buffer was KPP (20 mm, pH 7.2) containing NaCl (1 m), the elution buffer also contained imidazole (0.5 m) or EDTA (100 mm). After collection, the samples were dialyzed twice against the initial buffer and kept at -20 °C until use. Quantification of Phycocyanobilin and Protein—PCB was prepared as described before (25Storf M. Parbel A. Meyer M. Strohmann B. Scheer H. Deng M. Zheng M. Zhou M. Zhao K. Biochemistry. 2001; 40: 12444-12456Crossref PubMed Scopus (75) Google Scholar). PCB concentrations were determined spectroscopically using an extinction coefficient ϵ690 = 37,900 m-1·cm-1 in methanol/2% HCl (44Cole W.J. Chapman D.J. Siegelman H.W. Biochemistry. 1968; 7: 2929-2935Crossref PubMed Scopus (56) Google Scholar). Protein concentrations were determined with the protein assay reagent (Bio-Rad) according to the instructions given by the supplier, using bovine serum albumin as the standard (45Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar). SDS-PAGE—SDS-PAGE was performed with the buffer system of Laemmli (46Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). The gels were stained with Coomassie Brilliant Blue R for the protein, and with ZnCl2 for bilin chromophores (47Berkelman T. Lagarias J.C. Anal. Biochem. 1986; 156: 194-201Crossref PubMed Scopus (231) Google Scholar). The amounts of bilins in reconstituted phycobiliproteins were quantified by comparing their fluorescence intensity on the same SDS-PAGE. Gels were photographed, the negatives were scanned (model NUSCAN 700, Shanghai Zhongjing Computer Ltd., China), and the intensity was evaluated with Photoshop 6.0 (Adobe). Care was taken to avoid saturation. Complex Formation of CpeS with Other Proteins—CpeS with no His tag was incubated with His-tagged apo-proteins (CpcB(C155I) or PecB(C155I)), α84 lyases (CpcE, CpcF, PecE, and PecF), or CpeS at 4 °C overnight. The mixtures were then loaded on a Ni2+ affinity column, washed three times with 5 column volumes of initial buffer (see above), once with the same buffer but also containing 50 mm imidazole, and, finally, with the same buffer containing 0.5 m imidazole. The eluate from the last wash was analyzed by SDS-PAGE. Spectroscopy—UV-visible absorption spectra were recorded by a Lamda 25 spectrometer (PerkinElmer Life Sciences). Formation of the photochromic PVB-PecA (i.e. α-PEC) in the lyase reaction was monitored by the absorption at 570 nm and by double-difference spectroscopy of the reversible photoreaction of the PVB chromophore as previously described (48Zhao K.H. Scheer H. Biochim. Biophys. Acta. 1995; 1228: 244-253Crossref Scopus (58) Google Scholar). Fluorescence spectra were recorded with a spectrofluorometer (PerkinElmer Life Sciences model LS 45), and were not corrected. Excitation was done on the blue side of the visible absorption maxima (580-620 nm). The extinction coefficients of the reconstituted and biosynthesized PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), and PCB-PecB were determined based on the extinction coefficient of PCB in CPC in 8 m acidic urea (49Glazer A.N. Fang S. J. Biol. Chem. 1973; 248: 659-662Abstract Full Text PDF PubMed Google Scholar) (ϵ660 = 35,500 m-1cm-1). Fluorescence yields ΦF of the reconstituted and biosynthesized PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), and PCB-PecB were determined in KPP buffer (20 mm, pH 7.2), using the known fluorescence quantum yield (ΦF = 0.27) of CPC from Anabaena sp. PCC 7120 (50Cai Y.A. Murphy J.T. Wedemayer G.J. Glazer A.N. Anal. Biochem. 2001; 290: 186-204Crossref PubMed Scopus (70) Google Scholar) as standard. Lyase Activity Assay: Optimization of Reconstitution—PCB was reconstituted by incubating the desired apoprotein (CpcB(C155I), CpcB(C84S), CpcB, PecB(C155I), PecB(C84S), or PecB, 12-46 μm), with CpeS (12-40 μm) in KPP (20-1000 mm, pH 6.0-11.0) containing one or more of the following: Tris·HCl (0-100 mm), NaCl (0-500 mm), mercaptoethanol (ME, 0-5 mm), various metal ions (0-5 mm), EDTA (0-20 mm), Triton X-100 (TX100) or reduced TX100 (0-1% v/v) and glycerol (0-25% v/v). Then PCB (final concentration = 3-10 μm) was added as a concentrated solution ín Me2SO (0.3-1 mm), such that the final concentration of Me2SO was 1% (v/v). The mixture was incubated at 20-75 °C for 1-3 h in the dark. Reconstitution was assayed via the absorption and fluorescence of the mixture. Optimum conditions were as follows: KPP (500 mm, pH 7.5), PCB (5 μm), NaCl (150 mm), EDTA (1 mm), no Tris·HCl, ME, metal ions, TX100, or glycerol, incubation for 1 h at 37 °C. After centrifugation at 12,000 × g for 15 min, the supernatant was inspected by absorption and fluorescence spectroscopy. The sample was then purified by Ni2+-affinity chromatography (see above) and the imidazole (500 mm) eluate was dialyzed to remove imidazole. The dialysate was then inspected again by absorption and fluorescence spectroscopy. For kinetic tests, only purified proteins were used. Reconstitution of PCB with CpcB and PecB was followed by fluorescence (emission at 645 and 630 nm and excitation at 620 and 600 nm, respectively). The purified apo-proteins (CpcB(C155I) or PecB(C155I), 25 μm) and different amounts of PCB were incubated at 37 °C in the optimum reconstitution system (see above). At regular time intervals, the reaction was terminated by rapidly cooling the samples on ice to 0 °C, and then the product was analyzed spectroscopically without delay (11Zhao K.H. Su P. Böhm S. Song B. Zhou M. Bubenzer C. Scheer H. Biochim. Biophys. Acta. 2005; 1706: 81-87Crossref PubMed Scopus (63) Google Scholar, 27Zhao K.-H. Wu D. Wang L. Zhou M. Storf M. Bubenzer C. Strohmann B. Scheer H. Eur. J. Biochem. 2002; 269: 4542-4550Crossref PubMed Scopus (28) Google Scholar). Km, vmax, and kcat were calculated from Lineweaver-Burk plots, using Origin V7 (Origin Lab Corp.). Chromophore Cleavage Assay—CpeS (2 μm) was incubated for 3 h at 37 °C with donor chromoprotein (reconstituted and purified PCB-CpcB(C155I) or PCB-PecB(C155I), 1.0 μm)), PecA (4 μm), and PecE/F (4 μm). In the control experiments, CpeS was omitted under otherwise identical conditions. Chromophore transfer was assayed by the reversible photochemistry of reconstituted PVB-PecA (i.e. α-PEC (48Zhao K.H. Scheer H. Biochim. Biophys. Acta. 1995; 1228: 244-253Crossref Scopus (58) Google Scholar)). Chromopeptides from PCB-CpcB(C155I) and PCB-PecB(C155I)— Reconstituted chromoproteins, CpcB(C155I) or PecB(C155I), were purified by Ni2+ chromatography (see above) and dialyzed against KPP (20 mm, pH 7.2). The desired chromoprotein solution was acidified with HCl to pH 1.5, and pepsin was added to the sample (1:1, w/w). The mixture was incubated for 3 h at 37 °C and then fractionated on Bio-Gel P-60 (Bio-Rad) equilibrated with dilute HCl (pH 2.5). Colorless peptides and salts were eluted with the same solvent (51Zhao K.H. Haessner R. Cmiel E. Scheer H. Biochim. Biophys. Acta. 1995; 1228: 235-243Crossref Scopus (55) Google Scholar), and the adsorbed chromopeptides were eluted with acetic acid (30%, v/v) in dilute HCl (pH 2.5). The collected samples were dried with a rotary evaporator and subjected to HPLC (Waters 2695 system with model 2487 variable wavelength detector) on a Zorbax 300SB-C18 column (Agilent Technologies) using a gradient of KPP (100 mm, pH 2.1) and acetonitrile (80:20 to 60:40), as described by Storf (52Storf M. Chromophorbindung und Photochemie der α-Untereinheit des Phycoerythrocyanins aus Mastigocladus laminosus, Dissertation. Ludwig Maximilians Universität, München2004Google Scholar). For the controls, natural β-CPC and β-PEC were isolated from M. laminosus via isoelectric focusing (48Zhao K.H. Scheer H. Biochim. Biophys. Acta. 1995; 1228: 244-253Crossref Scopus (58) Google Scholar) and subjected to the same procedure. Expression and Purification of the Apoproteins and CpeS—Shen et al. (36Shen 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. Saint Adele, Québec, Canada2004: 14-15Google Scholar) have recently identified lyase activities for the products of a family of genes, cpcS, -U, and -V, and for the more distantly related cpcT from Synechococcus sp. PCC7002. The genome of Anabaena sp. PCC7120 contains a single gene homologous to cpcS, namely alr0617. This gene was expressed in E. coli, with and without N-terminal His and S tags. Incubation of PCB and CpcB with CpeS resulted in a rapid increase of absorption ∼619 nm and the emergence of a bright red fluorescence (λmax = 643 nm). Identical results were obtained with a mutant lacking the β-155 binding site, namely CpcB(C155I) (Fig. 1A), however, there was no such reaction with mutant CpcB(C84S) lacking the binding cysteine-β84 (see below). The affinity-purified product has the intense visible absorption and fluorescence typical of native biliproteins, with the position of the bands red-shifted compared with holo-β-CPC, peaking in the range associated with the cysteine-β84 chromophore of CPC (53Debreczeny M.P. Sauer K. Zhou J. Bryant D.A. J. Phys. Chem. 1995; 99: 8412-8419Crossref Scopus (65) Google Scholar, 54Sauer K. Scheer H. Biochim. Biophys. Acta. 19
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