Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase
2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês
10.1074/jbc.m809784200
ISSN1083-351X
AutoresNicolas Blot, Xianjun Wu, Jean‐Claude Thomas, Juan Zhang, Laurence Garczarek, Stephan Böhm, Jun‐Ming Tu, Ming Zhou, Matthias Plöscher, Lutz A. Eichacker, Frédéric Partensky, Hugo Scheer, Kai‐Hong Zhao,
Tópico(s)Light effects on plants
ResumoMost cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. To perform photosynthesis, the main energetic basis for life on earth, phototrophic organisms have to cope with large spatial and temporal variations of light conditions. A major evolutionary step in meeting this challenge was the development of light-harvesting complexes, the most variable part of the photosynthetic apparatus (1Glazer A.N. J. Biol. Chem. 1989; 264: 1-4Abstract Full Text PDF PubMed Google Scholar). By binding a large number of chromophores, these antennas can considerably enhance the photon absorption capacity of reaction centers that are responsible for the conversion of solar energy into chemical energy. Pigmented proteins associated with light-harvesting complexes also fill (at least partially) the large gap between the absorption bands of reaction center chlorophylls (e.g. ∼440 and 680 nm for chlorophyll a found in most oxygenic organisms). Antennas also transport the excitons with minimal loss and transduce high energy excitons into the low energy ones required by the reaction centers (1Glazer A.N. J. Biol. Chem. 1989; 264: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Green B. Parson W. Light-harvesting Antennas in Photosynthesis. Kluwer, Dordrecht, The Netherlands2003Crossref Google Scholar). They do not only vary among the different organisms but also with time within individual organisms, thereby providing the flexibility needed by the photosynthetic apparatus to work efficiently under varying ambient conditions. Cyanobacteria, which contribute a substantial fraction of global photosynthesis (3Garcia-Pichel F. Belnap J. Neuer S. Schanz F. Algol. Stud. 2003; 109: 213-228Crossref Google Scholar), evolved a particularly sophisticated and dynamic antenna complex, the phycobilisome (PBS) 4The abbreviations used are: PBS, phycobilisome; PC, phycocyanin; C-PC, C-type phycocyanin; PCB, phycocyanobilin; R-PC, R-phycocyanin (prefix R originally referred to as “rhodophytes,” but now designates spectral type); RpcA, apo-α-subunit of R-PC; RpcG, fused PUB lyase (EF-type); IEF, isoelectric focusing; PE, phycoerythrin; PEB, phycoerythrobilin; PEC, phycoerythrocyanin; PecA, apo-α-PEC; PecE, PecF, subunits of PVB:α-PEC lyase-isomerase; PUB, phycourobilin; PVB, phycoviolobilin; HPLC, high pressure liquid chromatography. (4Gantt E. Staehelin L.A. Arntzen C.J. Photosynthesis III: Photosynthetic Membranes and Light-harvesting Systems. Springer, Berlin1986: 260-268Google Scholar, 5Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 139-216Crossref Google Scholar). This extramembranous structure with a size of several MDa is mainly composed of the deeply colored and intensely fluorescing phycobiliproteins. A single PBS, generally composed of a central core and 6–8 radiating rods, contains hundreds of linear tetrapyrrole chromophores (phycobilins) that are covalently attached to their apoproteins. The remarkably wide diversity of PBSs found in nature is due to the large number of possible combinations of phycobiliproteins with various pigmentations that constitute the rods. Marine Synechococcus, the second most abundant oxyphototrophic organism on Earth after Prochlorococcus (3Garcia-Pichel F. Belnap J. Neuer S. Schanz F. Algol. Stud. 2003; 109: 213-228Crossref Google Scholar, 6Partensky F. Blanchot J. Vaulot D. Charpy L. Larkum A. Marine Cyanobacteria. Musée Océanographique, Monaco1999: 457-475Google Scholar), is the cyanobacterial group in which the largest diversity of PBS rod composition (and hence pigmentation) can be found (7Six C. Thomas J.C. Garczarek L. Ostrowski M. Dufresne A. Blot N. Scanlan D.J. Partensky F. Genome Biol. 2007; 8: R259Crossref PubMed Scopus (211) Google Scholar). This is particularly useful to enable this group to cope with the variety of light environments encountered in marine ecosystems. PBSs can, at least in part, be reversibly dissociated (8Canaani O. Lipschultz C.A. Gantt E. FEBS Lett. 1980; 115: 225-229Crossref Scopus (23) Google Scholar, 9Lipschultz C.A. Gantt E. Biochemistry. 1981; 20: 3371-3376Crossref PubMed Scopus (13) Google Scholar, 10Lundell D.J. Williams R.C. Glazer A.N. J. Biol. Chem. 1981; 256: 3580-3592Abstract Full Text PDF PubMed Google Scholar). Progress in understanding PBS assembly was slow, however, due to the complex and poorly understood post-translational modifications of the phycobiliproteins required prior to assembly. Only after maturation can phycobiliproteins form spontaneously trimers, which are then integrated into the PBSs by interaction with specific structural proteins, so-called linker polypeptides. These post-translational modifications reactions include the covalent attachment of 1–4 chromophores to each individual apoprotein (11Fairchild C.D. Glazer A.N. J. Biol. Chem. 1994; 269: 8686-8694Abstract Full Text PDF PubMed Google Scholar, 12Schluchter W.M. Glazer A.N. Peschek G.A. Löffelhardt W. Schmetterer G. The Phototrophic Prokaryotes. Kluwer/Plenum Press, New York1999: 83-95Crossref Google Scholar, 13Schluchter W.M. Bryant D.A. Smith A.G. Witty M. Heme, Chlorophyll, and Bilins. Humana Press, Totowa, NJ2002: 311-334Google Scholar, 14Storf M. Parbel A. Meyer M. Strohmann B. Scheer H. Deng M. Zheng M. Zhou M. Zhao K. Biochemistry. 2001; 40: 12444-12456Crossref PubMed Scopus (74) Google Scholar, 15Saunée N.A. Williams S.R. Bryant D.A. Schluchter W.M. J. Biol. Chem. 2008; 283: 7513-7522Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 17Shen G. Saunee 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 (80) Google Scholar, 18Scheer H. Zhao K.-H. Mol. Microbiol. 2008; 68: 263-276Crossref PubMed Scopus (137) Google Scholar, 19Zhao K.-H. Zhang J. Tu J.M. Böhm S. Plöscher M. Eichacker L. Bubenzer C. Scheer H. Wang X. Zhou M. J. Biol. Chem. 2007; 282: 34093-34103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Zhao K.-H. Su P. Tu J.M. Wang X. Liu H. Plöscher M. Eichacker L. Yang B. Zhou M. Scheer H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14300-14305Crossref PubMed Scopus (88) Google Scholar), methylation of an asparagine residue (21Miller C.A. Leonard H.S. Pinsky I.G. Turner B.M. Williams S.R. Harrison Jr., L. Fletcher A.F. Shen G. Bryant D.A. Schluchter W.M. J. Biol. Chem. 2008; 283: 19293-19300Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 22Shen G. Leonard H.S. Schluchter W.M. Bryant D.A. J. Bacteriol. 2008; 190: 4808-4817Crossref PubMed Scopus (15) Google Scholar, 23Thomas B.A. Bricker T.M. Klotz A.V. Biochim. Biophys. Acta. 1993; 1143: 104-108Crossref Scopus (14) Google Scholar), and cleavage of N-terminal methionine residue (5Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 139-216Crossref Google Scholar, 24Liu L.N. Chen X.L. Zhang Y.Z. Zhou B.C. Biochim. Biophys. Acta. 2005; 1708: 133-142Crossref PubMed Scopus (147) Google Scholar) (see UniProtKB/Swiss-Prot entry Q1XDQ2). Of the three major phycobilins, the red-absorbing phycocyanobilin (PCB) and the green-absorbing phycoerythrobilin (PEB) are generated by specialized reductases from biliverdin (Fig. 1), the cleavage product of the heme macrocycle (26Dammeyer T. Frankenberg-Dinkel N. Photochem. Photobiol. Sci. 2008; 7: 1121-1130Crossref PubMed Scopus (60) Google Scholar). Free PCB and PEB molecules are then attached by phycobilin lyases to specific binding sites on the phycobiliprotein subunits (18Scheer H. Zhao K.-H. Mol. Microbiol. 2008; 68: 263-276Crossref PubMed Scopus (137) Google Scholar). Biosynthesis of a third chromophore, phycourobilin (PUB), which has never been found in free form, was until now enigmatic. Phycobiliproteins from all marine cyanobacteria adapted to oceanic waters, including Synechococcus, Crocosphaera, and Trichodesmium, are particularly rich in PUB, probably making it the most abundant phycobilin in the ocean (7Six C. Thomas J.C. Garczarek L. Ostrowski M. Dufresne A. Blot N. Scanlan D.J. Partensky F. Genome Biol. 2007; 8: R259Crossref PubMed Scopus (211) Google Scholar, 27Swanson R.V. Ong L.J. Wilbanks S.M. Glazer A.N. J. Biol. Chem. 1991; 266: 9528-9534Abstract Full Text PDF PubMed Google Scholar, 28Ong L.J. Glazer A.N. J. Biol. Chem. 1991; 266: 9515-9527Abstract Full Text PDF PubMed Google Scholar, 29Ong L.J. Glazer A.N. Waterbury J.B. Science. 1984; 224: 80-83Crossref PubMed Scopus (83) Google Scholar, 30Subramaniam A. Carpenter E.J. Karentz D. Falkowski P.G. Limnol. Oceanogr. 1999; 44: 608-617Crossref Scopus (122) Google Scholar). Indeed, this chromophore absorbs efficiently blue-green light (λmax ∼495 nm), a wavelength range prevailing in open oceanic subsurface waters and which is only poorly absorbed by chlorophyll a. Thus, elucidating the PUB biosynthesis process has been a challenge for many years. So far, only three types of phycocyanin were known in marine Synechococcus spp., and none of them contained PUB. Among the 11 recently sequenced strains (31Dufresne A. Ostrowski M. Scanlan D.J. Garczarek L. Mazard S. Palenik B.P. Paulsen I.T. Tandeau de Marsac N. Wincker P. Dossat C. Ferriera S. Johnson J. Post A.F. Hess W.R. Partensky F. Genome Biol. 2008; 9: R90Crossref PubMed Scopus (250) Google Scholar), two (WH5701 and RS9917) have PBSs with rods entirely constituted of C-type phycocyanin (C-PC (7Six C. Thomas J.C. Garczarek L. Ostrowski M. Dufresne A. Blot N. Scanlan D.J. Partensky F. Genome Biol. 2007; 8: R259Crossref PubMed Scopus (211) Google Scholar)), a form frequently found in freshwater cyanobacteria (5Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 139-216Crossref Google Scholar). This C-PC binds PCB at all three available chromophore binding sites: α-84, β-82, and β-153. Several phycoerythrin-containing marine Synechococcus strains possess phycocyanin of the R-type that carry PEB either at β-153 only (R-PC III), as in WH7805, or both at α-84 and at β-153 (R-PC II), as in WH7803 (5Sidler W.A. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer, Dordrecht, The Netherlands1994: 139-216Crossref Google Scholar, 32Ong L.J. Glazer A.N. Stevens S.E. Bryant D.A. Light-Energy Transduction in Photosynthesis: Higher Plant and Bacterial Models. American Society of Plant Physiologists, Rockville, MD1988: 102-121Google Scholar, 33Ong L.J. Glazer A.N. J. Biol. Chem. 1987; 262: 6323-6327Abstract Full Text PDF PubMed Google Scholar). Using comparative genomics, candidate genes were recently retrieved that code for the different phycobilin lyases required for catalyzing the chromophorylation of these various phycocyanins (7Six C. Thomas J.C. Garczarek L. Ostrowski M. Dufresne A. Blot N. Scanlan D.J. Partensky F. Genome Biol. 2007; 8: R259Crossref PubMed Scopus (211) Google Scholar). For the C-PC, it was demonstrated in freshwater cyanobacteria (15Saunée N.A. Williams S.R. Bryant D.A. Schluchter W.M. J. Biol. Chem. 2008; 283: 7513-7522Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16Shen G. Schluchter W.M. Bryant D.A. J. Biol. Chem. 2008; 283: 7503-7512Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 17Shen G. Saunee 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 (80) Google Scholar, 18Scheer H. Zhao K.-H. Mol. Microbiol. 2008; 68: 263-276Crossref PubMed Scopus (137) Google Scholar, 19Zhao K.-H. Zhang J. Tu J.M. Böhm S. Plöscher M. Eichacker L. Bubenzer C. Scheer H. Wang X. Zhou M. J. Biol. Chem. 2007; 282: 34093-34103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Zhao K.-H. Su P. Tu J.M. Wang X. Liu H. Plöscher M. Eichacker L. Yang B. Zhou M. Scheer H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14300-14305Crossref PubMed Scopus (88) Google Scholar) that three phycobilin lyases, the heterodimeric CpcE/F, the monomeric CpcS (also sometimes found in heterodimer with CpcU), and the monomeric CpcT, are needed to catalyze the attachment of PCB at α-84, β-84, and β-155, respectively (note that the positions of the chromophore binding sites slightly differ between the α-phycocyanin of freshwater and marine cyanobacteria), and all three sequences have clear orthologs in the WH5701 and RS9917 genomes. WH7805 contains phycobilin lyase genes (called rpcE-F) that are homologous to cpcE-F, and it has been suggested that they could encode a phycocyanin α-84 PEB lyase (34Wilbanks S.M. Glazer A.N. J. Biol. Chem. 1993; 268: 1226-1235Abstract Full Text PDF PubMed Google Scholar). In WH7803, the cpcT gene is missing and replaced by rpcT, which has been proposed to encode a phycocyanin β-153 PEB lyase (7Six C. Thomas J.C. Garczarek L. Ostrowski M. Dufresne A. Blot N. Scanlan D.J. Partensky F. Genome Biol. 2007; 8: R259Crossref PubMed Scopus (211) Google Scholar). In four sequenced marine Synechococcus, including two oceanic strains with a particularly high PUB content, WH8102 and CC9605, and two type IV chromatic adapters, RS9916 and BL107, the rpcE-F operon is replaced by a single gene, rpcG (7Six C. Thomas J.C. Garczarek L. Ostrowski M. Dufresne A. Blot N. Scanlan D.J. Partensky F. Genome Biol. 2007; 8: R259Crossref PubMed Scopus (211) Google Scholar). This gene appears to encode a fusion protein, and its N and C termini show much higher homology to Nostoc (Anabaena) sp. PCC 7120 PecE and PecF, respectively, which form the heterodimeric phycoerythrocyanin α-84 PCB lyase-isomerase, than to CpcE/F or RpcE/F lyases from other marine Synechococcus. Moreover, the RpcG C terminus carries a short motif (NHCQGN) that has been assigned an isomerase function in the F-subunit of PecF (35Zhao K.H. Wu D. Zhou M. Zhang L. Böhm S. Bubenzer C. Scheer H. Biochemistry. 2005; 44: 8126-8137Crossref PubMed Scopus (28) Google Scholar). This motif is responsible for the concomitant isomerization of the A-ring of PCB to generate the PVB chromophore. The analogous isomerization starting from free PEB would thus be expected to generate a cysteine-bound PUB chromophore (Fig. 1). This hypothesis led us to explore the function and specificity of the putative lyase-isomerase RpcG from two marine Synechococcus strains by a heterologous approach in Escherichia coli and to investigate the implications for phycobiliprotein pigmentation. Preparation of Intact Phycobilisomes-Marine Synechococcus spp. strains WH8102 and RS9916 were grown in PCR-S11 medium under continuous white light as described previously (36Everroad C. Six C. Partensky F. Thomas J.C. Holtzendorff J. Wood A.M. J. Bacteriol. 2006; 188: 3345-3356Crossref PubMed Scopus (90) Google Scholar). Extraction of intact PBSs was carried out with 0.75 m phosphate buffer on a sucrose density gradient as described previously (37Six C. Thomas J.-C. Thion L. Lemoine Y. Zal F. Partensky F. J. Bacteriol. 2005; 187: 1685-1694Crossref PubMed Scopus (49) Google Scholar). PBSs used for purifying R-PC V were concentrated by ultracentrifugation, and pellets were either used immediately or kept frozen at -30 °C until use. Isolation of the Trichromatic Phycocyanin, R-PC V-PBS pellets were solubilized in Hepes buffer (10 mm, pH 7.2) containing NaCl (5 mm), Pefabloc (1 mm), EDTA (1 mm), and β-mercaptoethanol (1%, v/v). PBS dissociation was allowed for 90 min at 0 °C. Individual phycobiliproteins were separated by non-denaturing isoelectric focusing (IEF) using 6% polyacrylamide (30% acrylamide, 1.6% bisacrylamide) tube gels, 4 mm in diameter, containing a convenient mixture of ampholytes (4% Servalyt 4–6, 1% Servalyt 3–7). Protein (200 μg) was loaded on the gel, and the isoelectric focusing was achieved with NaOH (20 mm) as a cathode buffer and phosphoric acid (0.045%) as the anode buffer (36Everroad C. Six C. Partensky F. Thomas J.C. Holtzendorff J. Wood A.M. J. Bacteriol. 2006; 188: 3345-3356Crossref PubMed Scopus (90) Google Scholar). Voltage was gradually increased from 150 to 400 V by 50-V steps every 45 min. Phycocyanin was found focusing on the basic part of the gel, as a violet purple band, near various PE complexes that were all more acidic. The allophycocyanin components, focusing at lower acidic pH, were lost under these conditions. Bands of interest were cut out and kept frozen until further characterization. Subunit Isolation by Denaturing Isoelectric Focusing and Mass Spectrometry-Denaturing polyacrylamide gels were prepared in 4-mm tubes as described above, the only modification being the addition of urea (8 m). Bands containing native biliproteins obtained by non-denaturing IEF were cut into small pieces and incubated in situ, on the top of urea acrylamide gels, in urea (9 m) containing mercaptoethanol (1% v/v) for 45 min, before starting the IEF in the same conditions as described above. All operations were made under dim light or, when possible, in complete darkness. Colored bands were cut out at the end of electrophoresis and frozen until use. For mass spectrometric characterization of polypeptides, bands were cut out from IEF gels and fragmented into small pieces, denatured with 6% lithium dodecyl sulfate denaturation buffer, and loaded on lithium dodecyl sulfate-PAGE plate gels as described (37Six C. Thomas J.-C. Thion L. Lemoine Y. Zal F. Partensky F. J. Bacteriol. 2005; 187: 1685-1694Crossref PubMed Scopus (49) Google Scholar). The Coomassie Blue G 250-stained bands of phycobiliprotein subunits were analyzed by mass spectrometry using the facility at the “Unité de Phytopharmacie et Médiateurs Chimiques,” Institut Nationale de la Recherche Agronomique (INRA) Versailles, France. Details on mass spectrometry experimental procedures can be found elsewhere (36Everroad C. Six C. Partensky F. Thomas J.C. Holtzendorff J. Wood A.M. J. Bacteriol. 2006; 188: 3345-3356Crossref PubMed Scopus (90) Google Scholar). In-gel Spectroscopic Analyses of Separated Biliproteins-Absorption spectra were obtained with a model DW2 spectrophotometer (Aminco Chance, Bogart, GA) using fragments of acrylamide gels containing the separated biliproteins. Polyacrylamide ampholyte gel fragments, with or without 8 m urea, were used as a blank. For recording the absorption spectra of denatured α- and β-subunits of R-PC V, samples were soaked for 15 min in acidic urea (8 m, acidified with HCl to pH 3) before recording the spectra. Gene Cloning-Cloning and expression followed generally the standard procedures (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The genes cpcB and pecB were PCR-amplified from Fischerella sp. PCC7603 (Mastigocladus laminosus), subsequently producing cpcB(C84S), cpcB(C155I), pecB(C84A), and pecB(C155I) via site-directed mutation (39Zhao 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); cpcA, cpcE, cpcF, cpcS, cpcT, ho1, pcyA, pecA, pecE, pecF from Nostoc sp. PCC 7120 (19Zhao K.-H. Zhang J. Tu J.M. Böhm S. Plöscher M. Eichacker L. Bubenzer C. Scheer H. Wang X. Zhou M. J. Biol. Chem. 2007; 282: 34093-34103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 40Zhao K.H. Su P. Li J.A. Tu J.M. Zhou M. Bubenzer C. Scheer H. J. Biol. Chem. 2006; 281: 8573-8581Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); and cpeA1, cpeB, pebA, and pebB from Tolypothrix (Calothrix) sp. PCC 7601 and then subsequently producing cpeA(C139S) and cpeB(C48A/C59S/C165S) via site-directed mutation (20Zhao K.-H. Su P. Tu J.M. Wang X. Liu H. Plöscher M. Eichacker L. Yang B. Zhou M. Scheer H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14300-14305Crossref PubMed Scopus (88) Google Scholar). The genes cpeA2, rpcG, mpeA, and rpcA from Synechococcus sp. WH8102 and the genes pebA, pebB, ho1, rpcA, and rpcG from Synechococcus sp. RS9116 were PCR-amplified with the respective primers (supplemental Table S1), then cloned into pBluescript SK (Stratagene, Beijing, China) or TOPO-TA cloning vector (Invitrogen), and then subcloned into pET-30 (Novagen, Munich, Germany). For application of the multiplasmid expression-chromophorylation system in E. coli (20Zhao K.-H. Su P. Tu J.M. Wang X. Liu H. Plöscher M. Eichacker L. Yang B. Zhou M. Scheer H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14300-14305Crossref PubMed Scopus (88) Google Scholar), the above cloned genes were constructed into the corresponding duet plasmids (supplemental Table S2). Heterologous Gene Expression-Duet plasmids were transformed together into BL21(DE3) cells under the appropriate antibiotic selections (chloramphenicol for pACYC-derivative, streptomycin for pCDF-derivative, kanamycin for pCOLA-derivative or pET30-derivative, ampicillin for pETDuet-derivative, see supplemental Table S2). To test the chromophorylation of the various apoproteins (CpcA, CpcB(C84S), CpcB(C155I), PecA, PecB(C84A), PecB(C155I), CpeA1, CpeA(C139S), CpeB, CpeB(C48A/C59S/C165S), and CpeA2, MpeA, RpcA), the respective encoding plasmid was transformed together with PCB- or PEB-producing plasmids (pACYC-ho1-pcyA for PCB, pCDF-ho1-pebB plus pACYC-pebA for PEB) and one or two of the lyase-producing plasmids into E. coli BL21(DE3) (supplemental S2). In the control experiments, plasmids containing lyase genes were omitted from the transformations. For chromophorylation in E. coli, cells were grown at 16–20 °C, depending on the genes to be expressed. 18–24 h after induction with isopropyl-1-thio-β-d-galactopyranoside (1 mm), cells were collected by centrifugation, washed twice with doubly distilled water, and stored at -20 °C until use. Isolation of Heterologously Synthesized Chromoproteins-For isolation and purification of chromophorylated proteins, E. coli cells were suspended is Tris-HCl buffer (20 mm, pH 8) containing NaCl (300 mm) and imidazole (5 mm) and lysed by ultrasonication. The lysate was then centrifuged twice (10 min, 10,000 × g) to remove cell debris, and tagged proteins were isolated from the supernatant by Ni2+ chromatography as described previously (19Zhao K.-H. Zhang J. Tu J.M. Böhm S. Plöscher M. Eichacker L. Bubenzer C. Scheer H. Wang X. Zhou M. J. Biol. Chem. 2007; 282: 34093-34103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 41Zhao K.-H. Wu D. Zhang L. Zhou M. Böhm S. Bubenzer C. Scheer H. FEBS J. 2006; 273: 1262-1274Crossref PubMed Scopus (27) Google Scholar). If necessary, the affinity-enriched proteins were further purified by fast protein liquid chromatography (Amersham Biosciences, Shanghai, China) over a DEAE FF column developed with a gradient of 0–0.6 m NaCl in potassium phosphate buffer (20 mm, pH 7.0). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis-SDS-PAGE was performed with the buffer system of Laemmli (42Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3025) Google Scholar). The gels were stained with zinc acetate for bilin chromophores (43Berkelman T. Lagarias J.C. Anal. Biochem. 1986; 156: 194-201Crossref PubMed Scopus (229) Google Scholar) and subsequently with Coomassie Brilliant Blue for the protein detection. Spectroscopy-The chromophorylation of phycobiliproteins was analyzed by UV-visible absorption (model Lambda 25, PerkinElmer Life Sciences, Shanghai, China, or model UV-2401PC, Shimadzu, Duisburg, Germany), circular dichroism (model J-810 spectropolarimeter, JASCO, Shanghai, China), and fluorescence spectroscopy (PerkinElmer Life Sciences model LS45 or model LS-50B). Fluorescence of PCB-chromoproteins could be detected by the emission at 630 or 645 nm (19Zhao K.-H. Zhang J. Tu J.M. Böhm S. Plöscher M. Eichacker L. Bubenzer C. Scheer H. Wang X. Zhou M. J. Biol. Chem. 2007; 282: 34093-34103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 40Zhao K.H. Su P. Li J.A. Tu J.M. Zhou M. Bubenzer C. Scheer H. J. Biol. Chem. 2006; 281: 8573-8581Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), of PEB-chromoproteins at 575 nm (20Zhao K.-H. Su P. Tu J.M. Wang X. Liu H. Plöscher M. Eichacker L. Yang B. Zhou M. Scheer H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14300-14305Crossref PubMed Scopus (88) Google Scholar), of PUB-chromoproteins at 505 nm (44MacColl R. Eisele L.E. Williams E.C. Bowser S.S. J. Biol. Chem. 1996; 271: 17157-17160Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 45Yu M.H. Glazer A.N. Spencer K.G. West J.A. Plant Physiol. 1981; 68: 482-488Crossref PubMed Google Scholar), and of PVB-chromoproteins at 580 nm (46Zhao K.H. Haessner R. Cmiel E. Scheer H. Biochim. Biophys. Acta. 1995; 1228: 235-243Crossref Scopus (55) Google Scholar). PCB, PEB, and Protein Concentration Determinations-The covalently bound chromophores in phycobiliproteins were quantified by absorption spectroscopy in acidic urea (8 m, pH 1.5) (13Schluchter W.M. Bryant D.A. Smith A.G. Witty M. Heme, Chlorophyll, and Bilins. Humana Press, Totowa, NJ2002: 311-334Google Scholar) using the molar extinction coefficients of PCB at 662 nm (∈ = 35,500 m-1cm-1) (47Glazer A.N. Fang S. J. Biol. Chem. 1973; 248: 659-662Abstract Full Text PDF PubMed Google Scholar), of PEB at 550 nm (∈ = 42,800 m-1cm-1) (48Glazer A.N. Hixson C.S. J. Biol. Chem. 1975; 250: 5487-5495Abstract Full Text PDF PubMed Google Scholar), of PUB at 495 nm (∈ = 104,000 m-1cm-1) (49Glazer A.N. Hixson C.S. J. Biol. Chem. 1977; 252: 32-42Abstract Full Text PDF PubMed Google Scholar), and of PVB at 590 nm (∈ = 38,600 m-1cm-1) (50Bishop J.E. Rapoport H. Klotz A.V. Chan C.F. Glazer A.N. Füglistaller P. Zuber H. J. Am. Chem. Soc. 1987; 109: 875-881Crossref Scopus (57) Google Scholar). Protein concentrations were determined according to the Bradford protein assay (51Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215560) Google Scholar), using bovine serum albumin as standard. Analyses of Covalently Bound PUB or PVB-To determine the covalently bound PUB or PVB, the purified and dialyzed chromophorylated phycobiliproteins were digested with trypsin. The
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