Báo cáo khoa học: Chromophore attachment in phycocyanin Functional amino acids of phycocyanobilin – a-phycocyanin lyase and evidence for chromophore binding doc

Chromophore attachment in phycocyanin Functional amino acids of phycocyanobilin – a-phycocyanin lyase and evidence for chromophore binding Kai-Hong Zhao1, Dong Wu1, Ling Zhang1, Ming Zhou1, Stephan Bohm2, Claudia Bubenzer2 ă and Hugo Scheer2 College of Life Science and Technology, Huazhong University of Science and Technology, Hubei, China Department Biologie I Bereich Botanik, Universitat Munchen, Germany ă ă Keywords biliproteins; biosynthesis; cyanobacteria; photosynthesis; post-translational modification Correspondence K.-H Zhao, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, China Tel ⁄ Fax: +86 27 8754 1634 E-mail: kaihongzhao@tom.com H Scheer, Department Biologie I Bereich Botanik, Universitat Munchen, ă ă Menzinger Str 67, D-80638 Munich, Germany Fax: +49 89 17861 271 Tel.: +49 89 17861 295 E-mail: hugo.scheer@lmu.de (Received 10 November 2005, revised 17 January 2006, accepted 20 January 2006) doi:10.1111/j.1742-4658.2006.05149.x Covalent attachment of phycocyanobilin (PCB) to the a-subunit of C-phycocyanin, CpcA, is catalysed by the heterodimeric PCB : CpcA lyase, CpcE ⁄ F [Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA & Glazer AN (1992) Proc Natl Acad Sci USA 89, 7017–7021] CpcE and CpcF of the cyanobacterium, Mastigocladus laminosus PCC 7603, form a : complex Lyase-mutants were constructed to probe functional domains When in CpcE (276 residues) the N terminus was truncated beyond the R33YYAAWWL motif, or the C terminus beyond amino acid 237, the enzyme became inactive Activity decreases to 20% when C-terminal truncations went beyond L275, which is a key residue: the Km of CpcE(L275D) and (L276D) increased by 61% and 700%, kcat ⁄ Km decreased 3- and 83-fold, respectively The enzyme also lost activity when in CpcF (213 residues) the 20 N-terminal amino acids were truncated; truncation of 53 C-terminal amino acids inhibited complex formation with CpcE, possibly due to misfolding According to chemical modifications, one accessible arginine and one accessible tryptophan are essential for CpcE activity, and one carboxylate for CpcF Both subunits bind PCB, as assayed by Ni2+ affinity chromatography, SDS ⁄ PAGE and Zn2+-induced fluorescence The bound PCB could be transferred to CpcA to yield a-CPC The PCB transfer capacity correlates with the activity of the lyase, indicating that PCB bound to CpcE ⁄ F is an intermediate of the enzymatic reaction A catalytic mechanism is proposed, in which a CpcE ⁄ F complex binds PCB and adjusts via a salt bridge the conformation of PCB, which is then transferred to CpcA Phycobiliproteins are a homologous family of lightharvesting proteins present in cyanobacteria, red algae, and cryptophytes [1,2] They absorb light in the regions where chlorophyll absorbs poorly, and transfer excitation energy with high quantum efficiency to the photosynthetic reaction centres Directed energy transfer is determined by the spectroscopic properties and relative positions of the various chromophores present, Abbreviations APC, allophycocyanin; CHD, 1,2-cyclohexanedione; CPC, C-phycocyanin; CpcA, a subunit apoprotein of C-phycocyanin; CpcE(x-y) or CpcF(x-y), truncated CpcE or CpcF, respectively, extending from amino acid ‘‘x’’ to amino acid ‘‘y’’; CpcE, CpcF subunits of the heterodimeric PCB:CpcA lyase; CpcE ⁄ F, complex of CpcE and CpcF, DEPC, diethylpyrocarbonate; EDAC, 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide; IAA, iodoacetic acid; KPB, potassium phosphate buffer; NBS, N-bromosuccinimide; PC, phycocyanin; PCB, phycocyanobilin; PCMS, p-chloromercuriphenylsulfonic acid; PE, phycoerythrin; PEB, phycoerythrobilin; PEC, phycoerythrocyanin; PecE, PecF, subunits of PVB:PecA isomerase-lyase; PGO, phenylglyoxal; PLP, pyridoxal-5’-phosphate; PUB, phycourobilin; PVB, phycoviolobilin; TX100, Triton X-100 1262 FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS K.-H Zhao et al i.e linear tetrapyrroles (phycobilins) of which one to four are covalently attached to the subunits by thioether bonds to conserved cysteines Phycobiliproteins from cyanobacteria are heterohexamers (ab)3, which are organized by linker proteins to large antenna complexes, the phycobilisomes Some of the linkers also carry phycobilin chromophores [3] In cyanobacteria, four classes of biliproteins have been assigned on the basis of their visible absorptions and sequence homologies: phycocyanin (PC), phycoerythrin (PE), allophycocyanin (APC) and phycoerythrocyanin (PEC) They contain, alone or in combination, four different types of isomeric bilin chromophores: phycourobilin (PUB), phycoerythrobilin (PEB), phycobiliviolin (PVB), and phycocyanobilin (PCB) [4] C-phycocyanin (CPC) from Mastigocladus laminosus PCC 7603 studied in this work, carries three PCB at cysteines a-C84, b-C82, and b-C155 [5,6] The last step in phycobilin biosynthesis [7] is the addition of phycobilin to the apoproteins In vivo, the correct attachment of most chromophores is catalysed by binding site- and chromophore-specific lyases, of which only a few have hitherto been characterized [8–10] Since chromophore addition is autocatalytic in some biliproteins (phytochromes, ApcE) [11–15] and proceeds spontaneously, but more slowly and with less fidelity, also in the phycobiliproteins like CPC [16,17], a chaperone-like action has been proposed for these lyases [8,17] This view has been strengthened by the observation of low activities of chromophore binding to all binding sites studied, including a-C84 of PecA [16–18], by the site-selective effect of Triton X-100 (TX100) on chromophore binding to PecB, and by its inhibition of side reactions on binding to CpcA [17] The first and hitherto best studied lyase attaches PCB to the highly conserved a-C84 of CpcA from Synechococcus sp PCC7002 [19,20] In this and several other cyanobacteria, it is coded by two genes, cpcE and cpcF, which are located in the cpc operon downstream of the structural genes for the two CPC subunits A similar organization has been found for other biliprotein:a-C84 lyase genes, but other arrangements including isolated and fused genes have been reported [8,21,22] The lyase function of the proteins has been demonstrated in vitro for CpcE and CpcF from Synechococcus sp PCC7002, Anabaena sp PCC7120, Synechocystis sp PCC6803, M laminosus PCC7603, but has only been studied in some detail for Synechococcus sp PCC7002 [13,23,24] Mutants lacking cpcE and ⁄ or cpcF (or their homologues) produce significantly reduced amounts of CPC [20,25,26] Homologous lyases of the ‘E ⁄ F-type’ are involved in the attachment of PEB and PCB to C84 Chromophore binding by a-phycocyanin lyase of the a-subunits of CPE and PEC, respectively; the latter reaction involves a concomitant isomerization of PCB to PVB [9,18] In vitro, CpcE and CpcF produced in Escherichia coli jointly catalysed the correct attachment of PCB to CpcA-C84, while CpcE or CpcF alone were ineffective CpcE and CpcF can also transfer the bilin homo- and heterologously from a chromophorylated to a nonchromophorylated CpcA: this reaction was reversible and specific for the a-84 site [23] CpcE and CpcF from Nostoc sp PCC7120 can transfer PCB from chromophorylated CpcA to PecA, and even to apo-phytochrome AphA [13] Addition of the CpcE ⁄ F complex to a-CPC alters its absorption and dramatically reduces the fluorescence yield, no such changes are seen with b-CPC [27] Enzymes catalysing the biosynthesis of PCB (haem oxygenase and biliverdin reductase) were introduced together with the lyase (CpeE ⁄ F) and CpcA into E coli to generate a-CPC [24] There is currently no structure known for any of the biliprotein lyases CpcE and CpcF from different species show up to 60% identities, while they are only 20–40% homologous with other enzymes that are known or suggested as lyase for phycobilin addition, such as PecE and PecF [9], CpeY and CpeZ [21] The current study was initiated by the finding of conserved motifs in alignments among lyases from different species [28] We report truncations, site-directed mutations and chemical modifications which were guided by such sequence comparisons, and propose a model of action that involves transient covalent binding of the chromophore to the lyase Results Expression and purification of wild-type and mutant enzymes Full-length and truncated CpcE and CpcF proteins were expressed with N-terminal His- and S-tags using the pET-30a vector The proteins were generally well soluble (unlike those from Anabaena sp PCC 7120 [13]), and the yield of extracted protein was > 70% An exception was CpcF(1–160) which tended to precipitate (see below) The tags interfere with neither the functions of the lyase (CpcE ⁄ F), nor with the reactivity of the apoprotein (CpcA), but they facilitate protein purification and improve their solubilities [13,17,18] After expression in E coli, full-length CpcE, CpcF, and their mutants were purified and quantified by the Bradford method, and then their concentrations adjusted to 50 lm for further experiments FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS 1263 Chromophore binding by a-phycocyanin lyase K.-H Zhao et al Mutation of CpcE and CpcF The enzyme activities of the mutated subunits are compared with those of the wild-type subunits in Table 1, they were quantified by the fluorescence emission of chromophorylated CpcA at 640 nm [23] In these tests, a mutated subunit was always combined with the fulllength complementary one As mutations may affect the interactions among subunits, all enzyme activities of CpcE and CpcF were measured as before [28] in three ways: with the nonpurified proteins (supernatants of the disrupted E coli), with the subunits purified via Ni2+ affinity chromatography, and with the purified subunits which were first denatured together with the full-length complementary one in m urea and then slowly corenatured by dialysis against urea-free buffer (20 mm KPB, 0.5 m NaCl, pH 7.2) The individual subunits, CpcE or CpcF, did not show any enzyme activities, in agreement with previous studies using Synechococcus sp PCC 7002 [23] The full-length lyase showed highest activity when purified CpcE (276 residues) and CpcF (213 residues) were renatured jointly in a : mixture from m urea For CpcE(1–272) and CpcE(L275D), the supernatants showed higher activities than the purified proteins, which may be due to the deletion and sitedirected mutation that affect the interaction among the subunits, or some unknown factors in E coli CpcE(1–274) corenatured with CpcF showed somewhat higher activity than the supernatants and purified forms, indicating that the function lost by deletion of the two amino acids could be improved by CpcF The mutants were constructed according to a sequence comparison of PCB:CpcA lyases The N-terminal motif shown in Fig 1A is highly conserved in CpcE and PecE, therefore the truncated CpcE(42–276) was constructed to delete the motif Similarly, the truncation CpcE(1–272) was generated to remove the highly conserved C-terminal motif (i.e DSLL, in Fig 1B) In CpcE, deletion of 41 amino acids at the N terminus [CpcE(42–276)] and 39 amino acids at C terminus (CpcE(1–237)) abolished the enzymatic activity If judged from the Ni2+-column binding assay (see Experimental procedures) using bound His-tagged CpcF as a bait, CpcE(1–237) has lost the ability to form a complex with CpcF (data not shown), indicating that the amino acids in the region 238–276 are involved in the interaction of the two subunits CpcE(1–272) had only 17–28% activity of the wildtype This activity is retained if two more amino acids were removed in CpcE(1–272), but only if this subunit was not corenatured with CpcF Possibly, the deletion interferes with the refolding of CpcE Replacement of the conserved leucine-275 with the polar aspartate in CpcE(L275D) resulted only in a moderate decrease of activity Kinetic studies (Table 2) showed that the Km Table Comparisons of relative enzymatic activities of CpcE ⁄ F and their mutants, of covalent binding of PCB to CpcE and CpcF, and of their capacity for transferring PCB All test were done under standard reconstitution conditions, with the omissions of specific components given in the headings and footnotes Relative lyase activity [%]a Lyase subunits Non-purified Purified Co- renatured Subunit bound PCBb [%] Yield of a-CPC [lM] from PCB transferc,d PCB transfer [%]c CpcE CpcF CpcE(42–276) + CpcF CpcE(1–237) + CpcF CpcF(21–213) + CpcE CpcF(1–160)e + CpcE CpcF(10–213) + CpcE CpcE(1–272) + CpcF CpcE(1–274) + CpcF CpcE(L275D) + CpcF CpcE(L276D) + CpcF CpcF(I9K) + CpcE (CpcE + CpcF) 0 0 0 29 17 81 22 100 100 0 0 0 26 21 22 65 27 100 100 0 0 18 21 28 65 38 100 120 9.2 6.8 7.2 13.8 7.2 – 7.4 10.8 9.6 9.0 10.6 6.6 11.0 0 0 – 0.044 0.072 0.077 0.14 0.061 0.099 0.16 0 0 – 20 33 35 64 28 45 73 (–) (–) (0) (0) (0) – (0.038) (0.049) (0.061) (0.12) (0.061) (0.094) (0.16) a Purified CpcE ⁄ F ¼ 100% b No CpcA added, otherwise identical conditions as for reconstitutions Yields are given with respect to the concentrations of CpcE or CpcF (5 lM), the concentration of PCB was 10 lM c No PCB added, otherwise identical conditions as for reconstitutions d Values in parentheses are controls with added extra CpcE (5 lM) and CpcF (5lM) to test for free PCB; compared with the fluorescence brightness of the band on the Zn2+ SDS ⁄ PAGE, the lyase and mutants in these tests had 0.22 lM PCB bound e CpcF(1–160) has very low solubility, so PCB binding to the lyase could not be evaluated 1264 FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS K.-H Zhao et al Chromophore binding by a-phycocyanin lyase Table Kinetic analyses for the wild-type lyases and site-directed mutants Enzyme value increased by 61% and that kcat ⁄ Km decreased by 32% The same replacement at the neighbouring position L276, had much more drastic effects The activity of CpcE(L276D) decreased fourfold, the Km increased nearly eightfold, and kcat ⁄ Km decreased by almost two orders of magnitude Obviously, L276 is a vmax [pM S)1] 0.38 0.41 0.61 2.94 6.3 6.5 6.8 0.61 1.3 1.3 1.4 1.2 kcat ⁄ KM [s)1ỈlM)1] kcat [s)1] Wild-type CpcF(I9K) CpcE(L275D) CpcE(L276D) Fig (A, B) Comparison of conserved N- and C-terminal domains in CpcE and PecE from different organisms, and (C) of N-terminal amino acids in CpcF and PecF CLUSTAL W (1.8) multiple sequence alignment method was used The number in front of the sequence gives the accession code of the protein sequence in the Swiss-port database (A, B) CpcE Query: M laminosus PCC7603 (acc no AF506031, protein id AAM69288.2, note that the sequence has been updated on 12.7.2005); compared to CpcE from Anabaena sp PCC7120 (PO7125); Fremyella diplosiphon PCC7601 (P07126); Pseudanabaena sp PCC7409 (Q52448); Synechococcus sp PCC7002 (P31967); Synechocystis sp PCC6803 (P73638); Synechococcus elongatus (P50037), and to PecE of Anabaena sp PCC7120 (P35791); and M laminosus PCC7603 (P29729) (C) Query: CpcF from M laminosus PCC7603 (accession number AF506031, protein i.d AAM69289.2, note that the sequence has been updated on 12.7.2005) compared to CpcF from Anabaena sp PCC7120 (P29985; Synechococcus sp PCC7002 (P31968); Synechocystis sp PCC6803 (P72652); Synechococcus sp PCC7942 (Q44116); Pseudanabaena sp PCC7409 (Q52449); Synechococcus elongatus (P50038) and to PecF of M laminosus PCC7603 (P29730) KM [lM] ± ± ± ± 0.06 0.08 0.05 0.64 ± ± ± ± 0.5 0.6 1.2 0.04 · · · · 10)3 10)3 10)3 10)4 3.4 3.2 2.3 4.1 · · · · 10)3 10)3 10)3 10)5 critical residue, likely to be involved in the substrate affinity The truncated protein CpcF(20–213) was generated upon removal of the first ATG serving as initiation site, because there is a second ATG 60 bases downstream This product was inactive, in spite of a large degree of heterogeneity in the N-terminal region among the different lyases Therefore, the truncation mutant CpcF(10–213) was generated in order to investigate this region more closely CpcF(10–213) only lost activity in the supernatant form When purified and corenatured, the mutant showed 26% and 21% enzyme activity, respectively, indicating that amino acids 1–9 in CpcF are only moderately relevant for the activity In this region, only I9 shows high homology with other lyases However, the activity of the mutant CpcF(I9K) did not show any changes, which was verified by kinetic studies (Table 2) The C-terminally truncated CpcF(1–160) was mostly deposited in inclusion bodies (Fig 2B), and the soluble fraction of this mutant has lost activity It was partly regained, however, when it was corenatured with CpcE, indicating that the 53 amino acids in the C-terminal region of CpcF are important for CpcF folding PCB binding to CpcE and CpcF Work with the isomerizing lyase, PecE ⁄ F, had indicated that the chromophore is bound transiently to the lyase [28] Such binding was investigated now in more detail with CpcE ⁄ F Wild-type and mutant proteins of CpcE, CpcF and their : complexes were incubated with PCB under reconstitution conditions, but omitting the acceptor, CpcA They were then re-purified using a Ni2+ affinity column, where unbound PCB was removed in the m NaCl wash step, and dialysed against KPB (pH 7.2) The absorption spectra of these fractions under native (Fig 3A), and denaturing conditions (Fig 3B) showed that PCB could be bound by CpcE, CpcF, the CpcE ⁄ F complex, and also by their mutants (data not shown) Obviously, binding is strong enough to retain the chromophore under the FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS 1265 Chromophore binding by a-phycocyanin lyase K.-H Zhao et al A Transfer of enzyme-bound chromophore to CpcA B Fig SDS ⁄ PAGE of Ni2+ affinity column purified mutant proteins Lane assignments: (A) M, protein marker; 1, CpcE(42–276); 2, CpcE(L275D); 3, CpcE(1–274); 4, CpcE(1–237); 5, CpcE(1–272); 6, CpcE(L276D); (B) M, protein marker; 1, CpcF(10–213); 2, CpcF(21– 213); 3, CpcF(I9K); 4, CpcF(1–160); 5, CpcF(1–160) purified from inclusion bodies The last mutant was poorly soluble, when corenatured with CpcE, it showed a little activity (see text) The different mobilities irrespective of the similar sizes of CpcE(42–276) and CpcE(1–237) were reproducible high ionic strength conditions (1 m NaCl) used during chromatography Covalent binding was supported by the following observations: (a) denaturation with acidic urea (8 m, pH 2.0) caused a loss of the distinct peak at  650 nm on top of a broad background band > 600 nm The 650-nm peak, on top of a broad absorption, was recovered in 40–70% yield when the urea was dialysed out again, with the losses probably due to irreversible oxidation or denaturation Band narrowing and absorption increase are characteristic for chromophores bound to specific sites in native biliproteins, which are reversibly lost upon uncoupling of the chromophore by denaturation of the protein [4,8] The reversible loss of the distinct peak in chromophore-treated CpcE ⁄ F is reminiscent of such changes; (b) the small absorption decrease upon denaturation, the relatively broad background in Fig extending well beyond 700 nm, and the absence of fluorescence (see below) indicate, however, a less tight coupling between protein and chromophore, and a conformational heterogeneity of the latter This binding situation is rather different from the well-defined one typical for phycocyanin and phytochromes [6,29,30]; (c) PCB remained bound to the protein during SDS ⁄ PAGE, as shown by Zn2+-staining [31], even though the amount is small when compared to the fluorescence intensity of a CPC control (Fig 3D, Table 1) 1266 PCB bound to CpcE or CpcF has very low fluorescence (Fig 4C) This opened a way to test for the transfer of enzyme-bound chromophore to the final acceptor, CpcA, because the product, a-CPC, is strongly fluorescent CpcA was incubated, under standard reconstitution conditions, but in the absence of free PCB, with an aliquot of the samples shown in Fig 3A, which induced the fluorescence typical for a-CPC (Fig 4C) Obviously, bound PCB could be transferred from the lyase to CpcA to give the correct product, a-CPC As shown in Table 1, this capacity roughly parallels the enzymatic activities of the lyase and its mutants, indicating that the capacity of the lyase to transiently bind and subsequently transfer PCB is part of its enzymatic activity This is supported by another observation When PCB, CpcA, CpcE and CpcF were added simultaneously in the reconstitution system, there is generally a by-product obtained with maximum absorption at 640 nm and fluorescence at 660 nm, which arises from a spontaneous, nonenzymatic reaction [9,16,17] Its formation depends on the amount of PCB added, and is particularly pronounced at higher concentrations (Fig 4A) If, however, PCB (0.05–1 lm, i.e substoichiometric amounts with respect to the lyase) was first incubated with CpcE (0.8 lm) and CpcF (0.8 lm) for h, and then CpcA (5 lm) was added, no such nonnatural PCB-CpcA adduct was detectable even at high PCB concentrations (Fig 4B) Obviously, the nonenzymatic reaction was inhibited when PCB was preincubated with the lyase This nonenzymatic reaction was restored when CpcE and CpcF mutants were used that lost the transfer ability It is therefore concluded that binding of PCB by the lyase during the preincubation period inhibits the side reaction Chemical modifications of amino acids Arginine modification by 1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO) [32] resulted in inactivation of CpcE but not of CpcF (supplementary material Fig S1A and B) The semilogarithmic plots of remaining activity against reaction time are linear, indicating that the inactivations followed pseudo-first-order kinetics Second-order rate constants of 0.1 ± 0.02 and 0.7 ± 0.09 mm)1Ỉmin)1 were obtained from the linear plots of the first-order rate constants of inactivation against modifier concentrations, for the reactions with CHD and PGO, respectively The numbers of modified residues were obtained from plots of log(1 ⁄ t0.5) against log[PGO] or log[CHD] They resulted in straight lines FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS K.-H Zhao et al Chromophore binding by a-phycocyanin lyase A B C D Fig Binding of PCB to wild-type CpcE, CpcF and CpcE ⁄ F (A) Absorption spectra after incubation of the proteins indicated by the labels (all 10 lM) with PCB (10 lM) under reconstitution conditions (37 °C, h), subsequent purification by Ni2+ affinity column to remove unbound PCB, and by dialysis against KPB (pH 7.2), 12 h, in the dark (B) Absorption spectra of the same solutions after addition of M acidic urea (pH 2) (C) Absorption spectra after subsequent renaturation from M acidic urea (pH 2.0) by dialysis against KPB buffer (pH 7.2) See Experimental procedures for details (D) SDS ⁄ PAGE of proteins with bound PCB chromophore Lane assignments: M, protein marker; 1, CPC; 2, Histag-CpcE; 3, Histag-CpcF treated with PCB (Coomassie blue stain); 4, CPC; 5, Histag-CpcE; 6, Histag-CpcF treated with PCB (Zn2+-induced fluorescence) A B C 15 300 200 0.5 M PCB M PCB 100 650 700 750 Wavelength [nm] 800 Fluorescence intensity 400 Fluorescence intensity Fluorescence intensity 400 300 200 100 10 650 700 750 Wavelength [nm] 800 After Before 700 650 750 Wavelength [nm] 800 Fig (A) Fluorescence analysis of PCB transfer from CpcE ⁄ F to apo-CpcAA: PCB, CpcE (0.8 lM), CpcF (0.8 lM) and CpcA (5 lM) added together at the same time, and then incubated for 1.5 h (B) PCB was first incubated with CpcE (0.8 lM) and CpcF (0.8 lM) for h, then CpcA (5 lM) was added, and the sample incubated for another 1.5 h; PCB concentrations in (A) and (B): 0.05 lM (—); 0.1 lM (— —); 0.3 lM (– –); lM (– ỈỈ -); 0.8 lM (– Ỉ– Ỉ); lM (ỈỈỈỈ) (C) Fluorescence emission of PCB bound to CpcE ⁄ F (– –), and after adding CpcA to the system (—) All reactions were carried out under standard reconstitution conditions, see Experimental procedures for more details with slopes 1.09 and 0.89, respectively; it is therefore concluded that one accessible arginine residue is required for the catalytic activity of CpcE The modifi- cations may affect the lyase activity by interfering with the interactions of CpcE and CpcF This was tested by binding His-tagged CpcF to the Ni2+ affinity column, FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS 1267 Chromophore binding by a-phycocyanin lyase K.-H Zhao et al Fig Effect of chemical modifier on the formation of CpcE or CpcF complex SDS-containing gels of the fractions eluted with 500 mM imidazole from the Ni2+-chrelating columns (see Experimental procedures for details) Lane assignments: M, protein marker; 1, CpcE (modified by CHD) with His-tag-CpcF; 2, CpcE (modified by PGO) with His-tag-CpcF; 3, CpcE (modified by NBS) with His-tag-CpcF; 4, CpcE with His-tag-CpcF (modified by EDAC); 5, CpcE with His-tag-CpcF (modified by IAA) and analysing for a retention of untagged CpcE by SDS ⁄ PAGE (Fig 5) and by activity assays According to this criterion, the inactivating arginine modification did not interfere with complex formation between the two subunits, but reduced the enzymatic activity (Fig S1) Lysine residues were modified with pyridoxal-5’phosphate (PLP) [33] Treatment of CpcE and CpcF with an excess of the reagent for 30 resulted only in minor activity changes (96–98% versus control), indicating that none of the accessible lysines in CpcE and CpcF are required for the catalytic activity Carboxyl groups were modified with 1-ethyl-3-[3(dimethylamino)propyl] carbodiimide (EDAC) [34], resulting in inactivation of CpcF, but not of CpcE (Fig S2) The semilogarithmic plots of remaining activity against reaction time are linear, indicating that the inactivations followed pseudo-first-order kinetics A second-order rate constants of 0.6 ± 0.06 mm)1Ỉmin)1 was obtained from the linear plots of the first-order rate constants of inactivation against modifier concentrations The numbers of modified residues were obtained from plots of log(1 ⁄ t0.5) against log[EDAC], they resulted in straight lines with slopes 0.65 for the reactions with EDAC It is concluded that one accessible carboxyl group is required for the catalytic activity of CpcF This modification also did not affect the complex formation of CpcE and CpcF (Fig 5) Tryptophan residues were modified by N-bromosuccinimide (NBS) [35], it only affected the activity of CpcE There was a gradual decrease of activity, which was analysed for the number of critical residues, i, by the statistical method of Tsou [36] The data can be fitted to a straight line with i ¼ (Fig S3), suggesting that a single accessible tryptophan residue is critical for the activity of CpcE This modification of 1268 CpcE did not affect complex formation with CpcE (Fig 5) Histidine residues of CpcE were modified by DEPC [37], there is no histidine in CpcF of M laminosus In this case, untagged CpcE was used that was purified via ion-exchange column [28] DEPC had no effect on the activity of CpcE, even through all three histidine residues in CpcE were modified, as determined from the absorption increase at 242 nm [28] Cysteine residues were modified by p-chloromercuriphenylsulfonic acid (PCMS) and iodoacetic acid (IAA) [38] Reactions were carried out in KPB buffer containing m urea in two ways: Either CpcE and CpcF were modified separately, or the CpcE ⁄ F complex was modified In case of individual treatments, the treated subunit was combined with the complementary before they were corenatured PCMS had no effect on CpcE and reduced the activity of CpcF only slightly to 78% When CpcE and CpcF were modified together with PCMS, the activity still was 82% When IAA was used, as a more specific thiol reagent, to separately modify CpcE and CpcF, there were again only moderate losses of activity to 80% and 67%, respectively However, when they were treated together, the activity was reduced to 16% It is likely from these data, that accessible cysteine residues in both CpcE and CpcF play a role in the reaction, but are not essential There is no evidence that complex formation of CpcE and F involves an intersubunit disulfide bridge In the Ni2+affinity assay, the untagged subunit can always be removed by extensive washing, and at low concentrations the complex also dissociates during gel filtration (Superdex 200) Neither this nor any of the other modifications discussed below produced significant changes in the farUV CD-spectra (data not shown); according to this criterion the secondary structure (mainly a-helix) was retained after the modifications Discussion CpcE and CpcF, the two subunits of PCB:CpcA lyase, are involved in the PCB attachment to a-CPC at C84 [23] There are eight different pairs of CpcE ⁄ F of known sequence, they show large regions of high homology [39–41] The enzymology has been studied of CpcE ⁄ F from Synechococcus sp PCC7002 [23,27], but the amino acids that play a role in PCB attachment are not yet clear Also the bifunctional lyase, PecE ⁄ F, is homologous with CpcE ⁄ F, and some characteristic motifs were identified that distinguish in particular the F-subunits of the former and the latter [28] CpcF lacks, for example, the four contiguous histidines FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS K.-H Zhao et al of PecF, which caused a moderately strong binding to the Ni2+ affinity column and interferes with mutual binding assays using one His-tagged partner Complex formation of CpcE with CpcF, and PCB binding to them, could therefore be analysed with more confidence than for PecE ⁄ F, using Ni2+ affinity chromatography Also, the amounts of PCB bound by CpcE, CpcF and their mutants were larger than with PecE, PecF and their mutants, thus facilitating the quantitative analyses of PCB bound by CpcE, CpcF Several interesting N- and C-terminal motifs were noted when comparing the sequences PecE and PecF with those of CpcE and CpcF (Fig 1) For CpcE, both the N and C termini have conserved regions When the motif R33YYAAWWL near the N terminus was deleted (CpcE(41–273)), the enzyme lost its activity completely A 39-amino acid C-terminal truncation in CpcE also rendered the protein inactive; it also nearly lost the ability to form a complex (data not shown), indicating that this region is involved in the complex formation When the C-terminal motif D273SLL was removed in CpcE(1–272), there was still some activity left, but the mutant lost activity when CpcE(1–272) was denatured and corenatured with CpcF, indicating irreversible unfolding If the two residues D273 and S274 were maintained, the enzyme still had 28% relative activity: site-directed mutations of the two leucines (L275D, L276D) reduced the enzyme activity only moderately to 65% and 27%, respectively; these mutations also reduced the substrate affinity (Table 2) These regions were also important for PecE ⁄ F lyase-isomerase activity; truncations rendered the enzyme inactive but did not affect the stability of the proteins [28] The C terminus of CpcF shows only little homology for as much as 50 amino acids A truncation by 53 amino acids reduced the solubility of the protein, possibly due to misfolding, and most of the protein was deposited in E coli as inclusion bodies Only 18% relative activity was recovered by solubilization with urea (8 m) and corenaturation with CpcE, indicating that interaction with CpcE aided the re-folding The N terminus of CpcF showed more homology: a 10 amino acid truncation reduced the activity to 26%, and it was lost completely when 20 amino acids were truncated Among the 10 N-terminal amino acids, I9 is highly conserved among different CpcF, but its mutation to lysine resulted in no marked change of the enzyme activity (Table 2) There are four cysteines in CpcE and three in CpcF, of which only C99 of CpcE is highly conserved In reconstitution experiments in vitro, reducing reagents such as mercaptoethanol or dithiothreitol were not Chromophore binding by a-phycocyanin lyase required for enzyme activity, indicating that no disulfides are present that interfere with the enzymatic activity While the thiol group modification using PCMS proved ineffective, a more complex picture was obtained from modification with IAA When only one of the subunits was modified, the enzyme activity was retained, but modification of both subunits, CpcE and CpcF, in m urea reduced the activity to 16% These modifications were done in m urea to reach otherwise inaccessible cysteins, and the protein subsequently renatured They did not interfere noticeably with PCB binding, IAA therefore modifies residues that are otherwise involved in the catalytic activity (e.g PCB transfer) Both CpcE and CpcF bind PCB, as evidenced by absorption spectroscopy and chromatographic separation from unbound chromophore This binding is only moderately strong and reversible, as judged from the low amount of chromophore found on the SDS ⁄ PAGE purified proteins (Fig 3D) Covalent chromophore binding, albeit even weaker than with CpcE ⁄ F, has also been reported for PecE ⁄ F In the latter case, PCB bound to the enzyme was neither transferred to PecA to form the PCB adduct, nor transferred and concomitantly isomerized to PVB By contrast, chromophore transfer from CpcE ⁄ F to CpcA could now be demonstrated Furthermore, in mutants this transfer correlated with their lyase activities (Table 1) In combination, these results are evidence for a transient chromophore binding to the enzyme as part of the catalytic reaction A chaperone function of a-84 lyases had been suggested before as (at least part of) the enzymatic activity of a-84 lyases [17,42] The absorption spectral changes of PCB upon binding to CpcE ⁄ F are indicative of a conformational change, but at the same time indicate that the chromophore conformation is less restricted not yet extended as it is in a-CPC The lack of an intense fluorescence further suggests that the chromophore retains flexibility upon binding [29], which further supports a comparably weak binding, in a conformation that is intermediate between the cyclic one of the free chromophore, and its extended, rigid conformation in the a-CPC binding site There are 19 arginines, 13 lysines, three histidines and two tryptophans in CpcE, and 13 lysines, 10 arginines, one tryptophan and no histidine in CpcF According to chemical modification of these residues, only one accessible arginine and one tryptophan is involved in CpcE function, and one carboxyl group CpcF In the bifunctional lyase, PecE ⁄ F, a considerably larger number of critical amino acid residues have been identified by the same methods An additional histidine is required in the PecE subunit, and one tryptophan, one cysteine and one histidine in PecF [28] Of FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS 1269 Chromophore binding by a-phycocyanin lyase K.-H Zhao et al the latter, C121 and H122 are located in a region that has been related to the lyase function The only critical residue that is missing in the isomerizing lyase, is the essential carboxyl group in CpcF This may be related to the chromophore transfer capacity of CpcE ⁄ F, which is lacking in PecE ⁄ F Because the optimal pH for CpcE ⁄ F is 7.5–8.0, the carboxyl group in CpcF is expected to be present as a carboxylate anion Since the native PCB chromophore is protonated [43–45], a possible scenario is the formation of a salt-bridge between the carboxylate anion in CpcF and the protonated PCB, but this working hypothesis remains to be tested An alternative function for the carboxylate, i.e an intermolecular salt bridge with the essential Arg in CpcE, is not supported in view of the fact that the modification of the carboxyl of CpcF did not inhibit the CpcE ⁄ F complex formation (Fig 5) In summary, the two types of homologous lyases show some common features (heterodimeric complex, catalysis of attachment of phycobilin at C84 of a-subunit), but they differ in the manipulation of the chromophore not only by the isomerase capacity of PecE ⁄ F that is lacking in CpcE ⁄ F, but also in the chromophore transfer capacity that is present in CpcE ⁄ F, but absent in PecE ⁄ F Several residues have been identified that relate to the different functions However, a general catalytic model is still lacking Investigations of the new class of distantly related lyases recently identified are expected to further clarify the molecular basis of the variability and specificities of this class of enzymes Experimental procedures Deletion and site-directed mutants Truncated and site-directed mutants were prepared by the PCR method, using the following primers P1: 5¢-TGTCCCGGGGCATTGGTCATGACAGAAGCA-3¢, upstream; P2: 5¢-GGGCTCGAGCGGCAATTAAAGTGG GAAT-3¢, downstream; P3: 5¢-ATACCCGGGATACTCCT GACCATGACTGC-3¢, upstream; P4: 5¢-ACCCTCGAGT TATCTTGAGAGTGGAACAAA-3¢, downstream; P5: 5¢-ATGCCCGGGGGTAAGTTTCGCGTTCG-3¢, upstream; P6: 5¢-GGGCTCGAGTTACATCAAATTCATGACTCG-3¢, downstream; P7: 5¢-CCCCTCGAGCTTGCTACAATTAT GAATCCA-3¢, downstream; P8: 5¢-ACCCTCGAGTTATT TTCTACCTTGGCCAGC-3¢, downstream; P9: 5¢-TGTCC CGGGCAAATGACAGCAGCTGTA-3¢, upstream; P10: 5¢-AAACCCGGGCGCAGTGTAGCTGAAG-3¢, upstream; P11: 5¢-CCCCTCGAGCCCTTAAATTGGTTGTTGTA-3¢, downstream; P12: 5¢-ATACCCGGGATGACTGCCACTA CTCAACAATTAAAACGT-3¢, upstream; P13: 5¢-GGGCT CGAGCGGCGCTTACAAATCTGAATC-3¢, downstream, P14: 5¢-AGCCTCGAGCGCCTAGTCAAGTGAATCCAT CA-3¢, downstream All upstream primers have a SmaI site (CCCGGG) and the downstream primers have a XhoI site (CTCGAG), which ensure correct ligation of the fragments to pBluescript P1 and P2 were used to generate the full-length cpcE, P3 and P4 for full-length cpcF, P5 and P2 for cpcE(42–276), P1 and P6 for cpcE(1–272), P1 and P7 for cpcE(1–274), P1 and P8 for cpcE(1–237), P9 and P4 for cpcF(21–213), P10 and P4 for cpcF(10–213), P3 and P11 for cpcF(1–160), P12 and P4 for cpcF(I9K), P1 and P13 for cpcE(L275D), P1 and P14 for cpcE(L276D), In P12, P13 and P14, the site-directed mutations are underlined All mutations were verified by sequencing Materials and reagents 1,2-cyclohexanedione, PGO, NBS, PCMS and IAA were from Sigma (Beijing, P.R.C.); diethylpyrocarbonate (DEPC), PLP and EDAC were from Fluka (Beijing, P.R.C.) All other biochemicals and separation materials were of the highest purity available and obtained from the sources described previously [9,18] Recombinant proteins were purified as before [13] Full-length proteins Cloning and expression followed the standard procedures of Sambrook et al [46] The integral genes cpcA, cpcE and cpcF were PCR-amplified from M laminosus PCC7603 They were cloned first into pBluescript SK(+) (Stratagene, Shanghai, P.R.C.), and then subcloned into pET-30a (Novagen, Munich, Germany) Proteins without His- and S-tags were obtained by expressing pGEMEX containing the desired DNA [9] 1270 Expressions The pET-based plasmids were used to transform E coli BL21 (DE3) Cells were grown at 37 °C in Luria–Bertani medium containing kanamycin (30 lgỈml)1) When the cell density reached OD600 ¼ 0.5–0.7, isopropyl-thio-b-dgalactopyranoside (1 mm) was added h after induction, cells were collected by centrifugation, washed twice with doubledistilled water, and stored at )20 °C until use CpcA, CpcE, CpcF of M laminosus and all mutants were prepared using the methods described previously [18,47] SDS/PAGE SDS ⁄ PAGE was performed with the buffer system of Laemmli [48] The gels were stained with zinc acetate for bilin chromophores [31] and with Coomassie brilliant blue R for the protein The UV-induced fluorescence of protein-bound FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS K.-H Zhao et al bilins was recorded digitally with a camera The amounts of bilins bound by lyases and their mutants were quantitatively evaluated by comparing their scanned fluorescence intensity to that of a standard, i.e CPC, on the same SDS ⁄ PAGE, using photoshop 6.0 (Adobe, San Jose, CA, USA) Spectroscopy Enzyme reactions and amino acid modifications were followed by UV-visible spectrophotometry (Perkin-Elmer model Lamda 25) and fluorimetry (Perkin-Elmer LS55) The formation of chromophorylated-a-CPC was detected by the emission at 640 nm Far-uv CD spectra were recorded at 25 °C with a Dichrograph VI (ISA, Jobin Yvon, Munich, Germany) using mm cuvettes, five spectra were averaged and the data smoothed by 5-point averaging PCB and protein concentration determinations PCB was prepared as described before [18] PCB concentrations were determined spectroscopically in methanol ⁄ 2% HCl using e690 ¼ 37 900 m-1Ỉcm-1 [18] Protein concentrations were determined according to [49] using the protein assay reagent (Bio-Rad, Munich, Germany) according to the manufacturer’s instructions with BSA as standard Concentrations of overexpressed proteins in crude extract were determined by first assaying the total protein content by the Bradford method, and then the relative amount (%) by SDS ⁄ PAGE Phycobiliproteins CPC and a-CPC from M laminosus were prepared as before [17] Lyase activity assay Chromophore reconstitution with CpcA was assayed as described before [27] Either full-length CpcE was complemented with mutants of CpcF, or full-length CpcF was complemented with mutants of CpcE, using the following standard reaction conditions: potassium phosphate buffer (KPB, 15–20 mm, pH 7.2) containing NaCl (150–200 mm), MgCl2 (5 mm), CpcE and CpcF or their mutants (5 lm each), and His6-CpcA (5 lm) PCB (final concentration, lm unless stated otherwise) was added as a concentrated dimethylsulfoxide solution; the final concentration of dimethylsulfoxide was 1% (v ⁄ v) The mixture was incubated at 37 °C for h in the dark Products were quantified by fluorescence emission at 640 nm [27] The lyase reactions were carried out with three different preparations of each His-tagged CpcE, CpcF and their mutants: (1) nonpurified proteins, i.e the supernatants of the sonicated cells after Chromophore binding by a-phycocyanin lyase centrifugation; (2) proteins purified by Ni2+ chelating affinity chromatography as before [18]; (3) corenatured proteins, which were obtained by the following procedure: purified CpcE (or its mutants) and purified CpcF (or its mutants) were denatured separately with urea (8 m) at room temperature They were then mixed in equimolar amounts (5 lm) and renatured slowly by repeated dialysis against KPB (20 mm, pH 7.2) containing NaCl (0.5 m) at °C for h For kinetic tests, only purified proteins were used Either full-length CpcE was complemented with mutants of CpcF, or full-length CpcF was complemented with mutants of CpcE The purified subunits (5 lm), CpcA (5 lm) and different concentrations of chromophore substrate, PCB, were mixed in the reconstitution system (see above) and incubated at 20 °C At regular time intervals, the reaction was terminated by rapidly cooling the samples on ice to °C, then the product was quantified by the fluorescence emission at 640 nm The fluorescence was calibrated with a solution of a-CPC of known concentration Km, vmax and kcat were calculated from Lineweaver–Burk plots, using origin v6 (Origin Laboratory Corporation, Northampton, MA, USA) PCB binding to CpcE and CpcF A mixture of CpcE and CpcF (1 : 1), individual subunits, or their mutants was incubated in the reconstitution system, as described above, but using twice the standard concentration of PCB (10 lm) and omitting CpcA The products were purified by Ni2+ chelating chromatography, and analysed by absorption spectroscopy (300–800 nm), and by SDS ⁄ PAGE using Zn2+ staining [31] and Coomassie brilliant blue staining To check if bound PCB could be transferred to CpcA, the lyase CpcE ⁄ F with bound PCB was first purified by Ni2+ affinity chromatography to remove free PCB, and then dialysed against KPB (20 mm, pH 7.2) containing NaCl (0.5 m) at °C for 12 h in the dark The sample that has been freed of PCB was divided into three parts The first part of the sample was denatured with m acidic urea (pH 2.0), and its absorption spectrum recorded Then the sample was renatured against KPB (20 mm, pH 7.2) containing NaCl (0.5 m), and the absorption recorded again The second part of the sample was mixed with CpcA (one or both lyase subunits added when needed), and Mg2+(5 mm), and incubated at 37 °C for 1.5 h Then, fluorescence emission at 640 nm was measured as described above The third part was analysed by SDS ⁄ PAGE using Zn2+ and Coomassie brilliant blue staining as described above Complex formation of CpcE and CpcF Purified His-tagged CpcE or its mutants were corenatured (see Lyase activity assay) with untagged CpcF The mixtures were then loaded on a Ni2+ column, washed three FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS 1271 Chromophore binding by a-phycocyanin lyase K.-H Zhao et al times with five column volumes of KPB (20 mm, pH 7.2) containing NaCl (0.5 m), once with the same buffer containing in addition imidazole (50 mm), and finally with the same buffer containing 500 mm imidazole The eluate from the last wash was analysed by (a) SDS ⁄ PAGE, and (b) for enzymatic activity as described above, after dialysis against KPB buffer (pH 7.2) The1 : complex of His-tagged CpcF and untagged CpcE could be purified by this method The purified CpcE ⁄ F complex after Ni2+ affinity chromatography was further checked by size exclusion chromatography with Amersham Pharmacia FPLC system and a Superdex 200 column at °C The column was equilibrated in 0.1 m Tris buffer (pH ¼ 7.2) containing mm EDTA Fractions were collected and detected with SDS ⁄ PAGE Chemical modifications of amino acids Chemical modifications of arginine, lysine, carboxyl groups, tryptophan and histidine were performed as described before [28] The number of essential residues of a certain amino acid was determined by the kinetic method of [50] or by the statistical method of [36], as carried out for PEC lyase [28] Cysteine Purified CpcE and CpcF (5 lm) in KPB buffer (20 mm, pH 7.2) were modified by PCMS or IAA [38,51] For modification with PCMS, a stock solution (50 mm in doubledistilled water) was added in 2–5-fold molar excess over the sulfhydryl groups in the protein For modification with IAA, the purified enzyme was first transferred to KPB (20 mm, pH 7.2) containing urea (6 m), incubated with a fivefold excess of IAA under argon at room temperature in the dark At designated time intervals, the modification reaction was stopped by addition of mercaptoethanol (0.1 m) After dialysis against KPB (20 mm, pH 7.2, three times for h each), the enzyme activity was assayed as above 10 11 Acknowledgements HS and KHZ are grateful to Volkswagen Stiftung for a Partnership grant (I ⁄ 77900) The laboratory of KHZ is supported by National Natural Science Foundation of China (30270326, 30540070) and the Program for New Century Excellent Talents in University (NCET04–0717, P.R China), that of HS is supported by Deutsche Forschungsgemeinschaft (SFB 533, TP A1) 12 13 14 References Grossman AR, Schaefer MR, Chiang GG & Collier JL (1993) The phycobilisome, a light-harvesting complex 1272 responsive to environmental conditions Microbiol Rev 57, 725–749 Glazer AN (1994) Adaptive variations in phycobilisome structure Adv Mol Cell Biol 10, 119–149 Sidler WA (1994) Phycobilisome and phycobiliprotein structures In The Molecular Biology of Cyanobacteria (Bryant, DA, ed.), pp 139–216 Kluwer, Dordrecht Glazer AN (1985) Light harvesting by phycobilisomes Ann Rev Biophys Biophys Chem 14, 47–77 Frank G, Sidler W, Widmer H & Zuber H (1978) The complete amino-acid sequence of both subunits of C-phycocyanin from the cyanobacterium Mastigocladus laminosus Hoppe-Seyler’s Z Physiol Chem 359, 1491– 1507 Schirmer T, Bode W, Huber R, Sidler W & Zuber H (1985) X-Ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures J Mol Biol 184, 257–277 Beale SI (1994) Biosynthesis of cyanobacterial tetrapyrrole pigments: Hemes, chlorophylls and phycobilins In The Molecular Biology of Cyanobacteria (Bryant, DA, ed.), pp 519–558 Kluwer, Dordrecht Schluchter WM & Glazer AN (1999) Biosynthesis of Phycobiliproteins in Cyanobacteria In The Phototrophic Prokaryotes (Peschek, GA, Loffelhardt, W & Schmetă terer, G, eds), pp 83–95 Kluwer ⁄ Plenum Press, New York Zhao K-H, Deng M-G, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann B & Scheer H (2000) Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by PecE and PecF FEBS Lett 469, 9–13 Shen G, Saunee NA, Gallo E, Begovic Z, Schluchter WM & Bryant DA (2004) Identification of novel phycobiliprotein lyases in cyanobacteria In PS 2004 light-Harvesting Systems Workshop, book of abstracts p 14 Wu S-H & Lagarias JC (2000) Defining the bilin lyase domain: lessons from the extended phytochrome superfamily Biochemistry 39, 13487–13495 Hughes J, Lamparter T, Mittmann F & Hartmann E (1997) A prokaryotic phytochrome Nature 386, 663 Zhao K-H, Ran Y, Li M, Sun Y-N, Zhou M, Storf M, Kupka M, Bohm S, Bubenzer C & Scheer H (2004) ă Photochromic biliproteins from the cyanobacterium Anabaena sp PCC 7120: lyase activities, chromophore exchange and photochromism in phytochrome and phycoerythrocyanin Biochemistry 43, 11576–11588 Zhao KH, Su P, Bohm S, Song B, Zhou M, Bubenzer ă C & Scheer H (2005) Reconstitution of phycobilisome core-membrane linker, Lcm, by autocatalytic chromophore binding to ApcE Biochim Biophys Acta 1706, 81–87 FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS K.-H Zhao et al 15 Robben U, Lindner I, Gartner W & Schaffner K (2001) ¨ Analyse der Topologie der Chromophor-Bindungstasche von Phytochrom durch Variation des ChlromophorSubstitutionsmuster Angew Chemie 113, 1080–1082 16 Arciero DM, Bryant DA & Glazer AN (1988) In vitro attachment of bilins to apophycocyanin Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin J Biol Chem 263, 18343–18349 17 Zhao KH, Zhu JP, Song B, Zhou M, Storf M, Bohm S, ă Bubenzer C & Scheer H (2004) Non-enzymatic chromophore attachment in biliproteins: Conformational control by the detergent Triton X-100 Biochim Biophys Acta 1657, 131–145 18 Storf M, Parbel A, Meyer M, Strohmann B, Scheer H, Deng M, Zheng M, Zhou M & Zhao K (2001) Chromophore attachment to biliproteins: Specificity of PecE ⁄ PecF, a lyase ⁄ isomerase for the photoactive 31-Cys-a84phycoviolobilin chromophore of phycoerythrocyanin Biochemistry 40, 12444–12456 19 Swanson RV, Zhou J, Leary JA, Williams T, de Lorimier R, Bryant DA & Glazer AN (1992) Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment J Biol Chem 267, 16146–16154 20 Zhou J, Gasparich GE, Stirewalt VL, de Lorimier R & Bryant DA (1992) The cpcE and cpcF genes of Synechococcus sp PCC 7002 Construction and phenotypic characterization of interposon mutants J Biol Chem 267, 16138–16145 21 Kahn K, Mazel D, Houmard J, Tandeau de Marsac N & Schaefer MR (1997) A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis J Bacteriol 179, 998– 1006 22 Six C, Thomas J-C, Thion L, Lemoine Y, Zal F & Partensky F (2005) Characterization of two novel phycoerythrin-associated linker proteins in the marine cyanobacterium Synechococcus sp WH8102 J Bacteriol 187, 1685–1694 23 Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA & Glazer AN (1992) Phycocyanin a-subunit phycocyanobilin lyase Proc Natl Acad Sci USA 89, 7017–7021 24 Tooley AJ, Cai YA & Glazer AN (2001) Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-alpha subunit in a heterologous host Proc Natl Acad Sci USA 98, 10560–10565 25 Bhalerao RP & Gustafsson P (1994) Factors Influencing the Phycobilisome Rod Composition of the Cyanobacterium Synechococcus sp PCC7942 – effects of reduced phycocyanin content, lack of rod-linkers, and overexpression of the rod-terminating linker Physiol Plant 90, 187–197 26 Tandeau de Marsac N, Mazel D, Damerval T, Guglielmi G, Capuano V & Houmard J (1988) Photoregulation of gene expression in the filamentous Chromophore binding by a-phycocyanin lyase 27 28 29 30 31 32 33 34 35 36 37 38 39 40 cyanobacterium Calothrix sp PCC 7601: light-harvesting complexes and cell differentiation Photosynth Res 18, 99–132 Fairchild CD & Glazer AN (1994) Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase J Biol Chem 269, 8686–8694 Zhao KH, Wu D, Zhou M, Zhang L, Bohm S, Bubenă zer C & Scheer H (2005) Amino acid residues associated with enzymatic activity of the isomerizing phycoviolobilin-lyase PecE ⁄ F Biochemistry 44, 8126–8137 Scheer H (1982) Phycobiliproteins: Molecular aspects of photosynthetic antenna systems In Light Reaction Path of Photosynthesis (Fong, FK, ed.), pp 7–45 Springer, Berlin Braslavsky SE, Holzwarth AR & Schaffner K (1983) Solution conformations, photophysics, and photochemistry of bili-pigments – bilirubin and biliverdin dimethylesters and related linear tetrapyrroles Angew Chem Int Ed Engl 22, 656–674 Berkelman T & Lagarias JC (1986) Visualization of bilin-linked peptides and proteins in polyacrylamide gels Anal Biochem 156, 194–201 Suckau D, Mak M & Przybylski M (1992) Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping Proc Natl Acad Sci USA 89, 5630–5634 Bazaes S, Goldie H, Cardemil E & Jabalquinto AM (1995) Identification of reactive lysines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae FEBS Lett 360, 207–210 Hoare DG & Koshland JDE (1967) A method for the quantitative modification and estimation of carboxylic acid group in proteins J Biol Chem 242, 2447–2453 Spande TF & Witkop B (1967) Determination of the tryptophan content of proteins with N-bromosuccinimide Methods Enzymol 11, 498–506 Tsou CL (1962) Relation between modification of functional groups of proteins and their biological activity A graphical method for the determination of the number and type of essential groups Sci Sin 11, 1535–1558 Miles EW (1977) Modification of histidyl residues in proteins by diethylpyrocarbonate Methods Enzymol 47, 431–442 Lundblad RL (1991) Chemical Reagents for Protein Modification, 2nd edn CRC Press, Boca Raton Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S et al (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp strain PCC6803 II Sequence determination of the entire genome and assignment of potential proteincoding regions DNA Res 3, 109–136 Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, Ishikawa A, Kawashima FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS 1273 Chromophore binding by a-phycocyanin lyase 41 42 43 44 45 46 47 48 K.-H Zhao et al K, Kimura T et al (2001) Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp strain PCC 7120 DNA Res 8, 205–213 Nakamura Y, Kaneko T, Sato S, Ikeuchi M, Katoh H, Sasamoto S, Watanabe A, Iriguchi M, Kawashima K, Kimura T et al (2002) Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 DNA Res 9, 123–130 Schluchter WM & Bryant DA (2002) Analysis and reconstitution of phycobiliproteins: methods for the characterization of bilin attachment reactions In Heme, Chlorophyll, and Bilins (Smith, AG & Witty, M, eds), pp 311–334 Humana Press, Totowa, NJ, USA Adir N, Vainer R & Lerner N (2002) Refined structure of c-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 A: insights into the role of solvent molecules in thermal stability and co-factor structure Biochim Biophys Acta 1556, 168–174 Kneip C, Parbel A, Forstendorf H, Scheer H, Siebert F ă & Hildebrandt P (1998) FT-NIR-resonance raman spectroscopic study of the a-subunit of phycoerythrocyanin and phycocyanin from the cyanobacterium Mastigocladus laminosus J Raman Spectr 29, 939–944 Beuhler RJ, Pierce RC & Friedman L (1976) Reversible addition 3-athyliden mit R-SH J Biol Chem 251, 2405 ă 2411 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbour Laboratory Press, New York Zhao K-H, Wu D, Wang L, Zhou M, Storf M, Bubenzer C, Strohmann B & Scheer H (2002) Characterization of phycoviolobilin phycoerythrocyanina84-cystein-lyase-(isomerizing) from Mastigocladus laminosus Eur J Biochem 269, 4542–4550 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriopharge T4 Nature 227, 680–685 1274 49 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 50 Levy HM, Leber PD & Ryan EM (1963) Inactivation of myosin by 2,4-dinitrophenol and protection by adenosine triphosphatase and other phosphate compounds J Biol Chem 238, 3654–3659 51 Horowitz P & Criscimagna NL (1982) The specificity of active-site alkylation by iodoacetic acid in the enzyme thiosulfate sulfurtransferase Biochim Biophys Acta 702, 173–177 Supplementary material The following supplementary material is available online: Fig S1 Kinetics of inactivation of CpcE by CHD (A) and PGO (B) The insets show plots of log(1 ⁄ t0.5) of inactivation against log(modifier concentration) In (A), final CHD concentrations were (n) mm; (d) 10 mm; ( ) 15 mm; ( ) 30 mm; (r) 45 mm; in (B) final PGO concentrations were (n) 2.5 mm; (d) mm; ( ) 7.5 mm; ( ) 10 mm; (r) 15 mm Fig S2 Kinetics of inactivation of CpcF by EDAC The inset shows a plot of log(1 ⁄ t0.5) of inactivation against log(modifier concentration) Final EDAC concentrations were (n) 2.5 mm; (d) mm; ( ) 7.5 mm; ( ) 10 mm; (r)15 mm Fig S3 Relationship between residual enzyme activity and the number of tryptophan residues modified by NBS in CpcE Squares (n) are the experimental values, the lines are obtained from the equation of Tsou35 with i ¼ (); i ẳ (ặặặặ); i ẳ ( –) This material is available as part of the online atricle from http://www.blackwell-synergy.com FEBS Journal 273 (2006) 1262–1274 ª 2006 The Authors Journal compilation ª 2006 FEBS ... P5 and P2 for cpcE(4 2–2 76), P1 and P6 for cpcE( 1–2 72), P1 and P7 for cpcE( 1–2 74), P1 and P8 for cpcE( 1–2 37), P9 and P4 for cpcF(2 1–2 13), P10 and P4 for cpcF(1 0–2 13), P3 and P11 for cpcF( 1–1 60),... (1988) In vitro attachment of bilins to apophycocyanin Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C -phycocyanin J Biol Chem 263, 1834 3–1 8349... rigid conformation in the a-CPC binding site There are 19 arginines, 13 lysines, three histidines and two tryptophans in CpcE, and 13 lysines, 10 arginines, one tryptophan and no histidine in CpcF