:cystein-84 biliprotein lyase, a near- universal lyase for cysteine-84-binding sites in cyanobacterial

Kai-Hong Zhao*†, Ping Su‡, Jun-Ming Tu*§, Xing Wang‡, Hui Liu‡, Matthias Plo¨ scher§, Lutz Eichacker§, Bei Yang*, Ming Zhou†, and Hugo Scheer†§

Colleges of *Life Science and Technology and ‡Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China; and §Department Biologie I–Botanik, Universita¨t Mu¨ nchen, Menzinger Strasse 67, D-80638 Munich, Germany

Communicated by Elisabeth Gantt, University of Maryland, College Park, MD, July 3, 2007 (received for review March 12, 2007) , the light-harvesting complexes of and Of the biliprotein lyases, only the heterodimeric E/F-type has red , contain two to four types of that are hitherto been characterized in detail: it is specific for the , attached covalently to seven or more members of a family of homol- namely ␣-subunits of cyanobacterial (CPC) and ogous , each carrying one to four binding sites. the related phycoerythrocyanin (PEC) and for the binding site binding to apoproteins is catalyzed by lyases, of which only few have (cysteine-␣84) (12–14) and is often encoded by genes on the been characterized in detail. The situation is complicated by nonen- respective biliprotein operon (27, 28). The number of E/F-type zymatic background binding to some apoproteins. Using a modular lyases found in the genomes of sequenced strains of cyanobacteria, multiplasmidic expression-reconstitution in Escherichia coli however, is insufficient to account for the multitude of binding sites with low background binding, phycobilin:cystein-84 biliprotein lyase in the phycobiliproteins present in the phycobilisomes (12). More- (CpeS1) from Anabaena PCC7120, has been characterized as a nearly over, the formation of functional chromophorylated universal lyase for the cysteine-84-binding site that is conserved in all cores in the absence of EF-type lyases indicated the presence of biliproteins. It catalyzes covalent attachment of to all other lyases (27). The first direct evidence for other types of lyases subunits and to cysteine-84 in the ␤-subunits of was reported by Shen et al. (29), who identified a group of four C-phycocyanin and phycoerythrocyanin. Together with the known genes (cpcS, cpcT, cpcU, and cpcV)inSynechococcus PCC7002 that lyases, it can thereby account for chromophore binding to all binding encode lyases which attach phycocyanobilin (PCB; Fig. 1) to the sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it ␤-subunits of CPC. Shen et al. further indicated first, that these catalyzes the attachment of to cysteine-84 of both lyases had a broader specificity, and second, that the substrate subunits of C-. The only exceptions not served by CpeS1 specificity was controlled by the amounts of the different lyases among the cysteine-84 sites are the ␣-subunits from phycocyanin and present in mixtures. Homologous genes are ubiquitous in cyano- phycoerythrocyanin, which, by sequence analyses, have been defined bacteria (15, 16). Phycobilin:cystein-84 biliprotein lyase (CpeS1) of as members of a subclass that is served by the more specialized E/F Anabaena PCC7120 has subsequently been shown to catalyze the type lyases. regiospecific attachment of PCB to cysteine-84 of the ␤-subunits of CPC and PEC (16), whereas CpcT from PCC7002 biliprotein biosynthesis ͉ light-harvesting ͉ ͉ phycobilisome is regiospecific for the third binding site of CPC, namely cysteine- ␤155 (15). Together, the three lyases, CpcE/F (or PecE/F), CpcS, hycobilisomes, the extramembraneous light-harvesting anten- and CpcT in principle are sufficient for complete chromophore Pnas in cyanobacteria and , use four different types of binding (chromophorylation) to the three binding sites of CPC linear chromophores to harvest light in the green gap of and PEC. absorption (1–6). These are covalently Much less is known about the chromophorylation of other bound to seven or more proteins, each carrying one to four binding cyanobacterial phycobiliproteins (12, 29–32). All phycobilisomes sites. The chromophores are biosynthesized from the cyclic iron- tetrapyrrole, , by ring opening at C-5, followed by reduction Author contributions: K.-H.Z., M.Z., and H.S. designed research; P.S., J.-M.T., X.W., H.L., and, sometimes, also by isomerization (7–9). In the last step, these M.P., and B.Y. performed research; L.E. contributed new reagents/analytic tools; K.-H.Z., phycobilins are covalently attached to cysteines of the apoprotein M.P., M.Z., and H.S. analyzed data; and K.-H.Z. and H.S. wrote the paper. 1 via a thioether bond to C-3 on ring A (Fig. 1) and in some cases The authors declare no conflict of interest. 1 by an additional thioether bond to C-18 on ring D (6, 10–12). This Freely available online through the PNAS open access option. step, the binding to the apoprotein, is presently only poorly under- Abbreviations: APC, allophycocyanin; APB, APC B; CPC, cyanobacterial phycocyanin; CPE, stood; it involves a considerable number of binding sites and cyanobacterial phycoerythrin; CpeS1, phycobilin:cystein-84 biliprotein lyase; KPB, potas- chromophores, as well as the proper regulation and coordination of sium phosphate buffer; PCB, phycocyanobilin; PEB, phycoerythrobilin; PEC, phycoerythro- events.¶ cyanin. An increasing number of lyases has recently been identified that †To whom correspondence may be addressed. E-mail: [email protected] or hugo.scheer@ catalyze the chromophore addition and are specific not only for the lmu.de. chromophore but also for the apoprotein and the binding site ¶APC, apoproteins of ␣- (ApcA) and ␤-subunits (ApcB) are encoded by apcA1 and apcB; (12–16). Based on the capacity of several of the respective apopro- ␤-APC18, homologue of ␤-APC encoded by apcF; APB, encoded by apcD; ApcA2, homo- logue of ApcA1 in some cyanobacteria; CPC, apoproteins of ␣- (CpcA) and ␤-subunits teins to also bind the chromophores autocatalytically (17–21), a (CpcB) are encoded by cpcA and cpcB; CPE, apoproteins of ␣- (CpeA) and ␤-subunits (CpeB) chaperone-like function has been suggested (12). It enhances and are encoded by cpeA and cpeB; CpeS1, :Cys84- lyase encoded by guides the autocatalytic binding, which is generally of low fidelity, alr0617; CpeS2, CpeS1-like protein encoded by all5292; CpeT1, PCB:cysteine-␤155-phyco- possibly by conformational control of the chromophore (18). At the biliprotein lyase encoded by all5339; CpeT2, CpeT1-like protein encoded by alr0647; PEC, apoproteins of ␣- (PecA) and ␤-subunits (PecB) are encoded by pecA and pecB. If not stated same time, this autocatalytic binding interferes with the lyase otherwise, the amino acid positions of biliproteins refer to the consensus sequences shown analyses (22). The situation is somewhat similar to chromophore in SI Fig. 9. The numbering of the chromophore is shown in Fig. 1. binding in cytochromes c, which is autocatalytic under well con- This article contains supporting information online at www.pnas.org/cgi/content/full/ trolled conditions (23), but requires in situ a considerable number 0706209104/DC1. of proteins (24–26). © 2007 by The National Academy of Sciences of the USA

14300–14305 ͉ PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706209104 Downloaded by guest on September 28, 2021 ring synthesis of the chromophore PCB (i.e., and reductase), one of the His-tagged APC subunits as acceptor and one or more of the putative lyases. Free PCB is nonfluorescent under UV excitation but becomes fluorescent upon binding to native apoproteins (35–38). After induction, when chromophorylated subunits were formed, the E. coli cultures became brightly fluorescent [supporting information (SI) Fig. 4]. To assay the formed, the cells were broken and the supernatant analyzed by spectros- copy (Table 1); the products were then further analyzed after purification by Ni2ϩ chromatography. This E. coli system was superior to in vitro studies for poorly soluble proteins like CpeA, CpeB, ApcA2, and ApcB, and, furthermore, autocatalytic chro- mophore addition (17–20) was suppressed; it is generally Ͻ10% (Table 1). Possibly the most striking example was ApcA1, which is known to attach PCB autocatalytically in good yield (19), but, in the E. coli system, this background autocatalytic binding, in the absence of the lyase, was strongly reduced (Table 1). Moreover, the fluorescence emission of the autocatalytically bound product was red-shifted compared with that of the product of lyase-catalyzed binding, which indicates some chro- mophore oxidation to mesobiliverdin during autocatalytic binding. Fig. 1. Structures of free (Left) and bound (Right) PCB and PEB. The results obtained with the five APC subunits and the four putative lyases are summarized in Table 1. Because the fluores- cence yields of the chromoproteins differ (see below), quantitative contain a complex group of allophycocyanins (APC) that constitute comparison is possible only within a series of experiments with the the core, and many phycobilisomes contain that are same acceptor protein, e.g., within the columns of Table 1. There- BIOCHEMISTRY located in the distal parts of the rods and have a complex chro- fore, each column has been normalized to the amount of chro- mophore complement (6). Intrigued by the proposal of Shen et al. moprotein produced with the same acceptor protein in the presence (29) that the new group of lyases may singly, or in combination, have of CpeS1. Three results of this screening should be emphasized: (i) a broader substrate spectrum for PCB attachment, including an CpeS1 was capable of catalyzing the attachment of PCB to all of the APC subunit, we have now undertaken detailed analysis of the APC apoproteins tested, (ii) neither the CpeS1 homologue, CpeS2, homologous proteins from Anabaena PCC7120, which contains two nor the two CpeT homologues catalyzed PCB addition to APC cpeS-like genes (cpeS1 ϭ alr0617, CpeS2 ϭ all5292) and two subunits; (iii) the binding activity in the presence of the latter three cpeT-like genes (cpeT1 ϭ all5339 and cpeT2 ϭ alr0647) (33). The proteins was even below the background activity. It therefore enzymatic functions of the respective gene products have been appears that in Anabaena PCC7120, a single protein, CpeS1, can studied, in a multiplasmidic Escherichia coli system with low auto- catalyze the chromophore binding to all APC subunits. In the case catalytic background, for their ability to attach PCB to the various of sufficiently soluble apoproteins (ApcA1 and ApcF), this activity APC subunits and also to attach phycoerythrobilin (PEB; Fig. 1) to is fully supported by in vitro reconstitutions (see kinetic analyses C-phycoerythrin (CPE) subunits (CpeA and CpeB). Only one of below). the four putative lyases, CpeS1, showed any catalytic activity Characterization of products. The absorption and fluorescence emis- specific for the cysteine-84 sites (consensus sequence) but it showed sion data of the purified chromoproteins from these reconstitutions almost no discrimination to the protein substrate. A single protein, in E. coli catalyzed by CpeS1 are summarized in Fig. 2 and Table CpeS1, therefore, is capable of catalyzing chromophore binding to 2. Covalent chromophore attachment is proven (39) by Zn2ϩ- a surprisingly large number of apoprotein subunits. induced fluorescence of the SDS/PAGE bands (SI Fig. 5). The mass spectra (Table 3) confirm the attachment of PCB to the known Results binding sites (C84, consensus sequence) of all APC subunits, Biosyntheses of APC and Homologous Subunits. Screening of putative including ApcA2. After denaturation in acidic urea (8 M, pH 2.0), lyases. The APC lyase functions of CpeS1, phycobilin:cystein- the purified reconstituted chromoproteins (PCB-ApcA1, -ApcB, ␤155 biliprotein lyase (CpeT1), and of the homologous CpeS2 -ApcA2, -ApcD, and -ApcF) gave nearly identical spectra with and CpeT2 were screened in an E. coli system extended from that absorption maxima between 661 and 664 nm; the spectrum of of Tooley et al. (16, 34) to contain compatible plasmids confer- denatured PCB-ApcB is shown in SI Fig. 6. This, together with mass

Table 1. yield in E coli in both the absence and presence of CpeS1, CpeS2, CpeT1, or CpeT2 Relative yield of chromoprotein (%)

PEB-CpeA PEB-CpeB Product lyase PCB-ApcA1 PCB-ApcB PCB-ApcA2 PCB-ApcD PCB-ApcF (C139S) (C48A/C59S/C165S)

None 30 (Ϯ2) 16 (Ϯ3) 7 (Ϯ1) 7 (Ϯ1) 9 (Ϯ0) 2 (Ϯ0) 1 (Ϯ0) CpeS2 10 (Ϯ0) 7 (Ϯ0) 3 (Ϯ0) 6 (Ϯ1) 2 (Ϯ0) 8 (Ϯ0) 0 (Ϯ0) CpeT1 5 (Ϯ0) 11 (Ϯ0) 4 (Ϯ0) 2 (Ϯ0) 6 (Ϯ3) 6 (Ϯ0) 0 (Ϯ0) CpeS1 100 100 100 100 100 100 100 CpeT2 11 (Ϯ1) 8 (Ϯ1) 6 (Ϯ0) 3 (Ϯ0) 5 (Ϯ0) 7 (Ϯ0) 0 (Ϯ0)

Relative fluorescence yields were averaged from two independent measurements and normalized in each column to the yield obtained with CpeS1. With APC subunits, PCB was generated as chromophore; with phycoerythrin subunits, PEB was generated.

Zhao et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14301 Downloaded by guest on September 28, 2021 tinction coefficient and increased (ϩ15%) fluorescence yields of the former may reflect the different origin and measurement conditions. The strong absorption at 650 nm and relatively weak fluorescence emission at 663 nm of the reconstitution product of ApcD (Fig. 2, Table 2) agree well with those of the native ␣-subunit of APC B (APB) (45). The hitherto unreported CD spectrum is typical for a biliprotein. In this case, unlike the others, the absorption and fluorescence maxima of whole cells (SI Fig. 4) differ from those of the purified samples (Fig. 2): the absorption maximum of the E. coli cells was at 605 nm (PCB-ApcD605), with a long-wavelength shoul- der Ϸ650 nm, whereas the purified product absorbed at 650 nm (PCB-ApcD650) (Fig. 2). The origin of this shift is probably related to aggregation (unpublished work). In addition to the four ubiquitous apc genes, Anabaena PCC7120 contains a fifth gene, apcA2, that is highly homologous (70% amino acid identity) to that encoding the APC ␣-subunit, apcA1.Toour knowledge, the respective chromoprotein has never been isolated; it may be an APC-like protein (46). The heterologous reconstitu- tion product PCB-ApcA2 had absorption and fluorescence emis- sion maxima at 622 and 641 nm, respectively, that are very similar to those of PCB-ApcF. The strong fluorescence and the large Fig. 2. Absorption and fluorescence of reconstitution products. Absorption (thick lines) and fluorescence (thin lines) spectra of APC (A) and C-phycoerythrin intensity ratio of the Vis and UV absorptions are characteristic of subunits (B) reconstituted in E. coli in the presence of the lyase, CpeS1. Samples a native biliprotein; the CD signals, however, are of opposite sign were purified via Ni2ϩ affinity column in potassium phosphate buffer (20 mM, pH (SI Fig. 7), indicating a different chromophore conformation. 7.0) containing 0.5 M NaCl [last sample plus 10% glycerin (vol/vol) to improve The kinetics for PCB attachment to two substrates, solubility]. Reconstitutions in E. coli were done with genes from Anabaena ApcA1 and ApcF were studied in vitro, because these proteins are PCC7120, except cpeA/B and pebA/B, which are from Calothrix PCC7601 readily soluble after expression. From the linear Lineweaver–Burk Ϫ6 Ϫ1 plots, kinetic constants Km ϭ 2.7 Ϯ 0.4 ␮M, kcat ϭ 9.5⅐10 ⅐s , Ϫ5 Ϫ1 Km ϭ 2.4 Ϯ 0.1 ␮M, kcat ϭ 3.8⅐10 ⅐s were obtained with ApcA1 spectra data, is evidence for an intact cysteine-bound PCB devoid and ApcF, respectively. These values agree well with the range of significant amounts of the oxidation product, mesobiliverdin, obtained for PCB attachment to cysteine-84 of CPC and PEC by the often encountered with autocatalytic binding, which absorbs at E/F-type lyases (47, 48) or CpeS1 (16), thus further supporting both longer wavelengths (17, 40). The tryptic chromopeptides, in dilute the rather broad substrate specificity and the functional role as an HCl, absorb maximally at 656 nm (SI Fig. 6), which is again APC lyase. characteristic of the chromopeptides carrying an intact PCB chro- mophore (16, 17). Chromophore Binding to CPE Subunits. The surprisingly wide sub- The spectroscopic data of the reconstituted chromoproteins, strate spectrum of CpeS1 prompted a study of its activity (and that over the spectral range from 300 to 700 nm, agree both qualitatively the other three putative lyases) for phycoerythrin, which, with and quantitatively with those of the respective isolated native respect to the lyases, is the least-studied cyanobacterial biliprotein biliproteins. Fig. 2 (and SI Fig. 7) show that the products from (12, 31). The multiplasmidic E. coli system was modified for this ApcA1 and ApcB exhibited the characteristic absorption, fluores- study. The gene encoding the PCB oxidoreductase (pcyA) was cence emission, and CD spectra of the ␣- and ␤-subunits of APC replaced by two genes (peA/B), which encode the reductases (41–43) and showed complementary chromopeptide maps (SI Fig. converting biliverdin to PEB (49). Because Anabaena PCC7120 6). Likewise the absorption maxima (622 nm), CD [617(ϩ)/343(Ϫ) does not produce PEB, the respective genes, as well as cpeA and nm], and fluorescence maxima (645 nm) of PCB-ApcF are very cpeB, which encode the two CPE subunits, were taken from similar to the values reported for the native 16.2-kDa ␤-subunit of Calothrix PCC7601. Mastigocladus (PCC7603) (44); the somewhat lower (Ϫ19%) ex- The situation with CPE is more complex than with singly

Table 2. Quantitative absorption and fluorescence data of biliproteins obtained by CpeS1-catalyzed reconstitution Absorption Fluorescence

Ϫ1 Ϫ1 Biliprotein ␭max [nm] (QVis /uv) ␧Vis (M ⅐cm ) ␭max [nm] ⌽F

PCB-ApcA1 338/618 (4.3) 111,000 (Ϯ600) 641 0.37 (Ϯ0.01) PCB-ApcB 344/613 (2.4) 86,700 (Ϯ200) 640 0.20 (Ϯ0.01) PCB-ApcA2 365/622 (3.2) 71,800 (Ϯ800) 641 0.18 (Ϯ0.01)

PCB-ApcD600 410/602 (2.5) 69,000 (Ϯ500) 635 0.080 (Ϯ0.001) PCB-ApcD650 346/650 (2.3) 62,000 (Ϯ4,000) 663 0.074 (Ϯ0.002) PCB-ApcF 355/622 (2.4) 96,400 (Ϯ300) 645 0.19 (Ϯ0.01) PEB-CpeA(C139S) 361/564 (5.8) 104,400 (Ϯ700) 573 0.51 (Ϯ0.03) PEB-CpeB (C48A/C59S/C165S) 390/560 (5.2) 120,000 (Ϯ6,000) 574 0.63 (Ϯ0.01)

Data are given for the biosynthesized and purified products, PCB-ApcA1, -ApcB, -ApcA2, -ApcD, and -ApcF; PEB-CpeA(C139S) and -CpeB(C48A/C59S/C165S). Data were averaged from two independent experiments. Re- constitutions in E. coli were done with genes from Anabaena PCC7120, except cpeAB and pebAB, which are from Calothrix PCC7601; QVis/uv denotes the absorbance ratio of the visible and near-UV bands.

14302 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706209104 Zhao et al. Downloaded by guest on September 28, 2021 Table 3. Molecular weights (m/z) of chromopeptides from tryptic digestion peak (m/z)

Biliprotein Chromopeptide Ion Calc. Expt.

PCB-ApcA1 RPDVVSPGGNAYG QEMTATC(PCB)LR [M ϩ 4H]4ϩ 727.47 727.86 PCB-ApcA2 RPDIVSPGGNAYGQ DMTATC(PCB)LR [M ϩ 3H]3ϩ 969.96 970.09 PCB-ApcB YAAC(PCB)IR [M ϩ 2H]2ϩ 641.56 641.78 PCB-ApcD ALC(PCB)IR [M ϩ 2H]2ϩ 581.02 581.28 PCB-ApcF LAAC(PCB)LR [M ϩ 2H]2ϩ 616.56 616.78 PEB-CpeA(C139S) C(PEB)AR [M ϩ 2H]2ϩ 467.86 468.21 PEB-CpeB (C48A/C59S/C165S) MAAC(PEB)LR [M ϩ 2H]2ϩ 625.57 625.79

Calc., calculated; Expt., experimentally determined.

chromophorylated APC subunits. The ␣-subunit (CpeA) contains There is, however, one notable exception to this broad protein two cysteines for binding (C82 and C139), and the ␤-subunit specificity: the ␣-subunits of phycocyanin (CpcA) and PEC (PecA) (CpeB) contains four (C48, C59, C80, and C165). Therefore, six both have a cysteine-84-binding site, but here CpeS1 is inactive (16), mutants were generated by substitution of all but one of the and the sites are rather served by the site- and protein-specific potentially binding cysteines, which could then be individually EF-type lyases (12, 52). This exception and the presence of spe- probed (SI Tables 4 and 5). Note that this approach assumes an cialized indicate some special status of these subunits, independent attachment at the different sites, which may be incor- which may relate to the variety of chromophores attached in rect and requires further investigation. Again, with these mutants, different phyco(erythro)cyanins, namely PCB, phycoviolobilin, or only CpeS1 was enzymatically active, and the only enzymatically (6). One could argue that with PecA [and possibly formed PEB chromoproteins carried the chromophore at cysteine- R-phycocyanin (53)], the EF-type lyase has a second function as a 84, e.g., with CpeA(C139S) and CpeB(C48A/C59S/C165S) (Tables chromophore isomerase (14). However, this additional isomerase 1 and 2; see also SI Figs. 7 and 8). The activity with all other function is not required for CpcA, where CPC is attached in the

mutants, as well as the background activities, was particularly low conventional fashion by addition to the ⌬3,31 ethylidene double BIOCHEMISTRY with the CpeB; the absorption maxima of these products were also bond and with stereochemical and conformational properties very red-shifted to Ϸ640 nm. The biosynthesized and purified PEB similar to those of PCB bound to cysteine-84 of the CPC and PEC proteins had absorption and fluorescence maxima Ϸ560 and 575 ␤-subunits (54–56). This indicates a structural difference that, nm, respectively (Fig. 2B), typical of native phycoerythrin subunits among the cysteine-84-binding sites, distinguishes CpcA and PecA (50), and they also had typical CD spectra (51) (SI Fig. 7). from other biliproteins. Such a difference is supported by a se- SDS/PAGE (SI Fig. 5B) showed proteins of the expected size that quence analysis of the apoproteins (SI Fig. 9). In addition to the well strongly fluoresce in the presence of Zn2ϩ, as is expected for bilins known phylogenetic characteristics of the different types of bilip- covalently bound to their proteins (39). The attachment to the roteins (6) and the N-methylation of asparagine found only in correct site was further confirmed by mass spectrometry (Table 3). ␤-subunits (57), there are several sequence traits that set CpcA and We therefore conclude that CpeS1 also possesses CPE lyase PecA apart from all other subunits. Most of the respective amino activity; that it uses both subunits as substrates; and that the activity, acids map to the surface of the protein and cluster largely, but not in both cases, is restricted to a single site, cysteine-84. exclusively (see below), near the cysteine-84-binding site (Fig. 3). They include an insertion after amino acid 77 between helices b and Discussion The report of Shen et al. (29) on the CpcS/T/U/V-family was the first to suggest a second class of lyases, besides the well studied and highly specific EF-type, and further indicated first, that these lyases had a broader specificity and second, that the substrate specificity was controlled by the amounts of the different lyases present in mixtures. In this study, the first suggestion has been substantiated and even extended for one member, namely, CpeS1. The substrate specificity of CpeS1 is surprisingly broad; besides the already known activity with the ␤-subunits of CPC and PEC (16), not only can it attach PCB to all APC subunits but also PEB to both subunits of CPE. CpeS1 is therefore capable of attaching chromophores to cysteine-84 of members of all groups of cyanobacterial biliproteins. Particularly unexpected was the chromophorylation of CpeA at cysteine-84, because an E/F-type lyase had been suggested to be active on this protein (31). Although substrate specificity is broad, the binding site specificity is high, which is always cysteine-84. In APC proteins, this is the only binding site, but the same selectivity holds also for the proteins with multiple binding sites. For example, Fig. 3. Distinction of cysteine-84-binding sites served by EF-types layses and the CPE ␣ and ␤ subunits carry two and four binding sites, by CpeS1. Characteristic amino acids are shown, including the insertion in the respectively, but CpeS1 acts only on one, namely cysteine-84, that b-e loop, that distinguish ␣-subunits of C- and PECs from all other is homologous to the APC sites. A similar specificity had already biliprotein subunits (in purple), projected on the structure of C-phycocyanin from M. laminosus PCC7603 (54). Analysis by grouped alignment is shown in been shown for the CPC and PEC subunits, CpcB and PecB, which SI Fig. 9. The molecule (CPK style; no hydrogens shown) is viewed from the both carry two binding sites (16, 29). Thus, CpeS1 is apparently a ‘‘chromophore face’’ of the cysteine-84-binding site; the chromophore is (nearly) universal lyase with respect to the protein but is highly shown in blue, the sulfur of the binding cysteine-84 in yellow. This figure was specific for a single binding site, cysteine-84. prepared with DSViewer Pro V. 6 (Accelrys, San Diego, CA).

Zhao et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14303 Downloaded by guest on September 28, 2021 e [notation of Schirmer et al. (58)], the nearly buried tryptophane- however, is still less clear for those organisms producing phy- 128 near ring A of the chromophore (not visible in Fig. 3) and coerythrins, but the broad specificity of CpeS1 indicates that the several other unique replacements that differ in their polarity or respective sites may be served by other members of the S- and even charge. It is likely, therefore, that interactions of lyases with T-type lyases. Finally, an important and unresolved question is the apoproteins near the cysteine-84-binding site are different in the sequence of events in the multiple chromophore attachment CpcA and PecA from those of the homologous sites in all other to phycoerythrins and the ␤-subunits of phycocyanins and PEC, biliproteins. which can also be a point of regulation and contribute to mutual It is tempting to speculate on a functional difference of these two interference of lyases. We have evidence that the E. coli system groups of biliproteins that goes beyond the lyase specificity. The can be helpful, too, for investigating these questions, when ␣-84 sites of all cyanobacterial and red algal biliproteins are at the combined with studies in vitro and in the parent cyanobacteria. contact surface to the ␤-subunits in the trimers and show similar geometric and stereochemical properties; this argues against a Materials and Methods structural or photosynthetic function that sets CpcA and PecA apart Cloning. Cloning and expression generally followed standard pro- from all other subunits. The difference may be relevant, however, cedures (66). Full-length cpcA, pecA, cpeS1, ho1, and pcyA were to phycobilisome degradation. The ␣-subunit of EF-type lyases PCR-amplified from Anabaena PCC7120 or Mastigocladus lamin- share homologies to proteins involved in phycobilisome metabolism osus (Fischerella PCC7603), as described (14, 18, 61). For the (59, 60); they are, in fact, capable of chromophore detachment and construction of dual plasmids, ho1 and pcyA were cloned together transfer (47, 61), whereas no such activity has been found for CpeS1 in pACYCDuet (Novagen, Munich, Germany) to produce pHO1- (16). There is, moreover, another protein interaction that is specific PcyA. CpeS1, without His-tag, was obtained by expressing pGE- for these subunits: complex formation of ␣-CPC and ␣-PEC has MEX (Promega, Beijing, China) containing cpeS1 (16). The plas- been observed with NblA, which is implicated in biliprotein deg- mids containing apcA1, apcB, apcA2, apcD, apcF, cpeS2, cpeT1,or radation (62, 63). Interestingly, the binding motif, identified by cpeT2 from Anabaena PCC7120 and cpeA, cpeB, pebA,orpebB Bienert et al. (63), is also obvious in the grouped alignment (SI Fig. from Calothrix PCC7601 were constructed by using the primers 9; see in particular T22, Q25, R30, and S37), of which only R30 is P1–P24, shown in SI Table 4. They were cloned first into pBluescript seen at the bottom of the model shown in Fig. 3. The inactivity of (Stratagene, Beijing, China) and then subcloned into pET30a or the K53A mutant of NblA suggests electrostatic interactions with pETDuet (Novagen) and ho1 plus pebB constructed in pCDFDuet the biliprotein subunits, which may be unfavorable with most (Novagen) to produce pHO1-PebB. Mutants cpeA(C82S) and biliproteins, because of the presence of arginine-37, but more cpeA(C139S), cpeB(C48A/C59S/C80S), cpeB(C48A/C59S/C165S), favorable with CpcA and PecA that lack this residue. cpeB(C48A/C80S/C165S), and cpeB(C59S/C80S/C165S) were gen- The second part of the suggestion of Shen et al. (29), concerning erated from cpeA and cpeB from Calothrix PCC7601 with a a shifting specificity depending on the ratios of the STUV-type mutation kit from Takara Bio, Dalian, China (see SI Table 4 for the lyases, may have to be modified, at least for the system studied in mutation primers P25–P38). All molecular constructions were this paper. Of the four genes of this lyase family present in Anabaena verified by sequencing. PCC7120 (33), only one encodes the product, CpeS1, which has the catalytic capacity for chromophore attachment to cysteine-84. Expressions. The pET-based plasmids were expressed in E. coli CpeS2 and the two members of the CpeT family did not show any BL21(DE3) as before (67). The dual plasmids were transformed lyase activity (Table 1); further, they inhibited both autocatalytic together into BL21(DE3) cells under the appropriate antibiotic chromophore addition (Table 1) and enzymatic catalysis by CpeS1 selections (chloromycetin for pHO-PcyA, streptomycin for pCDF- (K.-H.Z., J. Zhang, J.-M.T., S. Bo¨hm, M.P., L.E., C. Bubenzer, H.S., derivative, kanamycin for pCOLA-derivative or pET30-derivative, X.W., and M.Z., unpublished work). This may indicate a regulatory ampicillin for pETDuet-derivative; see SI Table 5). To produce function, a notion supported by evidence of interaction among PCB-ApcA1, PCB-ApcB, PCB-ApcA2, PCB-ApcD, or PCB-ApcF, CpeS1 and the other three proteins; however, in view of the one of the plasmids pET-ApcA1, -B, -A2, -D, -F, or pCOLA- complexity of the E. coli system, this notion needs thorough ApcA1, -B, -A2, -D -F was used together with pHO1-PcyA, or investigation, especially in cyanobacteria. pCDF derivative, and/or a pETDuet derivative, containing cpeS1, What about the other members of the family? Does a similarly cpeT1, cpeS2,orcpeT2. In the control experiments, plasmids broad activity holds for CpeT/CpcT, which chromophorylates CpcB containing cpeS1, cpeT1, cpeS2,orcpeT2 were omitted from the (and PecB; unpublished work) at cysteine-155 (15)? Although transformations. For reconstitution in E. coli, cells were grown at several CPC-producing cyanobacteria have two pairs each of CpeS 20°C (APC) or 18°C (CPE) in LB medium containing kanamycin and CpeT homologues, those producing the PEB chromophore (20 ␮g⅐mlϪ1), chloromycetin (17 ␮g⅐mlϪ1), streptomycin (25 have even more members; for example, there are six cpeS and four ␮g⅐mlϪ1), and/or ampicillin (40 ␮g⅐mlϪ1). Twelve (APC) or 18 h cpeT homologues in Gloeobacter violaceus PCC7421 (64). Possibly, (CPE) after induction with isopropyl ␤-D-thiogalactoside (1 mM), lyases with similarly broad specificity as CpeS1 can be found for the cells were collected by centrifugation, washed twice with doubly other binding sites and for the occasional secondary attachment to distilled water, and stored at Ϫ20°C until use (16). ring D in phycoerythrins. Currently, very little is known about the Cells were lysed and tagged proteins isolated by Ni2ϩ- mode of action of any of these lyases; it also has to be separated chromatography, as described (16). If necessary, the affinity- from the autocatalytic capacities, albeit of low fidelity, of many of enriched proteins were further purified by FPLC (Amersham– the apoproteins (17–19). Consequently, we emphasize the advan- Amersham Pharmacia, Shangai, China) over a Superdex 75 column tages of the multiplasmidic E. coli system for these studies. Besides developed with buffer [50 mM potassium phosphate buffer (KPB)/ biotechnological applications, it offers rapid and flexible screening, 150 mM NaCl, pH 7.0], or with a DEAE FF column developed with including that of multiple protein attachment. Particularly advan- a gradient of 0–1 M NaCl in KPB (20 mM, pH 7.0). SDS/PAGE of tageous is the low background caused by spontaneous chro- proteins was performed with the buffer system of Laemmli (68). mophore addition, which is generally Ͻ10%. The gels were stained with Coomassie brilliant blue R for the In summary, CpeS1 fills a crucial gap; the attachment of all protein and with ZnCl2 for bilin chromophores (39). chromophores present in a cyanobacterial phycobilisome can be accounted for in combination with the two E/F-types lyases Spectroscopy and Enzyme Kinetic Assay. UV-VIS absorption spectra [cysteine-84 of CpcA and PecA (13, 14)], one T-type lyase were recorded with a Lamda 25 spectrometer (Perkin–Elmer, [cysteine-155 of CpcB and PecB (15)], and the autocatalytic Shangai, China). Fluorescence spectra were recorded with a LS 45 activity of the core-membrane linker, ApcE (65). The situation, spectrofluorimeter (Perkin–Elmer) and are not corrected. CD was

14304 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706209104 Zhao et al. Downloaded by guest on September 28, 2021 measured with a J-810 CD spectrometer (Jasco, Munich, mM, pH 2.1) and acetonitrile (80:20 to 60:40) (16). Natural APC Germany). was isolated via DEAE ion exchange chromatography from the same cyanobacterium, Anabaena PCC7120, according to Fu¨gli- Extinction Coefficients. Concentrations of the reconstituted and staller et al. (73) and then digested and purified as above. The biosynthesized biliproteins were determined by using the extinction resulting chromopeptides were used to identify those from the coefficient of PCB in CPC in 8 M acidic urea (␧660 ϭ 35,500 reconstituted samples. MϪ1⅐cmϪ1) (69) and of PEB in R-phycocyanin in 8 M acidic urea For mass spectrometry, chromoproteins (10 ␮M) were digested Ϫ1 Ϫ1 ␮ (␧560 ϭ 42,800 M ⅐cm ) (70). Fluorescence quantum yields, ⌽F, with trypsin (40 M)inKPB(100mM,pH7.0)for4hat37°C. were determined in KPB (50 mM, pH 7.2), using the known ⌽F After desalting with Sep-Pak cartridges (Model 583, Waters, Mil- (ϭ 0.27) of CPC from Anabaena PCC7120 (71) as standard. ford, MA), the digest was fractionated, as above, by HPLC column Enzyme kinetic assays were carried out as described (48), with diode array detection (model Tidas, J&M, Aalen, Germany), ϭ Km, vmax, and kcat were all calculated from Lineweaver–Burk using gradient A: B 80:20 to 60:40; solvent A: formic acid (0.1%, plots, using Origin V7 (Origin Lab Corporation, Munich, pH 2.0) and solvent B: acetonitrile containing 0.1% formic acid). Germany). The isolated chromopeptides were analyzed by mass spectrometry Reconstituted chromoproteins were dialyzed against KPB (20 in positive ion mode using a Q-Tof Premier mass spectrometer mM, pH 7.2). For HPLC analyses, the desired chromoprotein (WatersMicromass Technologies, Manchester, U.K.) with a nano- solution was acidified with HCl to pH 1.5 digested with pepsin ESI source. (1:1, wt/wt) for3hat37°C and then fractionated on Bio-Gel P-60 (Bio-Rad, Hercules, CA), equilibrated with dilute HCl (pH 2.5). We thank Robert J. Porra for valuable comments and Claudia Bubenzer, Colorless and salts were eluted with the same solvent Yu Chen, Ying Chi, and Xianjun Wu for experimental assistance. (72) and the adsorbed chromopeptides with acetic acid (30%, Support is acknowledged from the Volkswagen Stiftung for the Part- nership (Grant I/77900, to H.S. and K.-H.Z.), from the Deutsche vol/vol) in dilute HCl (pH 2.5). The collected samples were Forschungsgemeinschaft (Grant SFB 533 TPA1, to H.S.), the National subjected to HPLC (Waters 2695 system with model 2487 Natural Science Foundation of China (Grant 30670489, to K.-H.Z.), and variable wavelength detector) on a Zorbax 300SB-C18 column the Program for New Century Excellent Talents in University, P.R. (Agilent Technologies, Austin, TX) using a gradient of KPB (100 China (Grant NCET-04-0717, to K.-H. Z.).

1. Gantt B, Grabowski B, Cunningham FX (2003) in Light-Harvesting Antennas in Photo- 35. Scheer H (1981) Angew Chem Int Ed 20:230–250. BIOCHEMISTRY synthesis, eds Green B, Parson W (Kluwer, Dordrecht, The Netherlands), pp 307–322. 36. Ru¨diger W, Thu¨mmler F (1991) Angew Chem Int Ed Engl 30:1216–1228. 2. Glazer AN (1994) Adv Mol Cell Biol 10:119–149. 37. Braslavsky SE, Holzwarth AR, Schaffner K (1983) Angew Chem Int Ed Engl 3. Grossman AR, Schaefer MR, Chiang GG, Collier JL (1993) Microbiol Rev 57:725–749. 22:656–674. 4. MacColl R (1998) J Struct Biol 124:311–334. 38. Falk H (1989) The Chemistry of Linear Oligopyrroles and Bile Pigments (Springer, New 5. Scheer H (1982) in Light Reaction Path of Photosynthesis, ed Fong FK (Springer, York). Berlin), pp 7–45. 39. Berkelman T, Lagarias JC (1986) Anal Biochem 156:194–201. 6. Sidler WA (1994) in The Molecular Biology of Cyanobacteria, ed Bryant DA (Kluwer, 40. Arciero DM, Bryant DA, Glazer AN (1988) J Biol Chem 263:18343–18349. Dordrecht, The Netherlands), pp 139–216. 41. MacColl R (2004) Biochim Biophys Acta 1657:73–81. 7. Beale SI (1994) in The Molecular Biology of Cyanobacteria., ed Bryant DA (Kluwer, 42. Canaani OD, Gantt E (1980) Biochemistry 19:2950–2956. Dordrecht, The Netherlands), pp 519–558. 43. Gottschalk L, Lottspeich F, Scheer H (1993) Photochem Photobiol 58:761–767. 8. Frankenberg N, Lagarias JC (2002) in The Handbook, eds Kadish KM, 44. Ru¨mbeli R, Wirth M, Suter F, Zuber H (1987) Biol Chem Hoppe–Seyler 368:1–9. Smith KM, Guilard R (Academic, Amsterdam), pp 211–236. 45. Lundell DJ, Glazer AN (1981) J Biol Chem 256:12600–12606. 9. Frankenberg N, Mukougawa K, Kohchi T, Lagarias JC (2001) Cell 13:965–978. 46. Montgomery BL, Casey ES, Grossman AR, Kehoe DM (2004) J Bacteriol 186:7420– 10. Lagarias JC, Klotz AV, Dallas JL, Glazer AN, Bishop JE, Oconnell JF, Rapoport H 7428. (1988) J Biol Chem 263:12977–12985. 47. Fairchild CD, Glazer AN (1994) J Biol Chem 269:8686–8694. 11. Nagy JO, Bishop JE, Klotz AV, Glazer AN, Rapoport H (1985) J Biol Chem 48. Zhao KH, Wu D, Zhou M, Zhang L, Bo¨hm S, Bubenzer C, Scheer H (2005) 260:4864–4868. Biochemistry 44:8126–8137. 12. Schluchter WM, Glazer AN (1999) in The Phototrophic Prokaryotes, eds Peschek GA, 49. Dammeyer T, Frankenberg-Dinkel N (2006) J Biol Chem 281:27081–27089. Lo¨ffelhardt W, Schmetterer G (Kluwer/Plenum, New York), pp 83–95. 50. MacColl R, Guard-Friar D (1987) Phycobiliproteins (CRC, Boca Raton, FL). 13. Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA, Glazer AN (1992) Proc Natl 51. Sauer K, Scheer H (1988) Biochim Biophys Acta 936:157–170. Acad Sci USA 89:7017–7021. 52. Zhao K-H, Deng M-G, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann 14. Storf M, Parbel A, Meyer M, Strohmann B, Scheer H, Deng M, Zheng M, Zhou M, B, Scheer H (2000) FEBS Lett 469:9–13. Zhao K (2001) Biochemistry 40:12444–12456. 53. Six C, Thomas J-C, Thion L, Lemoine Y, Zal F, Partensky F (2005) J Bacteriol 15. Shen G, Saunee NA, Williams SR, Gallo EF, Schluchter WM, Bryant DA (2006) 187:1685–1694. J Biol Chem 281:17768–17778. 54. Schirmer T, Bode W, Huber R (1987) J Mol Biol 196:677–695. 16. Zhao KH, Su P, Li JA, Tu JM, Zhou M, Bubenzer C, Scheer H (2006) J Biol Chem 55. Adir N, Vainer R, Lerner N (2002) Biochim Biophys Acta 1556:168–174. 281:8573–8581. 56. Wang XQ, Li LN, Chang WR, Zhang JP, Gui LL, Guo BJ, Liang DC (2001) Acta 17. Arciero DM, Dallas JL, Glazer AN (1988) J Biol Chem 263:18350–18357. Crystallogr D 57:784–792. 18. Zhao KH, Zhu JP, Song B, Zhou M, Storf M, Bo¨hm S, Bubenzer C, Scheer H (2004) 57. Klotz AV, Glazer AN (1987) J Biol Chem 262:17350–17355. Biochim Biophys Acta 1657:131–145. 58. Schirmer T, Bode W, Huber R, Sidler W, Zuber H (1985) J Mol Biol 184:257–277. 19. Hu IC, Lee TR, Lin HF, Chiueh CC, Lyu PC (2006) Biochemistry 45:7092–7099. 59. Dolganov N, Grossman AR (1999) J Bacteriol 181:610–617. 20. Fairchild CD, Glazer AN (1994) J Biol Chem 269:28988–28996. 60. Balabas BE, Montgomery BL, Ong LE, Kehoe DM (2003) Mol Microbiol 50:781–793. 21. Isailovic D, Sultana I, Philipps GJ, Yeung ES (2006) Anal Biochem 358:38–50. 61. Zhao K-H, Ran Y, Li M, Sun Y-N, Zhou M, Storf M, Kupka M, Bo¨hm S, Bubenzer 22. Schluchter WM, Bryant DA (2002) in Heme, Chlorophyll, and Bilins, eds Smith AG, C, Scheer H (2004) Biochemistry 43:11576–11588. Witty M (Humana, Totowa, NJ), pp 311–334. 62. Luque I, Ochoa De Alda JA, Richaud C, Zabulon G, Thomas JC, Houmard J (2003) 23. Daltrop O, Allen JW, Willis AC, Ferguson SJ (2002) Proc Natl Acad Sci USA Mol Microbiol 50:1043–54. 99:7872–7876. 63. Bienert R, Baier K, Volkmer R, Lockau W, Heinemann U (2006) J Biol Chem 24. Thony-Meyer L (1997) Microbiol Mol Biol Rev 61:337–376. 281:5216–5523. 25. Page MD, Sambongi Y, Ferguson SJ (1998) Trends Biochem Sci 23:103–108. 64. Nakamura Y, Kaneko T, Sato S, Mimuro M, Miyashita H, Tsuchiya T, Sasamoto S, 26. Turkarslan S, Sanders C, Daldal F (2006) Mol Microbiol 60:537–541. Watanabe A, Kawashima K, Kishida Y, et al. (2003) DNA Res 10:137–145. 27. Bhalerao RP, Gustafsson P (1994) Physiol Plant 90:187–197. 65. Zhao KH, Su P, Bo¨hm S, Song B, Zhou M, Bubenzer C, Scheer H (2005) Biochim 28. Zhou J, Gasparich GE, Stirewalt VL, de Lorimier R, Bryant DA (1992) J Biol Chem Biophys Acta 1706:81–87. 267:16138–16145. 66. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual 29. Shen G, Saunee NA, Gallo E, Begovic Z, Schluchter WM, Bryant DA (2004) in PS (Cold Spring Harbor Lab Press, New York), 2nd Ed. 2004 Light-Harvesting Systems Workshop, eds Niederman RA, Blankenship RE, 67. Zhao K-H, Wu D, Wang L, Zhou M, Storf M, Bubenzer C, Strohmann B, Scheer H Frank H, Robert B, van Grondelle R (Saint Adele, Que´bec), pp 14–15. (2002) Eur J Biochem 269:4542–4550. 30. Wilbanks SM, Glazer AN (1993) J Biol Chem 268:1226–1235. 68. Laemmli UK (1970) Nature 227:680–685. 31. Kahn K, Mazel D, Houmard J, Tandeau de Marsac N, Schaefer MR (1997) J Bacteriol 69. Glazer AN, Fang S (1973) J Biol Chem 248:659–662. 179:998–1006. 70. Glazer AN, Hixson CS (1975) J Biol Chem 250:5487–5495. 32. Lundell DJ, Glazer AN, Delange RJ, Brown DM (1984) J Biol Chem 259:5472–5480. 71. Cai YA, Murphy JT, Wedemayer GJ, Glazer AN (2001) Anal Biochem 290:186–204. 33. Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, 72. Zhao KH, Haessner R, Cmiel E, Scheer H (1995) Biochim Biophys Acta 1228:235–243. Ishikawa A, Kawashima K, Kimura T, et al. (2001) DNA Res 8:205–213. 73. Fu¨glistaller P, Widmer H, Sidler W, Frank G, Zuber H (1981) Arch Microbiol 34. Tooley AJ, Cai YA, Glazer AN (2001) Proc Natl Acad Sci USA 98:10560–10565. 129:268–274.

Zhao et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14305 Downloaded by guest on September 28, 2021