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Proc Natl Acad Sci U S A. Sep 4, 2007; 104(36): 14300–14305.
Published online Aug 28, 2007. doi:  10.1073/pnas.0706209104
PMCID: PMC1955460

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


Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the β-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the α-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

Keywords: biliprotein biosynthesis, light-harvesting, photosynthesis, phycobilisome

Phycobilisomes, the extramembraneous light-harvesting antennas in cyanobacteria and red algae, use four different types of linear tetrapyrrole chromophores to harvest light in the green gap of chlorophyll absorption (16). These phycobilins are covalently bound to seven or more proteins, each carrying one to four binding sites. The chromophores are biosynthesized from the cyclic iron-tetrapyrrole, heme, by ring opening at C-5, followed by reduction and, sometimes, also by isomerization (79). In the last step, these phycobilins are covalently attached to cysteines of the apoprotein via a thioether bond to C-31 on ring A (Fig. 1) and in some cases by an additional thioether bond to C-181 on ring D (6, 1012). This step, the binding to the apoprotein, is presently only poorly understood; it involves a considerable number of binding sites and chromophores, as well as the proper regulation and coordination of events.

Fig. 1.
Structures of free (Left) and bound (Right) PCB and PEB.

An increasing number of lyases has recently been identified that catalyze the chromophore addition and are specific not only for the chromophore but also for the apoprotein and the binding site (1216). Based on the capacity of several of the respective apoproteins to also bind the chromophores autocatalytically (1721), a chaperone-like function has been suggested (12). It enhances and guides the autocatalytic binding, which is generally of low fidelity, possibly by conformational control of the chromophore (18). At the same time, this autocatalytic binding interferes with the lyase analyses (22). The situation is somewhat similar to chromophore binding in cytochromes c, which is autocatalytic under well controlled conditions (23), but requires in situ a considerable number of proteins (2426).

Of the biliprotein lyases, only the heterodimeric E/F-type has hitherto been characterized in detail: it is specific for the protein, namely α-subunits of cyanobacterial phycocyanin (CPC) and the related phycoerythrocyanin (PEC) and for the binding site (cysteine-α84) (1214) and is often encoded by genes on the respective biliprotein operon (27, 28). The number of E/F-type lyases found in the genomes of sequenced strains of cyanobacteria, however, is insufficient to account for the multitude of binding sites in the phycobiliproteins present in the phycobilisomes (12). Moreover, the formation of functional chromophorylated phycobilisome cores in the absence of EF-type lyases indicated the presence of other lyases (27). The first direct evidence for other types of lyases was reported by Shen et al. (29), who identified a group of four genes (cpcS, cpcT, cpcU, and cpcV) in Synechococcus PCC7002 that encode lyases which attach phycocyanobilin (PCB; Fig. 1) to the β-subunits of CPC. Shen et al. 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. Homologous genes are ubiquitous in cyanobacteria (15, 16). Phycobilin:cystein-84 biliprotein lyase (CpeS1) of Anabaena PCC7120 has subsequently been shown to catalyze the regiospecific attachment of PCB to cysteine-84 of the β-subunits of CPC and PEC (16), whereas CpcT from Synechococcus PCC7002 is regiospecific for the third binding site of CPC, namely cysteine-β155 (15). Together, the three lyases, CpcE/F (or PecE/F), CpcS, and CpcT in principle are sufficient for complete chromophore binding (chromophorylation) to the three binding sites of CPC and PEC.

Much less is known about the chromophorylation of other cyanobacterial phycobiliproteins (12, 2932). All phycobilisomes contain a complex group of allophycocyanins (APC) that constitute the core, and many phycobilisomes contain phycoerythrins that are located in the distal parts of the rods and have a complex chromophore complement (6). Intrigued by the proposal of Shen et al. (29) that the new group of lyases may singly, or in combination, have a broader substrate spectrum for PCB attachment, including an APC subunit, we have now undertaken detailed analysis of the homologous proteins from Anabaena PCC7120, which contains two cpeS-like genes (cpeS1 = alr0617, CpeS2 = all5292) and two cpeT-like genes (cpeT1 = all5339 and cpeT2 = alr0647) (33). The enzymatic functions of the respective gene products have been studied, in a multiplasmidic Escherichia coli system with low autocatalytic background, for their ability to attach PCB to the various APC subunits and also to attach phycoerythrobilin (PEB; Fig. 1) to C-phycoerythrin (CPE) subunits (CpeA and CpeB). Only one of the four putative lyases, CpeS1, showed any catalytic activity specific for the cysteine-84 sites (consensus sequence) but it showed almost no discrimination to the protein substrate. A single protein, CpeS1, therefore, is capable of catalyzing chromophore binding to a surprisingly large number of apoprotein subunits.


Biosyntheses of APC and Homologous Subunits.

Screening of putative lyases.

The APC lyase functions of CpeS1, phycobilin:cystein-β155 biliprotein lyase (CpeT1), and of the homologous CpeS2 and CpeT2 were screened in an E. coli system extended from that of Tooley et al. (16, 34) to contain compatible plasmids conferring synthesis of the chromophore PCB (i.e., heme oxygenase and biliverdin 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 (3538). After induction, when chromophorylated subunits were formed, the E. coli cultures became brightly fluorescent [supporting information (SI) Fig. 4]. To assay the chromoproteins formed, the cells were broken and the supernatant analyzed by fluorescence spectroscopy (Table 1); the products were then further analyzed after purification by Ni2+ chromatography. This E. coli system was superior to in vitro studies for poorly soluble proteins like CpeA, CpeB, ApcA2, and ApcB, and, furthermore, autocatalytic chromophore addition (1720) was suppressed; it is generally <10% (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 chromophore oxidation to mesobiliverdin during autocatalytic binding.

Table 1.
Chromoprotein yield in E coli in both the absence and presence of CpeS1, CpeS2, CpeT1, or CpeT2

The results obtained with the five APC subunits and the four putative lyases are summarized in Table 1. Because the fluorescence yields of the chromoproteins differ (see below), quantitative comparison is possible only within a series of experiments with the same acceptor protein, e.g., within the columns of Table 1. Therefore, each column has been normalized to the amount of chromoprotein produced with the same acceptor protein in the presence of CpeS1. Three results of this screening should be emphasized: (i) CpeS1 was capable of catalyzing the attachment of PCB to all of the APC apoproteins tested, (ii) neither the CpeS1 homologue, CpeS2, nor the two CpeT homologues catalyzed PCB addition to APC subunits; (iii) the binding activity in the presence of the latter three proteins was even below the background activity. It therefore appears that in Anabaena PCC7120, a single protein, CpeS1, can catalyze the chromophore binding to all APC subunits. In the case of sufficiently soluble apoproteins (ApcA1 and ApcF), this activity is fully supported by in vitro reconstitutions (see kinetic analyses below).

Characterization of products.

The absorption and fluorescence emission data of the purified chromoproteins from these reconstitutions in E. coli catalyzed by CpeS1 are summarized in Fig. 2 and Table 2. Covalent chromophore attachment is proven (39) by Zn2+-induced fluorescence of the SDS/PAGE bands (SI Fig. 5). The mass spectra (Table 3) confirm the attachment of PCB to the known binding sites (C84, consensus sequence) of all APC subunits, including ApcA2. After denaturation in acidic urea (8 M, pH 2.0), the purified reconstituted chromoproteins (PCB-ApcA1, -ApcB, -ApcA2, -ApcD, and -ApcF) gave nearly identical spectra with absorption maxima between 661 and 664 nm; the spectrum of denatured PCB-ApcB is shown in SI Fig. 6. This, together with mass spectra data, is evidence for an intact cysteine-bound PCB devoid of significant amounts of the oxidation product, mesobiliverdin, often encountered with autocatalytic binding, which absorbs at longer wavelengths (17, 40). The tryptic chromopeptides, in dilute HCl, absorb maximally at 656 nm (SI Fig. 6), which is again characteristic of the chromopeptides carrying an intact PCB chromophore (16, 17).

Fig. 2.
Absorption and fluorescence of reconstitution products. Absorption (thick lines) and fluorescence (thin lines) spectra of APC (A) and C-phycoerythrin subunits (B) reconstituted in E. coli in the presence of the lyase, CpeS1. Samples were purified via ...
Table 2.
Quantitative absorption and fluorescence data of biliproteins obtained by CpeS1-catalyzed reconstitution
Table 3.
Molecular weights (m/z) of chromopeptides from tryptic digestion

The spectroscopic data of the reconstituted chromoproteins, over the spectral range from 300 to 700 nm, agree both qualitatively and quantitatively with those of the respective isolated native biliproteins. Fig. 2 (and SI Fig. 7) show that the products from ApcA1 and ApcB exhibited the characteristic absorption, fluorescence emission, and CD spectra of the α- and β-subunits of APC (4143) and showed complementary chromopeptide maps (SI Fig. 6). Likewise the absorption maxima (622 nm), CD [617(+)/343(−) nm], and fluorescence maxima (645 nm) of PCB-ApcF are very similar to the values reported for the native 16.2-kDa β-subunit of Mastigocladus (PCC7603) (44); the somewhat lower (−19%) extinction coefficient and increased (+15%) 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 α-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 shoulder ≈650 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 α-subunit, apcA1. To our knowledge, the respective chromoprotein has never been isolated; it may be an APC-like protein (46). The heterologous reconstitution product PCB-ApcA2 had absorption and fluorescence emission maxima at 622 and 641 nm, respectively, that are very similar to those of PCB-ApcF. The strong fluorescence and the large intensity ratio of the Vis and UV absorptions are characteristic of a native biliprotein; the CD signals, however, are of opposite sign (SI Fig. 7), indicating a different chromophore conformation.

The enzyme kinetics for PCB attachment to two substrates, ApcA1 and ApcF were studied in vitro, because these proteins are readily soluble after expression. From the linear Lineweaver–Burk plots, kinetic constants Km = 2.7 ± 0.4 μM, kcat = 9.5·10−6·s−1, Km = 2.4 ± 0.1 μM, kcat = 3.8·10−5·s−1 were obtained with ApcA1 and ApcF, respectively. These values agree well with the range obtained for PCB attachment to cysteine-84 of CPC and PEC by the E/F-type lyases (47, 48) or CpeS1 (16), thus further supporting both the rather broad substrate specificity and the functional role as an APC lyase.

Chromophore Binding to CPE Subunits.

The surprisingly wide substrate spectrum of CpeS1 prompted a study of its activity (and that the other three putative lyases) for phycoerythrin, which, with respect to the lyases, is the least-studied cyanobacterial biliprotein (12, 31). The multiplasmidic E. coli system was modified for this study. The gene encoding the PCB oxidoreductase (pcyA) was replaced by two genes (peA/B), which encode the reductases converting biliverdin to PEB (49). Because Anabaena PCC7120 does not produce PEB, the respective genes, as well as cpeA and cpeB, which encode the two CPE subunits, were taken from Calothrix PCC7601.

The situation with CPE is more complex than with singly chromophorylated APC subunits. The α-subunit (CpeA) contains two cysteines for binding (C82 and C139), and the β-subunit (CpeB) contains four (C48, C59, C80, and C165). Therefore, six mutants were generated by substitution of all but one of the potentially binding cysteines, which could then be individually probed (SI Tables 4 and 5). Note that this approach assumes an independent attachment at the different sites, which may be incorrect and requires further investigation. Again, with these mutants, only CpeS1 was enzymatically active, and the only enzymatically formed PEB chromoproteins carried the chromophore at cysteine-84, e.g., with CpeA(C139S) and CpeB(C48A/C59S/C165S) (Tables 1 and and2;2; see also SI Figs. 7 and 8). The activity with all other mutants, as well as the background activities, was particularly low with the CpeB; the absorption maxima of these products were also red-shifted to ≈640 nm. The biosynthesized and purified PEB proteins had absorption and fluorescence maxima ≈560 and 575 nm, respectively (Fig. 2B), typical of native phycoerythrin subunits (50), and they also had typical CD spectra (51) (SI Fig. 7). SDS/PAGE (SI Fig. 5B) showed proteins of the expected size that strongly fluoresce in the presence of Zn2+, as is expected for bilins covalently bound to their proteins (39). The attachment to the correct site was further confirmed by mass spectrometry (Table 3). We therefore conclude that CpeS1 also possesses CPE lyase activity; that it uses both subunits as substrates; and that the activity, in both cases, is restricted to a single site, cysteine-84.


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 β-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, the CPE α and β subunits carry two and four binding sites, respectively, but CpeS1 acts only on one, namely cysteine-84, that is homologous to the APC sites. A similar specificity had already been shown for the CPC and PEC subunits, CpcB and PecB, which both carry two binding sites (16, 29). Thus, CpeS1 is apparently a (nearly) universal lyase with respect to the protein but is highly specific for a single binding site, cysteine-84.

There is, however, one notable exception to this broad protein specificity: the α-subunits of phycocyanin (CpcA) and PEC (PecA) both have a cysteine-84-binding site, but here CpeS1 is inactive (16), and the sites are rather served by the site- and protein-specific EF-type lyases (12, 52). This exception and the presence of specialized enzymes indicate some special status of these subunits, which may relate to the variety of chromophores attached in different phyco(erythro)cyanins, namely PCB, phycoviolobilin, or phycourobilin (6). One could argue that with PecA [and possibly R-phycocyanin (53)], the EF-type lyase has a second function as a chromophore isomerase (14). However, this additional isomerase function is not required for CpcA, where CPC is attached in the conventional fashion by addition to the Δ3,31 ethylidene double bond and with stereochemical and conformational properties very similar to those of PCB bound to cysteine-84 of the CPC and PEC β-subunits (5456). This indicates a structural difference that, among the cysteine-84-binding sites, distinguishes CpcA and PecA from other biliproteins. Such a difference is supported by a sequence analysis of the apoproteins (SI Fig. 9). In addition to the well known phylogenetic characteristics of the different types of biliproteins (6) and the N-methylation of asparagine found only in β-subunits (57), there are several sequence traits that set CpcA and PecA apart from all other subunits. Most of the respective amino acids map to the surface of the protein and cluster largely, but not exclusively (see below), near the cysteine-84-binding site (Fig. 3). They include an insertion after amino acid 77 between helices b and e [notation of Schirmer et al. (58)], the nearly buried tryptophane-128 near ring A of the chromophore (not visible in Fig. 3) and several other unique replacements that differ in their polarity or even charge. It is likely, therefore, that interactions of lyases with the apoproteins near the cysteine-84-binding site are different in CpcA and PecA from those of the homologous sites in all other biliproteins.

Fig. 3.
Distinction of cysteine-84-binding sites served by EF-types layses and by CpeS1. Characteristic amino acids are shown, including the insertion in the b-e loop, that distinguish α-subunits of C-phycocyanins and PECs from all other biliprotein subunits ...

It is tempting to speculate on a functional difference of these two groups of biliproteins that goes beyond the lyase specificity. The α-84 sites of all cyanobacterial and red algal biliproteins are at the contact surface to the β-subunits in the trimers and show similar geometric and stereochemical properties; this argues against a structural or photosynthetic function that sets CpcA and PecA apart from all other subunits. The difference may be relevant, however, to phycobilisome degradation. The α-subunit of EF-type lyases share homologies to proteins involved in phycobilisome metabolism (59, 60); they are, in fact, capable of chromophore detachment and transfer (47, 61), whereas no such activity has been found for CpeS1 (16). There is, moreover, another protein interaction that is specific for these subunits: complex formation of α-CPC and α-PEC has been observed with NblA, which is implicated in biliprotein degradation (62, 63). Interestingly, the binding motif, identified by Bienert et al. (63), is also obvious in the grouped alignment (SI Fig. 9; see in particular T22, Q25, R30, and S37), of which only R30 is seen at the bottom of the model shown in Fig. 3. The inactivity of the K53A mutant of NblA suggests electrostatic interactions with the biliprotein subunits, which may be unfavorable with most biliproteins, because of the presence of arginine-37, but more favorable with CpcA and PecA that lack this residue.

The second part of the suggestion of Shen et al. (29), concerning a shifting specificity depending on the ratios of the STUV-type lyases, may have to be modified, at least for the system studied in this paper. Of the four genes of this lyase family present in Anabaena PCC7120 (33), only one encodes the product, CpeS1, which has the catalytic capacity for chromophore attachment to cysteine-84. CpeS2 and the two members of the CpeT family did not show any lyase activity (Table 1); further, they inhibited both autocatalytic chromophore addition (Table 1) and enzymatic catalysis by CpeS1 (K.-H.Z., J. Zhang, J.-M.T., S. Böhm, M.P., L.E., C. Bubenzer, H.S., X.W., and M.Z., unpublished work). This may indicate a regulatory function, a notion supported by evidence of interaction among CpeS1 and the other three proteins; however, in view of the complexity of the E. coli system, this notion needs thorough investigation, especially in cyanobacteria.

What about the other members of the family? Does a similarly broad activity holds for CpeT/CpcT, which chromophorylates CpcB (and PecB; unpublished work) at cysteine-155 (15)? Although several CPC-producing cyanobacteria have two pairs each of CpeS and CpeT homologues, those producing the PEB chromophore have even more members; for example, there are six cpeS and four cpeT homologues in Gloeobacter violaceus PCC7421 (64). Possibly, lyases with similarly broad specificity as CpeS1 can be found for the other binding sites and for the occasional secondary attachment to ring D in phycoerythrins. Currently, very little is known about the mode of action of any of these lyases; it also has to be separated from the autocatalytic capacities, albeit of low fidelity, of many of the apoproteins (1719). Consequently, we emphasize the advantages of the multiplasmidic E. coli system for these studies. Besides biotechnological applications, it offers rapid and flexible screening, including that of multiple protein attachment. Particularly advantageous is the low background caused by spontaneous chromophore addition, which is generally <10%.

In summary, CpeS1 fills a crucial gap; the attachment of all chromophores present in a cyanobacterial phycobilisome can be accounted for in combination with the two E/F-types lyases [cysteine-84 of CpcA and PecA (13, 14)], one T-type lyase [cysteine-155 of CpcB and PecB (15)], and the autocatalytic activity of the core-membrane linker, ApcE (65). The situation, however, is still less clear for those organisms producing phycoerythrins, but the broad specificity of CpeS1 indicates that the respective sites may be served by other members of the S- and T-type lyases. Finally, an important and unresolved question is the sequence of events in the multiple chromophore attachment to phycoerythrins and the β-subunits of phycocyanins and PEC, which can also be a point of regulation and contribute to mutual interference of lyases. We have evidence that the E. coli system can be helpful, too, for investigating these questions, when combined with studies in vitro and in the parent cyanobacteria.

Materials and Methods


Cloning and expression generally followed standard procedures (66). Full-length cpcA, pecA, cpeS1, ho1, and pcyA were PCR-amplified from Anabaena PCC7120 or Mastigocladus laminosus (Fischerella PCC7603), as described (14, 18, 61). For the construction of dual plasmids, ho1 and pcyA were cloned together in pACYCDuet (Novagen, Munich, Germany) to produce pHO1-PcyA. CpeS1, without His-tag, was obtained by expressing pGEMEX (Promega, Beijing, China) containing cpeS1 (16). The plasmids containing apcA1, apcB, apcA2, apcD, apcF, cpeS2, cpeT1, or cpeT2 from Anabaena PCC7120 and cpeA, cpeB, pebA, or pebB from Calothrix PCC7601 were constructed by using the primers P1–P24, shown in SI Table 4. They were cloned first into pBluescript (Stratagene, Beijing, China) and then subcloned into pET30a or pETDuet (Novagen) and ho1 plus pebB constructed in pCDFDuet (Novagen) to produce pHO1-PebB. Mutants cpeA(C82S) and cpeA(C139S), cpeB(C48A/C59S/C80S), cpeB(C48A/C59S/C165S), cpeB(C48A/C80S/C165S), and cpeB(C59S/C80S/C165S) were generated from cpeA and cpeB from Calothrix PCC7601 with a mutation kit from Takara Bio, Dalian, China (see SI Table 4 for the mutation primers P25–P38). All molecular constructions were verified by sequencing.


The pET-based plasmids were expressed in E. coli BL21(DE3) as before (67). The dual plasmids were transformed together into BL21(DE3) cells under the appropriate antibiotic selections (chloromycetin for pHO-PcyA, streptomycin for pCDF-derivative, kanamycin for pCOLA-derivative or pET30-derivative, ampicillin for pETDuet-derivative; see SI Table 5). To produce PCB-ApcA1, PCB-ApcB, PCB-ApcA2, PCB-ApcD, or PCB-ApcF, one of the plasmids pET-ApcA1, -B, -A2, -D, -F, or pCOLA-ApcA1, -B, -A2, -D -F was used together with pHO1-PcyA, or pCDF derivative, and/or a pETDuet derivative, containing cpeS1, cpeT1, cpeS2, or cpeT2. In the control experiments, plasmids containing cpeS1, cpeT1, cpeS2, or cpeT2 were omitted from the transformations. For reconstitution in E. coli, cells were grown at 20°C (APC) or 18°C (CPE) in LB medium containing kanamycin (20 μg·ml−1), chloromycetin (17 μg·ml−1), streptomycin (25 μg·ml−1), and/or ampicillin (40 μg·ml−1). Twelve (APC) or 18 h (CPE) after induction with isopropyl β-d-thiogalactoside (1 mM), cells were collected by centrifugation, washed twice with doubly distilled water, and stored at −20°C until use (16).

Cells were lysed and tagged proteins isolated by Ni2+-chromatography, as described (16). If necessary, the affinity-enriched proteins were further purified by FPLC (Amersham–Amersham Pharmacia, Shangai, China) over a Superdex 75 column developed with buffer [50 mM potassium phosphate buffer (KPB)/150 mM NaCl, pH 7.0], or with a DEAE FF column developed with a gradient of 0–1 M NaCl in KPB (20 mM, pH 7.0). SDS/PAGE of proteins was performed with the buffer system of Laemmli (68). The gels were stained with Coomassie brilliant blue R for the protein and with ZnCl2 for bilin chromophores (39).

Spectroscopy and Enzyme Kinetic Assay.

UV-VIS absorption spectra were recorded with a Lamda 25 spectrometer (Perkin–Elmer, Shangai, China). Fluorescence spectra were recorded with a LS 45 spectrofluorimeter (Perkin–Elmer) and are not corrected. CD was measured with a J-810 CD spectrometer (Jasco, Munich, Germany).

Extinction Coefficients.

Concentrations of the reconstituted and biosynthesized biliproteins were determined by using the extinction coefficient of PCB in CPC in 8 M acidic urea (ε660 = 35,500 M−1·cm−1) (69) and of PEB in R-phycocyanin in 8 M acidic urea (ε560 = 42,800 M−1·cm−1) (70). Fluorescence quantum yields, ΦF, were determined in KPB (50 mM, pH 7.2), using the known ΦF (= 0.27) of CPC from Anabaena PCC7120 (71) as standard.

Enzyme kinetic assays were carried out as described (48), Km, vmax, and kcat were all calculated from Lineweaver–Burk plots, using Origin V7 (Origin Lab Corporation, Munich, Germany).

Reconstituted chromoproteins were dialyzed against KPB (20 mM, pH 7.2). For HPLC analyses, the desired chromoprotein solution was acidified with HCl to pH 1.5 digested with pepsin (1:1, wt/wt) for 3 h at 37°C and then fractionated on Bio-Gel P-60 (Bio-Rad, Hercules, CA), equilibrated with dilute HCl (pH 2.5). Colorless peptides and salts were eluted with the same solvent (72) and the adsorbed chromopeptides with acetic acid (30%, vol/vol) in dilute HCl (pH 2.5). The collected samples were subjected to HPLC (Waters 2695 system with model 2487 variable wavelength detector) on a Zorbax 300SB-C18 column (Agilent Technologies, Austin, TX) using a gradient of KPB (100 mM, pH 2.1) and acetonitrile (80:20 to 60:40) (16). Natural APC was isolated via DEAE ion exchange chromatography from the same cyanobacterium, Anabaena PCC7120, according to Füglistaller et al. (73) and then digested and purified as above. The resulting chromopeptides were used to identify those from the reconstituted samples.

For mass spectrometry, chromoproteins (10 μM) were digested with trypsin (40 μM) in KPB (100 mM, pH 7.0) for 4 h at 37°C. After desalting with Sep-Pak cartridges (Model 583, Waters, Milford, MA), the digest was fractionated, as above, by HPLC column with diode array detection (model Tidas, J&M, Aalen, Germany), using gradient A: B = 80:20 to 60:40; solvent A: formic acid (0.1%, pH 2.0) and solvent B: acetonitrile containing 0.1% formic acid). The isolated chromopeptides were analyzed by mass spectrometry in positive ion mode using a Q-Tof Premier mass spectrometer (WatersMicromass Technologies, Manchester, U.K.) with a nano-ESI source.

Supplementary Material

Supporting Information:


We thank Robert J. Porra for valuable comments and Claudia Bubenzer, Yu Chen, Ying Chi, and Xianjun Wu for experimental assistance. Support is acknowledged from the Volkswagen Stiftung for the Partnership (Grant I/77900, to H.S. and K.-H.Z.), from the Deutsche Forschungsgemeinschaft (Grant SFB 533 TPA1, to H.S.), the National Natural Science Foundation of China (Grant 30670489, to K.-H.Z.), and the Program for New Century Excellent Talents in University, P.R. China (Grant NCET-04-0717, to K.-H. Z.).


cyanobacterial phycocyanin
cyanobacterial phycoerythrin
phycobilin:cystein-84 biliprotein lyase
potassium phosphate buffer


The authors declare no conflict of interest.

APC, apoproteins of α- (ApcA) and β-subunits (ApcB) are encoded by apcA1 and apcB; β-APC18, homologue of β-APC encoded by apcF; APB, encoded by apcD; ApcA2, homologue of ApcA1 in some cyanobacteria; CPC, apoproteins of α- (CpcA) and β-subunits (CpcB) are encoded by cpcA and cpcB; CPE, apoproteins of α- (CpeA) and β-subunits (CpeB) are encoded by cpeA and cpeB; CpeS1, bilin:Cys84-phycobiliprotein lyase encoded by alr0617; CpeS2, CpeS1-like protein encoded by all5292; CpeT1, PCB:cysteine-β155-phycobiliprotein lyase encoded by all5339; CpeT2, CpeT1-like protein encoded by alr0647; PEC, apoproteins of α- (PecA) and β-subunits (PecB) are encoded by pecA and pecB. If not stated otherwise, the amino acid positions of biliproteins refer to the consensus sequences shown in SI Fig. 9. The numbering of the chromophore is shown in Fig. 1.

This article contains supporting information online at www.pnas.org/cgi/content/full/0706209104/DC1.


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