Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins.
ABSTRACT 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 beta-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 alpha-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.
Article: Cyanobacterial phycobilisomes[show abstract] [hide abstract]
ABSTRACT: Cyanobacterial phycobilisomes harvest light and cause energy migration usually toward photosystem II reaction centers. Energy transfer from phycobilisomes directly to photosystem I may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the four chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by complementary chromatic adaptation is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The linkers govern the assembly of the biliproteins into phycobilisomes, and, even if colorless, in certain cases they have been shown to improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Details of how linkers perform their tasks are still topics of interest. The transfer of excitation energy from bilin to bilin is considered, particularly for monomers and trimers of C-phycocyanin, phycoerythrocyanin, and allophycocyanin. Phycobilisomes are one of the ways cyanobacteria thrive in varying and sometimes extreme habitats. Various biliprotein properties perhaps not related to photosynthesis are considered: the photoreversibility of phycoviolobilin, biophysical studies, and biliproteins in evolution. Copyright 1998 Academic Press.Journal of Structural Biology 01/1999; 124(2-3):311-34. · 3.36 Impact Factor
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ABSTRACT: Allophycocyanin (APC) is one of the phycobiliproteins expressed in cyanobacteria. Phycobiliproteins contain a covalently bound chromophore, and thus, they are valuable as fluorescent probes. Biosynthesis of a functional phycobiliprotein is achieved by a bilin attachment process between the chromophore and apoprotein. Chromophore lyases are necessary to catalyze the chromophorylation of cyanobacterial phycobiliproteins, such as C-phycocyanin, and phycoerythrocyanin. To identify the lyase that catalyzes the chromophorylation of the APC alpha-subunit (ApcA), we searched the entire genomes of two cyanobacteria, Synechocystis sp. PCC6803 and Anabaena sp. PCC 7120; however, these genomes do not appear to encode an APC-specific chromophore lyase. In this study, chromophorylated ApcA (chromo-ApcA) was obtained via a spontaneous bilin attachment reaction. The absorption and fluorescence characteristics of chromo-ApcA were similar to those of the native APC alpha-subunit. The extent of chromophore attachment to apo-ApcA was comparable to that of the lyase-catalyzed reactions for other phycobiliproteins. These results indicate that ApcA has autocatalytic bilin:biliprotein lyase activity.Biochemistry 07/2006; 45(23):7092-9. · 3.38 Impact Factor
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ABSTRACT: Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.Proceedings of the National Academy of Sciences 09/1992; 89(15):7017-21. · 9.74 Impact Factor
Phycobilin:cystein-84 biliprotein lyase, a near-
universal lyase for cysteine-84-binding sites in
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)
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 homol-
been characterized in detail. The situation is complicated by nonen-
zymatic 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
allophycocyanin subunits and to cysteine-84 in the ?-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
as members of a subclass that is served by the more specialized E/F
biliprotein biosynthesis ? light-harvesting ? photosynthesis ? phycobilisome
chlorophyll absorption (1–6). 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 (7–9). In the last step, these
phycobilins are covalently attached to cysteines of the apoprotein
via a thioether bond to C-31on ring A (Fig. 1) and in some cases
by an additional thioether bond to C-181on ring D (6, 10–12). This
step, the binding to the apoprotein, is presently only poorly under-
stood; it involves a considerable number of binding sites and
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
(12–16). Based on the capacity of several of the respective apopro-
teins to also bind the chromophores autocatalytically (17–21), a
chaperone-like function has been suggested (12). It enhances and
guides the autocatalytic binding, which is generally of low fidelity,
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 con-
trolled conditions (23), but requires in situ a considerable number
of proteins (24–26).
hycobilisomes, the extramembraneous light-harvesting anten-
nas in cyanobacteria and red algae, use four different types of
Of the biliprotein lyases, only the heterodimeric E/F-type has
hitherto been characterized in detail: it is specific for the protein,
namely ?-subunits of cyanobacterial phycocyanin (CPC) and
the related phycoerythrocyanin (PEC) and for the binding site
(cysteine-?84) (12–14) 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,
in the phycobiliproteins present in the phycobilisomes (12). More-
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
?-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 cyano-
bacteria (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 ?-subunits of
CPC and PEC (16), whereas CpcT from Synechococcus PCC7002
is regiospecific for the third binding site of CPC, namely cysteine-
?155 (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
Much less is known about the chromophorylation of other
cyanobacterial phycobiliproteins (12, 29–32). All phycobilisomes
Author contributions: K.-H.Z., M.Z., and H.S. designed research; P.S., J.-M.T., X.W., H.L.,
M.P., and B.Y. performed research; L.E. contributed new reagents/analytic tools; K.-H.Z.,
M.P., M.Z., and H.S. analyzed data; and K.-H.Z. and H.S. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: APC, allophycocyanin; APB, APC B; CPC, cyanobacterial phycocyanin; CPE,
cyanobacterial phycoerythrin; CpeS1, phycobilin:cystein-84 biliprotein lyase; KPB, potas-
sium phosphate buffer; PCB, phycocyanobilin; PEB, phycoerythrobilin; PEC, phycoerythro-
†To whom correspondence may be addressed. E-mail: email@example.com or hugo.scheer@
¶APC, apoproteins of ?- (ApcA) and ?-subunits (ApcB) are encoded by apcA1 and apcB;
?-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
are encoded by cpeA and cpeB; CpeS1, bilin:Cys84-phycobiliprotein lyase encoded by
alr0617; CpeS2, CpeS1-like protein encoded by all5292; CpeT1, PCB:cysteine-?155-phyco-
biliprotein lyase encoded by all5339; CpeT2, CpeT1-like protein encoded by alr0647; PEC,
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/
© 2007 by The National Academy of Sciences of the USA
September 4, 2007 ?
vol. 104 ?
located in the distal parts of the rods and have a complex chro-
mophore complement (6). Intrigued by the proposal of Shen et al.
a broader substrate spectrum for PCB attachment, including an
APC subunit, we have now undertaken detailed analysis of the
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 auto-
catalytic background, for their ability to attach PCB to the various
C-phycoerythrin (CPE) subunits (CpeA and CpeB). Only one of
the four putative lyases, CpeS1, showed any catalytic activity
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-
?155 biliprotein lyase (CpeT1), and of the homologous CpeS2
of Tooley et al. (16, 34) to contain compatible plasmids confer-
ring 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 (35–38). 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 spectros-
copy (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 chro-
mophore addition (17–20) 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 chro-
mophore oxidation to mesobiliverdin during autocatalytic
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
comparison is possible only within a series of experiments with the
same acceptor protein, e.g., within the columns of Table 1. There-
fore, each column has been normalized to the amount of chro-
of CpeS1. Three results of this screening should be emphasized: (i)
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
Characterization of products. The absorption and fluorescence emis-
in E. coli catalyzed by CpeS1 are summarized in Fig. 2 and Table
2. Covalent chromophore attachment is proven (39) by Zn2?-
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
Fig. 1.Structures of free (Left) and bound (Right) PCB and PEB.
Table 1. Chromoprotein yield in E coli in both the absence and presence of CpeS1, CpeS2, CpeT1, or CpeT2
Relative yield of chromoprotein (%)
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 ?
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 chro-
mophore (16, 17).
The spectroscopic data of the reconstituted chromoproteins,
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, fluores-
cence emission, and CD spectra of the ?- and ?-subunits of APC
(41–43) 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 ?-subunit of
Mastigocladus (PCC7603) (44); the somewhat lower (?19%) ex-
tinction coefficient and increased (?15%) fluorescence yields of
the former may reflect the different origin and measurement
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
cells was at 605 nm (PCB-ApcD605), with a long-wavelength shoul-
der ?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).
acid identity) to that encoding the APC ?-subunit, apcA1. To our
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
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 ?M, kcat? 9.5?10?6?s?1,
Km? 2.4 ? 0.1 ?M, kcat? 3.8?10?5?s?1were obtained with ApcA1
and ApcF, respectively. These values agree well with the range
the rather broad substrate specificity and the functional role as an
Chromophore Binding to CPE Subunits. The surprisingly wide sub-
strate 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
The situation with CPE is more complex than with singly
(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
7.0) containing 0.5 M NaCl [last sample plus 10% glycerin (vol/vol) to improve
solubility]. Reconstitutions in E. coli were done with genes from Anabaena
PCC7120, except cpeA/B and pebA/B, which are from Calothrix PCC7601
Absorption and fluorescence of reconstitution products. Absorption
Table 2. Quantitative absorption and fluorescence data of biliproteins obtained
by CpeS1-catalyzed reconstitution
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/uvdenotes the absorbance ratio of the visible and near-UV bands.
www.pnas.org?cgi?doi?10.1073?pnas.0706209104Zhao et al.
chromophorylated APC subunits. The ?-subunit (CpeA) contains
two cysteines for binding (C82 and C139), and the ?-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 incor-
rect 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 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
in both cases, is restricted to a single site, cysteine-84.
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
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,
the CPE ? and ? 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 ?-subunits of phycocyanin (CpcA) and PEC (PecA)
and the sites are rather served by the site- and protein-specific
EF-type lyases (12, 52). This exception and the presence of spe-
cialized 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 ?3,31ethylidene double
bond and with stereochemical and conformational properties very
similar to those of PCB bound to cysteine-84 of the CPC and PEC
?-subunits (54–56). 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 se-
known phylogenetic characteristics of the different types of bilip-
roteins (6) and the N-methylation of asparagine found only in
?-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).
Table 3. Molecular weights (m/z) of chromopeptides from tryptic digestion
Peptide peak (m/z)
[M ? 4H]4?
[M ? 3H]3?
[M ? 2H]2?
[M ? 2H]2?
[M ? 2H]2?
[M ? 2H]2?
[M ? 2H]2?
Calc., calculated; Expt., experimentally determined.
by CpeS1. Characteristic amino acids are shown, including the insertion in the
biliprotein subunits (in purple), projected on the structure of C-phycocyanin
from M. laminosus PCC7603 (54). Analysis by grouped alignment is shown in
SI Fig. 9. The molecule (CPK style; no hydrogens shown) is viewed from the
‘‘chromophore face’’ of the cysteine-84-binding site; the chromophore is
shown in blue, the sulfur of the binding cysteine-84 in yellow. This figure was
prepared with DSViewer Pro V. 6 (Accelrys, San Diego, CA).
Distinction of cysteine-84-binding sites served by EF-types layses and
Zhao et al.PNAS ?
September 4, 2007 ?
vol. 104 ?
no. 36 ?
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
groups of biliproteins that goes beyond the lyase specificity. The
?-84 sites of all cyanobacterial and red algal biliproteins are at the
contact surface to the ?-subunits in the trimers and show similar
geometric and stereochemical properties; this argues against a
from all other subunits. The difference may be relevant, however,
to phycobilisome degradation. The ?-subunit of EF-type lyases
(59, 60); they are, in fact, capable of chromophore detachment and
for these subunits: complex formation of ?-CPC and ?-PEC has
been observed with NblA, which is implicated in biliprotein deg-
radation (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
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
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
(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,
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 (17–19). Consequently, we emphasize the advan-
tages 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 advan-
tageous is the low background caused by spontaneous chro-
mophore 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 phy-
coerythrins, 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 ?-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. Cloning and expression generally followed standard pro-
cedures (66). Full-length cpcA, pecA, cpeS1, ho1, and pcyA were
PCR-amplified from Anabaena PCC7120 or Mastigocladus lamin-
osus (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 pGE-
MEX (Promega, Beijing, China) containing cpeS1 (16). The plas-
mids 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
(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 gen-
erated from cpeA and cpeB from Calothrix PCC7601 with a
mutation primers P25–P38). All molecular constructions were
verified by sequencing.
Expressions. 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
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 ?g?ml?1), chloromycetin (17 ?g?ml?1), streptomycin (25
?g?ml?1), and/or ampicillin (40 ?g?ml?1). Twelve (APC) or 18 h
(CPE) after induction with isopropyl ?-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–
developed with buffer [50 mM potassium phosphate buffer (KPB)/
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 ZnCl2for bilin chromophores (39).
Spectroscopy and Enzyme Kinetic Assay. UV-VISabsorptionspectra
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
www.pnas.org?cgi?doi?10.1073?pnas.0706209104Zhao et al.
measured with a J-810 CD spectrometer (Jasco, Munich,
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 (?660 ? 35,500
M?1?cm?1) (69) and of PEB in R-phycocyanin in 8 M acidic urea
(?560? 42,800 M?1?cm?1) (70). Fluorescence quantum yields, ?F,
were determined in KPB (50 mM, pH 7.2), using the known ?F
(? 0.27) of CPC from Anabaena PCC7120 (71) as standard.
Enzyme kinetic assays were carried out as described (48),
Km, vmax, and kcatwere all calculated from Lineweaver–Burk
plots, using Origin V7 (Origin Lab Corporation, Munich,
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
(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 Fu ¨gli-
staller et al. (73) and then digested and purified as above. The
resulting chromopeptides were used to identify those from the
For mass spectrometry, chromoproteins (10 ?M) were digested
with trypsin (40 ?M) in KPB (100 mM, pH 7.0) for 4 h at 37°C.
After desalting with Sep-Pak cartridges (Model 583, Waters, Mil-
ford, 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-
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 Part-
nership (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.).
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