The enzyme also lost activity when in CpcF 213 resi-dues the 20 N-terminal amino acids were truncated; truncation of 53 C-terminal amino acids inhibited complex formation with CpcE, poss
Trang 1Functional amino acids of phycocyanobilin – a-phycocyanin lyase
and evidence for chromophore binding
Kai-Hong Zhao1, Dong Wu1, Ling Zhang1, Ming Zhou1, Stephan Bo¨hm2, Claudia Bubenzer2
and Hugo Scheer2
1 College of Life Science and Technology, Huazhong University of Science and Technology, Hubei, China
2 Department Biologie I – Bereich Botanik, Universita¨t Mu¨nchen, Germany
Phycobiliproteins are a homologous family of
light-harvesting proteins present in cyanobacteria, red algae,
and cryptophytes [1,2] They absorb light in the
regions where chlorophyll absorbs poorly, and transfer
excitation energy with high quantum efficiency to the photosynthetic reaction centres Directed energy trans-fer is determined by the spectroscopic properties and relative positions of the various chromophores present,
Keywords
biliproteins; biosynthesis; cyanobacteria;
photosynthesis; post-translational
modification
Correspondence
K.-H Zhao, College of Life Science and
Technology, Huazhong University of Science
and Technology, Wuhan 430074, Hubei,
China
Tel ⁄ Fax: +86 27 8754 1634
E-mail: kaihongzhao@tom.com
H Scheer, Department Biologie I – Bereich
Botanik, Universita¨t Mu¨nchen,
Menzinger Str 67, D-80638 Munich,
Germany
Fax: +49 89 17861 271
Tel.: +49 89 17861 295
E-mail: hugo.scheer@lmu.de
(Received 10 November 2005, revised 17
January 2006, accepted 20 January 2006)
doi:10.1111/j.1742-4658.2006.05149.x
Covalent attachment of phycocyanobilin (PCB) to the a-subunit of C-phy-cocyanin, CpcA, is catalysed by the heterodimeric PCB : CpcA lyase, CpcE⁄ F [Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA & Glazer
AN (1992) Proc Natl Acad Sci USA 89, 7017–7021] CpcE and CpcF of the cyanobacterium, Mastigocladus laminosus PCC 7603, form a 1 : 1 com-plex Lyase-mutants were constructed to probe functional domains When
in CpcE (276 residues) the N terminus was truncated beyond the R33YYAAWWL motif, or the C terminus beyond amino acid 237, the enzyme became inactive Activity decreases to 20% when C-terminal trun-cations went beyond L275, which is a key residue: the Kmof CpcE(L275D) and (L276D) increased by 61% and 700%, kcat⁄ Km decreased 3- and 83-fold, respectively The enzyme also lost activity when in CpcF (213 resi-dues) the 20 N-terminal amino acids were truncated; truncation of 53 C-terminal amino acids inhibited complex formation with CpcE, possibly due to misfolding According to chemical modifications, one accessible arginine and one accessible tryptophan are essential for CpcE activity, and one carboxylate for CpcF Both subunits bind PCB, as assayed by Ni2+ affinity chromatography, SDS⁄ PAGE and Zn2+-induced fluorescence The bound PCB could be transferred to CpcA to yield a-CPC The PCB transfer capacity correlates with the activity of the lyase, indicating that PCB bound
to CpcE⁄ F is an intermediate of the enzymatic reaction A catalytic mech-anism is proposed, in which a CpcE⁄ F complex binds PCB and adjusts via
a salt bridge the conformation of PCB, which is then transferred to CpcA
Abbreviations
APC, allophycocyanin; CHD, 1,2-cyclohexanedione; CPC, C-phycocyanin; CpcA, a subunit apoprotein of C-phycocyanin; CpcE(x-y) or CpcF(x-y), truncated CpcE or CpcF, respectively, extending from amino acid ‘‘x’’ to amino acid ‘‘y’’; CpcE, CpcF subunits of the heterodimeric PCB:CpcA lyase; CpcE ⁄ F, complex of CpcE and CpcF, DEPC, diethylpyrocarbonate; EDAC, 1-ethyl-3-[3-(dimethylamino)propyl]
carbodiimide; IAA, iodoacetic acid; KPB, potassium phosphate buffer; NBS, N-bromosuccinimide; PC, phycocyanin; PCB, phycocyanobilin; PCMS, p-chloromercuriphenylsulfonic acid; PE, phycoerythrin; PEB, phycoerythrobilin; PEC, phycoerythrocyanin; PecE, PecF, subunits
of PVB:PecA isomerase-lyase; PGO, phenylglyoxal; PLP, pyridoxal-5’-phosphate; PUB, phycourobilin; PVB, phycoviolobilin;
TX100, Triton X-100.
Trang 2i.e linear tetrapyrroles (phycobilins) of which one to
four are covalently attached to the subunits by
thio-ether bonds to conserved cysteines Phycobiliproteins
from cyanobacteria are heterohexamers (ab)3, which
are organized by linker proteins to large antenna
com-plexes, the phycobilisomes Some of the linkers also
carry phycobilin chromophores [3]
In cyanobacteria, four classes of biliproteins have
been assigned on the basis of their visible absorptions
and sequence homologies: phycocyanin (PC),
erythrin (PE), allophycocyanin (APC) and
phyco-erythrocyanin (PEC) They contain, alone or in
combination, four different types of isomeric bilin
chromophores: phycourobilin (PUB),
phycoerythrobi-lin (PEB), phycobiliviophycoerythrobi-lin (PVB), and phycocyanobiphycoerythrobi-lin
(PCB) [4] C-phycocyanin (CPC) from Mastigocladus
laminosusPCC 7603 studied in this work, carries three
PCB at cysteines a-C84, b-C82, and b-C155 [5,6]
The last step in phycobilin biosynthesis [7] is the
addition of phycobilin to the apoproteins In vivo, the
correct attachment of most chromophores is catalysed
by binding site- and chromophore-specific lyases, of
which only a few have hitherto been characterized
[8–10] Since chromophore addition is autocatalytic in
some biliproteins (phytochromes, ApcE) [11–15] and
proceeds spontaneously, but more slowly and with less
fidelity, also in the phycobiliproteins like CPC [16,17],
a chaperone-like action has been proposed for these
lyases [8,17] This view has been strengthened by the
observation of low activities of chromophore binding
to all binding sites studied, including a-C84 of PecA
[16–18], by the site-selective effect of Triton X-100
(TX100) on chromophore binding to PecB, and by its
inhibition of side reactions on binding to CpcA [17]
The first and hitherto best studied lyase attaches
PCB to the highly conserved a-C84 of CpcA from
Syn-echococcus sp PCC7002 [19,20] In this and several
other cyanobacteria, it is coded by two genes, cpcE
and cpcF, which are located in the cpc operon
down-stream of the structural genes for the two CPC
sub-units A similar organization has been found for other
biliprotein:a-C84 lyase genes, but other arrangements
including isolated and fused genes have been reported
[8,21,22]
The lyase function of the proteins has been
demon-strated in vitro for CpcE and CpcF from Synechococcus
sp PCC7002, Anabaena sp PCC7120, Synechocystis sp
PCC6803, M laminosus PCC7603, but has only been
studied in some detail for Synechococcus sp PCC7002
[13,23,24] Mutants lacking cpcE and⁄ or cpcF (or their
homologues) produce significantly reduced amounts of
CPC [20,25,26] Homologous lyases of the ‘E⁄ F-type’
are involved in the attachment of PEB and PCB to C84
of the a-subunits of CPE and PEC, respectively; the latter reaction involves a concomitant isomerization of PCB to PVB [9,18] In vitro, CpcE and CpcF produced
in Escherichia coli jointly catalysed the correct attach-ment of PCB to CpcA-C84, while CpcE or CpcF alone were ineffective CpcE and CpcF can also transfer the bilin homo- and heterologously from a chromophoryl-ated to a nonchromophorylchromophoryl-ated CpcA: this reaction was reversible and specific for the a-84 site [23] CpcE and CpcF from Nostoc sp PCC7120 can transfer PCB from chromophorylated CpcA to PecA, and even to apo-phytochrome AphA [13] Addition of the CpcE⁄ F complex to a-CPC alters its absorption and dramatic-ally reduces the fluorescence yield, no such changes are seen with b-CPC [27]
Enzymes catalysing the biosynthesis of PCB (haem oxygenase and biliverdin reductase) were introduced together with the lyase (CpeE⁄ F) and CpcA into
E colito generate a-CPC [24]
There is currently no structure known for any of the biliprotein lyases CpcE and CpcF from different spe-cies show up to 60% identities, while they are only 20–40% homologous with other enzymes that are known or suggested as lyase for phycobilin addition, such as PecE and PecF [9], CpeY and CpeZ [21] The current study was initiated by the finding of conserved motifs in alignments among lyases from different species [28] We report truncations, site-directed muta-tions and chemical modificamuta-tions which were guided by such sequence comparisons, and propose a model of action that involves transient covalent binding of the chromophore to the lyase
Results
Expression and purification of wild-type and mutant enzymes
Full-length and truncated CpcE and CpcF proteins were expressed with N-terminal His- and S-tags using the pET-30a vector The proteins were generally well soluble (unlike those from Anabaena sp PCC 7120 [13]), and the yield of extracted protein was > 70%
An exception was CpcF(1–160) which tended to precipitate (see below) The tags interfere with neither the functions of the lyase (CpcE⁄ F), nor with the reactivity of the apoprotein (CpcA), but they facilitate protein purification and improve their solubilities [13,17,18] After expression in E coli, full-length CpcE, CpcF, and their mutants were purified and quantified by the Bradford method, and then their
experiments
Trang 3Mutation of CpcE and CpcF
The enzyme activities of the mutated subunits are
com-pared with those of the wild-type subunits in Table 1,
they were quantified by the fluorescence emission of
chromophorylated CpcA at 640 nm [23] In these tests,
a mutated subunit was always combined with the
full-length complementary one As mutations may affect
the interactions among subunits, all enzyme activities
of CpcE and CpcF were measured as before [28] in
three ways: with the nonpurified proteins (supernatants
of the disrupted E coli), with the subunits purified via
Ni2+ affinity chromatography, and with the purified
subunits which were first denatured together with the
full-length complementary one in 8 m urea and then
slowly corenatured by dialysis against urea-free buffer
(20 mm KPB, 0.5 m NaCl, pH 7.2) The individual
subunits, CpcE or CpcF, did not show any enzyme
activities, in agreement with previous studies using
Synechococcussp PCC 7002 [23] The full-length lyase
showed highest activity when purified CpcE (276
resi-dues) and CpcF (213 resiresi-dues) were renatured jointly
in a 1 : 1 mixture from 8 m urea
For CpcE(1–272) and CpcE(L275D), the
superna-tants showed higher activities than the purified
proteins, which may be due to the deletion and
site-directed mutation that affect the interaction among
the subunits, or some unknown factors in E coli
CpcE(1–274) corenatured with CpcF showed some-what higher activity than the supernatants and purified forms, indicating that the function lost by deletion of the two amino acids could be improved by CpcF The mutants were constructed according to a sequence comparison of PCB:CpcA lyases The N-ter-minal motif shown in Fig 1A is highly conserved in CpcE and PecE, therefore the truncated CpcE(42–276) was constructed to delete the motif Similarly, the truncation CpcE(1–272) was generated to remove the highly conserved C-terminal motif (i.e DSLL, in Fig 1B) In CpcE, deletion of 41 amino acids at the N terminus [CpcE(42–276)] and 39 amino acids at C ter-minus (CpcE(1–237)) abolished the enzymatic activity
If judged from the Ni2+-column binding assay (see Experimental procedures) using bound His-tagged CpcF as a bait, CpcE(1–237) has lost the ability to form a complex with CpcF (data not shown), indica-ting that the amino acids in the region 238–276 are involved in the interaction of the two subunits CpcE(1–272) had only 17–28% activity of the wild-type This activity is retained if two more amino acids were removed in CpcE(1–272), but only if this subunit was not corenatured with CpcF Possibly, the deletion interferes with the refolding of CpcE Replacement of the conserved leucine-275 with the polar aspartate in CpcE(L275D) resulted only in a moderate decrease of activity Kinetic studies (Table 2) showed that the Km
Table 1 Comparisons of relative enzymatic activities of CpcE ⁄ F and their mutants, of covalent binding of PCB to CpcE and CpcF, and of their capacity for transferring PCB All test were done under standard reconstitution conditions, with the omissions of specific components given in the headings and footnotes.
Lyase subunits
Relative lyase activity [%]a
Subunit bound PCB b [%]
Yield of a-CPC [l M ] from PCB transfer c,d
PCB transfer [%] c
Non-purified Purified Co- renatured
CpcF(10–213) + CpcE 0 26 21 7.4 0.044 (0.038) 20
CpcE(1–272) + CpcF 29 21 0 10.8 0.072 (0.049) 33
CpcE(1–274) + CpcF 17 22 28 9.6 0.077 (0.061) 35
CpcE(L275D) + CpcF 81 65 65 9.0 0.14 (0.12) 64
CpcE(L276D) + CpcF 22 27 38 10.6 0.061 (0.061) 28
CpcF(I9K) + CpcE 100 100 100 6.6 0.099 (0.094) 45
(CpcE + CpcF) 100 100 120 11.0 0.16 (0.16) 73
a Purified CpcE ⁄ F ¼ 100% b No CpcA added, otherwise identical conditions as for reconstitutions Yields are given with respect to the con-centrations of CpcE or CpcF (5 l M ), the concentration of PCB was 10 l M c No PCB added, otherwise identical conditions as for reconstitu-tions d Values in parentheses are controls with added extra CpcE (5 l M ) and CpcF (5l M ) to test for free PCB; compared with the fluorescence brightness of the band on the Zn 2+ SDS ⁄ PAGE, the lyase and mutants in these tests had 0.22 l M PCB bound e CpcF(1–160) has very low solubility, so PCB binding to the lyase could not be evaluated.
Trang 4value increased by 61% and that kcat⁄ Km decreased
by 32% The same replacement at the neighbouring
position L276, had much more drastic effects The
activity of CpcE(L276D) decreased fourfold, the Km
increased nearly eightfold, and kcat⁄ Km decreased by
almost two orders of magnitude Obviously, L276 is a
critical residue, likely to be involved in the substrate affinity
The truncated protein CpcF(20–213) was generated upon removal of the first ATG serving as initiation site, because there is a second ATG 60 bases down-stream This product was inactive, in spite of a large degree of heterogeneity in the N-terminal region among the different lyases Therefore, the truncation mutant CpcF(10–213) was generated in order to investigate this region more closely CpcF(10–213) only lost activity in the supernatant form When purified and corenatured, the mutant showed 26% and 21% enzyme activity, respectively, indicating that amino acids 1–9 in CpcF are only moderately relevant for the activity In this region, only I9 shows high homology with other lyases However, the activity of the mutant CpcF(I9K) did not show any changes, which was veri-fied by kinetic studies (Table 2) The C-terminally truncated CpcF(1–160) was mostly deposited in inclu-sion bodies (Fig 2B), and the soluble fraction of this mutant has lost activity It was partly regained, how-ever, when it was corenatured with CpcE, indicating that the 53 amino acids in the C-terminal region of CpcF are important for CpcF folding
PCB binding to CpcE and CpcF Work with the isomerizing lyase, PecE⁄ F, had indica-ted that the chromophore is bound transiently to the lyase [28] Such binding was investigated now in more detail with CpcE⁄ F Wild-type and mutant proteins of CpcE, CpcF and their 1 : 1 complexes were incubated with PCB under reconstitution conditions, but omit-ting the acceptor, CpcA They were then re-purified using a Ni2+ affinity column, where unbound PCB was removed in the 1 m NaCl wash step, and dialysed against KPB (pH 7.2) The absorption spectra of these fractions under native (Fig 3A), and denaturing conditions (Fig 3B) showed that PCB could be bound
by CpcE, CpcF, the CpcE⁄ F complex, and also by their mutants (data not shown) Obviously, binding is strong enough to retain the chromophore under the
Fig 1 (A, B) Comparison of conserved N- and C-terminal domains
in CpcE and PecE from different organisms, and (C) of N-terminal
amino acids in CpcF and PecF CLUSTAL W (1.8) multiple sequence
alignment method was used The number in front of the sequence
gives the accession code of the protein sequence in the Swiss-port
database (A, B) CpcE Query: M laminosus PCC7603 (acc no.
AF506031, protein id AAM69288.2, note that the sequence has
been updated on 12.7.2005); compared to CpcE from Anabaena sp.
PCC7120 (PO7125); Fremyella diplosiphon PCC7601 (P07126);
Pseudanabaena sp PCC7409 (Q52448); Synechococcus sp.
PCC7002 (P31967); Synechocystis sp PCC6803 (P73638);
Syn-echococcus elongatus (P50037), and to PecE of Anabaena sp.
PCC7120 (P35791); and M laminosus PCC7603 (P29729) (C)
Query: CpcF from M laminosus PCC7603 (accession number.
AF506031, protein i.d AAM69289.2, note that the sequence has
been updated on 12.7.2005) compared to CpcF from Anabaena sp.
PCC7120 (P29985; Synechococcus sp PCC7002 (P31968);
Syn-echocystis sp PCC6803 (P72652); Synechococcus sp PCC7942
(Q44116); Pseudanabaena sp PCC7409 (Q52449); Synechococcus
elongatus (P50038) and to PecF of M laminosus PCC7603
(P29730).
Table 2 Kinetic analyses for the wild-type lyases and site-directed mutants.
Enzyme K M [l M ]
vmax [p M S)1]
kcat [s)1]
kcat⁄ K M
[s)1Æl M )1]
Wild-type 0.38 ± 0.06 6.3 ± 0.5 1.3 · 10)3 3.4 · 10)3 CpcF(I9K) 0.41 ± 0.08 6.5 ± 0.6 1.3 · 10)3 3.2 · 10)3 CpcE(L275D) 0.61 ± 0.05 6.8 ± 1.2 1.4 · 10)3 2.3 · 10)3 CpcE(L276D) 2.94 ± 0.64 0.61 ± 0.04 1.2 · 10)4 4.1 · 10)5
Trang 5high ionic strength conditions (1 m NaCl) used during
chromatography Covalent binding was supported by
the following observations: (a) denaturation with acidic
urea (8 m, pH 2.0) caused a loss of the distinct peak
at 650 nm on top of a broad background band
> 600 nm The 650-nm peak, on top of a broad
absorption, was recovered in 40–70% yield when the
urea was dialysed out again, with the losses probably
due to irreversible oxidation or denaturation Band
narrowing and absorption increase are characteristic
for chromophores bound to specific sites in native
bili-proteins, which are reversibly lost upon uncoupling of
the chromophore by denaturation of the protein [4,8]
The reversible loss of the distinct peak in
chromo-phore-treated CpcE⁄ F is reminiscent of such changes;
(b) the small absorption decrease upon denaturation,
the relatively broad background in Fig 3 extending
well beyond 700 nm, and the absence of fluorescence
(see below) indicate, however, a less tight coupling
between protein and chromophore, and a
conforma-tional heterogeneity of the latter This binding
situation is rather different from the well-defined one
typical for phycocyanin and phytochromes [6,29,30];
(c) PCB remained bound to the protein during
SDS⁄ PAGE, as shown by Zn2+-staining [31], even
though the amount is small when compared to the
fluorescence intensity of a CPC control (Fig 3D,
Table 1)
Transfer of enzyme-bound chromophore to CpcA PCB bound to CpcE or CpcF has very low fluores-cence (Fig 4C) This opened a way to test for the transfer of enzyme-bound chromophore to the final acceptor, CpcA, because the product, a-CPC, is strongly fluorescent CpcA was incubated, under stand-ard reconstitution conditions, but in the absence of free PCB, with an aliquot of the samples shown in Fig 3A, which induced the fluorescence typical for a-CPC (Fig 4C) Obviously, bound PCB could be transferred from the lyase to CpcA to give the correct product, a-CPC As shown in Table 1, this capacity roughly parallels the enzymatic activities of the lyase and its mutants, indicating that the capacity of the lyase to transiently bind and subsequently transfer PCB is part of its enzymatic activity
This is supported by another observation When PCB, CpcA, CpcE and CpcF were added simulta-neously in the reconstitution system, there is generally
a by-product obtained with maximum absorption at
640 nm and fluorescence at 660 nm, which arises from
a spontaneous, nonenzymatic reaction [9,16,17] Its formation depends on the amount of PCB added, and
is particularly pronounced at higher concentrations (Fig 4A) If, however, PCB (0.05–1 lm, i.e substo-ichiometric amounts with respect to the lyase) was first incubated with CpcE (0.8 lm) and CpcF (0.8 lm) for
1 h, and then CpcA (5 lm) was added, no such non-natural PCB-CpcA adduct was detectable even at high PCB concentrations (Fig 4B) Obviously, the nonenzy-matic reaction was inhibited when PCB was preincu-bated with the lyase This nonenzymatic reaction was restored when CpcE and CpcF mutants were used that lost the transfer ability It is therefore concluded that binding of PCB by the lyase during the preincubation period inhibits the side reaction
Chemical modifications of amino acids Arginine modification by 1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO) [32] resulted in inactivation
of CpcE but not of CpcF (supplementary material Fig S1A and B) The semilogarithmic plots of remain-ing activity against reaction time are linear, indicatremain-ing that the inactivations followed pseudo-first-order kinet-ics Second-order rate constants of 0.1 ± 0.02 and 0.7 ± 0.09 mm)1Æmin)1 were obtained from the linear plots of the first-order rate constants of inactivation against modifier concentrations, for the reactions with CHD and PGO, respectively The numbers of modified residues were obtained from plots of log(1⁄ t0.5) against log[PGO] or log[CHD] They resulted in straight lines
A
B
Fig 2 SDS ⁄ PAGE of Ni 2+ affinity column purified mutant proteins.
Lane assignments: (A) M, protein marker; 1, CpcE(42–276); 2,
CpcE(L275D); 3, CpcE(1–274); 4, CpcE(1–237); 5, CpcE(1–272); 6,
CpcE(L276D); (B) M, protein marker; 1, CpcF(10–213); 2, CpcF(21–
213); 3, CpcF(I9K); 4, CpcF(1–160); 5, CpcF(1–160) purified from
inclusion bodies The last mutant was poorly soluble, when
corena-tured with CpcE, it showed a little activity (see text) The different
mobilities irrespective of the similar sizes of CpcE(42–276) and
CpcE(1–237) were reproducible.
Trang 6with slopes 1.09 and 0.89, respectively; it is therefore
concluded that one accessible arginine residue is
required for the catalytic activity of CpcE The
modifi-cations may affect the lyase activity by interfering with the interactions of CpcE and CpcF This was tested by binding His-tagged CpcF to the Ni2+ affinity column,
800 750 700 650
0
100
200
300
400
A
1 M PCB 0.5 M PCB
Wavelength [nm]
B
800 750 700 650 Wavelength [nm]
100 200 300 400
0
C
800 750 700 650 Wavelength [nm]
0 5 10 15
After Before
Fig 4 (A) Fluorescence analysis of PCB transfer from CpcE ⁄ F to apo-CpcAA: PCB, CpcE (0.8 l M ), CpcF (0.8 l M ) and CpcA (5 l M ) added together at the same time, and then incubated for 1.5 h (B) PCB was first incubated with CpcE (0.8 l M ) and CpcF (0.8 l M ) for 1 h, then CpcA (5 l M ) was added, and the sample incubated for another 1.5 h; PCB concentrations in (A) and (B): 0.05 l M (—); 0.1 l M (— —); 0.3 l M
(– –); 0 5 l M (– ÆÆ -); 0.8 l M (– Æ– Æ); 1 l M (ÆÆÆÆ) (C) Fluorescence emission of PCB bound to CpcE ⁄ F (– –), and after adding CpcA to the system (—) All reactions were carried out under standard reconstitution conditions, see Experimental procedures for more details.
C
B A
D
Fig 3 Binding of PCB to wild-type CpcE, CpcF and CpcE ⁄ F (A) Absorption spectra after incubation of the proteins indicated by the labels (all 10 l M ) with PCB (10 l M ) under reconstitution conditions (37 C, 1 h), subsequent purification by Ni 2+
affinity column to remove unbound PCB, and by dialysis against KPB (pH 7.2), 12 h, in the dark (B) Absorption spectra of the same solutions after addition of 8 M acidic urea (pH 2) (C) Absorption spectra after subsequent renaturation from 8 M acidic urea (pH 2.0) by dialysis against KPB buffer (pH 7.2) See Experimental procedures for details (D) SDS⁄ PAGE of proteins with bound PCB chromophore Lane assignments: M, protein marker; 1, CPC; 2, Histag-CpcE; 3, Histag-CpcF treated with PCB (Coomassie blue stain); 4, CPC; 5, Histag-CpcE; 6, Histag-CpcF treated with PCB (Zn 2+ -induced fluorescence).
Trang 7and analysing for a retention of untagged CpcE by
SDS⁄ PAGE (Fig 5) and by activity assays According
to this criterion, the inactivating arginine modification
did not interfere with complex formation between the
two subunits, but reduced the enzymatic activity
(Fig S1)
Lysine residues were modified with
pyridoxal-5’-phosphate (PLP) [33] Treatment of CpcE and CpcF
with an excess of the reagent for 30 min resulted only
in minor activity changes (96–98% versus control),
indicating that none of the accessible lysines in CpcE
and CpcF are required for the catalytic activity
Carboxyl groups were modified with
1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDAC) [34],
resulting in inactivation of CpcF, but not of CpcE
(Fig S2) The semilogarithmic plots of remaining
activity against reaction time are linear, indicating
that the inactivations followed pseudo-first-order
kinetics A second-order rate constants of 0.6 ±
0.06 mm)1Æmin)1 was obtained from the linear plots
of the first-order rate constants of inactivation against
modifier concentrations The numbers of modified
res-idues were obtained from plots of log(1⁄ t0.5) against
log[EDAC], they resulted in straight lines with slopes
0.65 for the reactions with EDAC It is concluded
that one accessible carboxyl group is required for the
catalytic activity of CpcF This modification also did
not affect the complex formation of CpcE and CpcF
(Fig 5)
Tryptophan residues were modified by
N-bromosuc-cinimide (NBS) [35], it only affected the activity of
CpcE There was a gradual decrease of activity, which
was analysed for the number of critical residues, i, by
the statistical method of Tsou [36] The data can be
fitted to a straight line with i¼ 1 (Fig S3), suggesting
that a single accessible tryptophan residue is
crit-ical for the activity of CpcE This modification of
CpcE did not affect complex formation with CpcE (Fig 5)
Histidine residues of CpcE were modified by DEPC [37], there is no histidine in CpcF of M laminosus In this case, untagged CpcE was used that was purified via ion-exchange column [28] DEPC had no effect on the activity of CpcE, even through all three histidine residues in CpcE were modified, as determined from the absorption increase at 242 nm [28]
Cysteine residues were modified by p-chloromercuri-phenylsulfonic acid (PCMS) and iodoacetic acid (IAA) [38] Reactions were carried out in KPB buffer con-taining 6 m urea in two ways: Either CpcE and CpcF were modified separately, or the CpcE⁄ F complex was modified In case of individual treatments, the treated subunit was combined with the complementary before they were corenatured PCMS had no effect on CpcE and reduced the activity of CpcF only slightly to 78% When CpcE and CpcF were modified together with PCMS, the activity still was 82% When IAA was used, as a more specific thiol reagent, to separately modify CpcE and CpcF, there were again only moder-ate losses of activity to 80% and 67%, respectively However, when they were treated together, the activity was reduced to 16% It is likely from these data, that accessible cysteine residues in both CpcE and CpcF play a role in the reaction, but are not essential There
is no evidence that complex formation of CpcE and F involves an intersubunit disulfide bridge In the Ni2+ -affinity assay, the untagged subunit can always be removed by extensive washing, and at low concentra-tions the complex also dissociates during gel filtration (Superdex 200)
Neither this nor any of the other modifications dis-cussed below produced significant changes in the
far-UV CD-spectra (data not shown); according to this criterion the secondary structure (mainly a-helix) was retained after the modifications
Discussion
CpcE and CpcF, the two subunits of PCB:CpcA lyase, are involved in the PCB attachment to a-CPC at C84 [23] There are eight different pairs of CpcE⁄ F of known sequence, they show large regions of high homology [39–41] The enzymology has been studied
of CpcE⁄ F from Synechococcus sp PCC7002 [23,27], but the amino acids that play a role in PCB attach-ment are not yet clear Also the bifunctional lyase, PecE⁄ F, is homologous with CpcE ⁄ F, and some char-acteristic motifs were identified that distinguish in par-ticular the F-subunits of the former and the latter [28] CpcF lacks, for example, the four contiguous histidines
Fig 5 Effect of chemical modifier on the formation of CpcE or
CpcF complex SDS-containing gels of the fractions eluted with
500 m M imidazole from the Ni 2+ -chrelating columns (see
Experi-mental procedures for details) Lane assignments: M, protein
marker; 1, CpcE (modified by CHD) with His-tag-CpcF; 2, CpcE
(modified by PGO) with His-tag-CpcF; 3, CpcE (modified by NBS)
with His-tag-CpcF; 4, CpcE with His-tag-CpcF (modified by EDAC);
5, CpcE with His-tag-CpcF (modified by IAA).
Trang 8of PecF, which caused a moderately strong binding to
the Ni2+ affinity column and interferes with mutual
binding assays using one His-tagged partner Complex
formation of CpcE with CpcF, and PCB binding to
them, could therefore be analysed with more
confid-ence than for PecE⁄ F, using Ni2+ affinity
chromato-graphy Also, the amounts of PCB bound by CpcE,
CpcF and their mutants were larger than with PecE,
PecF and their mutants, thus facilitating the
quantita-tive analyses of PCB bound by CpcE, CpcF
Several interesting N- and C-terminal motifs were
noted when comparing the sequences PecE and PecF
with those of CpcE and CpcF (Fig 1) For CpcE,
both the N and C termini have conserved regions
When the motif R33YYAAWWL near the N terminus
was deleted (CpcE(41–273)), the enzyme lost its
activ-ity completely A 39-amino acid C-terminal truncation
in CpcE also rendered the protein inactive; it also
nearly lost the ability to form a complex (data not
shown), indicating that this region is involved in the
D273SLL was removed in CpcE(1–272), there was still
some activity left, but the mutant lost activity when
CpcE(1–272) was denatured and corenatured with
CpcF, indicating irreversible unfolding If the two
resi-dues D273 and S274 were maintained, the enzyme still
had 28% relative activity: site-directed mutations of
the two leucines (L275D, L276D) reduced the enzyme
activity only moderately to 65% and 27%,
respect-ively; these mutations also reduced the substrate
affin-ity (Table 2) These regions were also important for
PecE⁄ F lyase-isomerase activity; truncations rendered
the enzyme inactive but did not affect the stability of
the proteins [28]
The C terminus of CpcF shows only little homology
for as much as 50 amino acids A truncation by 53
amino acids reduced the solubility of the protein,
pos-sibly due to misfolding, and most of the protein was
deposited in E coli as inclusion bodies Only 18%
rel-ative activity was recovered by solubilization with urea
(8 m) and corenaturation with CpcE, indicating that
interaction with CpcE aided the re-folding The N
ter-minus of CpcF showed more homology: a 10 amino
acid truncation reduced the activity to 26%, and it
was lost completely when 20 amino acids were
trun-cated Among the 10 N-terminal amino acids, I9 is
highly conserved among different CpcF, but its
muta-tion to lysine resulted in no marked change of the
enzyme activity (Table 2)
There are four cysteines in CpcE and three in CpcF,
of which only C99 of CpcE is highly conserved In
reconstitution experiments in vitro, reducing reagents
such as mercaptoethanol or dithiothreitol were not
required for enzyme activity, indicating that no disul-fides are present that interfere with the enzymatic activ-ity While the thiol group modification using PCMS proved ineffective, a more complex picture was obtained from modification with IAA When only one of the sub-units was modified, the enzyme activity was retained, but modification of both subunits, CpcE and CpcF, in
6 m urea reduced the activity to 16% These modifica-tions were done in 6 m urea to reach otherwise inaccess-ible cysteins, and the protein subsequently renatured They did not interfere noticeably with PCB binding, IAA therefore modifies residues that are otherwise involved in the catalytic activity (e.g PCB transfer) Both CpcE and CpcF bind PCB, as evidenced by absorption spectroscopy and chromatographic separ-ation from unbound chromophore This binding is only moderately strong and reversible, as judged from the low amount of chromophore found on the SDS⁄ PAGE purified proteins (Fig 3D) Covalent chromo-phore binding, albeit even weaker than with CpcE⁄ F, has also been reported for PecE⁄ F In the latter case, PCB bound to the enzyme was neither transferred to PecA to form the PCB adduct, nor transferred and concomitantly isomerized to PVB By contrast, chro-mophore transfer from CpcE⁄ F to CpcA could now be demonstrated Furthermore, in mutants this transfer correlated with their lyase activities (Table 1) In com-bination, these results are evidence for a transient chromophore binding to the enzyme as part of the cat-alytic reaction A chaperone function of a-84 lyases had been suggested before as (at least part of) the en-zymatic activity of a-84 lyases [17,42] The absorption spectral changes of PCB upon binding to CpcE⁄ F are indicative of a conformational change, but at the same time indicate that the chromophore conformation is less restricted not yet extended as it is in a-CPC The lack of an intense fluorescence further suggests that the chromophore retains flexibility upon binding [29], which further supports a comparably weak binding, in
a conformation that is intermediate between the cyclic one of the free chromophore, and its extended, rigid conformation in the a-CPC binding site
There are 19 arginines, 13 lysines, three histidines and two tryptophans in CpcE, and 13 lysines, 10 argi-nines, one tryptophan and no histidine in CpcF According to chemical modification of these residues, only one accessible arginine and one tryptophan is involved in CpcE function, and one carboxyl group CpcF In the bifunctional lyase, PecE⁄ F, a consider-ably larger number of critical amino acid residues have been identified by the same methods An additional histidine is required in the PecE subunit, and one tryp-tophan, one cysteine and one histidine in PecF [28] Of
Trang 9the latter, C121 and H122 are located in a region that
has been related to the lyase function The only critical
residue that is missing in the isomerizing lyase, is the
essential carboxyl group in CpcF This may be related
to the chromophore transfer capacity of CpcE⁄ F,
which is lacking in PecE⁄ F Because the optimal pH
for CpcE⁄ F is 7.5–8.0, the carboxyl group in CpcF is
expected to be present as a carboxylate anion Since
the native PCB chromophore is protonated [43–45], a
possible scenario is the formation of a salt-bridge
between the carboxylate anion in CpcF and the
proto-nated PCB, but this working hypothesis remains to be
tested An alternative function for the carboxylate, i.e
an intermolecular salt bridge with the essential Arg in
CpcE, is not supported in view of the fact that the
modification of the carboxyl of CpcF did not inhibit
the CpcE⁄ F complex formation (Fig 5)
In summary, the two types of homologous lyases
show some common features (heterodimeric complex,
catalysis of attachment of phycobilin at C84 of
a-sub-unit), but they differ in the manipulation of the
chromo-phore not only by the isomerase capacity of PecE⁄ F
that is lacking in CpcE⁄ F, but also in the chromophore
transfer capacity that is present in CpcE⁄ F, but absent
in PecE⁄ F Several residues have been identified that
relate to the different functions However, a general
cat-alytic model is still lacking Investigations of the new
class of distantly related lyases recently identified are
expected to further clarify the molecular basis of the
variability and specificities of this class of enzymes
Experimental procedures
Materials and reagents
1,2-cyclohexanedione, PGO, NBS, PCMS and IAA were
from Sigma (Beijing, P.R.C.); diethylpyrocarbonate
(DEPC), PLP and EDAC were from Fluka (Beijing,
P.R.C.) All other biochemicals and separation materials
were of the highest purity available and obtained from the
sources described previously [9,18] Recombinant proteins
were purified as before [13]
Full-length proteins
Cloning and expression followed the standard procedures
of Sambrook et al [46] The integral genes cpcA, cpcE and
cpcF were PCR-amplified from M laminosus PCC7603
They were cloned first into pBluescript SK(+) (Stratagene,
Shanghai, P.R.C.), and then subcloned into pET-30a
(Nov-agen, Munich, Germany) Proteins without His- and S-tags
were obtained by expressing pGEMEX containing the
desired DNA [9]
Deletion and site-directed mutants
Truncated and site-directed mutants were prepared by
P1: 5¢-TGTCCCGGGGCATTGGTCATGACAGAAGCA-3¢, upstream; P2: 5¢-GGGCTCGAGCGGCAATTAAAGTGG GAAT-3¢, downstream; P3: 5¢-ATACCCGGGATACTCCT GACCATGACTGC-3¢, upstream; P4: 5¢-ACCCTCGAGT
5¢-ATGCCCGGGGGTAAGTTTCGCGTTCG-3¢, upstream; P6: 5¢-GGGCTCGAGTTACATCAAATTCATGACTCG-3¢,
GAATCCA-3¢, downstream; P8: 5¢-ACCCTCGAGTTATT TTCTACCTTGGCCAGC-3¢, downstream; P9: 5¢-TGTCC CGGGCAAATGACAGCAGCTGTA-3¢, upstream; P10: 5¢-AAACCCGGGCGCAGTGTAGCTGAAG-3¢, upstream; P11: 5¢-CCCCTCGAGCCCTTAAATTGGTTGTTGTA-3¢, downstream; P12: 5¢-ATACCCGGGATGACTGCCACTA CTCAACAATTAAAACGT-3¢, upstream; P13: 5¢-GGGCT CGAGCGGCGCTTACAAATCTGAATC-3¢, downstream, P14: 5¢-AGCCTCGAGCGCCTAGTCAAGTGAATCCAT CA-3¢, downstream
All upstream primers have a SmaI site (CCCGGG) and the downstream primers have a XhoI site (CTCGAG), which ensure correct ligation of the fragments to pBlue-script
P1 and P2 were used to generate the full-length cpcE, P3 and P4 for full-length cpcF, P5 and P2 for cpcE(42–276), P1 and P6 for cpcE(1–272), P1 and P7 for cpcE(1–274), P1 and P8 for cpcE(1–237), P9 and P4 for cpcF(21–213), P10 and P4 for cpcF(10–213), P3 and P11 for cpcF(1–160), P12 and P4 for cpcF(I9K), P1 and P13 for cpcE(L275D), P1 and P14 for cpcE(L276D), In P12, P13 and P14, the site-directed mutations are underlined All mutations were verified by sequencing
Expressions
The pET-based plasmids were used to transform E coli BL21 (DE3) Cells were grown at 37C in Luria–Bertani medium containing kanamycin (30 lgÆml)1) When the cell density reached OD600¼ 0.5–0.7, isopropyl-thio-b-dgal-actopyranoside (1 mm) was added 5 h after induction, cells were collected by centrifugation, washed twice with double-distilled water, and stored at )20 C until use CpcA, CpcE, CpcF of M laminosus and all mutants were pre-pared using the methods described previously [18,47]
SDS/PAGE
SDS⁄ PAGE was performed with the buffer system of Lae-mmli [48] The gels were stained with zinc acetate for bilin chromophores [31] and with Coomassie brilliant blue R for the protein The UV-induced fluorescence of protein-bound
Trang 10bilins was recorded digitally with a camera The amounts
of bilins bound by lyases and their mutants were
quantita-tively evaluated by comparing their scanned fluorescence
intensity to that of a standard, i.e CPC, on the same
SDS⁄ PAGE, using photoshop 6.0 (Adobe, San Jose, CA,
USA)
Spectroscopy
Enzyme reactions and amino acid modifications were
followed by UV-visible spectrophotometry (Perkin-Elmer
model Lamda 25) and fluorimetry (Perkin-Elmer LS55)
The formation of chromophorylated-a-CPC was detected
by the emission at 640 nm Far-uv CD spectra were
recor-ded at 25C with a Dichrograph VI (ISA, Jobin Yvon,
Munich, Germany) using 1 mm cuvettes, five spectra were
averaged and the data smoothed by 5-point averaging
PCB and protein concentration determinations
PCB was prepared as described before [18] PCB
concentra-tions were determined spectroscopically in methanol⁄ 2%
HCl using e690¼ 37 900 m-1
Æcm-1 [18] Protein concentra-tions were determined according to [49] using the protein
assay reagent (Bio-Rad, Munich, Germany) according to
the manufacturer’s instructions with BSA as standard
Con-centrations of overexpressed proteins in crude extract were
determined by first assaying the total protein content by the
Bradford method, and then the relative amount (%) by
SDS⁄ PAGE
Phycobiliproteins
CPC and a-CPC from M laminosus were prepared as
before [17]
Lyase activity assay
Chromophore reconstitution with CpcA was assayed as
des-cribed before [27] Either full-length CpcE was
comple-mented with mutants of CpcF, or full-length CpcF was
complemented with mutants of CpcE, using the following
standard reaction conditions: potassium phosphate buffer
(KPB, 15–20 mm, pH 7.2) containing NaCl (150–200 mm),
MgCl2 (5 mm), CpcE and CpcF or their mutants (5 lm
each), and His6-CpcA (5 lm) PCB (final concentration,
5 lm unless stated otherwise) was added as a concentrated
dimethylsulfoxide solution; the final concentration of
dimethylsulfoxide was 1% (v⁄ v) The mixture was incubated
at 37C for 1 h in the dark Products were quantified by
fluorescence emission at 640 nm [27] The lyase reactions
were carried out with three different preparations of each
His-tagged CpcE, CpcF and their mutants: (1) nonpurified
proteins, i.e the supernatants of the sonicated cells after
centrifugation; (2) proteins purified by Ni2+chelating affin-ity chromatography as before [18]; (3) corenatured proteins, which were obtained by the following procedure: purified CpcE (or its mutants) and purified CpcF (or its mutants) were denatured separately with urea (8 m) at room tempera-ture They were then mixed in equimolar amounts (5 lm) and renatured slowly by repeated dialysis against KPB (20 mm, pH 7.2) containing NaCl (0.5 m) at 4C for 4 h For kinetic tests, only purified proteins were used Either full-length CpcE was complemented with mutants of CpcF,
or full-length CpcF was complemented with mutants of CpcE The purified subunits (5 lm), CpcA (5 lm) and differ-ent concdiffer-entrations of chromophore substrate, PCB, were mixed in the reconstitution system (see above) and incubated
at 20C At regular time intervals, the reaction was termin-ated by rapidly cooling the samples on ice to 0C, then the product was quantified by the fluorescence emission at
640 nm The fluorescence was calibrated with a solution of a-CPC of known concentration Km, vmax and kcat were calculated from Lineweaver–Burk plots, using origin v6 (Origin Laboratory Corporation, Northampton, MA, USA)
PCB binding to CpcE and CpcF
A mixture of CpcE and CpcF (1 : 1), individual subunits,
or their mutants was incubated in the reconstitution system,
as described above, but using twice the standard concentra-tion of PCB (10 lm) and omitting CpcA The products were purified by Ni2+chelating chromatography, and ana-lysed by absorption spectroscopy (300–800 nm), and by SDS⁄ PAGE using Zn2+staining [31] and Coomassie brilli-ant blue staining
To check if bound PCB could be transferred to CpcA, the lyase CpcE⁄ F with bound PCB was first purified by Ni2+
affinity chromatography to remove free PCB, and then dia-lysed against KPB (20 mm, pH 7.2) containing NaCl (0.5 m)
at 4C for 12 h in the dark The sample that has been freed
of PCB was divided into three parts The first part of the sample was denatured with 8 m acidic urea (pH 2.0), and its absorption spectrum recorded Then the sample was rena-tured against KPB (20 mm, pH 7.2) containing NaCl (0.5 m), and the absorption recorded again The second part
of the sample was mixed with CpcA (one or both lyase sub-units added when needed), and Mg2+(5 mm), and incubated
at 37C for 1.5 h Then, fluorescence emission at 640 nm was measured as described above The third part was ana-lysed by SDS⁄ PAGE using Zn2+ and Coomassie brilliant blue staining as described above
Complex formation of CpcE and CpcF
Purified His-tagged CpcE or its mutants were corenatured (see Lyase activity assay) with untagged CpcF The mix-tures were then loaded on a Ni2+ column, washed three