By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF subunits of PVB-PEC-lyase, and by experiments with single subuni
Trang 1Characterization of phycoviolobilin
Kai-Hong Zhao1, Dong Wu1, Lu Wang1, Ming Zhou1, Max Storf2, Claudia Bubenzer2, Brigitte Strohmann2 and Hugo Scheer2
1
College of Life Science and Technology Huazhong University of Science and Technology, Wuhan, Hubei, China,
2
Botanisches Institut, Universita¨t Mu¨nchen, Germany
Cofactor requirements and enzyme kinetics have been
studied of the novel, dual-action enzyme, the isomerizing
phycoviolobilinphycoerythrocyanin-a84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses
both the covalent attachment of phycocyanobilin to PecA,
the apo-a-subunit of phycoerythrocyanin, and its
isomeri-zation to phycoviolobilin Thiols and the divalent metals,
Mg2+or Mn2+, were required, and the reaction was aided
by the detergent, Triton X-100 Phosphate buffer inhibits
precipitation of the proteins present in the reconstitution
mixture, but at the same time binds the required metal
Kinetic constants were obtained for both substrates, the
chromophore (Km¼ 12–16 lM, depending on [PecA],
kcat 1.2 · 10)4Æs)1) and the apoprotein (Km¼ 2.4 lMat
14 lMPCB, kcat¼ 0.8 · 10)4Æs)1) The kinetic analysis in-dicated that the reconstitution reaction proceeds by a sequential mechanism By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the pre-valent function of PecE in binding and PecF in isomerizing the chromophore
Keywords: chromophore; cyanobacteria; enzymology; pho-tosynthesis; phycobilin isomerization; phycobilin lyase; phycobiliprotein synthase; thiol addition
Phycobilisomes are the major photosynthetic antenna
complexes of cyanobacteria and red algae [1,2] They
harvest light in the green-gap of chlorophyll absorption,
and transfer excitation energy with high quantum efficiency
to the photosynthetic reaction centers, mainly photosystem (PS)II Phycobilisomes are composed of phycobiliproteins, which absorb light, and linker proteins, which organize the former into the phycobilisome and modulate their absorp-tions Some of the linkers also carry bilin chromophores Cyanobacterial and red-algal biliproteins are generally trimers of an a/b-heterodimer a- and b-subunits are closely related proteins, carrying 1–4 covalently bound chromoph-ores, the phycobilins Based on their absorption spectra properties, phycobiliproteins have originally been classified into three major groups: allophycocyanins, phycocyanins (PC), and phycoerythrins (PE) The former two carry mainly phycocyanobilin (PCB) chromophores with a single covalent bond linking C-31 to cysteine residues of the apoproteins, while PE is characterized by phycoerythrobilin (PEB) chromophores However, the type of chromophore
as well as the mode of binding can be considerably more complex [3,4] Urobilin chromophores are frequently found
in PE and PC from marine cyanobacteria Phycoerythro-cyanin (PEC) carries a phycoviolobilin chromophore PEC
is a light-harvesting component of the phycobilisome in some filamentous, N2-fixing cyanobacteria However, un-like the other biliproteins, PEC shows a photochemistry reminiscent of the sensory photoreceptors, phytochromes (see for example [5–9]), which has been attributed to the phycoviolobilin (PVB) chromophore [10,11]
Of the four cyanobacterial and red-algal phycobilins, PCB and PEB possess a D3,31-ethylidene group They are synthesized from haem by ring opening at C-5 of the tetrapyrrole and several reduction steps, and then attached
to the apoproteins by addition of a cystein thiol to the ethylidene group [3,4,12–14] PCB and PEB can add thiols spontaneously and reversibly, including cysteines of
Correspondence: K.-H Zhao, College of Life Science and Technology,
Huazhong University of Science and Technology, Wuhan 430074,
Hubei, P.R China.
Fax: +86 27 8754 1634, Tel.: +86 27 8754 1634,
E-mail: khzhao@163.com
Hugo Scheer, Botanisches Institut, Universita¨t Mu¨nchen, Menzinger
Str 67, D-80638 Mu¨nchen, Germany.
Fax: +49 89 17861 185, Tel.: +49 89 17861 295,
E-mail: scheer-h@botanik.biologie.uni-muenchen.de
Abbreviations: DDA xxx/yyy , amplitude of photochemical signal with
difference maxima at xxx and yyy nm, normalized to maximum
absorption (see ref [11] for details); DME, dimethylester; PCB,
phycocyanobilin; PEB, phycoerythrobilin; PEC, phycoerythrocyanin;
PVB-PEC-lyase, phycoviolobilin
phycoerythrocyanin-a84-cystein-lyase (isomerizing); PecA, apoprotein of a-PEC; PecE, PecF, subunits
of PVB-PEC-lyase; PUB, phycourobilin; PVB, phycoviolobilin;
(There are two terms for the chromophore in the literature:
phycobi-liviolin [41] and phycoviolobilin [11]; the latter is used because it is
analogous to the names of the major phycobilins, viz
phycocyano-and phycoerythrobilin.) PC, C-phycocyanin; PEC,
phycoerythro-cyanin; a-PEC, chromophorylated a-PEC; TX-100, Triton X-100.
Enzymes: phycoviolobilin phycoerythrocyanin-a84-cystein-lyase
(PVB-PEC-lyase; isomerizing) This name has been submitted to
ENZMES, as an enzyme of the subclass 4.4.1, as an alternative name
we proposed holo-a-phycoerythrocyanin synthase, in analogy to the
cytochrome synthase 4.4.1.17.
Note: the names of all chromophores refer to the free chromophores,
while the chromophores attached to the apoproteins are
characterized as addition products.
(Received 21 April 2002, revised 23 July 2002, accepted 26 July 2002)
Trang 2apo-biliproteins, forming a relatively stable thioether bond
[3,5,15–17] However, the regio- and stereo-specifically
correct attachment has been demonstrated only for the
phytochromes (see [14]) and for a single site in PC, b-84
[18,19] Attachment to the cys-a-84 of CpcA, the a-PC
apoprotein, is catalysed enzymatically by heterodimeric
lyases [20,21] Genes encoding homologous proteins are
known from many cyanobacteria, but the functions of most
are unknown Biliproteins are also known to have up to five
binding sites per monomer, and little is currently known
about the attachment to sites other than cys-a-84 (reviewed
in [22])
Little information is available about the biosynthesis of
the other two chromophores, PVB and phycourobilin
(PUB) These free chromophores have not yet been isolated
from any source, they are only known as protein-bound 31
-thiol adducts Due to the presence of a D2,3-double bond,
which precludes a second one at D3,31, their mode of
attachment to the apoproteins also must be different from
that of PCB and PEB For PVB, the problem has recently
been clarified by the identification of a new enzymatic
activity of the two lyase subunits (PecE, PecF), whose genes
are located on the pec operon In addition to catalysing the
covalent attachment of PCB to cys-a-84 of PecA, the PEC
a-subunit, they promote a concomitant isomerization
[23,24] The result of this intriguing double action is
a-PEC, with the correct 31-cys-PVB chromophore attached
to PecA A similar reaction sequence would lead from PEB
to the 31-cys-PUB chromophore present in many PE
However, no such enzyme is currently known, and the
PVB-PEC-lyase (PecE/F) (previously termed lyase-isomerase)
does not accept the PEB as substrate
Intrigued by its unusual photochemistry, we became
interested in protein engineering a-PEC One goal is to
establish the structural basis of the highly reversible
photochemistry of a-PEC and the protein dynamics related
to the transformation, the other is to evaluate the potential
of the relatively small chromoprotein as a photo switch The
isomerizing PVB-PEC-lyase is crucial to this project: it
allows us to modify separately both the prosthetic group, i.e
the chromophore, and the apoprotein of a-PEC in a directed
manner, and then to reconstitute the chromoprotein in vitro
The a-PEC syntase consists of two proteins, PecE and
PecF, whose genes are encoded in the pec-operon
down-stream from the structural (pecB, A) and linker genes
(pecC) Our previous experiments showed that under
catalysis of the crude extract of heterologously (Escherichia
coli) over-expressed PecE and PecF, PCB can be converted
to PVB, and bound covalently to apo-a-PEC to give native
a-PEC We now report on the preparation of the subunits of
PVB-PEC-lyase possessing His6-tags at the N terminus
(to facilitate purification) and on their enzymatic
char-acterization
M A T E R I A L S A N D M E T H O D S
Overexpression of His6-PecA, His6-PecE, and His6-PecF
The genes pecA, pecE, and pecF were cloned from
Mastigocladus laminosus (Fischerella spec.) with vector
pBluescript (Stratagene), yielding plasmids pBlu-pecA,
pBlu-pecE, and pBlu-pecF, respectively All constructions
were verified by sequencing These genes were subcloned
into vector pET30a (Novagen) using the EcoRV and HindIII restriction sites (pecA) or EcoRV and XhoI restriction sites (pecE, pecF) pBlu-pecA, pBlu-PecE and pBlu-PecF were cleaved with Smal I and XhoI, and the released genes were ligated to the large pET30a fragment Purification of His6-PecA, His6-PecE, and His6-PecF His-tagged PecA, E and F were purified separately by metal ion chelating affinity chromatography on chelating seph-arose (fast flow; Amersham Pharmacia Biotech AB, according to the supplier’s protocol) charged with Ni2+ The E coli [strain BL21(DE3)] cells containing recombinant pET30a were grown in Luria–Bertani medium at 37C, and harvested 5 h after induction with isopropyl thio-b-D -galactoside (IPTG) The cells (usually from 1 L of culture) were washed twice with distilled water, and then suspended
in 30 mL start buffer (20 mMpotassium phosphate buffer
pH 7.2 containing 0.5M NaCl) The suspension was sonicated (Branson model 450 W, 30 min, 45 W) to break the cells, and then centrifuged for 30 min at 12000 g The supernatant was loaded directly onto the Ni2+chelating affinity column After washing with 5 column vols of start buffer to remove untagged proteins, His6-PecA, His6-PecE,
or His6-PecF were eluted with stripping buffer (20 mM
potassium phosphate buffer, pH 7.2 containing 100 mM
EDTA, 0.5MNaCl) The eluent was dialysed three times against 50 mM potassium phosphate buffer pH 7.2, con-taining 0.5MNaCl, to remove Ni2+and EDTA Optimi-zation experiments showed that the latter buffer prevents the otherwise ready precipitation of the His-tagged proteins
An alternative protocol resulted in proteins which can be stored better and for longer: the His-tagged proteins were first dialysed against 50 mM potassium phosphate buffer containing 0.5MNaCl, pH 7.2, and then twice against the same buffer containing also 1 mM 2-mercaptoethanol Finally, His6-PecA was stored at)20 C; His6-PecE, and His6-PecF were mixed with an equal volume of glycerol before storing at)20 C These proteins did not show any loss of activity after storage for 1 year at )20 C A 1-L culture of E coli yielded routinely 100 mg of PecA,
50 mg of PecE or 30 mg of PecF
PCB preparation PCB was prepared as described before [23]
Typical reconstitution of PCB with His6-PecA under catalysis of His6-PecE, and His6-PecF
The reconstitution system consists of PCB, His6-PecA, His6 -PecE, His6-PecF, and 2-mercaptoethanol The enzyme reaction was carried out at 37C for times between
15 min and 3 h, or at room temperature for 1–12 h PCB was added in dimethylsulfoxide solution, to a final concen-tration of 1% (v/v) dimethylsulfoxide His6-PecA, His6 -PecE, and His6-PecF were added to final at concentrations
of 17–86, 6.6–33, and 8.9–44 lM, respectively The final concentration of the other components were: potassium phosphate buffer, 15–20 mM; NaCl, 150–200 mM; 2-merca-ptoethanol, 5 mM; glycerol, 10% (v/v) In addition, 2-mercaptoethanol and a divalent metal (Mn2+or Mg2) are necessary for the activity of His-PecE and His-PecF
Trang 3The optimum concentration of 2-mercaptoethanol is 5 mM.
The activating metals (Mg2+or Mn2+) had optimum
con-centrations of 5 and 3 mM, respectively Mn2+is favoured,
as it stabilized the PVB-PEC-lyase at 37C The pH of the
reconstitution system was adjusted to 7.5 by addition of
Tris/HCl (1M, pH 7.5) to an end concentration of 100 mM
(see Results) Finally, the following detergents at end
con-centrations of 1% (v/v) were shown to improve the reaction:
Triton X-100 (usually used), Nonidet P-40, or Tween-20
In this work the His6-PecA, His6-PecE, and His6-PecF
were used at 15–25 lM, and the chromophore substrate,
PCB, was used at 25 lM, unless stated otherwise At the low
concentrations used for the experiments, which allowed easy
monitoring by spectrophotometry, PCB is otherwise liable
to oxidation by air and precipitation, resulting in loss of the
chromophore and formation of by-products
Spectroscopy
The enzyme reaction was monitored with a UV-VIS
spectrophotometer (Perkin-Elmer model Lamda2) The
product absorption (Z-a-PEC) was monitored at 570 nm
For better characterization, the reversible photoreaction of
the reconstituted product was routinely quantitated by its
DDA as detailed in [11] Reconstituted His6-a-PEC has a
DDA of 100–110% (see Results), the measured DDA
therefore almost equals the absorption of the correctly
reconstituted product, a-PEC (127%, [10,24]) The
extinc-tion coefficient of His6-a-PEC was taken as that of the
untagged protein (e562¼ 1.0 · 105ÆM )1Æcm)1[11])
Intermediates of the enzyme reaction
Reconstitution reactions were carried out as above, but
stopped after 1 h at 37C by the addition of 0.2%
trifluroacetic acid (v/v) After further addition of 2-propanol
(70%, v/v), the mixture was centrifuged to remove any
precipitates The supernatants were injected into HPLC (RP
18 column) and analysed in stream with the diode-array
detector (J & M model Tidas)
PCB and protein concentration determinations
PCB concentration was determined spectroscopically using
an extinction coefficient e690¼ 37.9 mM )1Æcm)1 in
meth-anol/2% HCl The protein concentration was assayed with
protein assay reagent (Bio-Rad) according to the
instruc-tions given by the supplier using BSA as a standard
SDS/PAGE was performed according to Laemmli [25]
R E S U L T S A N D D I S C U S S I O N
Overexpression of His6-PecA, His6-PecE, and His6-PecF
All three genes could be overexpressed effectively in the
vector pET30a The over-expressed His-tagged proteins
required sonification of the cell suspension for relatively
long times (30 min to bring 90% into solution) After
centrifugation, the supernatant containing His6-PecA can
already be used for many reconstitution experiments [24],
this solution is also used for purification via Ni2+
chelating chromatography His6-PecE and His6-PecF are
more soluble After sonification of the respective E coli
cells, they resided quantitatively in the supernatant, which can be used for reconstitution [24] or subsequent purifi-cation
Purification and conservation of His6-PecA, His6-PecE, and His6-PecF
In previous experiments, the subunits of PVB-PEC-lyase, PecE and PecF were over-expressed using the pGEMEX vector [24] Attempts to purify crude extracts of the lyase (ammonium sulfate precipitates) proved difficult and resul-ted in loss of activity Therefore, no further attemp was made to improve the purification methods; instead we concentrated on over-expression by switching to the pET30a vector The resulting His6-PecA, His6-PecE, and His6-PecF were easily purified by Ni2+ chelating affinity chromatography [23] A 1-L culture yielded routinely
100 mg PecA, 50 mg PecE or 30 mg PecF, which by affinity chromatography were concentrated to 10 mgÆmL)1, without loss of activity
However, the proteins are liable to precipitation Solu-bility was greatly enhanced by using potassium phosphate buffer containing high concentrations of NaCl (0.5M) Phosphate is critical because it interacts with the cofactors,
Mg2+and Mn2+(see below), but this effect could be largely compensated by increasing the concentration of these ions
It is convenient to strip the His6-tagged proteins with potassium phosphate buffer (20 mM, pH 7.2) containing EDTA (100 mM), and NaCl (0.5M), then the eluent can be fractionated according to the colour of the eluent due to
Ni2+ In this case, exhaustive dialysis is necessary to remove any Ni2+, which quenches the enzyme (see below) The over-expressed His-tagged PecA, PecE, PecF are stable, if kept frozen at)20 C There was no loss of activity over
1 year Repetitive freezing and thawing should be avoided,
as it causes the purified proteins to precipitate, particularly His6-PecE and His6-PecF Very stable preparations were obtained by adding an equal volume of glycerol before freezing The purified His6-PecA can be also conserved by this method, but in this case care has to be taken keep the glycerol concentration < 10% (v/v), as more decreased the PVB-PEC-lyase activity
Optimal conditions for a-PEC reconstitution The enzyme reaction was shown previously to require several cofactors [23] These requirements were now tested
in more detail
Metal specificity Activation of the PVB-PEC-lyase is more effective with Mn2+ than with Mg2+ (Table 1), although without Mn2+ and even in the presence of 0–50 mMEDTA, the PVB-PEC-lyase had still 21% activity The optimum concentration of Mn2+ is 3 mM, that of
Mg2+is 5 mM Higher concentrations of Mn2+are critical Firstly, when Mn2+ was used as an activator, the concentration of 2-mercaptoethanol needed to be a little higher than that of Mn2+, otherwise the Mn2+was less effective (see below) Secondly, Mn2+ concentration of
25 mMcompletely inhibited the PVB-PEC-lyase By com-parison, the same concentration of Mg2+ (25 mM) still resulted in 62% of the maximum activity Thirdly, the metal ions differ in the effect of EDTA on the PVB-PEC-lyase
Trang 4It inhibited catalysis by Mn2+much more rapidly than by
Mg2+ Last but not least, when Mn2+ was used as an
activator, the Mn2+/EDTA complex accelerated the
oxi-dation of chromophore, resulting in rapid loss of
chromo-phore and complex reconstitution mixtures While this can
be compensated in analytical assays by an excess of the
lyase, it is problematic for preparative reconstitutions We
also found out that it was beneficial to prepare concentrated
stock solutions of Mn2+, to store them at)20 C, and to
add the correct amount of Mn2+ immediately before
starting the enzyme reaction Possibly, this prevents
oxida-tion and dimerizaoxida-tion of the metal ion, but this hypothesis
was not pursued in detail
Several other divalent metals were tested for catalysis
(Table 1) Besides Mn2+and Mg2+, only Ca2+activated
the isomerase activity of PVB-PEC-lyase, but was less
effective than the former All other metal ions tested (Fe2+,
Co2+, Ni2+, Cu2+, Zn2+) resulted in inactivation of
enzymatic isomerization Because the enzyme is moderately
active in the absence of the activating metals, Mg2+ or
Mn2+(Table 1), but the activity is completely lost in the
presence of the other metals tested, this indicates a genuine
inactivation by the latter Because Ni2+was used to bind
His6-tagged proteins during metal chelating
chromatogra-phy, any Ni2+ eluted in the process was removed from
isolated His6-tagged PecA, PecE, and PecF, by exhaustive
dialysis against the potassium phosphate/NaCl
reconstitu-tion buffer With some metals, unwanted side reacreconstitu-tions were
observed in addition: Fe2+, Cu2+, and Zn2+accelerated
chromophore oxidation; Co2+ formed a complex with 2-mercaptoethanol absorbing around 470 nm; and in the presence of Ni2+, a broad, unstructured absorption formed
in the 610–650 nm region
Both Mn2+ and Mg2+ are ubiquitous chelators of nucleotides and cofactors of many related enzymes The catalysis by Mn2+was therefore tested in the presence of ATPand or GTP(data not shown) However, neither increased the activity of catalysis by His6-PecE/His6-PecF Because metals can complex linear tetrapyrroles, the effect
of the metals was investigated on the absorption spectrum
of PCB: addition of Mn2+caused no change to the visible absorption spectrum, irrespective of the presence or absence
of the His6-PecE and His6-PecF (data not shown) As the chromophore spectrum is very sensitive to environmental changes (see for example the effect of Triton X-100 discussed below), these results suggest that Mn2+acted on the PVB-PEC-lyase, and not (or only transitorily) on the conformation of PCB
Thiols 2-mercaptoethanol or thiols like such as dithiothreitol are required for the isomerization reaction of the lyase: without, only the PCB addition product was formed, but no PVB-His6-a-PEC (Table 1) Also the spontaneous addition (no enzymes added) of PCB yielding the cys-a84-PCB-adduct, proceeds in the absence of 2-mercaptoethanol or other thiols However, too much 2-mercaptoethanol will cause the loss of chromophore, in a reaction requiring oxygen When
Mg2+was used as the activator, the optimal concentration
of 2-mercaptoethanol is 5 mM, with Mn2+it is 3 mM The effect of thiols is specific, they could not be replaced by other biological reductants such as NADPH or ascorbic acid (data not shown)
Other factors influencing activity NaCl is beneficial to the reconstitution by preventing the precipitation of the over-expressed proteins However, it proved inhibitory at high concentrations The activity of the His6-PecE and His6-PecF was not noticeably affected up to
250 mMNaCl, but decreased to 50% in 500 mM A similar optimum was found with potassium phosphate, which is needed to dissolve the purified His6-tagged proteins; but this buffer decreases the effect of the activators, Mn2+or Mg2+,
by 20% as compared with Tris/HCl buffer This is most probably due to formation of metal complexes To balance these effects, it proved best practice to use a mixed buffer system consisting of one volume of potassium phosphate (50 mM) containing NaCl (0.5M), and two volumes of Tris/ HCl (150 mM), resulting in final concentrations of 17, 170 and 100 mM, respectively
Under these conditions, the lyase has an optimal pH at around 7.5–7.8 (Fig 1) As the bilins become more liable to oxidation at higher pH [26], a pH £ 7.5 was favoured, and usually buffers of pH 7.5 were used in this work
Temperature The lyase requires relatively high tempera-tures (Table 2) With Mg2+, the reaction time at room temperature of 1 h, can be reduced to 10 min at 37C However, room temperature is recommended in the absence
of TX-100, because the proteins tend to precipitate at 37C
Table 1 Relative activities of the lyase, His 6 -PecE and His 6 -PecF,
depending on the presence and concentrations of 2-mercaptoethanol and
divalent metals Relative activities were determined by the type I
photochemical activity of the product according to Zhao et al [11].
Added cofactors (concentrations [mM]) Relative activity (%)
ME (0), or ME (0) and Mn 2+ (3) 0
ME (5), EDTA (5), Mg 2+ (5) 70
ME (5), EDTA (10), Mg 2+ (5) 21
ME (5), EDTA (5–10), Mn2+(3) 2a
ME (5), Mg2+(5) + Ca2+(5) 73
a Acceleration of chromophore oxidation by Mn 2+ –EDTA.
Trang 5In the presence of TX-100 (1% v/v), the temperature can be
increased without precipitation to 37C (Mg2+as
activa-tor), or even to 45C (Mn2+as activator) This temperature
stability is not surprising in view of the optimum growth of
M laminosus at 50–55C The data in Table 2 were
obtained after 60 min reaction time, the activities are given
as the amplitude of the reversible photochemistry of the
product, a-PEC, relative to that of the reaction at the
optimum temperature, 45C For longer reaction times
(180 min), the temperature optimum is reduced to 37C
(Mn2+), the activity at 45C being 20% less Apparently,
the lyase has a high transient at 45C, but also becomes
more rapidly inactivated than at 37C, probably by
precipitation Because of this inactivation, no experiments
were carried out at T > 45C At these temperatures,
photo-oxidative side-reactions also become prominent (data
not shown) In vivo, M laminosus cells can prevent
oxidation and protect the lyase from precipitation at
considerably higher temperatures, up to 55C
Detergents Although the His-tagged lyase as well as PecA
are well water soluble at temperatures £ 37 C, addition of
mild detergents [e.g 0.2–1% (v/v) Triton X-100, Nonidet
P-40, Tween-20] was beneficial, doubling reaction speed
There was also another beneficial effect: when the Triton
X-100 was present in the reaction system, the spontaneous
addition of PCB to PecA forming adducts was reduced, thereby increasing the proportion of isomerization product,
31-Cys-PVB-PecA (as demonstrated before [24], the PCB-PecA adduct cannot be transformed to a-PEC by His6-PecE and His6-PecF) Addition of Triton X-100 to the reaction mixture resulted in an absorption shift of the long wavelength band of PCB from 620 nm to 600 nm (Fig 2), irrespective of the presence of the lyase (PecE/F) and the structural protein, PecA This implies that the absorption change was due to the amphipathic property of Triton X-100 Possibly, Triton X-100 modifies the confor-mation of chromophore, to a form suitable for the PVB-PEC-lyase to act on, and unfavourable for PecA to bind spontaneously to PCB Changes of the conformational equilibria of bile pigments have been reported in a variety of environments [27,28], including lipids [29] It is possible that,
by analogy, PecE/F also changes the conformation of the bilin in the course of the addition reaction (see below) After optimization of the enzyme reaction, the reconsti-tution is accelerated by 10-fold as compared with the original conditions [23], and the amount of the spontaneous addition product, 31-Cys-PCB (kmax¼ 640 nm) is at the same time reduced Absorption spectra and light-induced changes of typical reconstitution mixtures are shown in Fig 3A and B, and those of a product purified by affinity chromatography in Fig 3C Note the relatively high absorption (580–600 nm) between the two major peaks,
Fig 1 The effect of different pH on the lyase/isomerase action of His 6
-PecE/F Except for the pH, the reaction was carried out under
opti-mized conditions (20 l M each PecA, PecE and PecF; 25 l M PCB,
5 m M ME, 3 m M Mn2+, see Materials and methods for details) The
product was assayed by the reversible photochemistry of the correct
product, 3 1 -Cys-PVB-PecA (a-PEC) according to Zhao et al [11].
Table 2 Temperature dependance of activity of His 6 -PecE and
His 6 -PecF All reactions were carried out in the optimized potassium
phosphate/Tris buffer system (see text) in the presence of Mn 2+
(3 m M ) and 2-mercaptoethanol (5 m M ) The yield of photoactive
a-PEC was assayed after 60 min.
Temperature (C) Relative activity (%)
Fig 2 Interaction of PCB with Triton X-100 Addition of Triton X-100 resulted in a blue shift of the absorption of PCB from 620 nm to
600 nm, both in the presence of His 6 -PecA, His 6 -PecE and His 6 -PecF (A), and in the absence of these proteins (B).
Trang 6which is not observed when the reconstitution is carried out
under catalysis of untagged PecE and PecF [24] This
absorption is seen only when His6-PecE was used, and in
particular when Triton X-100 was added to the reaction
mixture (Fig 3B) It is lost when the reaction mixture is
subjected to Ni2+ chelating chromatography (Fig 3C),
which shows that it derives from free chromophore(s)
Kinetics of enzymatic ligation/isomerization The kinetics of the formation of His-tagged a-PEC from PCB and His6-PecA, catalysed by His6-PecE and His6 -PecF, follows the Michaelis–Menten equation for each substrate, i.e PCB (Fig 4A) and His6-PecA (Fig 4B) The kinetic constants derived from these plots are summarized in Table 3 Like the only other phycobilin-lyase studied [30], the 31-Cys84-PecA:PCB lyase is a rather slow enzyme (kcat¼ 10)4)10)5Æs)1) with moderate affinity The Line-weaver–Burk plots with respect to PCB, obtained at different concentrations of His6-PecA, intersect within the limits of error at a common point which seems not to be located on the x-axis Such behaviour is typical for a sequential mechanism of the enzyme reaction with the two substrates bound one after the other [31,32]
Action of individual subunits, PecE and PecF
As shown before, both lyase subunits, PecE and PecF, are necessary for the reconstitution of PCB and PecA to yield
Fig 3 His 6 -PecE/F catalysed ligation and isomerization of PCB with
PecA Photochemistry [before (solid line) and after (dashed line)
saturating irradiation with 570 nm light) of the reconstitution mixture
(PCB plus His 6 -PecA, His 6 -PecE and His 6 -PecF) under otherwise
optimized conditions (see Fig 1 and Materials and methods), in the
absence (A) and presence (B) of Triton X-100 Note the relatively
strong absorption at 580–600 nm (arrow) between the two product
bands, which is increased in the presence of Triton X-100 It is lost after
Ni 2+ chelating chromatography (C).
Fig 4 Enzyme kinetics of the PVB-PEC-lyase Lineweaver–Burk plots of the ligation–isomerization reaction catalysed by His 6 -PecE and His 6 -PecF, for the two substrates, PCB (A) and His 6 -PecA (B) Other conditions were as described in Fig 1 At different concentrations of His -PecA, the corresponding linear fits do not intersect on the x-axis.
Trang 7the phycoviolobilin-bearing chromoprotein, a-PEC [23],
and neither of the two subunits alone could catalyse the
reconstitution effectively In this reconstitution, the enzyme
catalyses two reactions: the covalent binding of PCB to the
apo-protein, and its transformation to bound PVB It was
therefore interesting to see if and how the two subunits PecE
and PecF, which show a low degree of homology, function
in the absence of the other A careful inspection of the
absorption changes (Fig 5) indeed showed some
subunit-specific residual activities: PecE applied alone, increases the
spontaneous or auto(?) catalytic binding of PCB to PecA
by 25%, yielding, however, only 31-Cys84-PecA-PCB In
the presence of His6-PecF, this pure addition reaction of
PCB to His6-PecA was decreased 15% However, in this
case a small amount of the ligation/isomerization product,
His6-a-PECA, was formed (7% as compared to the
maximal yield of His6-a-PecA in the presence of His6-PecE
and His6-PecF) This may indicate that PecE is mainly
responsible for binding the chromophore to the apoprotein,
PecA, and PecF is mainly promoting the isomerization PCB
to PVB Interestingly, this model is supported by sequence
comparison between the respective subunits of the two
enzymes (Table 4): For the two organisms for which the
sequences are known, there is a significantly higher
homo-logy and Z-score for the E-subunits that for the F-subunits
If this functional distinction of the two subunits is correct,
the question arises as to the function of the F-subunit of the
phycocyanin lyases Possibly, it acts as an isomerase as well
in this case, but as one ensuring or chaperoning the
isomerization of improperly bound chromophores, for
example those having incorrect stereochemistry
It should be emphasized again, however, that the product
of the spontaneous addition reaction (31
-Cys84-PecA-PCB) bearing the PCB chromophore, can not be isomerized
to the PVB chromophore by the action of PecE and PecF,
either alone or in combination For some of the
phyto-chromes, a sequential ligation reaction is discussed
[14,33,34] The isomerization may therefore proceed at an
intermediate state
The concerted action of the lyase subunits is supported by
a physical interaction between them It had already been
shown that in case of the P C-Cys-a84 lyase, CpcE and CpcF
form a 1 : 1 complex [30] Gel filtration experiments with
PecE/F on HiPrep Sephacryl S-200 (Amersham Pharmacia
Biotech AB) proved inconclusive There were aggregates
(50–60, 80–90, 120–140 kDa) observed in the mixture of the
subunits, but both His6-PecE, and His6-PecF formed
homo-oligomers (e.g dimer, trimer, and tetramer), and the
resolution was insufficient to clearly distinguish
homo-from hetero-oligomers (data not shown) However, the
formation of complexes between the subunits, PecE and PecF; is supported by the following experiments In the first approach, His6-PecF was absorbed on a Ni2+chelating column in start buffer (0.5M NaCl, 20 mM potassium
Table 3 Enzymatic parameters for the ligation/isomerization of PCB to
His 6 -PecA, catalysed by His 6 -PecE/F under optimum conditions (see
text) Data were derived from the fits shown in Fig 4 [PecE] and
[PecF] were 8.6 l M , so k cat ¼ v max /8.6 · 10)6.
Substrate (S)
Co-substrate
(concentration) K S
m (l M )
v max
(n M Æs)1) k cat (s)1) PCB PecA (14 l M ) 2.4 0.65 0.76 · 10)4
PecA PCB (19 l M ) 12 1.1 1.3 · 10)4
PecA PCB (9.6 l M ) 14 0.97 1.1 · 10)4
PecA PCB (4.8 l M ) 16 0.95 1.1 · 10)4
Fig 5 Effect of individual lyase–isomerase subunits on the reaction of PCB with PecA (A) Compared to the spontaneous addition reaction (solid line), direct binding of PCB to His 6 -PecA without isomerization
to 3 1 -Cys-PVB, is increased by 25% in the presence of His 6 -PecE (dotted line), but no His 6 -a-PecA (k max ¼ 570 nm) is formed In the presence of His 6 -PecF (dashed line), the yield of PCB-His 6 -PecA is decreased by 15%, and a shoulder at 570 nm is clearly visible It was identified as 3 1 -Cys-PVB-PecA and quantitated by its reversible pho-tochemistry (B) Large amounts of the correct ligation–isomerization product 31-Cys-PVB-PecA were formed only in the presence of both PecE and PecF [solid line in (A)].
Table 4 Amino acid identities and Z-scores between the respective subunits of PCB-lyases and PVB-PEC-lyase from M laminosus (Fischerella PCC 7603 [35,36], D Wu, J.-P Zhu, H Scheer, K.-H Zhao, unpublished results, GenBank AF506031) and Anabena sp PCC7120 [37–40].
Amino acid identities [%] (Z-score) for comparison of
Trang 8phosphate, pH 7.2), and then the same amount of untagged
PecE [24] dissolved in the start buffer, was applied to the
preloaded column It was then first washed with the start
buffer, and subsequently with stripping buffer containing
EDTA (100 mM) and NaCl (0.5M) In the first washing
fractions with start buffer, 70% of the PecE (as judged by
SDS/PAGE) was eluted The remaining 30% of the PecE
stayed on the column during further washing, in spite of it
lacking a His-tag, and was eluted only with the stripping
buffer together with the majority of the His-tagged PecF
Independent support for the formation of complexes
between PecE and PecF comes also from reversible
denaturation experiments (Table 5): Denaturation in 8M
urea of the individual subunits, PecE or PecF, is largely
irreversible: if they are mixed together after dialysing out the
urea separately, they show only little activity This is also
true if either individually treated PecE is mixed with native
PecF, or vice versa By contrast, if the two subunits are
mixed in the denatured state and then the urea is dialysed
out from the mixture, the resulting product shows full
activity
C O N C L U S I O N S
Cofactor requirements and enzyme kinetics of
PVB-PEC-lyase from M laminosus have been studied The novel,
dual-action enzyme is responsible for the attachment and
isomerization of phycocyanobilin to PecA, the a-subunit
of phycoerythrocyanin Mercaptoethanol and the divalent
metals, Mg2+or Mn2+, were required, and the reaction was
aided by the detergent Triton X-100 The speed of the
reaction and the purity of the products was improved by
careful adjustment of the buffer, balancing in particular the
conflicting effects of potassium phosphate buffer, which
inhibits protein precipitation, but at the same time binds the
required metal These improvements will provide a basis for
the preparative reconstitution of the individually or jointly
modified reaction partners, viz the structural protein PecA
and the substrate chromophore, PCB
Kinetic experiments showed the enzyme to be rather
slow, comparable to a related mono-functional
PCB-phycocyanin lyase [30] Furthermore, they indicated that the reconstitution reaction proceeds by a sequential mech-anism, which has the characteristics that the enzyme reaction requires all of the substrates to be present before any product is released This is consistent with HPLC results detecting no chromophore other than the substrate PCB in the reaction mixture [23] Moreover, there is evidence, that PecE is responsible for chromophore binding, and PecF for the isomerization However, although PCB does bind covalently to His6-tagged PecA to form PCB-His6-tagged PecA, the latter is no substrate of the enzyme: it could not
be transformed to PVB-His6-tagged PecA (i.e His6-tagged a-PecA) under catalysis of PecE and/or PecF
By using a combination of untagged and His-tagged subunits, evidence was obtained for the interaction between PecE and PecF Experiments of this type are expected to guide the way to ternary and quaternary complexes of the unusual enzyme
The ligation mechanism of the chromophores to phyco-bilin and phytochrome apoproteins still remains largely unknown It is hoped that other isomerizing lyases leading
to biliproteins will be characterized in the future, in particular those yielding chromophores with a D2,3-double bond (phycourobilin, several cryptophytan proteins)
A C K N O W L E D G E M E N T S
The laboratory of K.H.Z is supported by Natural Science Foundation
of China (project number 39770175) K.H.Z is grateful to the DAAD, Bonn, Germany for a fellowship, and to the Alexander von Humboldt Foundation, Bonn, Germany for donation of a microcentrifuge subsequent to a postdoctoral fellowship The laboratory of H.S is supported by Deutsche Forschungsgemeinschaft (SFB 533, TPA1).
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