Tài liệu Báo cáo khoa học: Characterization of phycoviolobilin phycoerythrocyanin-a84-cysteinlyase-(isomerizing) from Mastigocladus laminosus pdf

Characterization of phycoviolobilin phycoerythrocyanin-a84-cystein-
lyase-(isomerizing) from
Mastigocladus laminosus
Kai-Hong Zhao
1
, Dong Wu
1
, Lu Wang
1
, Ming Zhou
1
, Max Storf
2
, Claudia Bubenzer
2
, Brigitte Strohmann
2
and Hugo Scheer
2
1
College of Life Science and Technology Huazhong University of Science and Technology, Wuhan, Hubei, China,
2
Botanisches Institut, Universita
¨
tMu
¨
nchen, Germany
Cofactor requirements and enzyme kinetics have been
studied of the novel, dual-action enzyme, the isomerizing
phycoviolobilin phycoerythrocyanin-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,
Mg
2+
or Mn
2+
, 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 (K
m
¼ 12–16 l
M
, depending on [PecA],
k
cat
 1.2 · 10
)4
Æs
)1
) and the apoprotein (K
m
¼ 2.4 l
M
at
14 l
M

PCB, k
cat
¼ 0.8 · 10
)4
Æs
)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-andb-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-3
1
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, N
2
-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 D3,3
1
-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:
Hugo Scheer, Botanisches Institut, Universita
¨
tMu
¨
nchen, Menzinger
Str. 67, D-80638 Mu
¨
nchen, Germany.
Fax: +49 89 17861 185, Tel.: +49 89 17861 295,
E-mail:
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)
Eur. J. Biochem. 269, 4542–4550 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03148.x
apo-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 3
1
-
thiol adducts. Due to the presence of a D2,3-double bond,
which precludes a second one at D3,3
1

, 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 3
1
-cys-PVB chromophore attached
to PecA. A similar reaction sequence would lead from PEB
to the 3
1
-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 His
6
-tags at the N terminus
(to facilitate purification) and on their enzymatic char-
acterization.
MATERIALS AND METHODS
Overexpression of His
6
-PecA, His
6
-PecE, and His
6
-PecF
The genes pecA, pecE,andpecF 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)orEcoRV and XhoI
restriction sites (pecE, pecF). pBlu-pecA, pBlu-PecE and
pBlu-PecF were cleaved with Smal IandXhoI, and the
released genes were ligated to the large pET30a fragment.

Purification of His
6
-PecA, His
6
-PecE, and His
6
-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 Ni
2+
.
The E. coli [strain BL21(DE3)] cells containing recombinant
pET30a were grown in Luria–Bertani medium at 37 °C, 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 m
M
potassium phosphate buffer
pH 7.2 containing 0.5
M
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 Ni
2+

chelating
affinity column. After washing with 5 column vols of start
buffer to remove untagged proteins, His
6
-PecA, His
6
-PecE,
or His
6
-PecF were eluted with stripping buffer (20 m
M
potassium phosphate buffer, pH 7.2 containing 100 m
M
EDTA, 0.5
M
NaCl). The eluent was dialysed three times
against 50 m
M
potassium phosphate buffer pH 7.2, con-
taining 0.5
M
NaCl, to remove Ni
2+
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 m
M

potassium phosphate buffer
containing 0.5
M
NaCl, pH 7.2, and then twice against the
same buffer containing also 1 m
M
2-mercaptoethanol.
Finally, His
6
-PecA was stored at )20 °C; His
6
-PecE, and
His
6
-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 His
6
-PecA under
catalysis of His
6
-PecE, and His
6
-PecF
The reconstitution system consists of PCB, His

6
-PecA, His
6
-
PecE, His
6
-PecF, and 2-mercaptoethanol. The enzyme
reaction was carried out at 37 °C 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. His
6
-PecA, His
6
-
PecE, and His
6
-PecF were added to final at concentrations
of 17–86, 6.6–33, and 8.9–44 l
M
, respectively. The final
concentration of the other components were: potassium
phosphate buffer, 15–20 m
M
; NaCl, 150–200 m
M
; 2-merca-
ptoethanol, 5 m
M
; glycerol, 10% (v/v). In addition,

2-mercaptoethanol and a divalent metal (Mn
2+
or Mg
2
)
are necessary for the activity of His
6
-PecE and His
6
-PecF.
Ó FEBS 2002 Enzymology of isomerizing phycoviolobilin lyase (Eur. J. Biochem. 269) 4543
The optimum concentration of 2-mercaptoethanol is 5 m
M
.
The activating metals (Mg
2+
or Mn
2+
)hadoptimumcon-
centrations of 5 and 3 m
M
, respectively. Mn
2+
is favoured,
as it stabilized the PVB-PEC-lyase at 37 °C. The pH of the
reconstitution system was adjusted to 7.5 by addition of
Tris/HCl (1
M
, pH 7.5) to an end concentration of 100 m
M

(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 His
6
-PecA, His
6
-PecE, and His
6
-PecF
were used at 15–25 l
M
, and the chromophore substrate,
PCB, was used at 25 l
M
, 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
TheenzymereactionwasmonitoredwithaUV-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 His
6
-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 His
6
-a-PEC was taken as that of the
untagged protein (e
562
¼ 1.0 · 10
5
Æ
M
)1
Æcm
)1
[11]).
Intermediates of the enzyme reaction
Reconstitution reactions were carried out as above, but
stopped after 1 h at 37 °C 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 e
690
¼ 37.9 m
M
)1

Æcm
)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].
RESULTS AND DISCUSSION
Overexpression of His
6
-PecA, His
6
-PecE, and His
6
-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 His
6
-PecA can
already be used for many reconstitution experiments [24],
this solution is also used for purification via Ni
2+
chelating chromatography. His
6
-PecE and His
6
-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 His
6
-PecA, His
6
-PecE,
and His
6
-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 His
6
-PecA, His
6
-PecE, and
His
6
-PecF were easily purified by Ni
2+
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 (0.5
M
).
Phosphate is critical because it interacts with the cofactors,
Mg
2+
and Mn
2+
(see below), but this effect could be largely
compensated by increasing the concentration of these ions.
It is convenient to strip the His
6
-tagged proteins with
potassium phosphate buffer (20 m
M
, pH 7.2) containing
EDTA (100 m
M
), and NaCl (0.5
M
), then the eluent can be
fractionated according to the colour of the eluent due to
Ni

2+
. In this case, exhaustive dialysis is necessary to remove
any Ni
2+
, 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
His
6
-PecE and His
6
-PecF. Very stable preparations were
obtained by adding an equal volume of glycerol before
freezing. The purified His
6
-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 Mn
2+
than with Mg
2+

(Table 1),
although without Mn
2+
andeveninthepresenceof
0–50 m
M
EDTA, the PVB-PEC-lyase had still 21% activity.
The optimum concentration of Mn
2+
is 3 m
M
,thatof
Mg
2+
is 5 m
M
. Higher concentrations of Mn
2+
are critical.
Firstly, when Mn
2+
wasusedasanactivator,the
concentration of 2-mercaptoethanol needed to be a little
higher than that of Mn
2+
, otherwise the Mn
2+
was less
effective (see below). Secondly, Mn
2+

concentration of
25 m
M
completely inhibited the PVB-PEC-lyase. By com-
parison, the same concentration of Mg
2+
(25 m
M
) still
resulted in 62% of the maximum activity. Thirdly, the metal
ions differ in the effect of EDTA on the PVB-PEC-lyase.
4544 K H. Zhao et al. (Eur. J. Biochem. 269) Ó FEBS 2002
It inhibited catalysis by Mn
2+
much more rapidly than by
Mg
2+
. Last but not least, when Mn
2+
wasusedasan
activator, the Mn
2+
/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 Mn
2+

,tostorethemat)20 °C, and to
add the correct amount of Mn
2+
immediately before
starting the enzyme reaction. Possibly, this prevents oxida-
tion and dimerization of the metal ion, but this hypothesis
was not pursued in detail.
Several other divalent metals were tested for catalysis
(Table 1). Besides Mn
2+
and Mg
2+
, only Ca
2+
activated
the isomerase activity of PVB-PEC-lyase, but was less
effective than the former. All other metal ions tested (Fe
2+
,
Co
2+
,Ni
2+
,Cu
2+
,Zn
2+
)resultedininactivationof
enzymatic isomerization. Because the enzyme is moderately
active in the absence of the activating metals, Mg

2+
or
Mn
2+
(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 Ni
2+
was used to bind
His
6
-tagged proteins during metal chelating chromatogra-
phy, any Ni
2+
eluted in the process was removed from
isolated His
6
-tagged PecA, PecE, and PecF, by exhaustive
dialysis against the potassium phosphate/NaCl reconstitu-
tion buffer. With some metals, unwanted side reactions were
observed in addition: Fe
2+
,Cu
2+
,andZn
2+
accelerated
chromophore oxidation; Co
2+
formed a complex with

2-mercaptoethanol absorbing around 470 nm; and in the
presence of Ni
2+
, a broad, unstructured absorption formed
in the 610–650 nm region.
Both Mn
2+
and Mg
2+
are ubiquitous chelators of
nucleotides and cofactors of many related enzymes. The
catalysis by Mn
2+
was therefore tested in the presence of
ATP and or GTP (data not shown). However, neither
increased the activity of catalysis by His
6
-PecE/His
6
-PecF.
Because metals can complex linear tetrapyrroles, the effect
of the metals was investigated on the absorption spectrum
of PCB: addition of Mn
2+
caused no change to the visible
absorption spectrum, irrespective of the presence or absence
of the His
6
-PecE and His
6

-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 Mn
2+
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-
His
6
-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
Mg
2+
was used as the activator, the optimal concentration
of 2-mercaptoethanol is 5 m
M
,withMn
2+
it is 3 m
M
.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
His
6
-PecE and His
6
-PecF was not noticeably affected up to
250 m
M
NaCl, but decreased to 50% in 500 m
M
. A similar
optimum was found with potassium phosphate, which is
needed to dissolve the purified His
6
-tagged proteins; but this
buffer decreases the effect of the activators, Mn
2+
or Mg
2+
,
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 m

M
) containing NaCl (0.5
M
), and two volumes of Tris/
HCl (150 m
M
), resulting in final concentrations of 17, 170
and 100 m
M
, 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 Mg
2+
, the reaction time at room
temperature of 1 h, can be reduced to 10 min at 37 °C.
However, room temperature is recommended in the absence
of TX-100, because the proteins tend to precipitate at 37 °C.
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) 21
ME (5), EDTA (5–50) 21
ME (5), EDTA (5), Mg
2+
(5) 70
ME (5), EDTA (10), Mg
2+
(5) 21
ME (5), EDTA (5–10), Mn
2+
(3) 2
a
ME (2.5), Mn
2+
(1.2) 55
ME (5), Mn
2+
(5) 64
ME (5), Mn
2+
(3) 100
ME (10), Mn
2+
(5) 87
ME (25), Mn
2+
(5) 71

ME (50), Mn
2+
(5) 42
ME (5), Mn
2+
(10) 13
ME (5), Mn
2+
(25) 0
ME (5), Mg
2+
(2.5) 58
ME (5), Mg
2+
(5) 80
ME (5), Mg
2+
(10) 74
ME (5), Mg
2+
(25) 51
ME (5), Mg
2+
(5) + Ca
2+
(5) 73
ME (5), Ca
2+
(5) 68
ME (5), Fe

2+
(5) 0
ME (5), Cu
2+
(5) 0
ME (5), Zn
2+
(5) 0
ME (5), Co
2+
(5) 0
ME (5), Ni
2+
(5) 0
a
Acceleration of chromophore oxidation by Mn
2+
–EDTA.
Ó FEBS 2002 Enzymology of isomerizing phycoviolobilin lyase (Eur. J. Biochem. 269) 4545
In the presence of TX-100 (1% v/v), the temperature can be
increased without precipitation to 37 °C(Mg
2+
as activa-
tor),orevento45°C(Mn
2+
as activator). This temperature
stability is not surprising in view of the optimum growth of
M. laminosus at 50–55 °C.ThedatainTable2were
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, 45 °C. For longer reaction times
(180 min), the temperature optimum is reduced to 37 °C
(Mn
2+
), the activity at 45 °C being 20% less. Apparently,
the lyase has a high transient at 45 °C, but also becomes
more rapidly inactivated than at 37 °C, probably by
precipitation. Because of this inactivation, no experiments
were carried out at T > 45 °C. 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 55 °C.
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,
3
1
-Cys-PVB-PecA (as demonstrated before [24], the PCB-
PecA adduct cannot be transformed to a-PEC by His
6
-PecE
and His
6

-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, 3
1
-Cys-PCB (k
max
¼ 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,
5m
M
ME, 3 m
M
Mn
2+
, 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 (%)
20 20
30 43
37 67
45 100
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).
4546 K H. Zhao et al. (Eur. J. Biochem. 269) Ó FEBS 2002
which is not observed when the reconstitution is carried out
under catalysis of untagged PecE and PecF [24]. This
absorption is seen only when His
6
-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 Ni
2+
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 His
6
-PecA, catalysed by His
6
-PecE and His
6
-
PecF, follows the Michaelis–Menten equation for each
substrate, i.e. PCB (Fig. 4A) and His
6
-PecA (Fig. 4B). The
kinetic constants derived from these plots are summarized in
Table 3. Like the only other phycobilin-lyase studied [30],
the 3
1
-Cys84-PecA:PCB lyase is a rather slow enzyme
(k
cat
¼ 10
)4
)10
)5
Æs
)1
) with moderate affinity. The Line-
weaver–Burk plots with respect to PCB, obtained at
different concentrations of His
6
-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
6
-PecA, the corresponding linear fits do not intersect on the x-axis.
Ó FEBS 2002 Enzymology of isomerizing phycoviolobilin lyase (Eur. J. Biochem. 269) 4547
the 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
ÔspontaneousÕ or auto(?) catalytic binding of PCB to PecA
by 25%, yielding, however, only 3
1
-Cys84-PecA-PCB. In
the presence of His
6
-PecF, this pure addition reaction of
PCB to His
6
-PecA was decreased 15%. However, in this
case a small amount of the ligation/isomerization product,

His
6
-a-PECA, was formed (7% as compared to the
maximal yield of His
6
-a-PecA in the presence of His
6
-PecE
and His
6
-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(Table4):Forthetwoorganismsforwhichthe
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 ÔspontaneousÕ addition reaction (3
1
-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
shownthatincaseofthePC-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 His
6
-PecE, and His
6
-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, His
6
-PecF was absorbed on a Ni
2+
chelating
column in start buffer (0.5
M
NaCl, 20 m
M

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
,sok
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 ÔcorrectÕ ligation–isomerization
product 3
1
-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
Organism CpcE/PecE CpcF/PecF
M. laminosus 47.5 (311) 34.5 (212)

Anabena spec 46.7 (387) 32 (286)
4548 K H. Zhao et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphate, 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 m
M
)andNaCl(0.5
M
). 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 8
M
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.
CONCLUSIONS
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, Mg
2+
or Mn
2+
, 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 His

6
-tagged PecA to form PCB-His
6
-tagged
PecA, the latter is no substrate of the enzyme: it could not
be transformed to PVB-His
6
-tagged PecA (i.e. His
6
-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).
ACKNOWLEDGEMENTS
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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