(Received for publication, November 28, 1994; and in revised form, January 27, 1995)
From the
Stilbene (STS) and chalcone (CHS) synthases are homodimeric,
related plant-specific polyketide synthases. Both perform a sequential
condensation of three acetate units to a starter residue to form a
tetraketide intermediate that is folded to the ring systems specific to
the different products. Protein cross-linking and site-directed
mutagenesis identified a subunit contact site in position 158, close to
the active site (Cys). This suggested that the active
site pockets may be neighboring, possibly alternating in the condensing
reactions rather than acting independently. This was investigated by
co-expression of active site mutants with differently mutated, inactive
proteins. With both STS and CHS, the heterodimers synthesized the end
products, indicating that each subunit performed all three
condensations. In co-action with a monomeric reductase, CHS also
synthesizes 6`-deoxychalcone, but with the chalcone as second product
when using plant preparations. The two enzymes expressed as single
species in Escherichia coli synthesized both products, and
both were also obtained with a CHS heterodimer containing a single
active site. The results showed that 6`-deoxychalcone synthesis
required no other plant factor and that the formation of two products
may be an intrinsic property of the interaction between dimeric CHS and
monomeric reductase.
Stilbene synthases (STS) ()occur in a limited number
of widely unrelated plants. They synthesize the backbone of the
stilbene phytoalexins that have antifungal properties and contribute to
the pathogen defenses. Chalcone synthases (CHS) synthesize naringenin
chalcone, the precursor for a large number of flavonoids. Both enzymes
are polyketide synthases performing complex reactions (Fig. 1).
These are most likely identical in the initial part that involves the
stepwise condensation of three acetate units from malonyl-CoA to a
starter residue to synthesize an enzyme-bound tetraketide intermediate.
The reactions correspond to the condensations in fatty acid and other
polyketide synthases(1, 2) . The tetraketides are
folded to new aromatic ring systems, and these are different in STS and
CHS (Fig. 1). The typical STS reaction also includes a
decarboxylation that is not performed by CHS (reviewed in (3) and (4) ).
Figure 1: Reactions of STS and CHS. Both enzymes perform with a starter CoA-ester three sequential condensation steps with acetate units from malonyl-CoA. The boxed part shows the postulated enzyme-bound tetraketide intermediates (three acetates added to the 4-coumaroyl residue) that are presumably identical in the STS and CHS reactions. The intermediates are folded to new aromatic ring systems, and these processes are different in the two enzymes. The formation of 6`-deoxychalcone requires the reduction of a specific carbonyl group at the tri- or tetraketide intermediate level. The reaction is performed by a specific NADPH-dependent reductase.
The similarities in the STS and CHS
reactions are reflected by extensive similarities in structure and
sequences. Both are dimers (5, 6, 7, 8, 9) of
identical subunits containing 388-400 amino acids, and the active
sites for the condensing reactions are in the same position
(Cys, Fig. 2)(10) . The sequences of all
known STS and CHS are
60% identical, and a recent analysis
suggested that STS evolved from CHS by a relatively small number of
exchanges in key amino acids(11) .
Figure 2:
Protein sequences. a, CHS from S. alba; b, STS from A. hypogaea; c, CHS/STS protein hybrid c1an (a protein fusion containing
the N-terminal 107 amino acids of the S. alba CHS and 288
amino acids of the A. hypogaea STS; the CHS part in c1an
carries three mutations toward the STS sequence, but the Q100E exchange
essential for STS activity is lacking). In b and c,
only the differences to the CHS are printed; dots indicate
sequence identity. The amino acids relevant for this work are in large bold letters, and their positions are indicated. The
numbering is based on the CHS from S. alba. Cys is the active site of the condensing
reaction.
Although three-dimensional structures are not yet available, the similarities in sequence and function suggest a common structure with some differences reflecting the different ring-folding to the end products. We became interested in the subunit interactions because these might well be an integral part of the reactions performed by STS and CHS. The first part of this work deals with the identification of a subunit contact site close to the active site of the condensing reaction, and with the experimental distinction between alternative reaction mechanisms suggested by the finding that the active sites of the two subunits may be close together in the dimer.
The second part analyzes the interaction between CHS and the monomeric reductase that leads to a reduced chalcone (6`-deoxychalcone) by reduction of a specific carbonyl group prior to formation of the chalcone ring system (Fig. 1)(12, 13, 14) . This product is the precursor of the isoflavonoid phytoalexins which are part of the plant defenses against pathogens, and the reductase is the key to their formation. One of the puzzling aspects of that interaction was that plant CHS preparations always produced both 6`-deoxychalcone and chalcone, and this was investigated with both enzymes expressed as single species in Escherichia coli and with CHS heterodimers containing a single active site for the condensing reaction.
The plasmids
pQ/STS (STS from Pinus sylvestris),
pQ/STS
(STS from A. hypogaea), pQ/CHS
(CHS from S. alba), and pQ/CHS/STS(c1an)[2]
had been used before(11, 17) . The mutant protein
CHS/STS(c1an)[2] contained the STS from A. hypogaea with the N-terminal first 107 amino acids replaced by the
corresponding sequence from the S. alba CHS. The protein had
no STS or CHS activity, but the CHS part contained all the mutations
necessary to obtain STS activity, except for the Q100E exchange that
had been shown to be essential(11) . The first 107 amino acids
are shown in Fig. 2. The plasmid pQ/CHS
(CHS from P. sylvestris) was provided by Sigrid Raiber in our group.
The pQE-6 expression plasmids for several proteins were constructed
from cDNAs cloned in the vectors pTZ19R or pKK233-2 and available
from previous work: RED (reductase from Glycine
max, soybean)(18) , and
STS
(C169S)[1],
STS
(H166Q)[3],
CHS
(C169S)[1](10) . The
CHS
(Q100E)[2] mutant in vector pTZ18R was
constructed by T. Lanz in our laboratory. The mutants
STS
(C35S), STS
(C158S),
CHS
(S158C), and
pQ/CHS
(K180Q)[3] were obtained by
site-directed mutagenesis with appropriate custom-synthesized
oligonucleotides. The mutagenesis was performed with the cDNAs cloned
in vector pTZ19R(19) , and single-stranded DNA was obtained
with helper phage M13K07 in E. coli strain RZ1032 (20) . The changes were verified by DNA sequence analysis with
the dideoxynucleotide chain termination
technique(21, 22) .
The recloning of the cDNAs from vector pTZ19R into expression vector pQE-6 had been described in detail(11) . The steps involved in all cases the introduction of an NcoI site into the start AUG by site-directed mutagenesis and the fusion of the protein-coding region start AUG via the NcoI site to the optimal promoter-translation configuration in the vector(16) .
Figure 3:
Construction of plasmids for co-expression
of two proteins. The plasmids are drawn in the linearized form
(digested with one of the restriction enzymes used in the cloning
procedure) to facilitate the presentation of the constructions. Thick lines, STS or CHS cDNA; thin lines, vector
sequences. The protein-coding regions of the cDNAs
(-x->) are labeled corresponding to the designation
in the plasmid names. N, NcoI, restriction site for
fusion of the cDNA protein-coding regions to the vector promoter (P> ). >Pri and Pri< in B,
position of the primers used for fragment amplification with polymerase
chain reaction. Only the restriction sites relevant for the cloning are
shown. B, BamHI; S, SmaI; Sa, SalI; Sc, SacI; X, XhoI; Xb, XbaI.
-, blunt-end sites;
*, site destroyed in the resulting plasmids (ligation of XhoI
and SalI). The ligation of SmaI with a filled-in XhoI site regenerated a XhoI
site.
The relative enzyme activities were not
based on total protein in the extracts because the rate of protein
expression varied between the mutants. Therefore, each enzyme extract
(soluble proteins after a centrifugation of 15 min at 15,000 g) was analyzed by immunoblotting with STS or CHS antiserum,
and the relative enzyme activity was based on the quantity of the
immunodecorated protein(10) .
The incubations (0.1 ml) contained the components
of the standard CHS assays and, in addition, 0.5 mM NADPH, 10
mM glucose 6-phosphate, and 0.35 unit of glucose-6-phosphate
dehydrogenase (NADPH regenerating system, Boehringer Mannheim). The pH
6 required for the co-action of the reductase with CHS (12) was
obtained by using 50 mM potassium phosphate (pH 6.0) as
buffer. The protein extracts were added in such amounts that less than
25% of the radioactive substrate (malonyl-CoA) was converted into the
products (linear assay conditions). The amounts established in a series
of trial experiments were 5 µg of protein for CHS,
20 µg for CHS heterodimers from co-expressed subunits, and 20
µg for RED
.
The incubations were stopped after 30
min at 30 °C, and the products were extracted into ethyl acetate
without prior treatment at pH 9 (no conversion of the chalcone into
flavanone, see CHS assay). Naringenin chalcone, naringenin flavanone,
and 6`-deoxychalcone were separated by TLC and quantified with a TLC
analyzer. The separation of the three substances was achieved by a
two-solvent system. The plates were developed for 4 cm with 15% acetic
acid, dried, and then further developed in chloroform/glacial acetic
acid/HO (10:9:1, v/v/v)(12) . Chalcone synthesis
was defined as the sum of radioactivity in naringenin chalcone and
flavanone.
Figure 4:
Immunoblots of STS and CHS after
incubations without(-) or with (+) BMH cross-linker. Lanes: 1, STS (STS A.
hypogaea); 2, mutant STS
(C35S); 3, mutant STS
(C158S); 4, CHS
(CHS S. alba); 5, mutant
CHS
(S158C). The sizes of the protein standard markers
are indicated at the left side.
No such cross-link was detected with the CHS from S.
alba (Fig. 4, lane 4+), and the same negative
results were obtained with the CHS and STS from P. sylvestris (not shown). This indicated that a cysteine specific for the STS
from A. hypogaea was responsible. The protein contained eight
cysteines, but six are conserved in all CHS and STS, and this left the
residues in position 35 and 158 as candidates (Fig. 2, only the
sequences for the A. hypogaea STS and S. alba CHS are
shown because these were used for the experiments). The two cysteines
were mutagenized separately to serine, resulting in the mutants
STS(C35S) and STS
(C158S). Both possessed
high enzyme activity (80 ± 5% of the parent protein), indicating
that these residues played no essential or important role in the STS
reaction. The cross-linking experiments showed that Cys158 was
responsible for the subunit linking of the STS, because
STS
(C35S) revealed the cross-linked protein (Fig. 4, lane 2+), but STS
(C158S)
did not (Fig. 4, lane 3+).
The six conserved
cysteines in the A. hypogaea STS had been mutagenized in
previous work(10) , and experiments with those mutants revealed
that the subunits were cross-linked by BMH in all cases (not shown).
Taken together, these experiments investigated all cysteines in the STS
from A. hypogaea. The modification of Cys was
the only one leading to the loss of cross-linking, indicating that no
other cysteine was involved, and that the two cysteines in this
position were cross-linked. We concluded that this amino acid was at or
very close to the interface of the two subunits.
The CHS from S.
alba contained a serine in position 158 (Fig. 2). It was
mutagenized to cysteine (CHS(S158C)) to test whether the
positions in STS and CHS were equivalent with respect to the closeness
of the subunits or not. The mutant protein was active (90 ± 8%
of the unmodified enzyme), and the subunits could now be cross-linked
by BMH (Fig. 4, lane 5+). The result indicated
that the amino acids in position 158 were at or close to the subunit
interface in both STS and CHS.
Other experiments with site-directed
mutagenesis ()indicated that Lys
, a residue
strictly conserved in all STS and CHS, was important not only for
activity of the enzymes (one example to be discussed later), but also
for the structure of both proteins. A direct proof for a role in
subunit interaction, however, was not possible because most mutations
of that amino acid led to insoluble protein aggregates, and, therefore,
no distinction could be made between a role in the initial folding of
the subunits or in dimerization. Nevertheless, the close neighborhood
of Cys
to positions 158 and 180 (Fig. 2) suggested
that the two active site pockets in the dimer may be close to each
other. One of the intriguing possibilities suggested by this hypothesis
was that the two active sites did not operate independently, but
cooperated during the three condensing reactions.
Figure 5:
Models for independent (1) or
cooperative (2) action of the two subunits in the three
sequential condensing reactions performed by STS and CHS. Each subunit (A and B) contains one active site (thiol group of
Cys). The starter residue (X) or the
intermediates with one or two acetate units (1-X or 2-1-X) are bound to the thiol group prior to the condensation
with acetate units from malonyl-CoA. 3-2-1-X corresponds to
the tetraketide intermediate shown in Fig. 1. The intermediates
are not modified in the standard STS or CHS reaction. The only known
exception is the biosynthesis of 6`-deoxychalcone that involves a
reduction of a specific carbonyl group prior to the CHS ring closure (Fig. 1).
The
features common to both models are that the starter group (X, e.g. a 4-coumaroyl-residue) is bound to the active site (thiol
group of Cys) prior to the condensing reaction, that
malonyl-CoA serves directly as donor of the acetate unit, and that the
product (1 - X) is released as CoA-ester. Two
repetitions of the steps led to the tetraketide intermediate which is
then folded either to a chalcone or a stilbene (see also Fig. 1). Model 1 assumes that each subunit performs all three
condensation reactions and that two end products are synthesized per
dimer and reaction cycle. Model 2 considers the possibility that the
subunits alternate in the condensing reactions and that one product is
synthesized per dimer. A close neighborhood of the active sites also
permits a model 2 variant (not shown), in which the starter residue is
bound to Cys
in subunit A and the malonyl residue is
transferred to Cys
in subunit B prior to the condensing
reaction. A role for other cysteines in malonyl residue binding was
excluded by the finding that Cys
was the only cysteine
essential for enzyme activity ( (10) and this work).
The two models could be distinguished with dimers containing only one active site. In that case, model 1 would predict an active enzyme (possibly with about 50% of the parent activity), while Model 2 would predict that such protein was either inactive or would at most release a product containing only one acetate unit added to the 4-coumaroyl residue. Interestingly, a substance synthesized via such single acetate addition (4-hydroxyphenylbutan-2-one) is known in nature(32) , and similar substances were also observed as by-products of CHS reactions under nonoptimal assay conditions(33) .
It has not
been possible so far to reassociate STS or CHS subunits to active
enzyme, and that precluded a biochemical approach. The techniques of
molecular biology, however, allowed a different strategy. It used
mutants in the active site cysteine (Cys), co-expression
with another mutant, and the functional analysis of the resulting
heterodimers containing a single active site.
The success of
the complementation between the amino acids in positions 169 and 100
led us to attempt a complementation between the much closer amino acids
in positions 169 and 166 by co-expression of the mutants
STS(C169S)[1] and
STS
(H166Q)[3] from plasmid
pQ/STS
[1 + 3]. The H166Q protein was a
previously used mutant (10, 34) of low STS activity (Table 2); the only difference to the parent enzyme was the
exchange of the histidine in position 166 to glutamine (Fig. 2),
a residue that is strictly conserved in all CHS. The results of the
co-expression revealed the same activities as with
STS
(H166Q)[3] alone (Table 2), and,
therefore, the interpretation was not as unambiguous as in the case
discussed before. Nevertheless, the data could be interpreted as
complementation, because all other possibilities (no or inactive
heterodimers) should have led to a significant reduction of the
apparent activity because of the presence of inactive homodimers.
This conclusion also
implied that a single active site in the heterodimers was sufficient
for CHS activity, but other explanations were not rigorously excluded
because of the residual activity of
CHS(Q100E)[2]. We investigated that point by
co-expression of CHS
(C169S)[1] and
CHS
(K180Q)[3] from plasmid
pQ/CHS
[1 + 3] (Table 2, Fig. 3C). As noted before, mutations of Lys
led in most cases to insoluble protein aggregates. The K180Q
exchange was one of the few resulting in an appreciable amount of
soluble protein (Table 2), but that required the lowering of the
temperature from 28 °C to 25 °C during protein expression. The
mutant revealed no significant enzyme activity. Table 2shows
that the co-expression of the two inactive proteins led to CHS
activity, indicating not only that the defect in position 180 was
complemented by the other subunit in the heterodimers, but also that a
single active site in a dimer was sufficient for CHS activity. Like
with STS, the K
for 4-coumaroyl-CoA revealed no
significant differences between heterodimer and parent (Table 2).
We
addressed these points in two types of experiments. (i) We employed
cloned proteins both for CHS and the reductase, thus eliminating the
heterogeneities in both enzymes and also the possible effects of other
plant factors; these experiments took advantage of the fact that the
reductase co-acts with CHS from all plants tested so far ((12) )(); (ii) we used a CHS heterodimer that
possessed only one active site und therefore could produce only one
product per reaction cycle.
The data from representative experiments
are summarized in Table 3. The soybean reductase
(RED) and the unmodified S. alba CHS
(CHS
) co-acted in the formation of 6`-deoxychalcone, but
the nonreduced chalcone was also produced. This showed for the first
time that no other plant factor was necessary for the formation of the
reduced chalcone, and that a single CHS species synthesized both
products. The experiments with the CHS heterodimer possessing a single
active site were performed with pQ/CHS
[1 +
3]. The co-incubation with the reductase also led to the formation
of both products, and the ratio was not largely different from that
observed with the unmodified CHS. A percentage of more than
35-45% 6`-deoxychalcone was also never obtained in the
experiments that investigated the optimal ratios between reductase and
CHS or its heterodimer. Those incubations tested widely different
ratios of the two enzymes, including a large excess of reductase.
Polyketide synthases are usually complexes of several different subunits or large proteins with domains for the different functions(1, 2) . STS and CHS appear to be exceptional because the complex reactions are performed by a single protein consisting of two comparably small, identical subunits, and the lack of protein sequence homology with other polyketide synthases indicated that they represent independent developments(10) . The results presented in this work demonstrated that each subunit was capable of performing all three condensation steps, and they provided the first definite evidence that malonyl-CoA was the direct donor of the acetate units. The complementation of defective subunits in the heterodimers indicated that the subunits cooperate in the formation of the products, but it is not possible yet to distinguish whether this is in stabilization of the enzyme-bound intermediates or in the ring closure or both.
The relative activities obtained by the co-expression of
inactive subunits were 22 ± 6% (STS[1 + 2]) and
15 ± 3% (CHS[1 + 3]) when compared with the fully
active enzymes (Table 2). The determinations were based on the
amount of immunoreactive protein in Western blots, and, in the
co-expression experiments, these included the inactive homodimers. The
mutants were expressed from identical promoter-translation start
configurations (Fig. 3A). It therefore seems a
reasonable assumption that they were synthesized at comparable rates
and available at about equimolar concentrations for dimerization.
Assuming no strong preference for either hetero- or homodimer
formation, the ratio of AA + BB and AB + BA dimers could have
been about 1:1, suggesting a correction factor of 2 for the specific
activity of the heterodimer. This would increase the activity in the
(STS[1 + 2]) co-expression to about 44% of the parent
enzyme, and that came reasonably close to the 50% expected for a dimer
possessing only one active site, but otherwise being fully functional.
The values obtained with (CHS[1 + 3]) were lower, and
that was likely a consequence of the reduced capability of the
CHS(K180Q) mutant to form dimers (Table 2).
No
basic differences between STS and CHS were detected in all of these
experiments, and it was therefore an interesting question whether it
would be possible to create a CHSSTS heterodimer functional in
both activities. This was tested by co-expression of
CHS
(K180Q) and CHS/STS(c1an) carrying an additional
C169S mutation, but no activity was detectable. A second attempt by
co-expression of CHS/STS(c1an) with CHS
(Q100E) showed
the residual CHS activity from the mutated CHS, but no stilbene
formation was observed. These experiments covered only a few
combinations, but it may well be possible that the protein
conformations guiding the ring closures to stilbenes and chalcones are
incompatible either with heterodimer formation or with the enzymic
function of such a dimer, in particular if the ring closure requires a
cooperation of the subunits. A mutual exclusion of CHS and STS activity
also seems to be suggested by previous results from a large number of
other mutagenesis experiments that failed to detect a protein with both
activities(11) .
The mechanisms of the complex interaction between the dimeric CHS and the monomeric reductase remain unexplained. The results with the CHS heterodimer containing only one active site offered no support for the hypothesis that the reductase reduced only one of the two intermediates synthesized by dimeric CHS, because in that case one might have expected that the 6`-deoxychalcone would represent a more dominant product than with the parent CHS. The formation of two products may be an intrinsic property of the interaction, and a more detailed analysis would require the three-dimensional protein structures.