From the Department Biologie I/Botanik, Ludwig-Maximilians Universität, Menzinger Str. 67, D-80638 München, Germany
Received for publication, March 19, 2002, and in revised form, October 23, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plant 4-coumarate:coenzyme A ligases, acyl-CoA
ligases, peptide synthetases, and firefly luciferases are grouped in
one family of AMP-binding proteins. These enzymes do not only use a
common reaction mechanism for the activation of carboxylate substrates but are also very likely marked by a similar functional architecture. In soybean, four 4-coumarate:CoA ligases have been described that display different substrate utilization profiles. One of these (Gm4CL1) represented an isoform that was able to convert
highly ring-substituted cinnamic acids. Using computer-based
predictions of the conformation of Gm4CL1, a peptide motif
was identified and experimentally verified to exert a critical
influence on the selectivity toward differently ring-substituted
cinnamate substrates. Furthermore, one unique amino acid residue
present in the other isoenzymes of soybean was shown to be responsible
for the incapability to accommodate highly substituted substrates. The
deletion of this residue conferred the ability to activate sinapate
and, in one case, also 3,4-dimethoxy cinnamate and was accompanied by a
significantly better affinity for ferulate. The engineering of the
substrate specificity of the critical enzymes that activate the common
precursors of a variety of phenylpropanoid-derived secondary
metabolites may offer a convenient tool for the generation of
transgenic plants with desirably modified metabolite profiles.
The well regulated secondary metabolism of plants can be conceived
as an adaptive network of specialized enzymes, responding to diverse
environmental and internal stimuli. This network ensures an efficient
partitioning of resources for the benefit of the plant. The
phenylpropanoid pathway of plants represents a particular example for
this concept. This pathway uses phenylalanine-derived intermediates for
the biosynthesis of compounds used as UV protectants, defense
chemicals, signaling compounds, and flower pigments, as well as for the
building units of one of the most important structural polymers,
lignin. One essential early step in the biosynthesis of
phenylpropanoids is the activation of differently ring-substituted cinnamic acids to the corresponding coenzyme A thioesters. This reaction is catalyzed by 4-coumarate:CoA ligase
(4CL,1 EC 6.2.1.12), an
enzyme that employs a two-step reaction mechanism related to other
adenylate-forming enzymes (1) such as fatty acyl-CoA synthetases,
peptide synthetases, and luciferases. In the first step, ATP forms an
adenylate intermediate with the carboxyl moiety of the respective
substrate from which, in the second step, the activated group is
transferred to an acceptor, mostly the sulfhydryl group of an
enzyme-bound cofactor, followed by the concomitant release of AMP.
Crystal structures are available for some members of the protein
family, allowing the structural modeling of plant 4CLs (2, 3).
Plant 4CLs have been characterized from a wide range of species,
showing different isoform distribution patterns. Some plants exhibit
only a single form, whereas others contain multiple isoforms. From
those containing multiple isoforms, these can exhibit similar or highly
divergent substrate specificities in discriminating differently
ring-substituted cinnamic acids. For example, in parsley (Petroselinum crispum) (4), potato (Solanum
tuberosum) (5), and loblolly pine (Pinus taeda)
(6), the multiple 4CL isogenes encode identical or very similar
proteins. Conversely, soybean (Glycine max) (7, 8), tobacco
(Nicotiana tabaccum) (9), aspen (Populus
tremuloides) (10), hybrid poplar (Populus trichocarpa x P. deltoides) (11), and Arabidopsis thaliana
(12) contain structurally divergent 4CL isoforms. Due to the pronounced
differences at the structural, enzymatic, and regulatory levels, the
four soybean isozymes deserve special attention (7, 8). The two genes
encoding isoforms 3 and 4 are very closely related and could not be
distinguished with respect to the enzymatic properties of the encoded
proteins. These isoforms were strongly up-regulated at the
transcriptional level by elicitation or infection (7, 8). In contrast,
the structurally diverse isoform 1 was demonstrated to be
down-regulated after elicitation, whereas isoform 2 showed nearly no
response (8). In addition to this rather unusual behavior, isoform 1 displayed an interesting biochemical feature: it represented the first
4CL for which a cDNA was isolated to date, where the encoded enzyme
was able to convert sinapate to its respective CoA ester (8), a step
potentially of critical importance for the synthesis of syringyl-type lignin.
Protein sequence alignments of 4CLs revealed the existence of conserved
peptide motifs (5, 7, 11, 12). Box I, described as a putative
nucleotide-binding motif, has been used as a signature for the
superfamily of adenylate-forming enzymes (13, 14), whereas the absolute
conservation of the box II motif seemed to be restricted to 4CLs (5).
The box I and II motifs have been the subject of recent investigations
using mutant 4CL isoforms from Arabidopsis (2, 3, 15).
Interestingly, the modification of highly conserved residues included
in each of these boxes, which have been postulated to be essential for
the enzymatic reaction, did not result in the total loss of activity
but showed rather subtle changes of the kinetic parameters of the
conversion of caffeate to the respective CoA ester (2). Putative
substrate-binding domains within the amino acid sequence have been
identified by domain-swapping approaches (15) and have been shown to
include specificity-conferring residues that were predicted to be in
direct contact with the ring substituent of the cinnamic acid
substrate, ferulate (3).
In our earlier report, we described the first cloned 4CL isoenzyme
capable of converting sinapate to its thiol ester (8). We now aimed to
pinpoint the major difference(s) in the structures of the soybean
isozymes that are responsible for the different substrate
specificities. In a first attempt, domain swapping between one isoform
using a narrow substrate range and one isoform with a broad substrate
range was used to confine the regions important for the binding of the
acid substrate. Second, structural comparisons have been conducted
using 4CL protein alignments in conjunction with crystal structures
derived from enzymes utilizing similar enzymatic mechanisms. This
allowed the deduction of regions or single amino acid residues with a
suspected importance in the enzymatic reaction toward the cinnamic acid
substrate. Site-directed mutagenesis was then used to generate modified
proteins, which were assayed for altered substrate specificities.
Intriguingly, the deletion of one single amino acid residue from
Gm4CL2 as well as from Gm4CL3, which is absent in
wild-type Gm4CL1, resulted in the generation of new
specificities and allowed the mutant enzymes to convert sinapate. The
possibility of converting 4CL isoforms into sinapoyl-synthesizing
enzymes may lead to valuable transgenic plants, where both lignin
production and composition may be customized by using inducible
promoters in conjunction with recombinant 4CL proteins expressing the
substrate specificity of choice.
Structural Analysis--
Amino acid sequences were
aligned and modeled using SWISS-Model (www.expasy.ch). The crystal
structure of luciferase (Protein Data Bank codes 1LCI, 1BA3) (16) was
used as a template for the prediction of the putative conformations of
Gm4CL1, Gm4CL3, as well as
Gm4CL3dVal367.
4CL Hybrids--
The plasmids pQE-30/Gm4CL1 and
pQE-31/Gm4CL3, described in (8), were used for the
generation of hybrids between the soybean 4CL isoforms 1 and 3. Restriction sites available at identical positions in both cDNAs or
generated by PCR at the respective positions were used to assemble
different portions of the cDNAs according to Fig. 1A and
to yield the chimeric proteins depicted in Fig. 1B.
Site-specific Mutagenesis--
The modification of single
nucleotide residues was performed using proof-reading PCR according to
Refs. 17 and 18. Briefly, for each mutation, a pair of oligonucleotides
was synthesized including the desired alterations surrounded by at
least eight nucleotides of the original sequence. The size of the
primers was adjusted to yield a melting temperature of 75-77 °C by
using the following formula: Tm = 81.5 + 0.41 × GC (%) Expression in E. coli and Enzyme Purification--
Heterologous
expression, enzymatic assays, and calculations of isoenzyme
specificities were performed as described (8). Isolation of recombinant
proteins was achieved by affinity dye chromatography combined with
immobilized metal chelate affinity chromatography. Briefly, crude
bacterial extracts were applied to reactive blue CL-6B (Sigma) in 50 mM Tris/HCl, pH 8, 14 mM Antisera and Bacterial Strains--
An antiserum raised
against parsley 4CL (20) was used in conjunction with anti-rabbit IgG
from goat coupled to alkaline phosphatase (Sigma) to verify the
expression from different constructs according to Ref. 8. E. coli DH5 Domain Swapping--
The plant 4CLs belong to a family of
AMP-binding enzymes utilizing a common two-step activation mechanism
for carboxylate substrates as diverse as fatty, acetic, amino,
or cinnamic acids or chlorobenzoate and luciferin (1, 13, 16, 21).
PheA, the phenylalanine-activating subunit of gramicidin S synthetase 1 from Bacillus brevis, which employs the same reaction
mechanism, has been crystallized in a ternary complex with the
substrates AMP and phenylalanine (22). Luciferase from the firefly
Photinus pyralis, another member of this enzyme family from
which a structure is available, showed a structure similar to PheA,
which allowed the presumptive assignment of the amino acid residues
implicated in the binding of the substrates when compared with PheA
(16). Since firefly luciferase and plant 4CLs share 31-33% identical amino acid residues, it was then possible to use the structure of the
firefly luciferase as a template to model the hypothetical three-dimensional conformation of Gm4CL1. All these proteins fold into a larger N-terminal domain and a smaller C-terminal domain
with the substrate-binding pocket being located near the transition of
the N-terminal domain to the C-terminal domain. This putative
substrate-binding region located to the central part of
Gm4CL1.
We tested this prediction by using a domain-swapping approach
exploiting the two soybean 4CL isoforms, Gm4CL1 and
Gm4CL3, which showed the most diverse substrate
specificities (8). Following the assembly of different hybrids from
Gm4CL1 and Gm4CL3 cDNAs (Fig.
1A), the derived cDNAs
were cloned into a bacterial expression vector (pQE30 or pQE31). As
summarized in Fig. 1B, nine chimeric proteins have been
generated, from which five were shown to be inactive. The hybrid
proteins H1 and H5, which each contained the highly conserved
C-terminal domain of the other form, showed no differences in substrate
specificities (Table I), excluding an
impact of this protein region on the utilization of the cinnamic acid
substrate. The replacement of the central region of
Gm4CL1 with the respective portion of Gm4CL3 (H3,
H4) resulted in a significant alteration of the substrate specificity.
The hybrid proteins H3 and H4 accepted only the same restricted
substrate range of cinnamic acid substrates as isoform 3 (Table I). The
failure to generate active hybrid proteins in the reverse combination
of isoforms 1 and 3 (H7, H8) may have been due to the N-terminal
extension present in Gm4CL3 as opposed to Gm4CL1,
which may have resulted in a structural perturbation that was not
tolerated by an isoform 1-type enzyme. The hybrid forms H2 and H6,
which were merged in the middle of the central region, as well as H9,
which contained only the central part of Gm4CL1 in the
backbone of Gm4CL3, all yielded inactive enzymes despite a
positive expression of all of these recombinant proteins (Fig.
1C).
Alanine Scanning--
The involvement of the central domain of 4CL
in the recognition of cinnamic acid substrates was surveyed in more
detail. The sequential superposition of the central protein regions of
PheA, which have been shown to build part of the active site cavity, in
the first step to luciferase and then to a computer-generated model of Gm4CL1, led to the selection of a protein
region of 4CL presumably involved in the binding of the cinnamic acid
substrate (Gm4CL1331-348, Fig.
2A). In addition, in the
vicinity of the selected amino acid sequence, a tripeptide (VPP), which
was strictly conserved between 4CLs (Fig. 2A, positions
284-286 in Gm4CL1), was observed. The residues
Ala236, Ile330, Cys331, and
Ala322, Ala301, Thr278 of PheA have
been found to line both sides of the specificity pocket for the
phenylalanine side chain, completed by Trp239 at the bottom
of the cavity (22). From these, four residues were located in or close
to the highly conserved peptides shown in the restricted alignment of
PheA with the soybean and Arabidopsis 4CLs (Fig.
2A). Accordingly, these residues represented promising candidates for a functional role in 4CLs as well.
Both regions of Gm4CL1 highlighted in Fig. 2A,
therefore, were chosen for an alanine-scanning mutagenesis approach to
attribute functional importance to specific amino acid side chains by
replacing consecutive residues with alanine. The substrate
specificities of the recombinant proteins were determined and compared
with the wild-type Gm4CL1 enzyme (Fig.
2B). One replacement (T331A) did not have any influence on
enzyme activity, whereas eight of the mutations displayed major effects
resulting in nearly complete loss of conversion of cinnamic acid
substrates (G333A, Y336A, G337A, M338A, T339A, E340A, G342A, L344A,
M348A) despite the positive expression of all of the recombinant
proteins (Fig. 2C). Strikingly, the modification of residues
284-286, as well as of residue 334, resulted in larger activities with
the highly substituted substrate sinapate when compared with the
respective reference values obtained with 4-coumaric acid. With the
exception of the mutants P285A and Q334A, this alteration of the enzyme
activity ratio was also observed with the artificial substrate
3,4-dimethoxy cinnamate (3,4-DMC).
Deletion of a Single Residue--
The alignment of soybean 4CL1
with other plant isoforms for which the substrate range was specified
revealed one crucial difference (Fig. 2A and data not
shown): the absence of a single valine residue (lacking between
Pro343 and Leu344 of Gm4CL1) in the
region, which was shown to build the phenylalanine-binding pocket in
PheA (22). According to the designation of functional side chain
residues, as identified by the alanine-scanning mutagenesis, this part
of the putative binding pocket showed only a moderate influence on the
selectivity for differently substituted cinnamic acids (Fig.
2B). Furthermore, Gm4CL1 represents the only
isoform that is able to activate sinapate and 3,4-DMC. We therefore
tested the impact of the valine deletion by engineering it into the two other isoforms (Gm4CL2 and 3) of soybean, which have been
shown to display intermediate and narrow substrate utilization ranges, respectively (8). The mutant enzymes were indeed able to convert sinapate, although the affinities were shown to be rather low (Km values of 1208 and 323 µM,
respectively, Table II) when compared
with Gm4CL1 (Km value of 4.7 µM). Gm4CL2dVal345 did not show
any further significant alteration of the catalytic efficiency, in
contrast to Gm4CL3dVal367. The latter mutant
form did not only gain the capacity to convert sinapate but also the
capacity to utilize the artificial substrate 3,4-DMC, showing a
slightly better relative conversion rate in comparison with sinapate.
Moreover, the efficiency of activating ferulic acid was greatly
enhanced when compared with the wild-type 4CL3 enzyme (Table II). In
the case of Gm4CL3, the deletion of one single residue
resulted in a considerable alteration of the functionality of the
isoenzyme, albeit accompanied by a decrease of the efficiency for
4-coumarate, the highly preferred substrate of the wild-type isoform,
as well as for caffeate.
The soybean 4-coumarate:CoA ligase 1 (1) (Gm4CL1)
cDNA (8) represents a valuable tool for investigations on the
active site of a plant 4CL isoform, which is capable of converting the highly ring-substituted cinnamic acids, sinapate and 3,4-DMC, to the
respective CoA thioesters. The aim of our present study was to
elucidate the critical difference, inherited by Gm4CL1, that
allows this enzyme to accommodate a great range of differently substituted substrates. The analyses were provoked and facilitated by
the availability of two other soybean 4CL cDNAs, coding for Gm4CL2 and Gm4CL3, which are distinguished from
Gm4CL1 by an intermediate and narrow substrate utilization
range, respectively (8). By successively pinpointing one region of the
primary sequence of the enzyme contributing to the active site, a
single amino acid residue was identified that, in Gm4CL2 and
Gm4CL3, prohibited the conversion of the highly substituted substrates.
One further important prerequisite for the design of our experiments
was structural information describing proteins executing similar
enzymatic reactions. 4CLs belong to the superfamily of AMP-binding
enzymes, which also includes PheA, the adenylation domain of gramicidin
S synthetase, and the firefly luciferase (13, 16, 21, 22). The
structures of these proteins have been determined, in the case of PheA
even as a ternary complex in the presence of the substrates AMP and
phenylalanine (16, 22). Based on the amino acid sequence alignment of
Gm4CL1 and the unliganded luciferase, a structural model was
created, which then was used to superpose PheA. In the predicted
conformation of Gm4CL1, the putative substrate-binding
cavity located to the central amino acid sequence including the amino
acid residues 284 to 348 of ligase 1. This prognosis was verified by
domain-swapping experiments (Fig. 1). The hybrid H4 revealed a
conversion of the broad substrate range to the restricted one, as
typified by Gm4CL3, after the substitution of a sequence of
just 288 amino acid residues in the center of the Gm4CL1
protein with the respective region of Gm4CL3. The
difficulties in generating active hybrid proteins experienced with the
majority of the constructs prevented more in-depth studies of the
localization of the regions that may be important for the determination
of the substrate range. This may have been due to the higher divergence
in similarity and size of the soybean isoforms 1 and 3 (58% identity
(8)) as compared with the Arabidopsis forms 1 and 2 (86%
identity (12)), for which a larger number of active hybrid enzymes have
been generated recently (15). The analyses of the substrate recognition
profiles of the chimeric proteins from Arabidopsis pointed
to the same region in the central part of the enzymes, as was found for
soybean. Moreover, the dissection of this inner part of the
Arabidopsis 4CLs resulted in the designation of two
substrate-binding domains (sbd I and sbd II), which independently were
able to confer a change in the substrate range (15). Included in these
domains are the regions that were analyzed by alanine-scanning
mutagenesis in the present study (Fig. 2A). The sequence of
Gm4CL1284-286 corresponds to a highly conserved
tripeptide in the alignment of the amino acid sequences of a large
number of the adenylate-forming enzymes (data not shown) and was
therefore chosen for closer inspection. According to the
structural data, this tripeptide
(Leu279-Pro280-Pro281 in PheA) is
in close proximity to the phenolic side chain of the aromatic acid
substrate (22). Interestingly, the exchange of each of the respective
positions in Gm4CL1 with alanine resulted in an increase of
the activity with more highly ring-substituted cinnamic acids with the
remarkable exception of the loss of 3,4-DMC activation by the mutation
P285A (Fig. 2B). This artificial substrate contains an
O-methyl group in para-position, which may bear a steric hindrance in preventing a productive enzyme-substrate complex in
the modified substrate groove. Although the participation of the
tripeptide in substrate binding cannot be firmly deduced without the
elucidation of the real structure of a 4CL in the presence of the acid
substrate, our data clearly support the computational model of
Gm4CL1, which places this peptide at one end of the substrate-binding pocket. The different ring substitutions of the
cinnamic acids implicate different spatial requirements for the
substrates, which may or may not fit into the groove built by this
stretch of amino acid residues, depending on their respective side
chains. This conclusion was corroborated by the recent description of a
substrate specificity-conferring amino acid in 4CL2 from Arabidopsis (3). The position Met293 of
At4CL2, which was speculated to correspond to
Thr278 of PheA, was shown to influence the spatial
arrangement of the substrate-binding pocket in such a way that only the
exchange with a smaller residue allowed the utilization of ferulate
(3). Regardless of minor displacements of the PheA amino acid sequence in relation to different plant 4CL protein alignments used in the
aforementioned publication (3), published recently elsewhere (15), or
presented in our study (Fig. 2A), the accumulated results indicate that in silico biology can be applied to approach
the questions posed in these studies.
According to the alignment of Fig. 2A, the second selected
region of Gm4CL1 (positions 331 to 348) corresponds to a
small amino acid motif of PheA, which includes residues contributing to
the fixation of both substrates (phenylalanine and AMP) by both main
chain carbonyl oxygens (Ala322, Gly324,
Ile330) as well as side chain interactions
(Asn321, Tyr323, Thr326,
Glu327) (22). The importance of this central part of the
polypeptide chain of Gm4CL1 is reflected by the loss of
activity observed for 9 out of 13 single mutant enzymes (Fig.
2B). Interestingly, the exchange at Q334A did not result in
a total loss of activity despite the importance of the respective
functionally analogous residue Asn321 of PheA. Here, the
side chain was proposed to interact both with the exocyclic nitrogen of
adenine as well as with N1 of the purine ring through a well
ordered water molecule (22). Moreover, the substrate specificity of the
mutant Q334A displayed a reversal of the pattern when compared with
that of the wild-type Gm4CL1 enzyme: caffeate and sinapate
were preferably converted, in contrast to coumarate, ferulate, and
3,4-DMC. This result could indicate a major influence on cinnamic acid
substrate discrimination rather than on AMP binding.
The most conspicuous difference of the central part of the polypeptide
chain between Gm4CL1 and all other known plant 4CL proteins
was the absence of a conserved valine residue between the positions
Pro343 and Leu344 of Gm4CL1 (Fig.
2A and data not shown). We substantiated the important
contribution of this amino acid to the substrate specificity by
engineering its absence into two other 4CL isoforms of soybean that
were known to accept only a subset of the ring-substituted cinnamic
acids as substrates (8). The single amino acid deletions had a profound
impact on the range of accommodated substrates, generating new
substrate specificities for Gm4CL2 and Gm4CL3 (Table II). The mutation of ligase 3, which efficiently converted only
4-coumarate and caffeate, to a form that not only accepted two
additional substrates (sinapate and 3,4-DMC) but also showed a drastic
improvement of the affinity for another (ferulate), illustrated the
relevance of this amino acid for substrate discrimination. Computer-based modeling suggested that the deletion of the single valine residue caused a displacement of the respective peptide loop of
Gm4CL3 and concomitantly resulted in a steric rearrangement of the orientation of the neighboring leucine side chain.
Interestingly, the exchange at L344A of Gm4CL1 resulted in
the total loss of enzyme activity (Fig. 2B). Furthermore,
recent evidence indicated the participation of additional neighboring
regions of the polypeptide chain, which allowed the mutant
Arabidopsis enzymes to convert ferulate (3, 15). Taken
together, these findings suggest that spatial restrictions of the
binding groove rather than specific interactions between the substrate
and amino acid residues of the polypeptide chain may be decisive
in determining the substrate specificity of distinct 4CL isoenzymes.
In summary, the comparison of enzymatically highly divergent isoforms
of 4CL from soybean enabled the prediction of functionally important
amino acid residues. Active mutant enzymes with single amino acid
exchanges revealed some of the structural principles responsible for
the ability to utilize differently substituted cinnamic acids to a
different extent. The principles detected for 4CL isozymes from soybean
as well as from other sources (2, 3) help to understand the catalytic
action of this important enzyme of the general phenylpropanoid pathway
of plants. They could be useful also for the design of novel 4CL
specificities. For example, the manipulation of the lignin composition
of trees used for paper production is an economically important trait
since a higher content of syringyl monolignols improves the commercial lignin degradation. Although recent discoveries concerning alternative pathways for the synthesis of monolignols provide interesting targets
for genetically modified crop plants (23-25), the utilization of 4CL
to preferentially activate a range of cinnamic acid substrates leading
to highly substituted lignin building units may also constitute a
valuable tool.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
675/number of bases
sequence deviation (%). For the amplification, 10 ng of
column-purified plasmid DNA (Genomed, Bad Oeynhausen, Germany) was used
in a total volume of 15 µl, including 1 µM of each
primer, 200 mM dNTPs, and one unit of Pfu
polymerase (Promega/Serva). In total, 18 cycles were conducted,
consisting each of 35 s at 94 °C, 45 s at 55 °C, and 12 min at 72 °C, preceded by a melting step at 94 °C for 2 min and followed by a final extension step at 72 °C for 10 min.
Subsequently, the parental template DNA was digested with
DpnI (Promega/Serva), and the amplified plasmids were
purified and transformed into Escherichia coli DH5
(19). The mutations were verified by sequencing.
-mercaptoethanol,
and 30% (v/v) glycerol (buffer A), the column was washed with buffer A
including 0.6 M NaCl, and proteins were eluted by
addition of 2 M NaCl. The eluate was applied to
nickel-nitrilotriacetic acid agarose (Qiagen), equilibrated previously
in buffer A including 2 M NaCl. After washing (buffer A, 1 M NaCl, 10 mM imidazole), the bound proteins
were eluted by raising the imidazole concentration to 200 mM.
was used for the propagation of recombinant plasmids,
and the strain SG13009 (Qiagen) was used for the expression of proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Construction of chimeric
4CL proteins. A, schematic representation of the coding
regions of Gm4CL1 and Gm4CL3, as present in the
respective expression vectors. Internal restriction sites are labeled
above the bars representing the open reading
frames. New sites generated by PCR are marked with arrows
below the bars. B, scheme of hybrid
proteins generated after fusion of different portions of the open
reading frames from Gm4CL1 (open boxes) and
Gm4CL3 (black boxes). The type of substrate
specificity of each chimeric protein is given to the right,
as deduced from the data shown in Table I. , no enzyme activity
detectable. The fusion points for the various chimeric proteins
are as follows: Hybrid H1:
Gm4CL1M1-G400::Gm4CL3Y425-P570;
H2:
Gm4CL1M1-A256::Gm4CL3A280-P570;
H3:
Gm4CL1M1-A131::Gm4CL3Y156-P570;
H4:
Gm4CL1M1-A131::Gm4CL3Y156-T444::Gm4CL1G421-N546;
H5:
Gm4CL3M1-T444::Gm4CL1G421-N546;
H6:
Gm4CL3M1-E294::Gm4CL1L272-N546;
H7:
Gm4CL3M1-K159::Gm4CL1I136-N546;
H8:
Gm4CL3M1-K159::Gm4CL1I136-G400::Gm4CL3Y425-P570;
H9:
Gm4CL3M1-E294::Gm4CL1L272-G400:: Gm4CL3Y425-P570.
In the above list of chimeric proteins, the subscript single-letter
codes represent the amino acids, and the subscript numbers represent
the position numbers. C, detection of the recombinant
chimeric Gm4CL proteins generated in E. coli by
Western blotting. Crude protein extracts (10 µg of protein each) were
separated on 10% SDS-polyacrylamide gels and transferred onto
nitrocellulose filters. For immunodetection, antiserum raised against
parsley 4CL (20) combined with goat anti-rabbit IgG conjugated to
alkaline phosphatase was used. The relative molecular masses of protein
standards are shown on the left.
Substrate specificities of chimeric 4CL enzymes generated by domain
swaps between Gm4CL1 and Gm4CL3
View larger version (28K):
[in a new window]
Fig. 2.
Alanine-scanning mutagenesis of
Gm4CL1. A, partial amino acid alignment of
soybean (Glycine max, Gm) and A. thaliana (At) 4CL isoforms 1, 2, and 3, respectively,
in comparison with PheA, the phenylalanine-activating subunit of
gramicidin S synthetase 1 from B. brevis. The denoted
numbering is according to each single polypeptide, as found
in the databases. B, enzyme activity profiles of different
recombinant Gm4CL1 proteins generated by site-directed
mutagenesis in the protein regions depicted in panel A in
comparison with the wild-type (wt) enzyme activity of
Gm4CL1 (4CL1/wt). The recombinant
proteins were expressed in E. coli, and the specific
activities were measured in crude extracts as described previously by
using 2 mM cinnamate or 0.5 mM of all other
cinnamic acid substrates, respectively (8). The conversion rates for
the cinnamic acids, as catalyzed by the recombinant proteins, are each
depicted in the order cinnamate, 4-coumarate, caffeate, ferulate,
sinapate, and 3,4-DMC (left to right),
respectively, and are denoted by different shading of the
columns. C, detection of the single-residue
mutant proteins and wild-type Gm4CL1 generated in E. coli by Western blotting, performed as described in the legend for
Fig. 1. The relative molecular masses of protein standards are shown on
the left.
Substrate specificities of deletion mutants of the soybean 4CL2 and
4CL3 isoenzymes in comparison to wildtype 4CL1
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank K. Hahlbrock (Köln, Germany) for providing the P. crispum 4CL antiserum and A. Mithöfer for critically reading the manuscript and valuable discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft (Grant Eb 62/11-3), the Fonds der Chemischen Industrie, and a fellowship of the state of Bavaria (to C. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 49-89-1782274;
E-mail: j.ebel@botanik.biologie.uni-muenchen.de.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M202632200
1 The abbrevations used are: 4CL, 4-coumarate:CoA ligase; 3,4-DMC, 3,4-dimethoxy cinnamate; 4-coumarate, 4-hydroxy cinnamate; caffeate, 3,4-dihydroxy cinnamate; ferulate, 3-methoxy, 4-hydroxy cinnamate; sinapate, 3,5-dimethoxy, 4-hydroxy cinnamate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Knobloch, K.-H., and Hahlbrock, K. (1975) Eur. J. Biochem. 52, 311-320[Abstract] |
2. | Stuible, H.-P., Büttner, D., Ehlting, J., Hahlbrock, K., and Kombrink, E. (2000) FEBS Lett. 467, 117-122[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Stuible, H.-P.,
and Kombrink, E.
(2001)
J. Biol. Chem.
276,
26893-26897 |
4. | Douglas, C. J., Hoffmann, H., Schulz, W., and Hahlbrock, K. (1987) EMBO J. 6, 1189-1195 |
5. |
Becker-André, M.,
Schulze-Lefert, P.,
and Hahlbrock, K.
(1991)
J. Biol. Chem.
266,
8551-8559 |
6. |
Zhang, X. H.,
and Chiang, V. L.
(1997)
Plant Physiol.
113,
65-74 |
7. |
Uhlmann, A.,
and Ebel, J.
(1993)
Plant Physiol.
102,
1147-1156 |
8. |
Lindermayr, C.,
Möllers, B.,
Fliegmann, J.,
Uhlmann, A.,
Lottspeich, F.,
Meimberg, H.,
and Ebel, J.
(2002)
Eur. J. Biochem.
269,
1304-1315 |
9. |
Lee, D.,
and Douglas, C. J.
(1996)
Plant Physiol.
112,
193-205 |
10. |
Hu, W.-J.,
Kawaoka, A.,
Tsai, C.-J.,
Lung, J.,
Osakabe, K.,
Ebinuma, H.,
and Chiang, V. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5407-5412 |
11. |
Allina, S. M.,
Pri-Hadash, A.,
Theilmann, D. A.,
Ellis, B. E.,
and Douglas, C. J.
(1998)
Plant Physiol.
116,
743-754 |
12. | Ehlting, J., Büttner, D., Wang, Q., Douglas, C. J., Somssich, I. E., and Kombrink, E. (1999) Plant J. 19, 9-20[CrossRef][Medline] [Order article via Infotrieve] |
13. | Babbitt, P. C., Kenyon, G. L., Martin, B. M., Charest, H., Slyvestre, M., Scholten, J. D., Chang, K.-H., Liang, P.-H., and Dunaway-Mariano, D. (1992) Biochemistry 31, 5594-5604[Medline] [Order article via Infotrieve] |
14. |
Hofmann, K.,
Bucher, P.,
Falque, L.,
and Bairoch, A.
(1999)
Nucleic Acids Res.
27,
215-219 |
15. | Ehlting, J., Shin, J. J. K., and Douglas, C. J. (2001) Plant J. 27, 455-465[CrossRef][Medline] [Order article via Infotrieve] |
16. | Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298[Medline] [Order article via Infotrieve] |
17. | Nelson, M., and McClelland, M. (1992) Methods Enzymol. 216, 279-303[Medline] [Order article via Infotrieve] |
18. | Papworth, C., Braman, J., and Wright, D. A. (1996) Strategies 9, 3-4 |
19. | Hanahan, D. (1983) J. Mol. Biol. 5, 557-580 |
20. |
Ragg, H.,
Kuhn, D. N.,
and Hahlbrock, K.
(1981)
J. Biol. Chem.
256,
10061-10065 |
21. | Fulda, M., Heinz, E., and Wolter, F. P. (1994) Mol. Gen. Genet. 242, 241-249[Medline] [Order article via Infotrieve] |
22. |
Conti, E.,
Stachelhaus, T.,
Marahiel, M. A.,
and Brick, P.
(1997)
EMBO J.
16,
4174-4183 |
23. |
Humphreys, J. M.,
Hemm, M. R.,
and Chapple, C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10045-10050 |
24. |
Li, L.,
Popko, J. L.,
Umezawa, T.,
and Chiang, V. L.
(2000)
J. Biol. Chem.
275,
6537-6545 |
25. |
Guo, D.,
Chen, F.,
Inoue, K.,
Blount, J. W.,
and Dixon, R. A.
(2001)
Plant Cell
13,
73-88 |