From the
Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes
the reaction connecting serine and cysteine/methionine metabolism,
plays a regulatory role in cysteine biosynthesis in plants. We have
isolated a cDNA clone encoding SATase by direct genetic complementation
of a Cys
Cysteine biosynthesis in plants is the most important pathway in
the sulfur cycle in nature, because the fixation of inorganic sulfur
into L-cysteine, the first organic sulfur-containing compound,
is performed through this biosynthetic pathway(1) . The cysteine
biosynthetic pathway involves several enzymatic reactions(2) .
Only two of these enzymes in plants, cysteine synthase (O-acetylserine(thiol)-lyase) (CSase)
Several lines of evidence in the literature (1, 12, 13) suggest that SATase and OAS are the
major regulatory factors in the biosynthesis of cysteine in plants. The
availability of OAS, of which the cellular concentration is strictly
controlled by SATase, regulates the biosynthetic flow of
cysteine(14, 15) . A recent study (16) involving
transgenic tobacco overexpressing CSase also supported the hypothesis
that the supply of OAS and the level of SATase activity are the
important factors for regulation of cysteine biosynthesis. Several
papers (17, 18, 19, 20) have described
characterization of SATase from plant sources in attempts to purify the
enzyme. Nevertheless, no cDNA clones have been isolated from plants,
probably because of the low level of enzyme protein and subsequent
difficulty in purification to homogeneity. From bacteria, in contrast,
the genes for SATase in the cys regulon were recently cloned
and sequenced(21, 22, 23) . SATase in Salmonella typhimurium is regulated by feedback inhibition by
the end product, L-cysteine, and forms a multi-enzyme complex,
the so-called ``cysteine synthase complex,'' with
CSase(24) . The aim of the present study is to clarify the
regulatory role of SATase in cysteine biosynthesis in plants by cloning
a plant enzyme, in comparison with a bacterial system.
For the
molecular cloning of SATase from plants, the problem was the low
quantity of enzyme protein, probably 100-fold less than that of CSase
on an activity basis(19, 20) . The strategy involving
protein purification, amino acid sequence determination, and subsequent
screening of a library with synthetic oligonucleotides, which we
employed previously for the cloning of CSases(3, 5) ,
seems not to be efficient. Alternatively, we succeeded in the isolation
of a CSase cDNA from Citrullus vulgaris (watermelon) by
genetic complementation in an Escherichia coli Cys
For RNA gel blots,
total RNA was isolated from cotyledons, hypocotyls, and roots of
16-day-old etiolated or green C. vulgaris seedlings by the
reported method(28) . Total RNA (40 µg) was denatured and
separated in a formaldehyde/agarose (1.2%) gel, followed by transfer to
a Hybond-N
The recombinant
SATase accumulated (up to 10-20% of the soluble proteins) in E. coli was purified to homogeneity. Briefly, cells (10 g)
were disrupted in buffer A (40 ml) containing 200 mM potassium
phosphate (pH 8.0), 250 mM sucrose, 10 mM
2-mercaptoethanol, and 0.5 mM EDTA by sonication. The
supernatant obtained on centrifugation at 10,000
RNA-blot hybridization
showed that a 1.5-kb transcript was expressed in a relatively
constitutive manner but preferentially in the hypocotyls of etiolated
seedlings (Fig. 3). This expression pattern suggested that the
pSAT2 clone presumably encodes an extra-chloroplastic isoform of SATase
(see below).
The SATase activity was
inhibited by L-cysteine, a final product of the biosynthetic
pathway, in a typical non-competitive manner. The K
In the present study, a cDNA clone encoding a plant SATase
was isolated from a watermelon library by direct functional rescue of a cysE mutation in E. coli. To the best of our
knowledge, this is the first cloning and identification of a SATase
gene from higher plants.
In bacteria, SATase and the reaction
product, OAS, formed through its enzymatic action have been assumed to
play key roles in the regulation of cysteine biosynthesis. In the plant
system, it has been suggested, by physiological studies, that the
availability of OAS is one of the major limiting factors for cysteine
biosynthesis(14, 15) . However, the regulatory function
of SATase has not been precisely elucidated so far, because of the lack
of molecular information of a plant SATase. Our present data clearly
indicate the important regulatory role of SATase, i.e. allosteric feedback inhibition by cysteine at a low concentration
(IC
Another
characteristic feature of the plant SATase revealed in the present
study is the formation of a high molecular mass multi-enzyme complex by
the recombinant SATase and CSase. This character was first reported for
a bacterial enzyme (24) and recently for plant enzymes (19, 20).
Through the formation of the multi-enzyme complex, the metabolic flow
of intermediates from serine to cysteine can more efficiently occur
preventing the diffusion of intermediary OAS. The possibility of
metabolic channeling by the multi-enzyme complex of bacterial enzymes
has been discussed previously by Cook and Wedding (49). It will be
interesting to elucidate the molecular mechanism underlying the
protein-protein interaction leading to the multi-enzyme complex and the
regulatory role in cysteine biosynthesis in further detail by using the
recombinant proteins.
It has been suggested that multiple forms of
SATase exist and are localized in subcellular compartments of plant
cells, like those of CSase(50, 51) . Recently, the
chloroplastic isoform was purified from spinach leaves(20) . The
present clone isolated from watermelon seedlings seems to encode a
cytoplasmic isoform for the following reasons: 1) the immunoreactive
protein was detected preferentially in etiolated seedlings to green
seedlings but not detected in chloroplastic stroma, 2) no transit
peptide-like sequence was identified in the deduced amino acid
sequence, and 3) the clone was isolated from a library constructed from
etiolated seedlings, from which we could isolate only the cytoplasmic
CSase cDNA(8) . Since the site for sulfate reduction to sulfide
is in chloroplasts(52) , chloroplasts are probably the major
compartment for de novo cysteine biosynthesis. In the future,
it will be necessary to clarify the subcellular network of cysteine
biosynthesis.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank
We thank Prof. A. Böck, Lehrstuhl für
Mikrobiologie der Universität München, München, Germany,
and Dr. B. Bachmann, Department of Biology, Yale University, New Haven,
Connecticut, for kindly supplying E. coli JM39/5 and JM15,
respectively.
Note Added in Proof-After submission of
this paper, the nucleotide sequence of a SATase from Arabidopsis
thaliana was deposited to the GenBank/EMBL Data Bank with
accession number L34076 by Ruffet et al. The deduced amino
acid sequence of A. thaliana clone showed 54% homology with
that of watermelon.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
mutation in Escherichia coli using
an expression library of Citrullus vulgaris (watermelon) cDNA.
The cDNA encodes a polypeptide of 294 amino acids (31,536 Da)
exhibiting 51% homology with that of E. coli SATase. DNA-blot
analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon. RNA hybridization analysis suggested the
relatively ubiquitous and preferential expression in the hypocotyls of
etiolated seedlings. Immunoblot analysis indicated the accumulation of
SATase predominantly in etiolated plants. L-Cysteine, an end
product of the cysteine biosynthetic pathway, inhibited the SATase in
an allosteric manner, indicating the regulatory function of SATase in
this metabolic pathway, whereas
-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed
partly through the cysteine biosynthetic pathway, showed no inhibitory
effect. A multi-enzyme complex was formed from recombinant proteins of
SATase and cysteine synthase (O-acetylserine(thiol)-lyase)
from watermelon, suggesting efficient metabolic channeling from serine
to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine.
(
)(EC 4.2.99.8), responsible for the terminal
reaction(3, 4, 5, 6, 7, 8, 9) ,
and ATP-sulfurylase, responsible for the initial assimilatory reaction
of sulfate(10, 11) , have been characterized by cDNA
cloning. Serine acetyltransferase (SATase) (EC 2.3.1.30), which
catalyzes the formation of O-acetyl-L-serine (OAS)
from L-serine and acetyl-CoA, is responsible for the entry
step from serine metabolism to cysteine biosynthesis. OAS then reacts
with hydrogen sulfide to yield L-cysteine through the action
of CSase.
auxotroph using an expression library(8) . In the present
study, we employed the same strategy, involving functional rescue of
the E. coli Cys
mutation, for the isolation
of cDNA encoding SATase from watermelon. In watermelon,
-(pyrazole-1-yl)-L-alanine (
-PA), a secondary
non-protein amino acid found in Curcurbitaceae plants, is produced on
the coupling OAS and pyrazole through the action of CSase (25, 26). The
isolated cDNA was overexpressed in E. coli, and the
recombinant SATase was purified for antibody preparation and catalytic
investigation. The regulatory roles of SATase were elucidated and
discussed in relation with the control of primary and secondary
metabolism.
cDNA Cloning and Sequencing
The expression cDNA
library in ZAPII (Stratagene, La Jolla, CA) was prepared from
8-day-old seedlings (cotyledons removed) of C. vulgaris cv.
Kinro (Sakata Co., Yokohama, Japan) as described previously(8) .
Recombinant pBluescript cDNAs were excised in vivo from the
ZAPII vector, and plasmid DNA was purified as
described(8) . A Cys
auxotroph, E. coli JM39/5 (F
, cysE51, recA56)(21) ,
was transformed with the plasmid library by electroporation using an
apparatus from Invitrogen (San Diego, CA) according to the
supplier's recommended protocol, and then plated on M9 agar
medium (27) containing carbenicillin (100 µg/ml) and
isopropyl-thio-
-D-galactopyranoside (500 µM)
at 37 °C for 2 days. The DNA sequence was determined by
single-strand sequencing after subcloning into an M13 phage vector on
both strands by the dideoxy method using a series of synthetic primers.
Nucleic Acid Hybridization Analysis
For DNA-blot
hybridization, total DNA was prepared from 10-14-day-old
etiolated C. vulgaris seedlings as described
previously(8) . DNA (20 µg) was digested with restriction
enzymes, separated in a 0.7% agarose gel, transferred to a nylon filter
(Hybond-N
), and then hybridized with the random
primer-labeled cDNA insert of pSAT2 as a
P-labeled probe.
The final wash of the filter was performed in 0.1
SSPE (27) and 0.1% SDS at 65 °C for 30 min.
filter. Hybridization was performed as
described (8) with the
P-labeled probe. The final
wash of the filter was performed in 0.1
SSPE and 0.1% SDS at 65
°C for 20 min.
Overexpression and Purification of Recombinant
SATase
NdeI sites were created on both ends of the
SATase coding region of pSAT2 by polymerase chain reaction engineering
using two synthetic primers: NdeI F,
5`-ACTCCCATATGCCAGTTGGTGAGCT-3`; NdeI R,
5`-ACGATCATATGACAAGGGCATAATG-3`. The engineered cDNA fragment was
inserted into the NdeI site of pET3a (29) (Novagen-Takara, Kyoto, Japan) to afford pSEY1, in which
the cDNA was placed under a strong 10 promoter in the sense
orientation. The cloned SATase gene was overexpressed in E. coli BL21 (DE3, pLysE), in which the gene for lysogenic T7 RNA
polymerase under the lacUV5 promoter is induced by
isopropyl-1-thio-
-D-galactopyranoside.
g (1440 mg of protein) was subjected to fractional precipitation
with ammonium sulfate (20-80% saturation). The precipitate
re-dissolved in buffer B containing 10 mM potassium phosphate
(pH 8.0), 10 mM 2-mercaptoethanol, and 0.5 mM EDTA
was desalted by passage through a Sephadex G-25 column and then applied
to a DEAE-Sepharose column. The absorbed proteins were eluted with a
linear gradient of NaCl (0-0.5 M) in buffer B. The
combined fractions containing SATase were divided into two parts, 12
and 58 mg, and further purified by preparative disc SDS-PAGE
(Nippon-Eido, Tokyo, Japan) for antibody preparation and gel filtration
chromatography on an Ultraspherogel SEC 3000 HPLC column (Beckman
Instruments Inc.) to study the catalytic properties, respectively.
Enzymatic Activity Assays
The SATase activity was
determined by two methods, either by monitoring of the decrease in
A due to the thioester bond of acetyl-CoA (30) or
by coupling reaction with CSase forming cysteine(31) , followed
by calorimetric assay. For the thioester-bond cleavage assay, the
standard mixture contained 50 mM Tris-HCl (pH 8.0), 0.1 mM acetyl-CoA, 1 mML-serine, and enzyme solution,
in a final volume of 600 µl. The reaction was initiated by the
addition of L-serine and carried out at 25 °C. The initial
velocity of the decrease in absorbance at 232 nm was monitored. For the
coupling reaction with CSase, the standard assay mixture contained 50
mM Tris-HCl (pH 8.0), 1 mM acetyl-CoA, 5 mML-serine, 1 mM Na
S, 5 mM
dithiothreitol, 0.7 units of recombinant CSase from C.
vulgaris(26) , and the enzyme solution, in a final volume
of 100 µl. The reaction was performed at 30 °C for 5-30
min, and the amount of L-cysteine was determined as described
(3).
Immunoblot Analysis
Western blotting and
immunostaining were carried out on an Immobilon P membrane (Millipore)
using phosphatase-labeled goat anti-rabbit IgG (Kirkegaard & Perry
Laboratories) and 5-bromo-4-chloro-3-indolylphosphate p-toluidine/nitro blue tetrazolium chloride (Life
Technologies, Inc.) as substrates. The rabbit primary antibody was
prepared against the recombinant SATase purified from E. coli extracts and used at 1:400 dilution.
Isolation of SATase cDNA
The strategy used for
the isolation of a cDNA clone encoding SATase was to screen an
expression library that functionally complemented the cysE mutation in E. coli. The Cys auxotrophic E. coli strains, JM39/5 and JM15
(F
, cysE50, tfr-8) mutated cysE locus, which are unable to grow on minimal medium lacking
cysteine, were used for direct functional rescue of the Cys
auxotroph. Approximately 1.5
10
independent
pBluescript clones divided into 17 pools (pool 1, comprising 5
10
clones, and pools 2-17, each comprising 0.65
10
clones) after being amplified 300-fold were
screened by transformation in E. coli JM39/5. On
transformation with pool 1, approximately 300 colonies appeared on
selection for cysteine prototrophy; pools 2-17 gave no positive
colonies. Restriction enzyme and cross-hybridization analyses indicated
that these positive clones were identical, because they resulted from
multiple representation of the same clone, which was designated as
pSAT2. This plasmid could complement E. coli JM15 as well as
JM39/5. The protein extract of E. coli JM39/5 harboring pSAT2
exhibited comparable SATase activity (cysteine formation dependent upon
serine and acetyl-CoA, 9.3 ± 0.6 nmol/min/mg protein) to that of
the positive control plasmid, pWT2(21) , carrying the cysE gene (14.7 ± 0.6 nmol/min/mg protein), confirming the
identity of the insert on pSAT2 encoding catalytically functional
SATase.
Features of the Deduced Amino Acid Sequence of
SATase
Sequence analysis of the cDNA clone revealed a 1140-base
pair insert containing an open reading frame encoding a polypeptide of
294 amino acids with a calculated molecular mass of 31,536 Da (Fig. 1). Comparison of the deduced amino acid sequence of pSAT2
with a protein data bank indicated a homology with the bacterial
proteins exhibiting SATase activity (Fig. 2). The cysE gene in E. coli(21, 22) , S.
typhimurium(23) , and Buchnera aphidicola(32) are assumed to encode SATase for cysteine
biosynthesis. The nifP product showing SATase activity in Azotobacter chroococcum is involved in nodulation(33) .
Higher homology was detected in the carboxyl half of the pSAT2 protein,
suggesting the presence of the catalytic domain in this part. In
particular, the region from Gly-209 to Val-263 (boxed in Fig. 2) is conserved in the sequences of other acetyltransferases
and related proteins, i.e.NodL proteins from Rhizobium meliloti(34) and Rhizobiumleguminosarum(35) , virgiamycin acetyltransferase
from Staphylococcus aureus(36) , chloramphenicol
acetyltransferases from E. coli(37) and Agrobacterium tumefaciens(38) , and galactoside
acetyltransferase (LacA protein) from E.
coli(39) . These facts suggested that this region is likely
the binding site for acetyl-CoA, which is conserved among
acetyltransferases.
Figure 1:
Nucleotide and deduced amino acid
sequences of a cDNA clone, pSAT2, encoding SATase from C.
vulgaris. Double underlines indicate putative
polyadenylation signals.
Figure 2:
Multiple alignment of deduced amino acid
sequences of three proteins exhibiting SATase activity and the
consensus sequence. Dashes indicate gaps in the sequences for
the best alignment. Asterisk indicates an identical amino acid
residue. Cv SAT, SATase from C. vulgaris; Ec
cysE, cysE product (SATase) of E. coli (21); AcnifP, NifP protein of A. chroococcum (33) possessing SATase activity. The boxed region is
conserved among several acetyltransferases other than SATase and, thus,
presumed to be the acetyl-CoA binding domain.
DNA and RNA Hybridization Analyses
On Southern
blot analysis of genomic DNA, hybridizing bands corresponding to 14 kb
on BamHI, 3.5 kb on EcoRI, 7 kb on EcoRV,
and 2.4 kb on HindIII digestion appeared. These results
indicated the presence of a single copy of the SATase gene, designated
as sat, in C. vulgaris.
Figure 3:
Northern blot analysis of total
watermelon RNA. Total RNA (40 µg) of cotyledons, hypocotyls, and
roots of etiolated or green seedlings was electrophoresed on an agarose
gel (1.2%), transferred to a nylon filter, and then hybridized with the P-labeled probe.
Purification of Recombinant SATase
The recombinant
SATase was overproduced in E. coli BL21 cells up to
10-20% of the soluble protein using a ET vector system with a
strong T7 promoter/polymerase. The SATase protein was purified by
ammonium sulfate precipitation and DEAE-Sepharose chromatography as the
initial two steps. For antibody preparation, the denatured protein was
finally purified by preparative disc SDS-PAGE to an apparent
homogeneity in a yield of 1.3 mg from 240 mg of crude E. coli protein. For catalytic studies, the non-denatured protein was
finally purified by gel filtration on an HPLC column to 95%
homogeneity, as judged on SDS-PAGE, in a yield of 18 mg, exhibiting
SATase activity of 14.2 units/mg from 1200 mg of crude protein of 1.3
units/mg.
Immunoblot Analysis of SATase
Western blot
analysis of partially fractionated protein extracts of watermelon
seedlings indicated that an immunoreactive SATase protein exhibiting
the same molecular mass as that of the recombinant protein on SDS-PAGE
was preferentially accumulated in etiolated seedlings, but with lower
abundance in green seedlings (Fig. 4). No immunoreactive protein
was detected in stromal proteins of chloroplasts. These findings
suggested that the present clone encodes an extra-chloroplastic,
presumably cytoplasmic, isoform of SATase.
Figure 4:
Expression analysis of the SATase protein
in seedlings of C. vulgaris by Western blotting. The SATase
fractions were partially purified by ammonium sulfate precipitation
(0-40%) and gel filtration on Ultraspherogel SEC3000 from
8-day-old etiolated and green seedlings. The stromal protein was
obtained from chloroplasts of green plants as described previously
(16). The proteins (50 µg) were separated by 12% SDS-PAGE,
transferred to a nylon filter, and then localized by immunostaining
using a rabbit antiserum raised against the recombinant SATase. The
standard protein was the purified recombinant enzyme from E. coli BL21.
Catalytic and Regulatory Properties
The catalytic
and regulatory properties of SATase were investigated using the
purified recombinant protein. The optimum pH was in the range of
7.6-8.0 in Tris buffer. The K values were determined to be 0.59 mM for L-serine and 0.13 mM acetyl-CoA, being comparable
with those reported previously for plant
SATases(18, 19, 20) .
values of L-cysteine were determined to be 3.7
µM for L-serine and 5.6 µM for
acetyl-CoA, as shown in Fig. 5. The concentration for 50%
inhibition (IC
) under the standard assay conditions was
2.9 µM, being of the same order for that of E. coli SATase (1.1 µM)(40) . This inhibitory effect
was specific to L-cysteine, only weak activities being
detected with D-cysteine (39% inhibition at 1 mM) and N-acetyl-L-cysteine (18% inhibition at 1
mM). L-cystine, DL-homocysteine, L-methionine, glutathione, and SO
ion showed neither an inhibitory nor a stimulating effect up to 1
mM. Interestingly,
-PA, which is also an end product of
this biosynthetic pathway, exhibited no inhibitory action at 1
mM. CoA-SH showed inhibitory activity in a competitive manner
as to acetyl-CoA (K
= 64
µM).
Figure 5:
Determination of the K and K values of recombinant SATase from double reciprocal plots. The
kinetic studies were carried out by monitoring the decrease in A232 due
to cleavage of the thioester bond of acetyl-CoA as described under
``Materials and Methods.'' The K values of L-cysteine were determined in the absence and presence of L-cysteine at 5 µM and 10 µM, as
indicated.
Formation of a Multi-enzyme Complex with CSase
The
formation of a multi-enzyme complex of SATase and CSase, both from
watermelon, was detected by using the recombinant proteins expressed in E. coli (Fig. 6). CSase and SATase alone formed
homotropic complexes of molecular masses of 40-60 kDa and
150-210 kDa, respectively. After mixing and disruption of E. coli cells accumulating the two proteins, the elution
positions of the activities of both CSase and SATase shifted to higher
molecular mass positions of
450-550 kDa on gel filtration
chromatography (Fig. 6, A and B). This
multi-enzyme complex exhibited immunoreactive bands against both CSase
and SATase antibodies (Fig. 6C). Other fractions not
shown in Fig. 6C also contained minor amounts of
immunoreactive proteins, for example, SATase in fraction 2 in AB-2 and
CSase in fraction 4 in AB-3, being comparable with the elution pattern
of enzymatic activity. However, it was evident that the high molecular
mass complex was formed from two recombinant proteins. This complex of
CSase and SATase was not dissociated in the presence of OAS (2
mM), unlike in the case of the bacterial enzymes(24) .
Figure 6:
Formation of a multi-enzyme complex from
recombinant SATase and CSase. Protein extracts of E. coli were
prepared through cell disruption by sonication in buffer A, followed by
centrifugation at 15,000 g for 15 min. A-1, the protein extracts of E. coli BL21
harboring pCEN1 (an overexpression vector for CSase from watermelon)
(26) were separated by gel filtration on an Ultraspherogel SEC3000 HPLC
column. B-1, the peak fractions were assayed for CSase and
SATase activities. A-2 and B-2, extracts of E.
coli harboring pSEY1 (an overexpression vector for SATase). A-3 and B-3, mixed extracts of E. coli (pCEN1) and E. coli (pSEY1). C, the peak
fractions for each activity, fr.4 (AB-1), fr.3 (AB-2), and fr.2 (AB-3),
were analyzed by SDS-PAGE/Western blotting and immunostaining using
anti-spinach CSase A (3) and anti-SATase sera. The antisera were not
cross-reactive toward bacterial CSase and
SATase.
= 2.9 µM), as illustrated in Fig. S1. The cellular concentration of cysteine is normally
10 nmol/g fresh weight(16, 41) . Thus, the feedback
inhibition at this physiological concentration certainly plays a
central role in regulation, since mRNA expression of the sat gene seemed rather constitutive (Fig. 3).
Figure S1:
Scheme 1Regulation of the biosynthesis of
cysteine and -PA in watermelon. SATase plays a central role in the
regulation of cysteine biosynthesis through allosteric feedback
inhibition by cysteine, but this mechanism does not operate for the
control of
-PA biosynthesis.
In watermelon,
-PA, a secondary non-protein amino acid generally found in
Curcurbitaceae plants(42) , is formed from OAS and pyrazole
through the action of CSase(25, 26) . Thus,
-PA is
also regarded as an end product of the cysteine biosynthetic pathway.
-PA, however, exhibited no inhibitory effect on SATase activity
even at a high concentration (1 mM), in contrast with the
potent inhibitory action of L-cysteine. This means that the
formation of cysteine is strictly controlled by allosteric inhibition
toward SATase, whereas this control mechanism does not operate for the
biosynthesis of
-PA (Fig. S1). In fact, the cellular
concentration of
-PA was reported to be as high as
10
µmol/g tissue of watermelon seeds (43, 44) and
cucumber seedlings (45). The availability of pyrazole from
1,3-diaminopropane is postulated to be a limiting factor for
-PA
formation(46) . These findings regarding the differences in
regulation of the biosynthesis of cysteine and
-PA may reflect one
of the general aspects for the evolutionary and regulatory relations of
primary and secondary metabolism in higher plants. The biological
functions of
-PA in plants have not been clearly determined;
however, binding to glutamic acid receptors in animal brains was
recently demonstrated(47) . This may imply that
-PA acts as
a deterrent compound against predators, which is suggested to be a
general function of plant secondary metabolites(48) .
/EMBL Data Bank with accession number(s)
D49535.
-PA,
-(pyrazole-1-yl)-L-alanine; PAGE, polyacrylamide gel
electrophoresis; SATase, serine acetyltransferase; HPLC, high
performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.