©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of a Plant Serine Acetyltransferase Playing a Regulatory Role in Cysteine Biosynthesis from Watermelon (*)

Kazuki Saito (§) , Hiroyuki Yokoyama , Masaaki Noji (¶) , Isamu Murakoshi

From the (1)Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal Resources, Chiba University, Chiba 263, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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)()(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.

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 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.


MATERIALS AND METHODS

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.

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 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.

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 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 NaS, 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.


RESULTS

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.

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).


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) .

The SATase activity was inhibited by L-cysteine, a final product of the biosynthetic pathway, in a typical non-competitive manner. The K 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.




DISCUSSION

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 = 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) .

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.


FOOTNOTES

*
This research was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan and from the Iyaku-shigen Foundation (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) D49535.

§
To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263, Japan. Tel./Fax: 81-43-290-2905.

Present address: RIKEN (The Institute of Physical and Chemical Research), Tsukuba Life Science Center, Tsukuba 305, Japan.

The abbreviations used are: CSase, cysteine synthase (O-acetylserine(thiol)-lyase); kb, kilobase pair(s); OAS, O-acetyl-L-serine; -PA, -(pyrazole-1-yl)-L-alanine; PAGE, polyacrylamide gel electrophoresis; SATase, serine acetyltransferase; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

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.


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