Tài liệu Báo cáo khoa học: Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon doc

Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon Kentaro Takahara, Kinya Akashi and Akiho Yokota Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan Drought in the presence of strong light is a major environmental stress that reduces plant productivity [1]. To adapt to this adverse condition, numerous bio- chemical and physiological tolerance mechanisms are expressed in plant cells [2]. One such response involves accumulation of small organic metabolites, such as mannitol, proline and glycine betaine, which are collec- tively referred to as compatible solutes [3]. Compatible solutes are thought to play important roles in drought tolerance in plants, acting as mediators of osmotic adjustment, stabilizers of subcellular structures, and scavengers of active oxygen radicals [4]. The mecha- nisms of proline, mannitol, and glycine betaine accu- mulation are highly regulated through activation of biosynthesis and ⁄ or suppression of catabolism [3–5]. Wild watermelon plants, which inhabit the Kalahari Desert, Botswana, exhibit high drought ⁄ strong-light stress tolerance [6]. They are able to maintain their photosynthetic apparatus during prolonged periods of drought in strong light, suggesting the presence of Keywords citrulline; drought/strong-light stress; glutamate N-acetyltransferase; thermostability; wild watermelon Correspondence A. Yokota, Nara Institute of Science and Technology, Graduate School of Biological Sciences, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan Fax: +81 743 72 5569 Tel: +81 743 72 5560 E-mail: yokota@bs.naist.jp (Received 12 July 2005, revised 18 August 2005, accepted 23 August 2005) doi:10.1111/j.1742-4658.2005.04933.x Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild water- melon glutamate N-acetyltransferase (CLGAT) that catalyses the trans- acetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutam- ate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 °C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought ⁄ strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought ⁄ strong-light stress. Abbreviations AOD, acetylornithine deacetylase; CLGAT, Citrullus lanatus glutamate N-acetyltransferase; DRIP-1, drought-induced polypeptide 1; DTT, dithiothreitol; GAT, glutamate N-acetyltransfease; GFP, green fluorescence protein. FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5353 mechanisms that allow them to tolerate oxidative stress arising from excess light energy absorbed by the leaves. Drought ⁄ strong-light stresses result in an accumulation of a novel compatible solute, citrulline, in the leaves [6]. The concentration of citrulline in the stressed leaves reaches up to 30 mm, compared to only 0.6 mm in unstressed leaves [6]. Among known compatible sol- utes, citrulline is one of the most efficient scavengers for hydroxyl radicals [7]. These findings suggest that citrulline functions as a hydroxyl radical scavenger in the presence of strong light. In addition to citruline, concentration of arginine increases from 0.3 mm in unstressed conditions to 7mm under drought ⁄ strong-light stress in the leaves of wild watermelon [6]. Although arginine is the final product of the arginine biosynthetic pathway, wild watermelon plants accumulate larger quantities of citrulline, an intermediate in this pathway, during drought ⁄ strong-light stress. Arginine is a key meta- bolite in regulation of this pathway in plants [8]. How- ever, the mechanism of accumulation for these metabolites in wild watermelon remains unclear. Regulation of citrulline and arginine synthesis has been studied extensively in prokaryotes and Saccharo- myces cerevisiae [9,10]. The pathway starts with acety- lation of glutamate into N-acetylglutamate, which is then converted into N-acetylornithine by three con- secutive enzymatic steps, namely, phosphorylation, reduction, and transamination (Fig. 1). In the fifth step, N-acetylornithine is converted into ornithine, which is used for synthesis of citrulline and arginine in the urea cycle. Two different enzymes are known to be required for catalysis of this fifth step; one is acetylorni- thine deacetylase (AOD, EC 3.5.1.16), which catalyses deacetylation of N-acetylornithine yielding ornithine and acetate [11]. This linear pathway is regulated by arginine-induced feedback-inhibition of N-acetyl- glutamate synthase, the first-step enzyme in the pathway [12]. AOD is found in Enterobacteriaceae such as Escherichia coli [9,13]. The second enzyme is glutamate N-acetyltransferase (GAT, EC 2.3.1.35), which catalyses transfer of the acetyl group from N-acetylornithine into glutamate yielding ornithine and N-acetylglutamate. Glutamate N-acetyltransferase therefore recycles the acetyl moiety of N-acetyl- ornithine, regenerating N-acetylglutamate in citrulline and arginine biosynthesis. This enzyme is functional in all other microorganisms characterized so far, such as Bacillus subtilis and S. cerevisiae [14,15]. In this acetyl- recycling pathway, both the first- and the second-step enzymes, N-acetylglutamate synthase and N-acetylglut- amate kinase, respectively, are inhibited by arginine. Glutamate N-acetyltransferase is also weakly inhibited by arginine [16,17]. As a result, the concentration of cit- rulline and arginine is kept low through these feedback inhibitions in the microorganisms examined so far. How wild watermelon is able to accumulate high levels of citrulline is therefore an intriguing question. In plants, knowledge on the pathway of citrulline and arginine biosynthesis is still fragmentary [8,18]. The genome project revealed that both GAT and AOD-homologous genes exist in Arabidopsis thaliana [19]. Our previous study showed that a novel protein, drought-induced polypeptide 1 (DRIP-1), which shares sequence homology with bacterial AOD, is strongly induced by drought ⁄ strong-light stress in wild water- melon [6]. However, the catalytic property of DRIP-1 remains to be determined, and it is not known whether DRIP-1 contributes to massive accumulation of citrul- line in wild watermelon. As a first step to understand the mechanism of citrul- line and arginine accumulation in wild watermelon, we focused on the fifth step of citrulline biosynthesis, at which point DRIP-1 was expected to function as AOD. However, GAT activity, not AOD activity, was detected in wild watermelon leaves in which DRIP-1 Fig. 1. The pathway of citrulline and arginine biosynthesis. AGS, N-acetylglutamate synthase; AGK, N-acetylglutamate kinase; AGPR, N-acetylglutamate 5-phosphate reductase; AOAT, N-acetylornithine transaminase; GAT, glutamate N-acetyltransferase; AOD, N-acetyl- ornithine deacetylase; OCT, ornithine carbamoyltransferase; ASS, argininosuccinate synthase and ASL, argininosuccinate lyase. Wild watermelon glutamate N-acetyltransferase K. Takahara et al. 5354 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS had been strongly induced. This paper reports the purification and characterization of GAT from wild watermelon leaves, and discusses its function during drought ⁄ strong-light stress on the basis of its two unique enzymatic properties; thermotolerance and insensitivity to inhibition by downstream products, cit- rulline and arginine. Results The enzyme involved in catalysis of the fifth step of citrulline biosynthesis in wild watermelon leaves During citrulline biosynthesis, N-acetylornithine is con- verted into ornithine by AOD and ⁄ or GAT (Fig. 1). To examine contribution of these two enzymes to cit- rulline synthesis in wild watermelon leaves, we assayed their activities in extracts of wild watermelon leaves during progression of drought ⁄ strong-light stress. At each time point investigated, AOD activity was below the detection limit (< 0.02 nmolÆmin )1 Æmg protein )1 ; Fig. 2A). Although bacterial AODs are activated by a divalent metal ion such as Co 2+ or Zn 2+ [20], no AOD activity was detected in extracts from wild watermelon leaves even if these metal ions were inclu- ded in the reaction mixture (data not shown). In a pos- itive control experiment, we could detect similar AOD activity in the extract of E. coli to that reported in the literature [11], demonstrating that the assay procedures were valid for detecting AOD activity. The undetect- able AOD activity in extracts from wild watermelon leaves is in contrast to the strong expression of DRIP- 1 during stress (Fig. 2). On the contrary, GAT activity was detected in leaves of wild watermelon (Fig. 2A). The specific activ- ity of GAT in unstressed leaves was approximately 3.2 nmolÆmin )1 mg protein )1 , and this did not change significantly during stress. This constant GAT activity did not correlate with the induction of DRIP-1 protein during drought ⁄ strong-light stress (Fig. 2B). Purification of GAT from wild watermelon leaves To characterize the GAT in detail, it was purified from wild watermelon leaves (Table 1). The purification procedure was developed by taking advantage of the thermal stability of the GAT activity in crude extracts. After centrifugation of the total leaf extract to remove cell debris, the supernatant was heated at 70 °C for 10 min and centrifuged. Negligible loss of enzymatic activity and 24-fold purification were achieved. Heat treatment was followed by six chromatography steps comprising hydrophobic interaction, anion and cation exchange, gel filtration, and hydroxyapatite chromato- graphies, resulting in more than a 7000-fold purifica- tion of GAT. When analysed by SDS ⁄ PAGE, two polypeptides of  27 kDa were detected in the sample (Fig. 3A). Protein sequencing analysis revealed that the N-terminal amino acid sequences of the small (a) and large (b) polypeptide were XATNEAANYLPEAP and XMLGVVTTDAVVACDVWRKMVQISVDRSFNQI TVD, respectively; X represents unidentified amino A B Fig. 2. Enzymatic activities of AOD and GAT and accumulation of DRIP-1 protein during the progressing drought ⁄ strong-light stress in wild watermelon leaves. (A) Changes in AOD (j) and GAT (h) activity. Data points represent means from three independent experiments and vertical bars are SD. (B) Immunoblot analysis of DRIP-1 in the total soluble proteins (20 lg per lane) isolated from plants before (0 day) and after 1, 2, 3 and 5 days of drought ⁄ strong-light treatment. Table 1. Purification of GAT from wild watermelon leaves. Step Protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Purification (fold) Crude extract 5500 37 0.00067 1 Heat treatment 280 46 0.017 24 Butyl sepharose 15 35 0.23 340 Mono Q (pH 8.0) 0.94 11 1.2 1700 Sephadex 200 0.24 4.6 1.9 2800 Mono Q (pH 7.0) 0.13 3.4 2.6 3900 Mono S 0.07 3.0 4.3 6400 Hydroxyapatite < 0.02 0.94 > 4.7 > 7000 K. Takahara et al. Wild watermelon glutamate N-acetyltransferase FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5355 acids. The N-terminal amino acid sequence of the a peptide was identical to a portion of the amino acid sequence predicted from a watermelon EST clone (accession number AI563351). Cloning of watermelon GAT cDNA To isolate a full-length cDNA clone of the GAT, gene- specific primers were designed from the sequence of watermelon EST AI563351 and used for 5¢- and 3¢-RACE. The cloned cDNA encoded a protein composed of 460 amino acids. The amino acid sequence had homology to At2g37500 from A. thaliana (70% identity), B. subtilis GAT (38%) and S. cerevisiae GAT (26%). Two regions of the deduced sequence (residues 27–42 and 239–273) were identical to the N-terminal amino acid sequences of the a and b pep- tides determined by Edman sequencing, respectively (Fig. 4); the enzyme was designated CLGAT (Citrullus lanatus glutamate acetyltransferase). The first 26-amino acid sequence at the N-terminus of CLGAT was pre- dicted to function as a chloroplast transit peptide using the chlorop program [21]. Genomic Southern blot analysis indicated that there are two copies of the GAT gene in wild watermelon (data not shown). To confirm that the cDNA cloned above was that of the GAT purified in this study, we screened a cDNA library (5 · 10 6 primary plaques) pre- pared from the mixture of stressed and unstressed leaves using the EST clone mentioned above as a probe. Sequences of all nine clones isolated from the library were identical with the EST and the RACE-derived clone described above, indicating that only one type of GAT mRNA is transcribed in wild watermelon leaves. Citrullus lanatus glutamate acetyltransferase (CLGAT) possessed the conserved AT(M ⁄ L)L motif for the GAT family, where the precursor polypeptide is self-cleaved between alanine and threonine residues (Fig. 4, asterisk [22,23]);. In fact, the N-terminal resi- due of the b peptide from purified CLGAT matched the second residue of this conserved motif. These results strongly suggest that the precursor peptide of CLGAT in wild watermelon is also self-cleaved at the ATML sequence in a manner similar to those in other GATs reported so far. The molecular masses of the a and b peptides calculated from the cDNA were 21.3 and 23.5 kDa, respectively – lower than those deter- mined by SDS ⁄ PAGE (Fig. 3A). However, MALDI- MS analysis of CLGAT detected two major peaks at m ⁄ z 21.3 and 23.5 kDa, which corresponded to the masses deduced from the cDNA (data not shown). The molecular mass of native GAT was determined by gel filtration chromatography as  90 kDa (Fig. 3B), suggesting that CLGAT is a heterotetramer composed of two each of the a and b subunits. Analysis of the N-terminal transit peptide of CLGAT Analysis of the CLGAT cDNA predicted a chloroplast transit peptide upstream from the N-terminus of CLGAT. To examine whether the precursor of CLGAT is imported into the chloroplasts, we prepared a plasmid in which the first 26 codons of the CLGAT A B Fig. 3. Molecular mass of GAT purified from leaves of wild water- melon. (A) SDS ⁄ PAGE of GAT. The peak fraction obtained after hydroxylapatite chromatography was analysed by PAGE (12% w ⁄ v, polyaclylamide) and detected by silver staining. (B) Molecular mass of the native form of wild watermelon GAT was determined from the plot between the e lution volumes of the enzyme and marker proteins in Superdex 200 gel chromatography. Thy, thyroglobulin; Fer, ferritin; Ald, aldolase; Ova, ovalbumin. Wild watermelon glutamate N-acetyltransferase K. Takahara et al. 5356 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS cDNA were fused in-frame to the coding sequence of green fluorescent protein (GFP). This fusion protein was transiently expressed in tobacco leaves and the pattern of GFP fluorescence was analysed by confocal microscopy. Chlorophyll autofluorescence was used as the chloroplast marker. When the putative GAT transit sequence–GFP fusion protein was expressed in the leaves, green fluorescence was superimposed on the chlorophyll autofluorescence giving yellow tint, but this was undetectable in the cytoplasm (Fig. 5A). In contrast, when nonfusion GFP was introduced into the cells, the fluorescence was detected both in the cyto- sol and nucleus but was excluded from chloroplasts (Fig. 5B). These observations strongly suggest that the Fig. 4. Comparison of the amino acid sequence of CLGAT (C; accession no. AB212224) with those from At2g37500 from A. thaliana (A), B. subtilis GAT (B) and S. cerevisiae GAT(S) (accession nos. AAC98066, NP389002, and NP012464, respectively). Consensus identical (black) and similar (grey) amino acids are shaded. The sequences of the two N-terminal sequences of the purified CLGAT subunits determined by Edman sequencing are indicated by lines above the alignment. The conserved motif involved in self-catalysed cleavage is marked by aster- isks. Filled and open arrows indicate the predicted cleavage sites of the watermelon GAT precursor polypeptide that occur as a result of stromal processing peptidase and self-catalysed cleavage, respectively. K. Takahara et al. Wild watermelon glutamate N-acetyltransferase FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5357 precursor of CLGAT can be targeted to the chloro- plasts, by the N-terminal transit sequence. Kinetic analysis of GAT purified from wild watermelon The enzyme activity of CLGAT was maximal at pH 7.0 (Fig. 6A). The K m for the forward reaction at pH 7.0 was 3.4 mm for N-acetylornithine and 17.8 mm for glutamate (Fig. 6B,C). To examine the regulation of CLGAT by downstream products, activity was meas- ured at a physiologically relevant concentration of citrulline or arginine. Citrullus lanatus glutamate acetyl- transferase activities in the presence of 30 mm citrulline or 7 mm arginine were 100 and 98% of the original activities, respectively, showing that CLGAT is not inhibited by these downstream products. In this study, the effect of ornithine was not determined because the concentration of ornithine in wild watermelon leaves is very low and constant before or after drought ⁄ strong light stress, whereas concentrations of citrulline and arginine in the leaves increased greatly during the stress [6]. Surprisingly, CLGAT activity was maximum at 70 °C (Fig. 6D). To determine its thermostability, the enzyme was incubated for 30 min at various tempera- tures between 30 and 90 °C and residual activity was determined (Fig. 6E). Citrullus lanatus glutamate acetyl- transferase retained 98% of the original activity after incubation at 70 °C, and still showed about 15% of the original activity after incubation at 80 °C. The thermotolerance of CLGAT observed above prompted us to examine the leaf temperature under drought conditions in the presence of strong light at an atmospheric temperature of 35 °C. Under unstressed conditions, the rate of leaf transpiration was about 450 mmol H 2 OÆm )2 Æs )1 , and the leaf tem- perature was about 30 °C (Fig. 7). In contrast, drought ⁄ strong-light stress for five days decreased the rate of transpiration to  15 mmol H 2 OÆm )2 Æs )1 , and raised the leaf temperature to  44 °C. Discussion Citrulline is the most efficient hydroxyl radical scaven- ger among all known compatible solutes, and its role in oxidative stress resistance in wild watermelon has been suggested [7]. In microorganisms examined so far, the concentration of citrulline is kept low as a result of rigid regulations such as feedback inhibition and tran- scriptional regulation [8,9,24]. In contrast, wild water- melon is unique for the massive accumulation of citrulline in the leaves in response to drought ⁄ strong- light stress. However, knowledge of the mechanism of this accumulation is limited. It was previously sugges- ted that the stress-induced AOD homologue, DRIP-1, is involved in citrulline accumulation [25], but its func- tion remains unclear. Unexpectedly, it is revealed in this study that AOD activity was not detected in stressed leaves where DRIP-1 was expressed at a high level. Instead, GAT activity was detected in leaves, suggesting that the fifth step of the citrulline pathway A B Fig. 5. CLGAT is a chloroplast protein the import of which depends on its N-terminal transit peptide. Fluorescence microscopy pictures of tobacco leaf cell transiently expressing a CLGAT-GFP fusion (A) or GFP alone as a control (B). Wild watermelon glutamate N-acetyltransferase K. Takahara et al. 5358 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS is mainly catalysed by GAT in wild watermelon. CLGAT was subsequently purified and identified from wild watermelon. This is the first report dealing with GAT purified from plant sources. Under drought ⁄ strong-light conditions, plants close their stomata to avoid loss of water, thereby increasing leaf temperature [26]. In this study, the temperature of wild watermelon leaves increased from 30 to 44 °C under experimental stress conditions (Fig. 7). Under natural desert conditions, leaf temperature of stressed wild watermelon plants rises up to 60 °C [27]. In this study, the optimum temperature of CLGAT was revealed to be 70 °C, which was comparable to that reported from the thermophilic microorganism B. stearo- thermophilus (Table 2). Moreover, CLGAT retained about 15% of its original activity after incubation at 80 °C for 30 min, whereas GAT from B. stearothermo- philus completely lost its activity after incubation at 75 °C for 30 min [28]. These results suggest that CLGAT has adapted to the thermogenic condition of leaf tissue under drought in the presence of strong light. In stressed leaves of watermelon, citrulline and arginine accumulate massively to about 30 and 7 mm, respectively [6], raising the question as to what extent biosynthetic enzymes are inhibited by the downstream products. The present results revealed that CLGAT was not inhibited by either citrulline or arginine. This is in contrast to GATs from other organisms: GAT from Thermus aquaticus ZO5 is strongly inhibited (K i ¼ 1.75 mm [29]); and that from S. cerevisiae is moderately inhibited by arginine (10% inhibition with 5mm arginine [30]). Therefore, CLGAT can function without a loss of activity in the presence of high con- centrations of citrulline and arginine. We did not examine whether CLGAT is inhibited by ornithine, another downstream product in the pathway. Although several microbial GATs have been shown to be inhibited by ornithine [28], the K i for ornithine in Fig. 6. Kinetic analysis of CLGAT. (A) The pH dependency of CLGAT activity. Enzyme activity was determined between pH 4.2 and 7.8 in citrate ⁄ sodium phosphate buffer (j) and Tris ⁄ HCl buffer (n). Activity is expressed as a percentage of the maximal activity. (B and C) Saturation kinetics of CLGAT for N-acetylornithine (B) and glutam- ate (C). The concentration of N-acetyl- ornithine was varied from 0.5 to 15 m M with glutamate fixed at 10 mM in (B), and in (C) that of glutamate was changed between 0.5 and 30 m M with N-acetylornithine fixed at 10 m M. Double reciprocal plot of CLGAT activity against the concentration of sub- strates are shown in the insets. The fitted linear regression lines and parameters are also presented. The K m was estimated based on these parameters. (D) Tempera- ture dependency of CLGAT activity. Enzyme activity was measured by incubating the reaction mixtures at 20–90 °C. Activity is expressed as percentage of the maximal activity. (E) Thermostability of CLGAT. The enzyme was incubated for 30 min at the indicated temperatures and residual enzyme activity was measured at 30 °C. Values represent the mean ± SE from three independent measurements. K. Takahara et al. Wild watermelon glutamate N-acetyltransferase FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5359 these GATs is between 1 and 3 mm, much higher than the physiological concentration of ornithine (approxi- mately 0.1 mm) in the leaves of wild watermelon under drought and strong light stress [6]. The kinetics parameters obtained in this study enabled us to discuss the role of CLGAT in citrulline accumulation. Although CLGAT activity was unchanged during drought stress in the presence of strong light, an elevated leaf temperature from 30 to 44 °C would enhance the CLGAT reaction by about two times that under unstressed conditions as shown in Fig. 6D. Moreover, an increased concentration of glutamate, a substrate for CLGAT, from 2.5 to 7.5 mm under drought ⁄ strong-light conditions [6] would further elevate the CLGAT reaction by about 2.6-fold. As CLGAT catalyses not only the fifth step, but also the first step of citrulline biosynthesis, these estimations suggest that the influx of glutamate carbon skeletons into the urea cycle is increased about fivefold during drought ⁄ strong-light stress. Thus, CLGAT can effectively participate in the citrulline biosynthesis under drought conditions through its unique proper- ties, namely, high thermostability and insensitivity to inhibitions induced by the downstream products such as citrulline and arginine. A GFP-localization assay (Fig. 5) suggested that CLGAT is a chloroplastic enzyme. In S. cerevisiae, ornithine biosynthetic enzymes including GAT are localized in mitochondria [15,30,31], and ornithine is exported to the cytosol to be converted into citrulline and arginine [31,32]. In plants, information on the localization of citrulline and arginine biosynthetic enzymes is scarce, but it has been reported that ornith- ine carbamoyltransferase, an enzyme required for the sixth step in the citrulline and arginine pathway, is localized in chloroplasts in Canavalia lineate [33], rais- ing the possibility that citrulline biosynthesis in plants is catalysed in chloroplasts. In fact, cDNAs for all six citrulline biosynthetic enzymes in Arabidopsis were predicted as having chloroplast-targeting sequences using the various programs [18]. A B Fig. 7. (A) Change in the stomatal conductance (h) and leaf tem- perature (d) of wild watermelon plants during drought. Data points represent the means from three independent experiments and vertical bars are the SD. (B) Visualization of the thermal distri- bution in wild watermelon plants. Images 1 and 2 represent photographs of wild watermelon plants watered daily and subjec- ted to drought for 5 days, respectively, and images 3 and 4 show their thermal distributions analysed using an infrared thermal camera, respectively. Table 2. Comparison of the kinetic parameters for GAT from wild watermelon, S. cerevisiae and B. stearothermophilus. ND, not deter- mined. Wild watermelon S. cerevisiae a B. stearothermophilus b K m glutamate (mM) 17.8 7.2 19.2 K m N-acetylornithine (mM) 3.4 1.0 2.3 Temperature for maximum activity (°C) 70 ND 75 The pH for maximum activity 7.0 7.5 8.0 Activity in the presence of 5 m M arginine c 100 < 90 ND a [15, 30]. b [28]. c Expressed as a percentage of the maximum activity. Wild watermelon glutamate N-acetyltransferase K. Takahara et al. 5360 FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS The CLGAT enzyme was composed of two subunits, a and b, derived from a single precursor polypeptide (Fig. 3). The N-terminal amino acid sequence of the b subunit from purified CLGAT coin- cided with the cleavage site of the conserved motif, ATML, in the sequence predicted from its cDNA. This suggests that the CLGAT precursor is self- cleaved as proposed for other GATs generating two subunits that assemble as an a2b2 heterotetramer [22,23,28]. Although such self-cleavage has been sug- gested in several proteins other than GAT in plants [34], CLGAT is the first example of the self-cleavage for a chloroplastic protein. The function of DRIP-1 remains to be determined. In this study, no AOD activity was detected in wild watermelon leaves in which abundant DRIP-1 accumu- lation was seen (Fig. 2). Moreover, recombinant DRIP-1 expressed in E. coli had no detectable AOD activity (data not shown). One possible explanation for this discrepancy is that DRIP-1 requires a cofactor or activator for its catalysis, although we tested Co 2+ and Zn 2+ in the present enzymatic assays. The second possibility is that DRIP-1 possesses a different func- tion unrelated to AOD. In this study, we demonstrated that CLGAT, which catalyses the first and fifth steps of the citrul- line biosynthetic pathway, can contribute effectively to citrulline biosynthesis under drought conditions owing to its high thermostability and insensitivity to the inhibition induced by citrulline and arginine. However, many different enzymes are involved in the metabolism of citrulline (Fig. 1), and their kinetic properties and ⁄ or expression pattern remain to be examined. Comprehensive analysis of the citrulline biosynthesis pathway is required to fully understand the mechanism of citrulline accumulation in wild watermelon. Experimental procedures Materials N-Acetylornithine was purchased from Sigma (St. Louis, MO, USA). Other chemicals and reagents were purchased from Nakalai (Kyoto, Japan). Plant material Wild watermelon (C. lanatus L. sp. no. 101117-1) was grown in a growth chamber (16 ⁄ 8 h light ⁄ dark regime at temperatures of 35 ⁄ 25 °C, 50 ⁄ 60% humidity and 800 lmol photonsÆm )2 Æs )1 ) in 500-mL paper pots. Soil for horti- culture was purchased from PROTOLEAF (Tokyo, Japan). Plants were watered daily at 9 a.m. (1 h after the start of the light period). Two-week-old plants with fully expanded fourth leaves were used in the experiments. Drought treat- ment was started by stopping watering. For GAT purifica- tion, the plants were grown in a greenhouse at a temperature between 25 and 35 °C for 2 months from June to August in 2002. Analysis of leaf transpiration and temperature Transpiration of attached fourth leaves was measured at a light intensity of 800 lmol photonsÆ m )2 Æs )1 at 35 °C using a porometer (type AP4; AT delta-T device, Cambridge, UK). Leaf temperature was measured using a thermometer (model AP-320, 0.25K-J1M1, Anritu meter, Tokyo), and thermal images were obtained using an infrared camera (TVS-8500, Nippon Avionics Ltd, Tokyo, Japan). Data were collected around 15:00 (7 h after the start of the light period). Enzyme assays Acetylornithine deacetylase and GAT activity were measured by quantifying the production of ornithine from N-acetylornithine using the colorimetric ninhydrin procedure as described previously [11,15]. The AOD assay mixture (200 lL) contained 100 mm potassium phosphate buffer pH 7.0, 6 mm N-acetylornithine, 0.5 mm metal salt (CoCl 2 or ZnCl 2 ) and the enzyme. The GAT assay mixture (200 lL) contained 100 mm potassium phosphate buffer (pH 7.0), 6 mm N-acetylorni- thine, 6 mm glutamate and the enzyme. The reaction was started by adding N-acetylornithine and stopped by adding 600 lL of ninhydrin reagent (0.4 m citric acid ⁄ 1% ninhydrin in 2-methoxyethanol, 1 : 2 v ⁄ v). After boiling for 10 min, 200 lL4m NaOH was added to the mixture and light absorbance at 470 nm was measured. One unit is defined as the amount of enzyme that forms 1 lmol ornithineÆmin )1 . Purification of GAT from wild watermelon leaves All purification steps were carried out at 0–4 °C, and col- umn chromatographies were performed with FPLC system (Amersham Biosciences, Uppsala, Sweden). Wild water- melon leaves (300 g) were homogenized with a blender in 300 mL extraction medium containing 100 mm potassium phosphate buffer pH 8.0, 1 mm EDTA, 10 mm 2-mercapto- ethanol, 1 mm phenylmethylsulfonyl fluoride and 1% (w ⁄ v) polyvinylpolypyrrolidone. After filtering through six layers of gauze, the resulting homogenate was centrifuged at 12 000 g for 30 min. Unless stated otherwise the centrifuga- tion described below was carried out in the same way. N-Acetylornithine was added to the supernatant to give a K. Takahara et al. Wild watermelon glutamate N-acetyltransferase FEBS Journal 272 (2005) 5353–5364 ª 2005 FEBS 5361 final concentration of 10 m m. The mixture was then warmed in a water bath at 70 °C for 10 min with gentle stirring. The mixture was promptly cooled in an ice bath until the temperature dropped below 4 °C and centrifuged. The supernatant was brought to 20% saturation by adding (NH 4 ) 2 SO 4 powder, incubated on ice with gentle stirring for 30 min and centrifuged. The supernatant was applied to a Butyl-Sepharose Fast Flow column (1.6 cm i.d. · 10 cm; Amersham Bioscience) equilibrated with buffer A contain- ing 20 mm potassium phosphate buffer pH 7.0, 1 mm EDTA, 10 mm 2-mercaptoethanol, 1 mm dithiothreitol (DTT) and 20% (v ⁄ v) glycerol, and 20% saturation of (NH 4 ) 2 SO 4 . The enzyme was eluted with a linear gradient of 30–0% saturation of (NH 4 ) 2 SO 4 in buffer A. Active fractions were collected and applied to a Sephadex G-25 column (5 cm i.d. · 20 cm) equilibrated with buffer B con- taining 5 mm potassium phosphate buffer (pH 8.0), 1 mm EDTA, 10 mm b-mercaptoethanol, 1 mm DTT, and 20% (v ⁄ v) glycerol. Active fractions were pooled and applied to a Mono Q HR column (0.5 cm i.d. · 5 cm; Amersham Bioscience) equilibrated with buffer B. The column was developed with a 0–200 mm linear gradient of KCl in buffer B. Active fractions were pooled and concentrated with Centriplus YM-10 (Amicon, Bevery, MA, USA). The enzyme solution was applied to a Sephadex 200 column (1.6 cm i.d. · 60 cm; Amersham Bioscience) equilibrated with 20 mm potassium phosphate buffer pH 7.0, 1 mm EDTA, 10 mm 2-mercaptoethanol, 1 mm DTT, 150 mm KCl and 20% (v ⁄ v) glycerol and developed with the same buffer. Active fractions were loaded onto a Mono Q HR column (0.5 cm i.d. · 5 cm; Amersham Bioscience) then a Mono S HR column (0.5 cm i.d. · 5 cm; Amersham Bio- science). In these chromatographies GAT activity was recovered in the flow-through fractions. Active fractions were applied to a Sephadex G-25 column (1.6 cm i.d. · 10 cm) equilibrated with buffer C containing 5 mm potassium phosphate buffer pH 7.0, 10 mm 2-mercapto- ethanol, 1 mm DTT, and 20% (v ⁄ v) glycerol. Active frac- tions were applied to a hydroxyapatite column (0.7 cm i.d. · 5.2 cm; Bio-Rad, Hercules, CA, USA) equilibrated with buffer C. The column was developed with a 5–100 mm linear gradient of potassium phosphate in buffer C. Aliqu- ots from the active fractions were used for SDS ⁄ PAGE on a 12.5% polyacrylamide gel, and proteins were visualized by silver staining with a commercial kit (Daiichikagaku Chemical, Osaka, Japan). Proteins were measured according to the Bradford method [35], using BSA as a standard. For analysis of the N-terminal amino acid sequence, CLGAT subunits were separated by SDS ⁄ PAGE and electroblotted onto polyvinyldene difluoride membranes. Blotted proteins were stained with Coomassie Brilliant R-250. Stained regions were cut from the membrane and used for sequencing with an automated protein sequencer (Model 492, Applied Biosystems, Foster City, CA, USA). For analysis of MALDI-TOF MS data 10 lL of purified CLGAT ( 0.5 mgÆmL )1 ) were mixed with 1 lL matrix solution containing 10 mgÆ mL )1 a-cyano-4-hydroxycinnamic acid and 50% (v ⁄ v) acetonitrile. The mixture was loaded onto a sample plate and the solvent was removed by evapor- ation. The molecular mass of CLGAT was determined by MALDI-TOF MS (Autoflex II, Bruker Daltonics, Billerica, MA, USA), calibrated with insulin (5734 Da), cyto- chrome c (12361 Da), myoglobin (16952 and 8476 Da) and ubiquitin (8566 Da; all Sigma) as standard proteins. Western blotting Proteins from SDS ⁄ PAGE were transferred to polyvinyl- dene difluoride membranes (Sequi-Blot PVDF membrane; Bio-Rad) using a semidry blotting apparatus (NA-1512, Ni- hon-Eido, Tokyo, Japan). The specific antibody for DRIP- 1 [25] was used at a dilution of 1 : 500 in buffer containing 30 mm Tris ⁄ HCl buffer pH 7.5, 200 mm NaCl and 5% (w ⁄ v) skim milk. Immunoreactive proteins were detected with an Immunostaining HRP-1000 kit (Konica, Tokyo, Japan) according to the manufacturer’s instructions. cDNA cloning Total RNA was prepared from 3 g wild watermelon leaves subjected to drought stress for 1 day using TRIzol (Invitro- gen, Carlsbad, CA, USA). Poly(A) + RNA was isolated from the total RNA using a mRNA purification kit (Amer- sham Bioscience) according to the manufacturer’s instruc- tions. The sequence of the watermelon EST clone (accession number AI563351) was used to design four GAT-specific primers; CLGAT5a (5¢-GGCATCAACAT- CACAAGCAACAAGTGCAAG-3¢), CLGAT5b (5¢-TCCT- CCATCAATCTGCTTCCATGGACCATC-3¢), CLGAT3a (5¢-GCTGTGGCTACGAATGAGGCCGCC-3) and CLGAT3b (5¢-AAGGGAGAGAAACCTGACCTTGCACTTG-3¢). The 5¢-RACE was performed using Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions, using CLGAT5a for the first PCR and CLGAT5b for the second PCR. For 3¢-RACE, single-stranded cDNA was synthesized using First-Strand cDNA Synthesis Kit (Amersham Bioscience) with a NotI-d(T) 18 bifunctional primer (5¢-TAA- CTGGAAGAATTCGCGGCCGCAGGAAT (18) -3¢). The single- stranded cDNA was used for PCR w ith the primers Not1 (5¢-AACTGGAAGAATTCGCGGCCGC-3¢) and CLGAT3a. An aliquot from t he first PCR products was subjected to the second round of PCR using primers Not2 (5¢-GAA GAAT- TCGCGGCCGCAGG-3¢) and CLGAT5b. Amplified products were cloned into the plasmid vector pBC (Stratagene, La Jolla, CA, USA). Sequencing was carried out using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with a DNA sequencer (model 3100, Applied Biosystems). Wild watermelon glutamate N-acetyltransferase K. 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Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon Kentaro Takahara, Kinya Akashi. massive accumulation of citrul- line in wild watermelon. As a first step to understand the mechanism of citrul- line and arginine accumulation in wild watermelon,