Báo cáo khoa học: Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana

Isolation and Characterization of a D-cysteine Desulfhydrase Protein from Arabidopsis thaliana

Tác giả: Anja Riemenschneider, Rosalina Wegele, Ahlert Schmidt and Jutta Papenbrock

Lĩnh vực: Sinh học thực vật, Hóa sinh

Nội dung tài liệu: Nghiên cứu này tập trung vào việc phân lập và đặc tính hóa một protein D-cysteine desulfhydrase (D-CDes) từ loài thực vật Arabidopsis thaliana. Protein này được xác định là có khả năng phân hủy D-cysteine thành pyruvate, H2S và NH3. Nghiên cứu đã xác định trình tự gen mã hóa, biểu hiện và tinh sạch protein tái tổ hợp, đồng thời phân tích hoạt tính enzyme của nó. Vị trí nội bào của protein cũng được xác định là trong ty thể. Biểu hiện gen, mức độ protein và hoạt tính enzyme đã được nghiên cứu dưới các điều kiện khác nhau như sự lão hóa của cây, chu kỳ sáng/tối và nồng độ sulfate. Các chức năng tiềm năng của protein D-CDes trong thực vật cũng được thảo luận.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 19 November 2004, revised 3 January 2005, accepted 11 January 2005)
• In several organisms D-cysteine desulfhydrase (D-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes D-cysteine into pyruvate, H2S, and NH3. A gene encoding a putative D-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has D-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5′-phosphate binding site. The purified recombinant mature protein had a Km for D-cysteine of 0.25 mm. Only D-cysteine but not L-cysteine was converted by D-CDes to pyruvate, H2S, and NH3. The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5′-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded D-CDes protein in the mitochondria. D-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; D-CDes protein levels remained almost unchanged in the same plants whereas specific D-CDes activity was highest in senescent plants. In plants grown in a 12-h light/12-h dark rhythm D-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of D-CDes RNA and increased protein levels, the D-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsis D-CDes protein are discussed.
• It is well documented that, in general, amino acids are used in the L-form, and enzymes involved in their metabolism are stereospecific for the L-enantiomers. However, D-amino acids are widely distributed in living organisms [1]. Examples of the natural occurrence of D-amino acids include D-amino acid-containing natural peptide toxins [2], antibacterial diastereomeric peptides [3], and the presence of D-amino acids at high concentrations in human brain [4]. In plants D-amino acids were detected in gymnosperms as well as mono- and dicotyledonous angiosperms of major plant families. Free D-amino acids in the low percentage range of 0.5-3% relative to their L-enantiomers are principle constituents of plants [5]. The functions of D-amino acids and their metabolism are largely unknown. Various pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyse elimination and replacement reactions of amino acids have been purified and characterized [6].
• Abbreviations
• D-cysteine desulfhydrase from a higher plant
• However, most act specifically on L-amino acids. Only a few PLP enzymes that act on D-amino acids have been found such as D-serine dehydratase [7], 3-chloro-D-alanine chloride-lyase [8], and D-cysteine desulfhydrase (D-CDes) [9-11]. The Escherichia coli D-CDes (EC 4.1.99.4) is capable of catalysing the transformation of D-cysteine into pyruvate, H2S, and NH3 [9,10]. A similar activity was detected in several plant species, such as Spinacia oleracea, Chlorella fusca, Cucurbita pepo, Cucumis sativus and in suspension cultures of Nicotiana tabacum [11-14]. In all publications cited, the D-CDes activity could be clearly separated from L-CDes activity by demonstrating different pH optima for the enzyme activity [11], different sensitivity to inhibitors [14], and different localization in the cell [14]. Both CDes protein fractions were separated by conventional column chromatography, however, because of low protein stability and low yields neither of the proteins could be purified to homogeneity from plant material [11,12].
• The D-CDes protein from E. coli is a PLP-containing enzyme. It catalyses the α,β-elimination reaction of D-cysteine and of several D-cysteine derivatives, and also the formation of D-cysteine or D-cysteine-related amino acids from β-chloro-D-alanine in the presence of various thiols or from O-acetyl-D-serine and H2S [9,10]. The physiological role of bacterial D-CDes is unknown. Studies indicated that E. coli growth is impaired in the presence if micromolar amounts of D-cysteine [15]. To assess the role of D-CDes in adaptation to D-cysteine, a gene was cloned from E. coli corresponding to the ORF yedO at 43.03 min on the genetic map of E. coli [16] (protein accession number D64955). The amino acid sequence deduced from this gene is homologous to those of several bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. However, the E. coli YedO protein did not use ACC as substrate, but exhibited D-CDes activity. YedO mutants exhibited hypersensitivity or resistance, respectively, to the presence of D-cysteine in the culture medium. It was suggested that D-cysteine exerts its toxicity through an inhibition of threonine deaminase. On the other hand, the presence of the yedo gene stimulates cell growth in the presence of D-cysteine as sole sulfur source because the bacterium can utilize H2S released from D-cysteine as sulfur source. Consequently, the yedo expression was induced by sulfur limitation [16].
• In the Arabidopsis genome, a gene homologous to yedo has been identified [16] (At1g48420). To date ACC deaminase activity has not been demonstrated for plants. Therefore the tentative annotation as an ACC deaminase is probably not correct and the deduced protein might be a good candidate for the first D-CDes enzyme in higher plants of which the sequence is known. The putative D-CDes encoding cDNA was amplified by RT/PCR from Arabidopsis, the protein was expressed in E. coli, and the purified protein was analysed enzymatically. It was shown to exhibit D-CDes activity with the products pyruvate, H2S, and NH3. The nuclear-encoded protein was transported into mitochondria. Expression analysis revealed higher D-CDes mRNA and protein levels in older plants, during the light phase in a diurnal light/dark rhythm and under sulfate limitation.
• Results
• In silico characterization and isolation of the Arabidopsis protein homologous to Yedo from E. coli
• The existence of D-CDes activity was demonstrated in different plant species a long time ago and it could be shown that at least part of the activity was PLP dependent [12,14,17]. However, the respective encoding gene(s) had not been identified in any plant species because the putative D-CDes protein from spinach could not be purified to sufficient homogeneity for amino acid sequencing (data not shown). Recently, a protein with D-CDes activity and its respective gene, called yedo, were isolated from E. coli [16]. Consequently, the sequenced Arabidopsis genome [18] was screened for homologues to the E. coli yedo gene. The highest identities at both the nucleotide and the amino acid levels revealed a sequence that had been annotated based on sequence homologies to several bacterial proteins such as ACC deaminase (EC 3.5.99.7), an enzyme activity not identified in plants to date. The putative D-CDes encoding Arabidopsis gene is located on chromosome 1 (At1g48420, DNA ID NM_103738, protein ID NP_175275). The corresponding EST clone VBVEE07 from Arabidopsis, ecotype Columbia (available from the Arabidopsis stock Resource center, DNA Stock Center, The Ohio State University) was not complete at the 5′ end. The complete coding region of 1203 bp was obtained by RT/PCR from RNA isolated from 3-week-old Arabidopsis plants.
• The respective D-CDes protein consists of 401 amino acids including the initiator methionine and excluding the terminating amino acid. The protein has a predicted molecular mass of 43.9 kDa and a pI of 7.2. It contains relatively high amounts of the sulfur amino acids cysteine (four residues) and methionine (10 residues). According to several programs predicting the intracellular localization of proteins in the cell
• (http://www.expasy.ch/tools), the protein might possess an N-terminal extension (in PSORT, a probability of 0.908 for mitochondria; PREDATOR, mitochondrial score of 0.965; MITOPROT, 0.9547 probability of export to mitochondria). In PSORT a protease cleavage site between amino acids 19 and 20 counting from the start methionine was predicted, indicating a presequence of 19 amino acids. The mature protein would have a molecular mass of 41.7 kDa and a pI of 6.34.
• The YedO protein from E. coli and the D-CDes from Arabidopsis showed an overall identity of 36% and a similarity of 50%. The BLASTP program in its default positions was used to identify eukaryotic protein sequences revealing sequence similarities to the Arabidopsis D-CDes protein. The resulting phylogenetic tree including the YedO protein sequence is shown (Fig. 1). Two proteins closely related to Arabidopsis D-CDes were detected in the plant species Oryza sativa and Betula pendula. The YedO protein from E. coli showed higher similarities to the plant D-CDes protein than to related proteins from several yeast species (for clarity only representative sequences from three species are shown). The respective protein from Hansenula saturnus was already crystallized and a model of its 3D structure determined [19]. Interestingly, both Arabidopsis and Oryza contain a second protein revealing a lower sequence similarity to the true D-CDes proteins. Their function is unknown so far.
• All enzymes aligned belong to the PLP-dependent protein family (PALP, PF00291, http://pfam.wustl.edu/hmmsearch.shtml). Members of this protein family catalyse manifold reactions in the metabolism of amino acids. In addition to the PLP-binding site a number of other PROSITE (http://expasy.hcuge.ch/sprot/prosite.html) patterns and rules were detected in the D-CDes protein sequence, such as N-glycosylation, tyrosine sulfation, phosphorylation, myristylation, and amidation sites, all of them are characterized by a high probability of occurrence.
• Enzyme activity of the recombinant protein
• The recombinant Arabidopsis D-CDes proteins including and excluding the targeting peptide were expressed in E. coli and already 2 h after induction the proteins accumulated up to 5% of the total E. coli protein (Fig. 2). The D-CDes proteins were purified by nickel affinity chromatography under native conditions to about 95% homogeneity as demonstrated by loading
• Fig. 2. SDS/PAGE analysis of E. coli carrying Arabidopsis cDNA encoding the mature D-CDes protein cloned into the pQE-30 expression vector. SDS/PAGE was performed according to Laemmli (1970). Samples were denatured in the presence of 56 mm DTT and 2% SDS, heated for 15 min at 95 °C, and centrifuged. Aliquots of the supernatant were loaded onto SDS-containing gels. Lanes described from the left to the right: M, protein marker (Roth); 0 h, protein extract of transformed E. coli strain XL1-blue shortly before induction of the culture with IPTG; 2 h, transformed E. coli strain XL1-blue protein extract 2 h after induction with IPTG; P, protein purified by Ni2+-affinity chromatography (10 µg). The molecular masses of the marker proteins are given in kDa on the left.
• the purified protein fraction on an SDS-containing gel and subsequent Coomassie- and silver-staining. The Coomassie-stained SDS gel visualizing the purified mature D-CDes protein is shown in Fig. 2. The purified recombinant D-CDes proteins including and excluding the targeting peptide were dialysed overnight against 20 mM Tris/HCl pH 8.0 and used for enzyme assays.
• The pH optimum for the D-CDes reaction was determined to pH 8.0, in contrast to L-CDes activity with an optimum of pH 9.0 [20]. The purified D-CDes proteins were as heat labile as other proteins as demonstrated by incubation experiments in 100 mM Tris/HCl pH 8.0, for 15 min at elevated temperature and subsequent enzyme activity analysis. They lost activity at 50 °C and no activity was left at 60 °C. However, the D-CDes protein including the targeting peptide was very sensitive to freezing. One freeze-thaw cycle led to a loss of activity of 75%. Several complex dialysing buffers including glycerol, PLP, dithiothreitol and EDTA did not increase the stability of the protein after freezing. The results are in agreement with earlier stability problems during conventional column purification [17]. The mature D-CDes protein that had been expressed without the targeting peptide was more stable with respect to freezing and was therefore used for most of the enzyme assays.
• The Km value for D-cysteine was determined to 0.25 mm. D-Cysteine concentrations higher than 2 mm reduced the enzyme activity by substrate inhibition as observed previously for the E. coli protein [9]. The catalytic constant kcat was determined to 6.00 s⁻¹. The molecular mass for the recombinant protein was calculated excluding the His6-tag (41.7 kDa). The catalytic efficiency was determined to be 24 mm⁻¹.s⁻¹. The enzyme activity using L-cysteine as substrate showed only about 5% of the D-CDes activity indicating a high specificity for D-cysteine.
• In previous experiments it was demonstrated that the E. coli D-CDes protein catalysed the β-replacement reaction of O-acetyl-D-serine with sulfide to form D-cysteine [10]. Therefore it was tested whether the Arabidopsis D-CDes protein exhibits O-acetyl-D-serine(thiol)lyase or O-acetyl-L-serine(thiol)lyase activity, this was not the case. β-chloro-D-alanine and β-chloro-L-alanine were used in the O-acetyl-L-serine(thiol)lyase (OAS-TL) assay instead of O-acetyl-L/D-serine and the formation of cysteine was determined; the D-CDes protein did not reveal any activity in this assay. The protein was also tested for β-cyanoalanine synthase activity by using D-cysteine and cyanide as substrates; the D-CDes protein did not show any β-cyanoalanine synthase activity.
• Because originally the protein was identified as an ACC deaminase the recombinant D-CDes protein was used to determine this enzyme activity according to Jia et al. [21]. The recombinant protein did not show any ACC deaminase activity. Plant extracts of the soluble protein fraction did not exhibit ACC deaminase activity either.
• As mentioned above the D-CDes protein contains a PLP-binding site and was grouped into the PALP family. The absorption spectrum of the purified D-CDes protein determined between 250 and 470 nm revealed a small shoulder at 412 nm (data not shown), indicating the presence of the cofactor PLP. The ratio A280: A412 was ≈ 21.4: 1. A molar ratio of PLP (A412) to protein (A280) of 2:1 would suggest that there was one molecule of PLP associated with one protein molecule. The protein preparation was not completely pure as seen in Fig. 2. However, the ratio indicates that not all D-CDes protein molecules contained the PLP cofactor. Addition of pyridoxine and thiamine to the protein expression medium or to the dialysis buffer did not increase the protein/PLP cofactor ratio. To obtain further evidence for the involvement of PLP in the reaction, experiments with specific inhibitors for PLP proteins were performed. The inhibitors aminooxy acetic acid (AOA) and hydroxylamine were applied in the concentration range 10 µm to 5 mm to determine the 150 concentration using the purified D-CDes protein in the H2S-releasing assay. At the higher inhibitor concentrations the activity was completely blocked. The 150 for AOA was determined to 30.5 µm and for hydroxylamine to 15.9 µm. The results underline the identification of the D-CDes protein as PLP dependent. In former experiments