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• W2150689878 abstract "Article1 March 1997free access Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria Fabrice Rébeillé Corresponding Author Fabrice Rébeillé Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author David Macherel David Macherel Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Jean-Marie Mouillon Jean-Marie Mouillon Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Jérome Garin Jérome Garin Laboratoire de Chimie des Protéines, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Roland Douce Roland Douce Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Fabrice Rébeillé Corresponding Author Fabrice Rébeillé Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author David Macherel David Macherel Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Jean-Marie Mouillon Jean-Marie Mouillon Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Jérome Garin Jérome Garin Laboratoire de Chimie des Protéines, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Roland Douce Roland Douce Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France Search for more papers by this author Author Information Fabrice Rébeillé 1, David Macherel1, Jean-Marie Mouillon1,2, Jérome Garin2 and Roland Douce1 1Laboratoire de Physiologie Cellulaire Végétale, CNRS URA no. 576, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France 2Laboratoire de Chimie des Protéines, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, Cédex 9, France The EMBO Journal (1997)16:947-957https://doi.org/10.1093/emboj/16.5.947 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In pea leaves, the synthesis of 7,8-dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase enzyme, which represented 0.04–0.06% of the matrix proteins. The enzyme had a native mol. wt of 280–300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8-dihydropteroate synthase domain of the protein was Mg2+-dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated Mr of 56 454 Da. Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated Mr of the mature protein was 53 450 Da. Southern blot experiments suggested that a single-copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8-dihydropteroate synthesis in higher plant cells. Introduction One-carbon metabolism in cells is mediated by a variety of tetrahydrofolate polyglutamate derivatives (Cossins, 1984; McGuire and Coward, 1984). As a result, a number of pathways such as those involved in the metabolisms of methionine, serine, glycine, purine or thymidylate are dependent on an endogenous supply of these coenzymes (McGuire and Bertino, 1981; Appling, 1991). Plants and microorganisms, in contrast to animals, are able to synthesize tetrahydrofolate from 6-hydroxymethyl-7,8-dihydropterin de novo. This pathway requires the sequential operation of five enzymes: a 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a 7,8-dihydropteroate synthase (DHPS) (EC 2.5.1.15), a dihydrofolate synthetase (DHFS) (EC 6.3.2.12), a dihydrofolate reductase (DHFR) (EC 1.5.1.3) and a folylpolyglutamate synthetase (FPGS) (EC 6.3.2.17). In microorganisms, a lot of attention has been given to the first steps of this synthesis because they represent potential targets for antimicrobial agents. This is the case for DHPS, the target of sulfonamide drugs which are p-aminobenzoic acid (p–ABA) analogs that are recognized by DHPS as alternate substrates (Shiota, 1984; Hong et al., 1995). In bacteria, such as Bacillus subtilis and Streptococcus pneumoniae, HPPK and DHPS are part of a cluster of genes involved in folate biosynthesis (Slock et al., 1990; Lacks et al., 1995). In Escherichia coli, HPPK is a monofunctional protein with a compositional mol. wt of ∼18 kDa (Talarico et al., 1992). In S.pneumoniae, the situation is different and HPPK is part of a bifunctional protein also supporting dihydroneopterin aldolase (DHNA) activity (Lopez and Lacks, 1993). DHNA catalyzes the conversion of 7,8-dihydroneopterin into 6-hydroxymethyl-7,8-dihydropterin, substrate of the HPPK activity. In this organism, HPPK/DHNA is a tetrameric protein made up of 31 kDa subunits. DHPS in prokaryotes is a monofunctional protein, distinct from HPPK, with a native mol. wt of ∼70–100 kDa (Lopez et al., 1987) and is made up of identical subunits of 30–34 kDa (Lopez et al., 1987; Dallas et al., 1992; Kellam et al., 1995). In contrast, HPPK and DHPS activities were always associated in the eukaryotes studied so far. In the protozoa Plasmodium falciparum and Toxoplasma gondii, the DHPS enzyme is bifunctional, also supporting HPPK activity (Allegra et al., 1990; Triglia and Cowman, 1994). In P.falciparum, the corresponding gene encodes a protein with an apparent Mr of 83 kDa but the observed size on SDS–-PAGE gels is 68 kDa (Triglia and Cowman, 1994). In T.gondii, the primary structure of the protein was not determined, but the reported molecular weight of the native enzyme [125 kDa (Allegra et al., 1990)] is different from that of P.falciparum [200 kDa (Walter and Königk, 1974)]. The situation is even more complex in the sporozoa Pneumocystis carinii, where the enzyme is at least a trifunctional polypeptide supporting DHNA, HPPK and DHPS activities (Volpe et al., 1993). Surprisingly, the size of this trifunctional protein was similar to the size of the bifunctional enzyme of P.falciparum (Volpe et al., 1993). Taken as a whole, these data indicate that the proteins supporting HPPK and DHPS activities may vary greatly from one species to another. Folate metabolism in plants has been studied intensively by several authors, and much information regarding the nature of folates and the regulation of enzymes involved in C1 metabolism is now available (for reviews, see Cossins, 1984, 1987). However, there is much less data concerning the initial steps of folate synthesis. In an early work, Okinaka and Iwai (1970a) purified the DHPS from whole pea seedlings. The enzyme appeared as a bifunctional protein, also supporting HPPK activity, with an apparent native Mr of 180 kDa. However, the protein was not characterized further and its primary structure was not determined. Fractionation studies suggested that a large part (73–78%) of the DHPS activity was present in mitochondria, but the cross-contamination of the various fractions was not examined (Okinaka and Iwai, 1970b). In a recent study, we presented evidence indicating that mitochondria contain all the enzymes involved in 6-hydroxymethyl-7,8-dihydropterin to tetrahydrofolate conversion, thus confirming the important role that these organelles play in folate synthesis (Neuburger et al., 1996). In the present study, we have purified the HPPK/DHPS from pea leaf mitochondria and determined some of its kinetic properties. In a second step, we have isolated a cDNA clone encoding the entire protein to allow the comparison of plant HPPK/DHPS with those from other sources and as a prerequisite to study the regulation of the protein synthesis. Results Cellular distribution of DHPS Cell fractionation experiments reported in the early work of Okinaka and Iwai (1970b) indicated that 73% of the DHPS acitivity was found in a mitochondrial fraction and 25% in a soluble fraction. However, as stated by these authors, it was not certain that the activity found in the soluble fraction was dependent on a DHPS isoenzyme because of a possible contamination from the mitochondrial fraction. Therefore, we measured DHPS and HPPK activities in highly purified cell organelles and in a cytosol-enriched fraction where contamination from the other cell compartments was estimated through the activities of marker enzymes (Table I). The purpose of this approach is not to calculate the intracellular distribution of HPPK and DHPS because the methods used to obtain the different fractions are only qualitative, but simply to assess whether or not these activities are present in a given compartment. As previously reported (Neuburger et al., 1996), and shown in Table I, significant HPPK and DHPS activities were detected only in mitochondria. Indeed, the very low DHPS activity found in the cytosol fraction could be accounted for totally by the small mitochondrial contamination which represented ∼0.8–1% of the proteins. It must be pointed out that the HPPK activity reported here was estimated through DHPS activity, according to several authors (Okinaka and Iwai, 1970c; Talarico et al., 1992; Lopez and Lacks, 1993). Separate control experiments indicate that this reaction, in contrast to the DHPS-catalyzed reaction, has an absolute requirement for ATP. Thus, estimations of the ATP-dependent global activity (HPPK + DHPS) do reflect the presence of a HPPK activity and not simply the ability of DHPS to convert 6-hydroxymethyl-7,8-dihydropterin into 7,8-dihydropteroate. The results presented in Table I suggest that higher plant mitochondria are a major site for 7,8-dihydropteroate synthesis. In order to strengthen this hypothesis, the mitochondrial DHPS protein was purified and the corresponding cDNA clone was isolated. Table 1. DHPS and HPPK activities in the different pea leaf cell compartments Enzyme Mitochondria Chloroplasts Cytosol Fumarase 51 000 ± 3000 ND 420 ± 120 PRK ND 27 000 ± 2400 10 200 ± 2400 PEPc ND ND 6000 ± 800 DHPS 16 ± 3 ND 0.08 ± 0.04 HPPK + DHPS 1.8 ± 0.3 ND ND HPPK activity was estimated in association with DHPS activity (see Materials and methods). The results are the average of five determinations and are expressed as nmol/h/mg protein. Fumarase, phosphoribulokinase (PRK) and phosphoenolpyruvate carboxylase (PEPc) were marker enzymes for mitochondria, chloroplasts and cytosol respectively. ND: not detected Purification and biochemical properties of the HPPK/DHPS The DHPS from higher plant mitochondria was purified in three steps (Table II). During the course of purification, the HPPK activity always co-eluted with the DHPS activity, which strongly suggests that these two activities are supported by a common protein. Thus, this enzyme catalyzes the first two reactions involved in folate synthesis: Table 2. Purification steps for HPPK/DHPS from pea leaf mitochondria Fractions Total protein (mg) HPPK + DHPS DHPS Yield (%) Total act. (nmol/h) Sp. act. (nmol/h/mg) Total act. (nmol/h) Sp. act. (nmol/h/mg) Matrix 630 1010 1.6 9200 14.6 100 Superdex 200 160 768 4.8 8000 50 87 Mono Q 7.4 525 71 5500 743 60 Folate agarose 0.137 383 2800 3500 25 500 38 As shown in Table II, the yield of recovery of the HPPK/DHPS was ∼30–40%, but this protein represented only 0.04–0.06% of the soluble proteins of the mitochondrial matrix. The global (HPPK + DHPS) activity was always lower than DHPS activity alone, a result previously observed with the pea seedling enzyme (Okinaka and Iwai, 1970a). It is not certain whether the global activity reported here reflects the maximal HPPK activity, which prevents any comparison between the two maximal rates. The measurement of HPPK activity alone requires a new assay method, the development of which is currently in progress. The apparent molecular weight of the native protein, determined either by gel filtration (Figure 1) or by PAGE in non-denaturing conditions (Figure 2A), was ∼ 280–300 kDa. This result is not in agreement with the previous work of Okinaka and Iwai (1970a) who reported an Mr of 180 kDa. SDS–PAGE analysis (Figure 2B) indicated that HPPK/DHPS was made up of identical subunits of ∼53 kDa. Mass spectrometry analysis of the protein, using a laser desorption technique, indicated a single peak of 53.65 kDa (result not shown), thus confirming the purity of our protein and the SDS–PAGE molecular mass determination. Figure 1.Separation by gel filtration of the HPPK and DHPS activities. Soluble proteins from purified mitochondria were loaded on a Superdex 200 (Pharmacia) column as described in Materials and methods. DHPS activity (□); HPPK activity (○). To facilitate the graph reading, the DHPS activity was reduced three times. The arrows mark the position of the peaks of the P-protein (200 kDa), the L-protein (120 kDa), the T-protein (45 kDa) and the H-protein (15 kDa) of the glycine cleavage system (Bourguignon et al., 1988) and the DHFR (140 kDa) (Neuburger et al., 1996). The inset is the plot of the molecular weight of the markers (logarithm scale) versus the fraction number. The arrow indicates the position of the HPPK/DHPS. The calculated value from this curve is 280 kDa. Download figure Download PowerPoint Figure 2.Electrophoretic analysis of purified HPPK/DHPS from pea leaf mitochondria. (A) Electrophoresis in non-denaturing conditions. Lane 1, 6 μg of the purified enzyme recovered from the folate agarose column; lanes 2 and 3, molecular weight standards. (B) SDS–PAGE. Lane 1, molecular weight standards; lane 2, 10 μg of the purified enzyme recovered from the folate agarose column. Download figure Download PowerPoint Preliminary studies of the kinetic properties of HPPK/DHPS are shown in Table III. The Km values for 6-hydroxymethyl-7,8-dihydropterin and p-ABA were very low, <1 μM, but the Km value for 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate was much higher (30–40 μM). The maximal DHPS activity was obtained for temperatures close to 50°C and the optimum pH, as often observed for folate enzymes, was ∼9. The catalytic properties of the DHPS domain of the bifunctional protein (Equation 2) were studied in more detail. As shown in Figure 3, when the initial rate of the reaction was measured in the presence of variable and limiting concentrations of each of the two substrates, we obtained a family of reciprocal plots intersecting at a unique point on the x-axis. On the basis of the rapid equilibrium hypothesis (Segel, 1975), these results are indicative of a random bireactant system where binding of one substrate does not change the affinity of the other. Interestingly, 7,8-dihydropteroate, the product of this reaction, appeared as a potent competitive inhibitor for either 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (Ki = 7–11 μM) or p-;ABA (Ki = 5–8 μM) (Figure 4), suggesting a strong feed-back inhibition. The presence of Mg2+ was required for maximal DHPS activity (result not shown). Indeed, omission of Mg2+ from the buffer medium resulted in a 50–60% decrease in the activity, and a complete loss of activity was obtained in the presence of 1 mM EDTA. At the moment, we still do not know whether Mg2+-bound substrates, such as Mg2+-6-hydroxymethyl-7,8-dihydropterin-pyrophosphate, are the true substrates of the reaction or if Mg2+ is required at the level of the catalytic pocket. This question is under investigation. Figure 3.Kinetic studies of the DHPS reaction. (A) Reciprocal plots showing the effect of varying p-ABA concentrations at different fixed concentrations of 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate. The fixed 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate concentrations were: ○ 300 μM, □ 50 μM, ▿ 20 μM, • 10 μM. (B) Reciprocal plots showing the effect of varying 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate concentrations at different fixed concentrations of p-ABA. The fixed p-ABA concentrations were: ▪ 4 μM, • 2 μM, ▿ 1.25 μM, □ 0.7 μM, ○ 0.6 μM. Download figure Download PowerPoint Figure 4.Inhibitory effect of 7,8-dihydropteroate upon DHPS activity. (A) Competitive inhibition of 7,8-dihydropteroate versus 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate in the presence of a saturating concentration of p-ABA (10 μM). 7,8-dihydropteroate concentrations were: □ 50 μM, ▿ 10 μM, ○ 0 μM. (B) Competitive inhibition of 7,8-dihydropteroate versus p-ABA in the presence of a saturating concentration of 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (100 μM). 7,8-dihydropteroate concentrations were: ▿ 50 μM, □ 5 μM, ○ 0 μM. Download figure Download PowerPoint Table 3. Kinetic constants, optimal temperature and optimal pH for the HPPK/DHPS of pea leaf mitochondria Kinetic parameters Km for 6-hydroxymethyl-7,8-dihydropterin (μM) 0.7 Km for ATP (μM) 70 Km for 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (μM) 30 Km for p-ABA (μM) 0.6 Optimal temperature (°C) 50 Optimal pH 9 Primary structure of the HPPK/DHPS From the purified HPPK/DHPS enzyme, the N-terminal part (12 amino acids) and three internal sequences were determined by microsequencing (see Materials and methods and Figure 5). These sequences were compared with those present in protein data banks, which led to the prediction that internal sequences 1 and 2 belonged respectively to HPPK and DHPS. We selected the amino acid sequences DMGRTDG (internal sequence 1) and AHIINDV (internal sequence 2) to design degenerate oligonucleotides which were tailed at their 5′ end with unrelated GC-rich sequences. cDNAs obtained by reverse transcription of pea leaf mRNAs were then combined with these oligonucleotides in a two-step PCR protocol (see Materials and methods and Figure 6). This procedure allowed the correct amplification of the desired sequence in spite of the degeneracy and low Tm of the primers. A 700 bp PCR product was obtained and further amplified using the non-degenerate GC-rich sequences as primers. This PCR product was sequenced, and it appeared to encode the HPPK/DHPS region boarded by the internal sequences 1 and 2 (see Figure 5). Thus, our RT-PCR approach using GC-rich tailed oligonucleotides appeared to be straightforward because it limited the non-specific amplifications generally observed with degenerate oligonucleotides, and allowed a direct sequencing of the PCR product without tedious subcloning. Specific oligonucleotides derived from this first nucleotide sequence were used to amplify a 430 bp DNA fragment which was, in turn, used as a probe to screen a λgt10 cDNA library prepared from green leaf mRNA. A single positive clone, containing a 2.1 kb insert, was picked out of 120 000 phages and purified. The digestion of phage DNA with EcoRI yielded two fragments of ∼1.8 and 0.3 kbp which were subcloned in pBluescript to yield pBSDH1 and pBSDH2 phagemids. The sequence analysis of pBSDH1 and pBSDH2 revealed that the isolated cDNA had a total length of 2159 nucleotides and contained an open reading frame (ORF) of 1545 bp starting at the ATG translation initiation start site at position 156 and ending at the TGA stop codon at position 1701 (Figure 5). The sequence analysis of pBSDH2 indicated that it contained the downstream region of the cDNA ending with a 56 nucleotide poly(A) tail. We do not know at present whether the poly(T) motif observed at the 5′ end of the cDNA was representative of a non-coding region of the mRNA or whether it was the result of an artefact during the library construction. Figure 5.Nucleotide sequence and deduced amino acid sequence of the HPPK/DHPS cDNA. Initiation (ATG) and stop (TGA) translation codons are indicated in bold. Annealing regions of primers DM and AH (see Materials and methods) are overlined with dotted lines. The N-terminal and the three internal sequences (named I-1, I-2, I-3) determined by microsequencing of the purified HPPK/DHPS are marked in bold and underlined. Numbers indicate nucleotide residues. The sequence was registered in the EMBL Nucleotide Sequence Database under the accession No.Y08611. Download figure Download PowerPoint Figure 6.Schematic representation of the cloning strategy to obtain the HPPK/DHPS cDNA. R.T., reverse transcriptase; DM and AH were the degenerated primers constructed from the internal sequences (black boxes) of the protein; X1 and X2 were the unrelated GC-rich sequences intended to increase the Tm of the whole primer. See Materials and methods for details. Download figure Download PowerPoint As also shown in Figure 5, the three amino acid sequences, previously determined by microsequencing of the purified HPPK/DHPS, were recognized on the translated sequence, thus confirming the identity of this cDNA. The deduced amino acid sequence corresponds to a polypeptide of 515 residues with a calculated Mr of 56 454 Da. Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicates that the HPPK/DHPS is synthesized as a cytosolic precursor containing a 28 amino acid putative transit peptide. As there is only one methionine before the first amino acid (Phe) of the mature protein, and because the reading frame is interrupted upward in the nucleotide sequence, it is likely that the ATG 156 encodes the real initiation codon. The transit peptide was classified as mitochondrial as it exhibits characteristics shared with other mitochondrial transit peptides such as charge transition, a high content of serine and basic residues and no acidic residues (Von Heijne, 1986; Gavel and Von Heijne, 1990). This is in agreement with the mitochondrial origin of the purified protein. The mature protein is predicted to consist of 487 amino acid residues, giving an Mr of 53.43 kDa. The 1836 bp fragment obtained by digestion of the phage DNA with EcoRI (pBSDH1) was used in a Northern hybridization experiment carried out with total RNA prepared from dark- or light-grown leaves. A single transcript of 2.2 kb was detected after several days of exposure in both cases, with a higher intensity for etiolated leaves (Figure 7A). This shows that the length of the cDNA which we isolated fitted very well with that of the transcript. In addition, this figure also indicates that the steady-state level of the transcript encoding HPPK/DHPS was low and not promoted by light, at least at the developmental stage used in this analysis. Figure 7.(A) Northern blot analysis of pea leaf RNA. Samples of denatured total RNA (10 μg) from light-grown (L) and dark-grown (D) leaves were separated on a 1.2% agarose gel, transferred to a nitrocellulose membrane and hybridized with the 1.8 kbp 32P-labeled cDNA probe (see Materials and methods). The blot was washed with 0.5× SSC/0.1% SDS at 65°C. Transcript sizes are indicated on the left. (B) Southern blot analysis of pea genomic DNA. A genomic DNA gel blot of pea DNA (10 μg) digested with EcoRI (EI), HindIII (H) and EcoRV (EV) was hybridized with the 1.8 kbp 32P-labeled cDNA probe as described in Materials and methods. The blot was washed with 2× SSC/0.1% SDS at 60°C. Arrowheads indicate the position of the two minor bands observed with HindIII and EcoRV. Approximate fragment sizes are indicated on the right. Download figure Download PowerPoint The 1836 bp 32P-labeled probe was used in a Southern hybridization experiment with pea nuclear DNA digested with several restriction endonucleases (Figure 7B). After washing at moderate stringency (see Materials and methods), only one band was seen with EcoRI fragments whereas two bands could be detected with HindIII and EcoRV fragments. HindIII and EcoRV enzymes had restriction sites on the HPPK/DHPS cDNA at positions 1647 and 943, respectively. Therefore, each of these enzymes produced two restriction fragments which hybridized with the 32P-labeled probe. These results strongly suggest that a single-copy gene codes for the HPPK/DHPS protein in pea, unless the mitochondrial protein had poor homology with isoenzymes possibly present in the other cell compartments. This last hypothesis is unlikely, however, since strong homologies were always observed among the various HPPK and DHPS reported so far. In addition, it is unlikely that two forms of HPPK/DHPS derive from the same gene because there is no start codon in the region coding for the N-terminal part of the mature protein, as is the case in human folylpolyglutamate synthetase (Freemantle et al., 1995). Comparison of the plant mitochondria HPPK/DHPS with other HPPK and DHPS The predicted amino acid sequence of the plant mitochondria HPPK/DHPS was aligned with HPPK and DHPS of either bacteria (E.coli) (Dallas et al., 1992; Talarico et al., 1992), protozoa (P.falciparum) (Triglia and Cowman, 1994) or sporozoa (P.carinii) (Volpe et al., 1992) (Figure 8A and B). As shown in Figure 8A, the N-terminal part of the pea enzyme showed a good identity with HPPK from E.coli (∼34% amino acid identity), whereas the C-terminal part of the protein presented numerous domains identical to the E.coli DHPS (∼36% amino acid identity). Most of these common regions were also found in all HPPK and DHPS studied so far (Lopez et al., 1987, 1990; Slock et al., 1990; Dallas et al., 1992; Talarico et al., 1992; Volpe et al., 1992; Ballantine et al., 1994; Brooks et al., 1994; Triglia and Cowman, 1994), indicating highly conserved domains which are possibly involved in substrate binding and catalysis. Unfortunately, there is presently no data available for the corresponding proteins in algae or fungi. Interestingly, the plant enzyme resembles a fusion of the two bacterial enzymes with a small spacer of 10–15 amino acids (Figure 8A). Indeed, addition of the molecular mass of the two bacterial enzymes gives a value of 48 kDa, which is roughly similar to the 53 kDa of the mature pea protein. This situation is not seen with HPPK/DHPS of other eucaryotes. In P.carinii, the central and C-terminal part of the enzyme, containing the HPPK and DHPS activities respectively, presented a strong homology with the pea enzyme (∼36% amino acid identity) and the bacterial enzymes (Volpe et al., 1993). These two regions were also comparable in size with either the plant protein or their bacterial counterparts (Figure 8B). However, as shown in Figure 8B, the N-terminal part of the P.carinii HPPK/DHPS is responsible for DHNA activity, an apparently unique feature among the known HPPK/DHPS. The pea leaf mitochondria HPPK/DHPS is also very different from the P.falciparum enzyme. In this latter case, the N-terminal part of the protein (HPPK domain) contains two large insertions of 92 amino acids and the C-terminal part (DHPS domain) a smaller insertion of 32 amino acids (Triglia and Cowman, 1994), thus conferring on the P.falciparum protein a relatively high molecular weight. Figure 8.Comparison of the predicted amino acid sequence of pea leaf mitochondria HPPK/DHPS with HPPK and DHPS primary sequences from several organisms. (A) Comparison between the pea protein and the two monofunctional HPPK (Talarico et al., 1992) and DHPS (Dallas et al., 1992) proteins of E.coli. Boxed areas indicate amino acid identity, dots indicate spaces inserted into the sequence to provide optimal homology, and numbers indicate amino acid residues. (B) Schematic comparison of the size of HPPK and DHPS from various organisms. Numbers indicate the first and the last amino acid of the proteins, black boxes indicate the main conserved regions of the different catalytic domains. Download figure Download PowerPoint Discussion Our preliminary studies indicate that plant DHPS catalyses a Mg2+-dependent reaction that can be described as a random bireactant system, according to the rapid equilibrium hypothesis of Michaelis-Menten (Segel, 1975). Interestingly, 7,8-dihydropteroate was a competitive inhibitor of the two substrates of the pea leaf mitochondrial enzyme, suggesting that it interacts with both 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate- and p-ABA-binding sites. Furthermore, the l" @default.
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• W2150689878 date "1997-03-01" @default.
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• W2150689878 title "Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria" @default.
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