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Originally published In Press as doi:10.1074/jbc.M412475200 on December 7, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5242-5248, February 18, 2005
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Analysis of the Vitamin B6 Biosynthesis Pathway in the Human Malaria Parasite Plasmodium falciparum*

Carsten Wrenger{ddagger}, Marie-Luise Eschbach{ddagger}, Ingrid B. Müller{ddagger}§, Dirk Warnecke¶, and Rolf D. Walter{ddagger}||

From the {ddagger}Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany and the Biozentrum Klein Flottbek und Botanischer Garten, University of Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany

Received for publication, November 4, 2004 , and in revised form, November 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin B6 is an essential cofactor for more than 100 enzymatic reactions. Mammalian cells are unable to synthesize vitamin B6 de novo, whereas bacteria, plants, fungi, and as shown here Plasmodium falciparum possess a functional vitamin B6 synthesis pathway. P. falciparum expresses the proteins Pdx1 and Pdx2, corresponding to the yeast enzymes Snz1-p and Sno1-p, which are essential for the vitamin B6 biosynthesis. An involvement of PfPdx1 and PfPdx2 in the de novo synthesis of vitamin B6 was shown by complementation of pyridoxine auxotroph yeast cells. Both plasmodial proteins act together in the glutaminase activity with a specific activity of 209 nmol min–1 mg–1 and a Km value for glutamine of 1.3 mM. Incubation of the parasites with methylene blue revealed by Northern blot analysis an elevated transcriptional level of pdx1 and pdx2, suggesting a participation of these proteins in the defenses against singlet oxygen. To be an active cofactor, vitamin B6 has to be phosphorylated by the pyridoxine kinase (PdxK). The recombinant plasmodial PdxK revealed Km values for the B6 vitamers pyridoxine and pyridoxal and for ATP of 212, 70, and 82 µM, respectively. All three enzymes expose a stage-specific transcription pattern within the trophozoite stage that guarantees the concurrent expression of Pdx1, Pdx2, and PdxK for the indispensable provision of vitamin B6. The occurrence of the vitamin B6 de novo synthesis pathway displays a potential new drug target, which can be exploited for the development of new chemotherapeutics against the human malaria parasite P. falciparum.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyridoxal 5'-phosphate (PLP)1 is the active cofactor of more than 100 vitamin B6-dependent enzymes and essential for their catalytic reactions such as amino acid decarboxylation, elimination, and amino transfer (1). PLP is produced from its precursor pyridoxine and the B6 vitamers pyridoxal and pyridoxamine. Whereas almost all bacteria, fungi, and plants possess their own vitamin B6 biosynthesis, mammals do not synthesize pyridoxine and entirely depend on the uptake of this indispensable nutrient from their diet. Two different pathways are currently known to synthesis pyridoxine de novo: the Escherichia coli-like pathway and the fungi-like pathway. In E. coli the biosynthetic pathway of vitamin B6, consisting of the pdx family (Pdx A, B, C, F, H, J, and GapA), has been the subject of various studies and is therefore well understood (2–5). It is known that in E. coli two different branches have to be amalgamated by the enzymes PdxA and PdxJ for the de novo synthesis of pyridoxine 5'-phosphate (PNP) (6). The substrate of one branch is D-erythrose-4-phosphate, while 1-deoxy-D-xylulose-5-phosphate (DOXP) was found to be the precursor of the other branch (3, 6). Recently a distinctly different synthesis pathway has been identified in the fungi Cercospora nicotianae and Saccharomyces cerevisiae as well as in the plant Arabidopsis thaliana and in some bacteria like Mycobacterium tuberculosis or Bacillus subtilis (7). Originally this pathway was assigned to be involved in detoxification of singlet oxygen (1O2) (7). However, the analysis of fungi mutants, deficient in SOR1 (singlet oxygen resistance) and therefore sensitive for singlet oxygen, demonstrated that the product of this gene was also participating in pyridoxine biosynthesis (8, 9). In C. nicotianae the Sor1-p or Pdx1-p enzyme (also named as Snz1-p in S. cerevisiae or YaaD in B. subtilis) corresponds to the highly conserved enzyme family Snz in yeast. In S. cerevisiae another protein, Sno1-p (Pdx2-p in C. nicotianae or YaaE in B. subtilis), is a member of the preserved Sno-p protein family. The Sno1-p protein is coregulated during growth and nutrient limitation with Snz1-p and interacts with Snz1-p as was shown by the yeast two-hybrid system (10). An involvement of Pdx2-p in vitamin B6 de novo synthesis was confirmed by complementation of mutants deficient in pyridoxine biosynthesis (11). Neither the precise enzymatic reaction, catalyzed by Pdx1-p and Pdx2-p (Snz1-p and Sno1-p in yeast), nor its substrates are known. However, Pdx2-p possesses glutamine amidotransferase activity (11, 12). To become the active cofactor, vitamin B6 has to be phosphorylated by the pyridoxine/pyridoxal kinase (PdxK), a reaction that occurs in all organisms known so far. Pyridoxine kinases are members of the ribokinase superfamily (13–16). They have been characterized in several organisms such as Homo sapiens, A. thaliana, B. subtilis, and the parasite Trypanosoma brucei (17-20).

Malaria is one of the most serious infectious diseases in the world (WHO, Communicable Disease Report). In the most deadly agent, Plasmodium falciparum, anti-malarial drugs are losing efficacy due to drug resistance. For this reason there is an urgent necessity to identify novel targets within the parasite's metabolism for the development of new chemotherapeutics (21). Very recently, Cassera et al. (22) provided evidence by metabolic labeling experiments for an active de novo biosynthesis of pyridoxine in P. falciparum.

Here we report the identification of genes encoding proteins that are similar to the vitamin B6 de novo synthesis enzymes Snz1-p and Sno1-p in yeast. Additionally, an open reading frame was identified within the plasmodial genome data base referring to a pyridoxine kinase that is responsible for the activation of vitamin B6. Biochemical analysis of these plasmodial proteins substantiates the presence of a functional vitamin B6 biosynthesis pathway that can be exploited as a potential new drug target in the human malaria parasite P. falciparum.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Restriction enzymes were purchased from New England Biolabs. Oligonucleotides were from Qiagen. The cloning vectors pASK-IBA7 and pASK-IBA3, Strep-Tactin-Sepharose, anhydrotetracycline, and desthiobiotine were from IBA (Institut für Bioanalytik). [{alpha}-32P]dATP (3000 Ci mmol–1) was from Amersham Biosciences. Glutamate dehydrogenase, acetylpyridine adenine dinucleotide (APAD), and NADP+, 6-diazo-5-oxo-L-norleucine (DON), pyridoxine, pyridoxal, and pyridoxamine were from Sigma.

Cloning of the Pdx1, Pdx2, and PdxK—The open reading frame encoding for Pdx1 was amplified by PCR using P. falciparum 3D7 genomic DNA and the sense and antisense oligonucleotides PfPdx1-IBA3-S (5'-GCGCGCGGTCTCGAATGGAAAATCATAAAGATGATGC-3'), PfPdx1-IBA3-AS (5'-GCGCGCGGTCTCAGCGCTTTGTGGTGTTAAAAATTTGGTGTG-3'). The open reading frames encoding Pdx2 and PdxK were amplified by PCR from a P. falciparum cDNA library as template using the sequence-specific antisense and sense oligonucleotides PfPdx2-IB-A3-AS (5'-GCGCGCGGTCTCAGCGCTTGAATATTTGTAATTTTTAACCTTC-3') PfPdx2-IBA3-S (5'-GCGCCGCGGTCTCGAATGTCAGAAATAACTATAGGGG-3') PfPdxK-IBA7-NcoI-AS (5'-GCGCCCATGGGCAAAAAAAACAGGCTCTTC-3'), PfPdxK-IBA7-SacII-S (5'-GCGCCCGCGGTATGAAGAAGGAAAATATTATC-3') PfPdxK-IBA3-NcoI-AS (5'-GCGCCCATGGGCAAAAAAAACAGGCTCTTC-3') and PfPdxK-IBA3-S-acII-S (5'-GCGCCCGCGGTATGAAGAAGGAAAATATTATCTCC-3'). The PCRs for the plasmodial constructs were performed using Pfu polymerase (Invitrogen) and the following PCR program: 3 min at 95 °C for 1 cycle followed by 35 cycles of 1 min 95 °C, 1.5 min at 42 °C, 3 min at 60 °C. The generated PCR products were digested either with BsaI (PfPdx1, PfPdx2) or with SacII and NcoI (PdxK) and cloned into the expression plasmids pASK-IBA3 and pASK-IBA7 previously digested with the same enzymes, resulting in the expression constructs PfPdx1-IBA3, PfPdx2-IBA3, PfPdxK-IBA3, and PfPdxK-IBA7. The plasmids pASK-IBA7 and pASK-IBA3 encode for an N-terminal or C-terminal Strep-Tag, respectively, that allows one-step purification of the recombinant fusion proteins using Strep-Tactin-Sepharose (23). For cloning into the yeast expression vector pYES2/NT C (Invitrogen), the open reading frames pdx1 and pdx2 were amplified by PCR using the sense and antisense primers PfPdx1-pYES-S (5'-GCGCGGATCCATGGAAAATCATAAAGATGATGC-3'), PfPdx1-pYES-AS (5'-GCGCCTCGAGTTGTGGTGTTAAAAATTTGGTGTG-3'), and PfPdx2-pYES-S (5'-GCGCGGATCCATGTCAGAAATAACTATAGGGG-3'), PfPdx2-pYES-AS (5'-GCGCCTCGAGTGAATATTTGTAATTTTTAACCTTC-3') on the previously cloned E. coli expression constructs PfPdx1-IBA3 and PfPdx2-IBA3. The PCR products were digested with the restriction enzymes BamHI and XhoI and cloned with the same enzyme-digested pYES2/NT C vector, which resulted in the constructs PfPdx1-pYES2/NT and PfPdx2-pYES2/NT. The nucleotide sequences of all PCR fragments and clones were verified by automated nucleotide sequencing using the automatic sequencer ABI 377 (Bio-Rad). Nucleotide and amino acid analyses were performed with the help of Generunner.

Expression and Purification of the PfPdx1, PfPdx2, and PfPdxK—E. coli BLR (DE3) (Stratagene) were transformed with the cloned P. falciparum Pdx1, Pdx2, and PdxK constructs. Single colonies were picked and grown overnight in Luria-Bertani medium. The bacterial culture was diluted 1:50 and grown at 37 °C until the OD600 reached 0.5. The expression was initiated with 200 ng ml–1 of anhydrotetracycline, and the cells were grown for 4 h at 37 °C before harvest. The cell pellet was resuspended in 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA containing 0.1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 100,000 x g for 1 h at 4 °C. The recombinant Strep-Tag fusion protein was purified according to the manufacturer's recommendation (IBA). The eluate of the affinity chromatography was analyzed by SDS-PAGE, and the protein was revealed by Coomassie Blue staining (24). The concentration of the purified recombinant protein was determined according to Bradford (25).

Glutaminase Assay—The glutaminase activity was assayed in two steps, according to Dong et al. (12), by measuring the formation of glutamate, which is subsequently converted to 2-oxoglutarate by glutamate dehydrogenase with APAD or NADP+ as co-substrate. The enzyme reaction was performed in 50 mM Tris-HCl, pH 8 in the presence of 1–10 mM glutamine in a total volume of 300 µl at 30 °C for 10 min. The enzymatic reaction was stopped by boiling for 1 min. A 50 mM Tris-HCl, pH 8 buffer containing 1 mM EDTA, 500 µM APAD or NADP+, and 7 units of glutamate dehydrogenase were added to a final volume of 1 ml and incubated for 90 min at 30 °C. Finally, the samples were centrifuged for 1 min at 14,000 x g, and the absorbance of the supernatant was determined at 363 or 340 nm in the presence of APAD or NADP+, respectively. The specific activity was calculated with the molar extinction coefficient of APADH (reduced form of APAD) of 8900 M–1 cm–1 and 6220 M–1 cm–1 of NADPH (26). Inhibition of the glutaminase activity was performed in the presence of DON at concentrations of 5 and 10 mM. The inhibitor and 150 µg of the equimolar enzyme complex (Pdx1 and Pdx2) were incubated in 50 mM Tris-HCl, pH 8 at a total volume of 1 ml at 30 °C. At varied time points, aliquots were removed and combined with the ingredients of the standard glutaminase assay.

Complementation Assay and Western Blot Analysis—A double mutant of S. cerevisiae (genotype: BY4741; Mat a; his3{Delta} 1; met15{Delta} 0; ura3{Delta} 0; leu2{Delta} 0; YMR095c::kanMX4; YMR096w::LEU2) was generated by SRD (Scientific Research and Development), which led to a deletion of the genes SNO1 and SNZ1 and resulted in vitamin B6 auxotrophy (12). This mutant, deficient in pyridoxine biosynthesis, was transformed with the constructs, PfPdx1-pYES2/NT and PfPdx2-pYES2/NT for expression of PfPdx1 and PfPdx2 as N- and C-terminal His fusion proteins in yeast mutants. The complementation of the vitamin B6 auxotrophy was assayed by cultivation in the following minimal medium: D-raffinose, 20 g; (NH4)2SO4, 1.02 g; KH2PO4, 0.875 g; K2HPO4, 0.125 g; CaCl2·H2O, 0.02 g; NaCl, 0.01 g; MgSO4, 7··H2O, 0.05 g; CuSO4 5·H2O, 40 µg; MnSO4·H2O, 400 µg; FeCl3 6·H2O, 200 µg; ZnSO4 7·H2O, 400 µg; Na2MoO4 2·H2O, 200 µg; KI, 100 µg; H3BO3, 500 µg; biotin, 2 µg; inositol, 10 mg; nicotinic acid, 0.2 mg, calcium pantothenate 0.2 mg; histidine, 20 mg; methionine, 20 mg; tryptophan, 20 mg; and agar, 20 g per liter modified according to Dong et al. (12). For induction of the expression, 2% galactose was added to the minimal medium. Growth was mediated at 30 °C for 4 days. The recombinant expression of PfPdx1 and PfPdx2 within the yeast mutant was analyzed by a Western blot. Hybridization of the blot was performed with a monoclonal anti-HIS peroxidase-coupled antibody (Invitrogen) at a dilution of 1:5000. The blot was developed by the usage of the ECL+ detection system (Amersham Biosciences) according to the manufacturer's recommendations.

Enzyme Assay for PfPdxK—Pyridoxine kinase activity was measured according to Kwok and Churchich (27). The change in absorbance was followed in a double beam spectrophotometer UVICON 933 (BIO-TEK Kontron) by the formation of pyridoxal 5'-phosphate, which has an absorption maximum at 388 nm with an extinction coefficient of 4900 M–1 cm–1. The enzyme assay was performed in a total volume of 1 ml at 30 °C with 70 mM potassium phosphate buffer, pH 6.5 containing 400 µM pyridoxal, 3 mM ATP, and 10 mM MgCl2. Kinetic analysis of PfPdxK was carried out in the presence of either 0–600 µM pyridoxal at an ATP concentration of 1 mM or 0–400 µM ATP at a concentration of 600 µM pyridoxal. For analysis of the substrate specificity, the standard assay was executed in a total volume of 150 µl using 400 µM pyridoxal or 400 µM pyridoxamine or 400 µM pyridoxine and 500 µM 250 nCi of [{alpha}-32P]dATP. For determining the metal specificity, the standard assay was performed with 0.05, 0.1, 0.5, 1, and 10 mM of the following metals: MgCl2, ZnCl2, MnCl2, NiCl2, and CaCl2. The reaction was incubated at 30 °C for 20 min, stopped by boiling, and the reaction products were separated by thin-layer chromatography (PEI cellulose F, Merck) using a solvent consisting of 0.5 M LiCl and 1 M formic acid. The ADP spots were visualized by exposure on x-ray films (Retina). Additional kinetic studies were performed in the presence of 0–600 µM pyridoxine and 500 µM ATP containing radioactively labeled ATP, which were also analyzed by thin-layer chromatography and quantified by a liquid scintillation analyzer (TRI-CARB 2000CA, United Instruments Packard). The results were analyzed using GraphPad PRISM 4 (GraphPad software), and the apparent Km values were derived from reciprocal Lineweaver-Burk plots.

Analysis of the Expression of the Plasmodial pdx1, pdx2, and pdxK Genes within the Erythrocytic Stages and the Effect of Oxidative Stress—P. falciparum 3D7 was cultivated according to Trager and Jensen (28) in human A+ erythrocytes, RPMI 1640 medium containing 10 mM glucose and 0.5% Albumax II (Invitrogen). For stage-specific analysis, the parasites were synchronized with 5% sorbitol according to Lambros and Vanderberg (29). The highly synchronized cells were harvested 12 ± 4 h (rings), 26 ± 4 h (trophozoites), and 40 ± 4 h (schizonts) after infection. For analysis of oxidative stress, the cells were treated with 1.2 and 6 nM methylene blue for 3 h. The IC50 value for methylene blue in P. falciparum was reported to be ~4 nM (30). At the beginning of the incubation with methylene blue the culture was light-induced, which stimulates the production of singlet oxygen. Isolation of total RNA was performed using saponin-lysed parasites (31), that were immediately transferred into TRIzol (Invitrogen) according to the manufacturer's instructions, and the obtained RNA was analyzed by Northern blotting (32). 25 µg of total RNA of each parasite stage or stressed and nonstressed parasites were separated on a 1.5% agarose gel containing 5 mM guanidine thiocyanate. Subsequently, the RNA was blotted overnight onto a positively charged nylon membrane (Roche Applied Science) before hybridizing the blot with radiolabeled pdx1, pdx2, pdxK probes and, as a loading control, with 18 S rRNA probe using standard procedures (24). After washing the blots twice in 1x SSC, 0.1% SDS for 15 min, they were exposed to x-ray films (Retina) overnight to obtain a detectable signal.

Molecular Size of P. falciparum PdxK—The molecular size and the oligomeric state of P. falciparum PdxK were assessed by subjecting the affinity-purified protein to fast protein liquid chromatography on a calibrated Superdex S-200 (1 cm x 30 cm) equilibrated with 70 mM potassium phosphate buffer, pH 6.5 containing 200 mM KCl and 10 mM MgCl2.

Accession Numbers of the Enzymes Used in This Work—Accession numbers are: PfPdx1 NP_703871 [GenBank] and P. falciparum pyridoxine kinase (PfPdxK) NP_703820 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Analyses of the Plasmodial Pdx1, Pdx2, and PdxK Sequences—To identify plasmodial genes involved in a hypothetical vitamin B6 biosynthesis pathway TBLASTN searches were performed in the Plasmodium genome data base (www.plasmodb.org) using the deduced amino acid sequence of the S. cerevisiae open reading frames SNZ1and SNO1 and the open reading frame of the human pdxK. The genes encoding for Pdx1, Pdx2, and PdxK were found in the P. falciparum genome. The pdx1 gene resides on chromosome 6 in P. falciparum. The size of the pdx1 open reading frame is 906 bp, and the gene does not contain introns. The deduced polypeptide consists of 301 amino acids with a predicted molecular mass of 33 kDa. The protein sequence is well conserved and shows identities of 53% to corresponding proteins of S. cerevisiae (AC: Q03148 [GenBank] ), Methanosacina acetivorans (AC: Q8TQH6), and B. subtilis (AC: P37527 [GenBank] ). In S. cerevisiae Snz1-p (corresponding to Pdx1 in P. falciparum) is encoded by a gene family consisting of 3 members that share sequence identities of at least 80% to each other. However, they are encoded on different chromosomes (10). BLAST searches within the Plasmodium genome data base revealed no gene duplication in P. falciparum, and pdx1 appeared to be a single copy gene.

In E. coli, two proteins (PdxA and PdxJ) act together for the ring closure of pyridoxine (6). In S. cerevisiae Snz1-p and Sno1-p are responsible for the de novo synthesis of vitamin B6. A homologue counterpart to Sno1-p was found in the P. falciparum genome on chromosome 11. The open reading frame of pdx2 consists of 660 bp, and the deduced amino acid sequence encodes for a polypeptide of 219 amino acids with a calculated molecular mass of 24 kDa. The pdx2 gene is predicted to be a single copy gene, which is disrupted by six introns with sizes of 76, 146, 150, 96, 167, and 148 bp, respectively. The protein seems to belong to the SNO glutamine amidotransferase family. This family possesses the same conserved amino acid residue as in all other known Pdx2-p/Sno1-p proteins like the motifs GGEST and FHPE (11). Additionally, the plasmodial Pdx2 protein displays the motif WGTCA that is highly conserved among fungal Pdx2-p/Sno1-p proteins. Furthermore the motifs GVLALQG and FIRAP, also well preserved in fungi and are substituted by GVLSLQG and CIRAP, respectively. The amino acid sequence of PfPdx2 shares identities of 31, 26 and 29% to the Sno1-p/Pdx2 proteins of S. cerevisiae (AC: NP 013813), M. acetivorans (AC: NP 616499), and A. thaliana (AC: NP 568922), respectively.

To obtain a functional cofactor, pyridoxal needs to be phosphorylated in a reaction that is catalyzed by PdxK. Pyridoxal/pyridoxine kinases are metal-dependent enzymes, that generally accept all three vitamin B6 derivates (pyridoxine, pyridoxamine, and pyridoxal) as substrates. The open reading frame of the plasmodial PdxK contains 1494 bp and encodes for a polypeptide of 497 amino acids with a predicted molecular mass of 57.2 kDa. The genomic sequence of pdxK is interrupted by two introns of 142 and 139 bp (33). The plasmodial PdxK shows 14, 12, and 14% identities to pyridoxal kinases from H. sapiens (AC: O00764 [GenBank] ), E. coli (AC: NP 416913), and A. thaliana (AC: Q8W1X2). The amino acid residues known to be involved in pyridoxal binding are conserved in the PfPdxK. Ser-12, Thr-47, Tyr-84, and Asp-235 in the mammalian pyridoxal kinase are at the respective positions Ser-11, Thr-46, Tyr-83, and Asp-430 in the plasmodial counterpart. The amino acid residues Tyr-84 and Val-19 are involved in stabilizing the pyridoxal ring within the active site (34). However, Val-19 in the mammalian pyridoxal kinase is substituted by Cys-18 in PfPdxK. The amino acid residues participating in ATP binding, either directly or via cations, are Thr-186, Ser-187, Asn-150, Glu-153, Asp-118, Tyr-127, and Thr-148 (34). They appear to be conserved at their respective positions in the plasmodial protein. It has been reported that Asp-118, Tyr-127 (Asp-323 and Tyr-328 in the PfPdxK), and the connecting 10 amino acid residues are responsible for the prevention of spontaneous ATP hydrolysis (35). However, the link between these two conserved amino acid residues is restricted to 6 amino acid residues in the plasmodial enzyme. Interestingly, the PfPdxK sequence is broken up by a spacer consisting of 207 amino acid residues. Long insertions have also been identified in several plasmodial proteins such as {gamma}-glutamylcysteine synthetase and the bifunctional ODC/AdoMetDC (36–38). All of these insertions are characterized by a high abundance of charged amino acids like Asp, Asn, Glu, Gln, Ser, and Lys. The insertion of the PfPdxK is dominated by the amino acids Asn, Asp, and His, which represent 20, 6, and 16%, respectively. Interestingly, these amino acid residues are conserved in the motifs Met-Asn-Gly-His or Thr-Asn-Asp-His within the spacer. The function of these insertions in plasmodial proteins is still unknown.

Further analysis of the deduced amino acid sequence of all three enzymes was performed with the bioinformatic tools provided on the ExPaSY web site (//c.expasy.org/tools/). This revealed that the Plasmodium proteins possess neither a mitochondrial nor an apicoplastidial targeting sequence, proposing a cytosolic localization of Pdx1, Pdx2, and PdxK.

Biochemical Characterization of Pdx1 and Pdx2—The plasmodial Pdx1 and Pdx2 Strep-Tag fusion proteins were recombinantly expressed as soluble polypeptides with an appreciable yield of 1.5 mg liter–1 and 1 mg liter–1 of bacterial culture, respectively. The proteins were purified by affinity chromatography, and the purity was confirmed by SDS-PAGE. The molecular mass of PfPdx1 and PfPdx2 including the Strep-Tag was approximately 35 and 24 kDa, respectively, which correlates well with the predicted protein sizes (Fig. 1). It has been discussed that Sno1-p or YaaE (Pdx2 in P. falciparum) in B. subtilis, and Haemophilus influencae possesses glutaminase activity (39). The plasmodial Pdx2 enzyme did not reveal detectable glutaminase activity. Additionally, the plasmodial Pdx1 protein also did not exhibit glutaminase activity. However, performing the glutaminase enzyme assay in the presence of an equimolar mixture of PfPdx1 and PfPdx2, glutaminase activity was detected with a specific activity of 209 ± 13 nmol min–1 mg–1 (n = 3). The glutamine hydrolysis mediated by the complex of PfPdx1 and PfPdx2 follows Michaelis-Menten kinetics with a Km value for glutamine of 1.3 ± 0.1 mM (n = 3), which is in a comparable range to the Sno1-p/Snz1-p enzyme complex in S. cerevisiae (12). For the verification of the glutaminase activity of the PfPdx1 and PfPdx2 complex, the inhibitor DON was employed. DON is an irreversible inhibitor of glutamine amidotransferases (40) and is indeed capable of deactivating the plasmodial enzyme complex. After 10 min of preincubation at an inhibitor concentration of 10 mM, approximately half of the glutaminase activity was abolished (data not shown).



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  		FIG. 1.
SDS-PAGE analysis of the plasmodial Pdx1, Pdx2, and PdxK. P. falciparum Pdx1, Pdx2, and PdxK were recombinantly expressed in E. coli BLR (DE3) and subsequently purified as described under "Experimental Procedures." A, PfPdx1; B, PfPdx2; C, PfPdxK. Sizes of the molecular mass standards are shown.

 
Complementation Assay of {Delta}sno1/{Delta}snz1-deficient S. cerevisiae Cells—Previously Dong et al. (12) reported for S. cerevisiae, that neither deletion of SNO1 nor deletion of SNZ1 resulted in auxotrophy for pyridoxine. Only the generation of a yeast {Delta}sno1/{Delta}snz1 double mutant led to pyridoxine auxotrophy. To prove the participation of PfPdx1 and PfPdx2 in the vitamin B6 biosynthesis, the open reading frames were cloned into the yeast expression vector pYES2-NT as described under "Experimental Procedures." Subsequently both constructs and the empty expression vector were transformed into the {Delta}sno1/{Delta}snz1-deficient yeast strain. The empty vector did not support growth on minimal medium without addition of vitamin B6 (Fig. 2, A and B). In contrast, the transformation of the PfPdx1 and PfPdx2 expression constructs resulted in a successful complementation of the {Delta}sno1/{Delta}snz1 double mutant on minimal medium agar plates (Fig. 2A), which suggests an involvement of PfPdx1 and PfPdx2 in the vitamin B6 de novo synthesis. The recombinant expression of both plasmodial proteins in the yeast double mutant was confirmed by Western blot analysis (Fig. 2C).



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  		FIG. 2.
Complementation of {Delta}sno1/{Delta}snz1-deficient S. cerevisiae by Pfpdx1 and Pfpdx2. The {Delta}sno1/{Delta}snz1-deficient yeast cells were transformed with the PfPdx1-pYES2/NT (1) and PfPdx2-pYES2/NT (2) yeast expression constructs carrying the plasmodial open reading frames pdx1 and pdx2, respectively. As a control, the empty expression vector pYES2/NT (3), which was also transformed into the yeast mutant, and the nontransformed {Delta}sno1/{Delta}snz1 double mutant (4) were grown on minimal media with (A) and without (B) pyridoxine at 30 °C for 4 days. The nontransformed {Delta}sno1/{Delta}snz1 double mutant was deficient in uracil biosynthesis, because of the lack of the vector pYES2/NT and therefore unable to grow on this medium. C, Western blot analysis of the recombinant expression of PfPdx1 (1), PfPdx2 (2) and the empty expression vector pYES2/NT (3) in the yeast double mutant were performed with the monoclonal anti-His-HRP antibody at a dilution of 1:5000. Sizes of the molecular mass standards are shown.

 
Expression of PfPdx1 and PfPdx2 under Oxidative Stress Conditions—Northern blot analysis of P. falciparum total RNA shows that the transcription levels of pdx1 and pdx2 are not significantly elevated when the parasites were exposed to oxidative stress conferred by incubation at 1.2 nM methylene blue, a singlet oxygen producer (Fig. 3). In contrast, incubation of the parasites at 6 nM methylene blue led to an up-regulated transcription level (Fig. 3). This result indicates that the synthesis of vitamin B6 in the human malaria parasite P. falciparum may participate in the protection against singlet oxygen.



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  		FIG. 3.
Expression levels of methylene blue-stressed P. falciparum. Northern blot analyses of total P. falciparum RNA was performed with 1.2 nM (lane 2) and 6 nM (lane 3) methylene blue-stressed parasites and compared with unstressed parasites (lane 1). The blot was probed with a pdx1(A) and pdx2(B)-specific radiolabeled probe and subsequently re-probed with the loading control (18 S rRNA) (C). Transcription sizes are shown on the right.

 
Biochemical Characterization of PdxK—The pdxK gene was cloned into the expression vectors pASK-IBA7 and pASK-IBA3, which allows purification of the recombinant protein because of an N- or C-terminal Strep-Tag, respectively. Expression of PdxK as a N-terminal fusion protein was undetectable, whereas expression of the PfPdxK cloned into pASK-IBA3 resulted in a soluble C-terminal fusion protein with a yield of 1.7 mg liter–1 bacterial culture. Affinity chromatography on Strep-Tactin-Sepharose resulted in a homogenous polypeptide that was used for further kinetic analyses (Fig. 1). The PfPdxK monomer has a molecular mass of about 58 kDa, including the Strep-Tag, which is in good agreement with the theoretical mass of 57 kDa. To evaluate the oligomeric state of PfPdxK, the recombinant protein was applied onto gel filtration using a calibrated Superdex S-200 column. The protein eluted at a molecular mass of ~110 kDa, suggesting a dimeric conformation of the plasmodial enzyme, as previously reported for other pyridoxal kinases (17). The plasmodial enzyme accepts all vitamers as substrates. The specific activities of the plasmodial pyridoxine kinase for pyridoxine, pyridoxal, and pyridoxamine were found to be 112, 50, and 30 nmol min–1 mg–1 with kcat values of 0.11, 0.05, and 0.03 s–1, respectively (Table I). In the presence of 5 mM EDTA, activity of PfPdxK could not be observed. The enzyme revealed the highest specific activity in the presence of 10 mM Mg2+ ions. However, the enzyme also accepted Zn2+ ions. The highest activity was found at a zinc concentration of 500 µM, but the activity was decreased to 30% of the activity in presence of 10 mM Mg2+ ions. Incubation of the enzyme in the reaction mixture containing Cu2+, Ca2+, Ni2+, and Mn2+ ions did not result in detectable activity. The Km value for pyridoxine was determined to be 70 µM, which is about 3x higher than that reported for the human enzyme (27) (Table I). Interestingly, the Km value for pyridoxal was calculated to be 212 µM, which is about 3x higher than the Km value for pyridoxine and ~3x lower than that testified for the PdxK of A. thaliana (19) (Table I). The Km value of ATP was found to be 82 µM, which is in the same range as that of the enzyme from A. thaliana (19) (Table I).


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  		TABLE I
Kinetic parameters of the plasmodial PdxK

The kinetic parameters of PfPdxK were determined as described under "Experimental Procedures." The results represent the mean values of at least three independent experiments.

 
Expression Pattern of pdx1, pdx2, and pdxK at Different Developmental Erythrocytic Stages of P. falciparum—The expression of pdx1, pdx2, and pdxK throughout the erythrocytic life cycle of P. falciparum was assessed by Northern blotting (Fig. 4). The stage-specific blot was hybridized with pdx1-, pdx2-, and pdxK-specific probes. Highly intense hybridization signals were obtained in the trophozoite stage (26 ± 4 h) and at lower intensity in the schizont stage (40 ± 4 h) for pdx1 and pdxK with transcription sizes of 2.4 kb and 2.7 kb, respectively (Fig. 4). The pdx2 gene is mainly transcribed within the ring stage (12 ± 4 h) with a size of about 2.3 kb. However, pdx2 transcription also occurs in the trophozoite stage (Fig. 4). These results clearly indicate a concurrent expression pattern of pdx1 and pdx2 in the trophozoite stage for the required functional interaction of these proteins, which guarantees the provision of the substrate for PfPdxK.



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  		FIG. 4.
Expression pattern of pdx1, pdx2, and pdxK within the developmental stages of P. falciparum. Stage-specific Northern blot analyses were performed by hybridization with radiolabeled pdx1, pdx2, and pdxK probes (A–C). The blot was stripped and reprobed with the loading control (18 S rRNA) (D). R, ring stage; T, trophozoite stage; S, schizont stage. Transcription sizes are shown on the right.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Currently, two different vitamin B6 biosynthetic pathways are known (8). Whereas the E. coli biosynthetic pathway consisting of the pdx protein family (Pdx A, B, C, F, H, J, and GapA) has been well characterized (2–5), the de novo synthesis pathway of the fungi C. nicotianae and S. cerevisiae as well as some Archaebacteria, Eubacteria and A. thaliana has only recently been identified (8, 19). Interestingly, the enzymes involved in the fungi pathway do not share any sequence similarities to the E. coli Pdx proteins. However, a functional complementation of an E. coli PdxJ deletion mutant expressing the pdx1 gene of C. nicotianae has been reported, suggesting a similar enzyme reaction (41). Nevertheless, the molecules, needed for the ring closure in the E. coli vitamin B6 synthesis pathway, differ from those in the yeast pathway. In yeast, Tanaka et al. (42) have shown that the nitrogen of the pyridoxine is derived from glutamine, whereas glutamate represents the source of the nitrogen atom in the E. coli pyridoxine biosynthesis pathway. Consistent with these findings, Galperin and Koonin (39) hypothesized that SnzB-p (Sno1-p in yeast) possesses glutamine amidotransferase activity. Interestingly, in S. cerevisiae glutaminase activity was only barely detectable for the Sno1-p enzyme alone, whereas the Sno1-p/Snz1-p enzyme complex showed a much enhanced activity (12). Very recently, evidence for an active vitamin B6 biosynthesis pathway in P. falciparum was shown by metabolic labeling experiments (22). Sequence analyses of parasite genes potentially involved in vitamin B6 synthesis revealed indeed the presence of two open reading frames (pdx1 and pdx2), whose deduced amino acid sequences possess a high degree of similarity to respective yeast Snz1-p and Sno1-p proteins. Snz1-p and Sno1-p (Pdx1 and Pdx2 in P. falciparum) belong to the Snz-p and Sno-p protein families, respectively. Both families consist of three highly identical constituents, Snz1/2/3-p and Sno1/2/3-p. Interestingly, BLAST searches within the Plasmodium genome data base with PfPdx1 and PfPdx2 homologues of other organisms discovered no further homologous genes in P. falciparum, which emphasizes the existence of PfPdx1 and PfPdx2 as single copy genes.

To verify the involvement of the plasmodial Pdx1 and Pdx2 proteins in vitamin B6 biosynthesis, successful complementation of a S. cerevisiae sno1/snz1 double mutant deficient in pyridoxine biosynthesis with the plasmodial pdx1 and pdx2 open reading frames has been performed. These results substantiate indeed a participation of PfPdx1 and PfPdx2 in the vitamin B6 de novo synthesis. Interestingly, functional complementation was only achieved with the {Delta}sno1/{Delta}snz1 double mutant. Consistent with our findings, Dong et al. (12) reported that only a sno1/snz1 double knock-out was auxotroph for vitamin B6, whereas single knock-outs of the yeast genes SNO1 and SNZ1 were capable of growing on minimal medium without vitamin B6. The authors proposed that this result is caused by a possible complementation by the other members, Sno2/3-p and Snz2/3-p, of the Sno-p or Snz-p protein families. However, further investigations are required to appraise the precise functions of both Snz-p and Sno-p proteins.

In accordance with the glutaminase activity reported for the yeast enzymes, the two plasmodial proteins were tested for their ability to act as glutaminases (12). P. falciparum Pdx1 and Pdx2 also revealed glutaminase activity with a specific activity of 209 nmol min–1 mg–1 and a Km value of 1.3 mM, which is in the same range as the yeast enzyme complex with 480 nmol min–1 mg–1 and 3.4 mM, respectively (12). Interestingly, PfPdx1 and PfPdx2 are enzymatically active as a complex as it has been proposed for the corresponding B. subtilis enzymes YaaD and YaaE, respectively (43). PfPdx2 alone did not show any detectable glutaminase activity. Consistent with these findings yeast two-hybrid analyses demonstrated an interaction of Snz1-p and Sno1-p (10). Furthermore, glutaminase activity of the PfPdx1/PfPdx2 complex is susceptible for the irreversible inhibitor DON as was shown for the yeast enzyme complex (12). Other components that are necessary for the synthesis of the pyridoxine ring are currently not known. It has been suggested that in addition to glutamine a pentulose and a triose are required for ring closure. 2'-Hydroxypyridoxol, identified to be an intermediate of vitamin B6 biosynthesis, was proposed to be convertible to pyridoxal (44, 45). However, the de novo synthesis of pyridoxal needs further clarification.

Originally it was believed that the genes encoding for Pdx1-p (Sor1-p, singlet oxygen resistance) were solely involved in the detoxification of singlet oxygen (1O2) generated during the synthesis of cercosporin, a potent photosensitizer enabling C. nicotianae to parasitize plants (7). Afterward, additional investigations demonstrated that Sor1-p plays an imperative role in the de novo syntheses of vitamin B6 (8). This result was completely unanticipated and was confirmed by the ability of vitamin B6 in quenching of 1O2 (8). Additionally it has been reported that the YaaD in B. subtilis, the counterpart to PfPdx1, is an oxidative stress-inducible protein, implicated in cellular stress response (46). Yeast mutants lacking either the gene SNZ1 or SNO1 reveal sensitivity to methylene blue, a singlet oxygen producer (9), whereas S. cerevisiae cells carrying deletions of the other members of the SNZ or SNO gene families were not susceptible for methylene blue (10). In order to prove a potential involvement of vitamin B6 synthesis in Plasmodium, the parasites were stressed with methylene blue. Northern blot analysis revealed increased transcriptional levels of PfPdx1 and PfPdx2, suggesting a participation of the plasmodial proteins in the cellular stress response for singlet oxygen.

To be an effective cofactor, vitamin B6 has to be phosphorylated to pyridoxal 5'-phosphate. This reaction is catalyzed by the enzyme PdxK (6). BLAST searches for bacteria, plant, or mammalian PdxK homologues in the P. falciparum genome data base resulted in an identification of a PdxK counterpart. The gene encoding PfPdxK was cloned, recombinantly expressed, and examined for its kinetic parameters. Interestingly, only expression of a C-terminal-tagged fusion protein resulted in appreciable amounts of PfPdxK, whereas recombinant expression of an N-terminal fusion protein was not detectable. The active plasmodial pyridoxine kinase displays a Km value for ATP of 82 µM, which is about 4x higher than the Km value reported for the human counterpart, but within the same range as the PdxK of A. thaliana (17, 19). The PfPdxK accepts all B6 vitamers as substrates although with different specificity. Pyridoxine supports the highest specific activity of PfPdxK, followed by pyridoxal. The specific activity for pyridoxal is two times lower than that for pyridoxine and about 12 or 20x lower than the activities reported for PdxK of A. thaliana or of T. brucei and of the mammalian PdxK, respectively (17–19), but is in the same range to the PdxK of B. subtilis (20). The lowest specific activity, which is a nearly half of that for pyridoxal, was detected when pyridoxamine was used as the substrate. The Km values for pyridoxine and pyridoxal are 3 and 5x higher than those determined for the T. brucei or mammalian pyridoxine kinase, respectively (17, 18). However, the Km value for pyridoxal of the plasmodial enzyme is about 3x lower than that of the enzyme of A. thaliana (19). In summary the PfPdxK prefers pyridoxine as the substrate and also accepts pyridoxal and pyridoxamine to a lesser extent. Conversely the human pyridoxal kinase favors pyridoxal as the substrate, while the usage of pyridoxine and pyridoxamine as substrates decreases its activity by up to 70% (17). The divergence in the substrate preference and especially the kinetic parameters between the mammalian PdxK and the PfPdxK possibly refer to the protein structure. Comparison of both protein sequences reveal, with the exception of the plasmodial-specific insertion of 207 amino acid residues, two major differences: 1) The substitution of Val-19 in the human PdxK to Cys-18 in the PfPdxK, and 2) the contraction of the loop between the respective amino acid residues Asp-118 and Tyr-127 in the human enzyme. Analyses of the crystal structure of mammalian pyridoxal kinase have shown that the Val-19 residue is responsible for adjusting the pyridoxal ring structure within the active site (34). Because of the exchange of valine to cysteine in the plasmodial enzyme the accessibility of pyridoxine into the active site may be higher than for pyridoxal or pyridoxamine. Further experiments are currently underway to evaluate this amino acid exchange in the plasmodial PdxK.

For prevention of premature ATP hydrolysis, ribokinases and adenine kinases possess a lid structure covering the active site. After binding of the first substrate, the lid starts to rotate and the active site becomes accessible for ATP binding (13, 14). Crystal structure analyses of the human pyridoxal kinase revealed a loop of 10 amino acids between the residues Asp-118 and Tyr-127, which has been reported to act as a lid for the active site (35). Interestingly, this loop is only conserved in the mammalian PdxK, whereas the yeast and bacterial counterparts as well as the plasmodial PdxK possess a reduced loop, suggesting a different covering mechanism for the prevention of premature ATP hydrolysis.

Northern blot analysis of the three genes involved in vitamin B6 biosynthesis in P. falciparum shows a concurrent expression pattern of PfPdx1, PfPdx2, and PfPdxK in the trophozoite stage, which is in agreement with the microarray data performed by Le Roch et al. (47) and Bozdech et al. (48). Moreover, all three plasmodial proteins do not possess a potential targeting sequence to either the apicoplast or the mitochondrion, suggesting a cytosolic location of the proteins for the de novo synthesis of pyridoxal by PfPdx1 and PfPdx2 and the subsequent phosphorylation by PfPdxK to pyridoxal 5'-phosphate, the essential cofactor. Mammalian cells have to assimilate vitamin B6 from exogenous sources via specialized energy-dependent transporters, which have not yet been identified (49). Recently, the gene encoding a vitamin B6 membrane transporter in S. cerevisiae has been reported (50). BLAST search analyses within the plasmodial genome data base, however, could not detect any similar polypeptides to such a transporter, suggesting that P. falciparum relies upon the de novo synthesis of vitamin B6.

Further analysis of the enzymes responsible for the yeast-like vitamin B6 biosynthesis by employing in vivo gene replacement techniques will address the role of Pdx1, Pdx2, and PdxK for the essential cofactor provision in the human malaria parasite P. falciparum.


   FOOTNOTES
 
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Wa 395/10. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be 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 GenBankTM/EBI Data Bank with accession number(s) AJ871406 [GenBank] (PfPdx2). Back

§ Conducted in partial fulfillment of the requirement for a Ph.D. from the University of Hamburg. Back

|| To whom correspondence should be addressed. Tel.: 49-40-42818-420; Fax: 49-40-42818-418; E-mail: walter{at}bni-hamburg.de.

1 The abbreviations used are: PLP, pyridoxal 5'-phosphate; PdxK, pyridoxal kinase; PfPdxK, plasmodial pyridoxal kinase; PN, pyridoxine; DON, 6-diazo-5-oxo-L-norleucine; APAD, acetylpyridine adenine dinucleotide. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Eva Liebau for critical reading of the manuscript and helpful discussions.



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 DISCUSSION
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Vitamin B6 biosynthesis in higher plants
PNAS, September 20, 2005; 102(38): 13687 - 13692.
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 J. Biol. Chem.Home page
T. Raschle, N. Amrhein, and T. B. Fitzpatrick
On the Two Components of Pyridoxal 5'-Phosphate Synthase from Bacillus subtilis
J. Biol. Chem., September 16, 2005; 280(37): 32291 - 32300.
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 J. Biol. Chem.Home page
J. Zhu, J. W. Burgner, E. Harms, B. R. Belitsky, and J. L. Smith
A New Arrangement of ({beta}/{alpha})8 Barrels in the Synthase Subunit of PLP Synthase
J. Biol. Chem., July 29, 2005; 280(30): 27914 - 27923.
[Abstract] [Full Text] [PDF]

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