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• W2053828364 abstract "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. 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. Pyridoxal 5′-phosphate (PLP) 1The 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.1The 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. 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 (1Percudani R. Peracchi A. EMBO Rep. 2003; 4: 850-854Crossref PubMed Scopus (356) Google Scholar). 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 (2Roa B.B. Connolly D.M. Winkler M.E. J. Bacteriol. 1989; 171: 4767-4777Crossref PubMed Google Scholar, 3Lam H.M. Tancula E. Dempsey W.B. Winkler M.E. J. Bacteriol. 1992; 174: 1554-1567Crossref PubMed Google Scholar, 4Lam H.M. Winkler M.E. J. Bacteriol. 1992; 174: 6033-6045Crossref PubMed Google Scholar, 5Hill R.E. Himmeldirk K. Kennedy I.A. Pauloski R.M. Sayer B.G. Wolf E. Spenser ID. J. Biol. Chem. 1996; 271: 30426-30435Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). 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) (6Yang Y. Zhao G. Man T.K. Winkler M.E. J. Bacteriol. 1998; 180: 4294-4299Crossref PubMed Google Scholar). 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 (3Lam H.M. Tancula E. Dempsey W.B. Winkler M.E. J. Bacteriol. 1992; 174: 1554-1567Crossref PubMed Google Scholar, 6Yang Y. Zhao G. Man T.K. Winkler M.E. J. Bacteriol. 1998; 180: 4294-4299Crossref PubMed Google Scholar). 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 (7Ehrenshaft M. Jenns A.E. Chung K.R. Daub M.E. Mol. Cell. 1998; 1: 603-609Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Originally this pathway was assigned to be involved in detoxification of singlet oxygen (1O2) (7Ehrenshaft M. Jenns A.E. Chung K.R. Daub M.E. Mol. Cell. 1998; 1: 603-609Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). 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 (8Ehrenshaft M. Bilski P. Li M.Y. Chignell C.F. Daub M.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9374-9378Crossref PubMed Scopus (250) Google Scholar, 9Osmani A.H. May G.S. Osmani S.A. J. Biol. Chem. 1999; 274: 23565-23569Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). 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 (10Padilla P.A. Fuge E.K. Crawford M.E. Errett A. Werner-Washburne M. J. Bacteriol. 1998; 180: 5718-5726Crossref PubMed Google Scholar). An involvement of Pdx2-p in vitamin B6de novo synthesis was confirmed by complementation of mutants deficient in pyridoxine biosynthesis (11Ehrenshaft M. Daub M.E. J. Bacteriol. 2001; 183: 3383-3390Crossref PubMed Scopus (70) Google Scholar). 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 (11Ehrenshaft M. Daub M.E. J. Bacteriol. 2001; 183: 3383-3390Crossref PubMed Scopus (70) Google Scholar, 12Dong Y.X. Sueda S. Nikawa J. Kondo H. Eur. J. Biochem. 2004; 271: 745-752Crossref PubMed Scopus (60) Google Scholar). 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 (13Mathews I.I. Erion M.D. Ealick S.E. Biochemistry. 1998; 37: 15607-15620Crossref PubMed Scopus (180) Google Scholar, 14Sigrell J.A. Cameron A.D. Mowbray S.L. J. Mol. Biol. 1999; 290: 1009-1018Crossref PubMed Scopus (66) Google Scholar, 15Campobasso N. Mathews I.I. Begley T.P. Ealick S.E. Biochemistry. 2000; 39: 7868-7877Crossref PubMed Scopus (60) Google Scholar, 16Schumacher M.A. Scott D.M. Mathews I.I. Ealick S.E. Roos D.S. Ullman B. Brennan R.G. J. Mol. Biol. 2000; 298: 875-893Crossref PubMed Scopus (87) Google Scholar). They have been characterized in several organisms such as Homo sapiens, A. thaliana, B. subtilis, and the parasite Trypanosoma brucei (17Kerry J.A. Rohde M. Kwok F. Eur. J. Biochem. 1986; 158: 581-585Crossref PubMed Scopus (52) Google Scholar, 18Scott T.C. Phillips M.A. Mol. Biochem. Parasitol. 1997; 88: 1-11Crossref PubMed Scopus (25) Google Scholar, 19Lum H.K. Kwok F. Lo S.C. Planta. 2002; 215: 870-879Crossref PubMed Scopus (33) Google Scholar, 20Park J.H. Burns K. Kinsland C. Begley T.P. J. Bacteriol. 2004; 186: 1571-1573Crossref PubMed Scopus (41) Google Scholar). 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 (21Newton P. White N. Annu. Rev. Med. 1999; 50: 179-192Crossref PubMed Scopus (115) Google Scholar). Very recently, Cassera et al. (22Cassera M.B. Gozzo F.C. D'Alexandri F.L. Merino E.F. Del Portillo H.A. Peres V.J. Almeida I.C. Eberlin M.N. Wunderlich G. Wiesner J. Jomaa H. Kimura E.A. Katzin A.M. J. Biol. Chem. 2004; 279: 51749-51759Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) 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. 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). [α-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 (23Wrenger C. Lu ̈ersen K. Krause T. Mu ̈ller S. Walter R.D. J. Biol. Chem. 2001; 276: 29651-29656Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). 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 × 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 (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual,2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 18.47-18.59Google Scholar). The concentration of the purified recombinant protein was determined according to Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211734) Google Scholar). Glutaminase Assay—The glutaminase activity was assayed in two steps, according to Dong et al. (12Dong Y.X. Sueda S. Nikawa J. Kondo H. Eur. J. Biochem. 2004; 271: 745-752Crossref PubMed Scopus (60) Google Scholar), 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 × 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 (26Van Kuilenburg A.B. Elzinga L. Van den Berg A.A. Slingerland R.J. Van Gennip A.H. Anticancer Res. 1994; 14: 411-415PubMed Google Scholar). 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; his3Δ 1; met15Δ 0; ura3Δ 0; leu2Δ 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 (12Dong Y.X. Sueda S. Nikawa J. Kondo H. Eur. J. Biochem. 2004; 271: 745-752Crossref PubMed Scopus (60) Google Scholar). 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. (12Dong Y.X. Sueda S. Nikawa J. Kondo H. Eur. J. Biochem. 2004; 271: 745-752Crossref PubMed Scopus (60) Google Scholar). 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 (27Kwok F. Churchich J.E. J. Biol. Chem. 1979; 254: 6489-6495Abstract Full Text PDF PubMed Google Scholar). 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 [α-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 (28Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6029) Google Scholar) 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 (29Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2759) Google Scholar). 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 (30Vennerstrom J.L. Makler M.T. Angerhofer C.K. Williams J.A. Antimicrob. Agents Chemother. 1995; 39: 2671-2677Crossref PubMed Scopus (190) Google Scholar). 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 (31Umlas J. Fallon J.N. Am. J. Trop. Med. Hyg. 1971; 20: 527-529Crossref PubMed Scopus (37) Google Scholar), that were immediately transferred into TRIzol (Invitrogen) according to the manufacturer's instructions, and the obtained RNA was analyzed by Northern blotting (32Kyes S. Pinches R. Newbold C. Mol. Biochem. Parasitol. 2000; 105: 311-315Crossref PubMed Scopus (226) Google Scholar). 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 (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual,2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 18.47-18.59Google Scholar). After washing the blots twice in 1× 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 × 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 and P. falciparum pyridoxine kinase (PfPdxK) NP_703820. 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), Methanosacina acetivorans (AC: Q8TQH6), and B. subtilis (AC: P37527). 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 (10Padilla P.A. Fuge E.K. Crawford M.E. Errett A. Werner-Washburne M. J. Bacteriol. 1998; 180: 5718-5726Crossref PubMed Google Scholar). 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 (6Yang Y. Zhao G. Man T.K. Winkler M.E. J. Bacteriol. 1998; 180: 4294-4299Crossref PubMed Google Scholar). 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 (11Ehrenshaft M. Daub M.E. J. Bacteriol. 2001; 183: 3383-3390Crossref PubMed Scopus (70) Google Scholar). 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 (33Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. Paulsen I.T. James K. Eisen J.A. Rutherford K. Salzberg S.L. Craig A. Kyes S. Chan M.S. Nene V. Shallom S.J. Suh B. Peterson J. Angiuoli S. Pertea M. Allen J. Selengut J. Haft D. Mather M.W. Vaidya A.B. Martin D.M. Fairlamb A.H. Fraunholz M.J. Roos D.S. Ralph S.A. McFadden G.I. Cummings L.M. Subramanian G.M. Mungall C. Venter J.C. Carucci D.J. Hoffman S.L. Newbold C. Davis R.W. Fraser C.M. Barrell B. Nature. 2002; 419: 498-511Crossref PubMed Scopus (3370) Google Scholar). The plasmodial PdxK shows 14, 12, and 14% identities to pyridoxal kinases from H. sapiens (AC: O00764), 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 (34Li M.H. Kwok F. Chang W.R. Liu S.Q. Lo S.C. Zhang J.P. Jiang T. Liang D.C. J. Biol. Chem. 2004; 279: 17459-17465Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (34Li M.H. Kwok F. Chang W.R. Liu S.Q. Lo S.C. Zhang J.P. Jiang T. Liang D.C. J. Biol. Chem. 2004; 279: 17459-17465Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 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 (35Li M.H. Kwok F. Chang W.R. Lau C.K. Zhang J.P. Lo S.C. Jiang T. Liang D.C. J. Biol. Chem. 2002; 277: 46385-46390Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). 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 γ-glutamylcysteine synthetase and the bifunctional ODC/AdoMetDC (36Lu ̈ersen K. Walter R.D. Mu ̈ller S. Mol. Biochem. Parasitol. 1999; 98: 131-142Crossref PubMed Scopus (41) Google Scholar, 37Mu ̈ller S. Da'dara A. Lu ̈ersen K. Wrenger C. Das Gupta R. Madhubala R. Walter R.D. J. Biol. Chem. 2000; 275: 8097-8102Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 38Birkholtz L.M. Wrenger C. Joubert F. Wells G.A. Walter R.D. Louw A.I. Biochem. J. 2004; 377: 439-448Crossref PubMed Scopus (36) Google Scholar). All of these insertions are char" @default.
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• W2053828364 title "Analysis of the Vitamin B6 Biosynthesis Pathway in the Human Malaria Parasite Plasmodium falciparum" @default.
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