Apicomplexan Parasites Possess Distinct Nuclear-encoded, but Apicoplast-localized, Plant-type Ferredoxin-NADP+ Reductase and Ferredoxin*

In searching for nuclear-encoded, apicoplast-localized proteins we have cloned ferredoxin-NADP+ reductase from Toxoplasma gondii and a [2Fe-2S] ferredoxin from Plasmodium falciparum. This chloroplast-localized redox system has been extensively studied in photosynthetic organisms and is responsible for the electron transfer from photosystem I to NADP+. Besides this light-dependent reaction in nonphotosynthetic plastids (e.g. from roots), electrons can also flow in the reverse direction, from NADPH to ferredoxin, which then serves as an important reductant for various plastid-localized enzymes. These plastids possess related, but distinct, ferredoxin-NADP+ reductase and ferredoxin isoforms for this purpose. We provide phylogenetic evidence that the T. gondii reductase is similar to such nonphotosynthetic isoforms. Both the P. falciparum[2Fe-2S] ferredoxin and the T. gondiiferredoxin-NADP+ reductase possess an N-terminal bipartite transit peptide domain typical for apicoplast-localized proteins. The recombinant proteins were obtained in active form, and antibodies raised against the reductase recognized two bands on Western blots ofT. gondii tachyzoite lysates, indicative of the unprocessed and native form, respectively. We propose that the role of this redox system is to provide reduced ferredoxin, which might then be used for fatty acid desaturation or other biosynthetic processes yet to be defined. Thus, the interaction of these two proteins offers an attractive target for drug intervention.

[1]  M. Vanoni,et al.  Glutamate synthase: a complex iron-sulfur flavoprotein , 1999, Cellular and Molecular Life Sciences (CMLS).

[2]  R. Baker,et al.  Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase. , 1994, The Journal of biological chemistry.

[3]  S. Brunak,et al.  SHORT COMMUNICATION Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites , 1997 .

[4]  J. Wyche,et al.  Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA. , 1991, BioTechniques.

[5]  J. Blanchard,et al.  The Non‐Photosynthetic Plastid in Malarial Parasites and Other Apicomplexans is Derived from Outside the Green Plastid Lineage 1 , 1999, The Journal of eukaryotic microbiology.

[6]  Irwin W. Sherman,et al.  Malaria : parasite biology, pathogenesis, and protection , 1998 .

[7]  D. Roos,et al.  The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes , 2000, The Journal of cell biology.

[8]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[9]  U. Heinemann,et al.  Adrenodoxin: Structure, stability, and electron transfer properties , 2000, Proteins.

[10]  Geoffrey I. McFadden,et al.  Plastid in human parasites , 1996, Nature.

[11]  M. Hirasawa,et al.  Ferredoxin-dependent chloroplast enzymes. , 1991, Biochimica et biophysica acta.

[12]  M. Havaux,et al.  Reduction of the plastoquinone pool by exogenous NADH and NADPH in higher plant chloroplasts. Characterization of a NAD(P)H-plastoquinone oxidoreductase activity , 1998, Biochimica et biophysica acta.

[13]  A. Arakaki,et al.  Plant‐type ferredoxin‐NADP+ reductases: a basal structural framework and a multiplicity of functions , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  R. Peyronnet,et al.  On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions. , 1988, European journal of biochemistry.

[15]  D. Tew,et al.  Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. , 1993, The Biochemical journal.

[16]  H. Sticht,et al.  The structure of iron-sulfur proteins. , 1998, Progress in biophysics and molecular biology.

[17]  G. McFadden,et al.  Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway , 2000, The EMBO journal.

[18]  D. Roos,et al.  Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  F. Seeber.  Consensus sequence of translational initiation sites from Toxoplasma gondii genes , 1997, Parasitology Research.

[20]  R. Lill,et al.  A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  B. Neel,et al.  Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. , 1993, Analytical biochemistry.

[22]  K. Cline,et al.  Import and routing of nucleus-encoded chloroplast proteins. , 1996, Annual review of cell and developmental biology.

[23]  J. Palmer,et al.  A Plastid of Probable Green Algal Origin in Apicomplexan Parasites , 1997, Science.

[24]  Roderic D. M. Page,et al.  TreeView: an application to display phylogenetic trees on personal computers , 1996, Comput. Appl. Biosci..

[25]  G. Cross,et al.  Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure. , 1993, Molecular and biochemical parasitology.

[26]  T. Hase,et al.  Comparison of the Electrostatic Binding Sites on the Surface of Ferredoxin for Two Ferredoxin-dependent Enzymes, Ferredoxin-NADP+ Reductase and Sulfite Reductase* , 1999, The Journal of Biological Chemistry.

[27]  H. Böhme,et al.  Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis. , 1998, Biochimica et biophysica acta.

[28]  D. Roos,et al.  Origin, targeting, and function of the apicomplexan plastid. , 1999, Current opinion in microbiology.

[29]  R. Baker,et al.  Protein expression using ubiquitin fusion and cleavage. , 1996, Current opinion in biotechnology.

[30]  G. Sheldrick,et al.  Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca. , 1999, Structure.

[31]  K. Strimmer,et al.  Quartet Puzzling: A Quartet Maximum-Likelihood Method for Reconstructing Tree Topologies , 1996 .

[32]  J. Felsenstein.  Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. , 1996, Methods in enzymology.

[33]  E. Heinz,et al.  Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[34]  P. Karplus,et al.  A productive NADP+ binding mode of ferredoxin–NADP + reductase revealed by protein engineering and crystallographic studies , 1999, Nature Structural Biology.

[35]  M. Waterman,et al.  Regulation of Steroidogenic and Related P450s , 1995 .

[36]  Radhey S. Gupta.  Protein Phylogenies and Signature Sequences: A Reappraisal of Evolutionary Relationships among Archaebacteria, Eubacteria, and Eukaryotes , 1998, Microbiology and Molecular Biology Reviews.

[37]  J. Remington,et al.  Biology of Toxoplasma gondii. , 1993, AIDS.

[38]  H. Lichtenthaler,et al.  Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. , 1999, Science.

[39]  R. Wilson,et al.  Extrachromosomal DNA in the Apicomplexa. , 1997, Microbiology and molecular biology reviews : MMBR.

[40]  D. Mattei,et al.  Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte. , 1994, The Biochemical journal.

[41]  David S. Roos,et al.  A plastid organelle as a drug target in apicomplexan parasites , 1997, Nature.

[42]  M. Emes,et al.  NONPHOTOSYNTHETIC METABOLISM IN PLASTIDS. , 2003, Annual review of plant physiology and plant molecular biology.

[43]  D. Roos,et al.  Apicomplexan plastids as drug targets. , 1999, Trends in microbiology.

[44]  H. Sakakibara,et al.  Differential interaction of maize root ferredoxin:NADP(+) oxidoreductase with photosynthetic and non-photosynthetic ferredoxin isoproteins. , 2000, Plant physiology.

[45]  P. Karplus,et al.  Structure-function relations for ferredoxin reductase , 1994, Journal of bioenergetics and biomembranes.

[46]  M. Hasegawa,et al.  Gene transfer to the nucleus and the evolution of chloroplasts , 1998, Nature.

[47]  D. Smith,et al.  Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. , 1988, Gene.

[48]  J. Boothroyd,et al.  Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii. , 1996, Gene.

[49]  John Shanklin,et al.  DESATURATION AND RELATED MODIFICATIONS OF FATTY ACIDS1. , 1998, Annual review of plant physiology and plant molecular biology.

[50]  J. Boothroyd,et al.  [1] Forward and reverse genetics in the study of the obligate, intracellular parasite Toxoplasma gondii , 1995 .