Hydrodynamic characterization of the SufBC and SufCD complexes and their interaction with fluorescent adenosine nucleotides.

Bacteria, as well as the plastid organelles of algae and higher plants, utilize proteins of the suf operon. These are involved in Fe-S cluster assembly, particularly under conditions of iron limitation or oxidative stress. Genetic experiments in some organisms found that the ATPase SufC is essential, though its role in Fe-S biogenesis remains unclear. To ascertain how interactions with other individual Suf proteins affect the activity of SufC we coexpressed it with either SufB or SufD from Thermotoga maritima and purified the resulting SufBC and SufCD complexes. Analytical ultracentrifuge and multiangle light-scattering measurements showed that the SufBC complex exists in solution as the tetrameric SufB(2)C(2) species, whereas SufCD exists as an equilibrium mixture of SufCD and SufC(2)D(2). Transient kinetic studies of the complexes were made using fluorescent 2'(3')-O-(N-methylanthraniloyl-(mant) analogues of ATP and ADP. Both SufBC and SufCD bound mantATP and mantADP much more tightly than does SufC alone. Compared to the cleavage step of the mantATPase of SufC alone, that of SufBC was accelerated 180-fold and that of SufCD only fivefold. Given that SufB and SufD have 20% sequence identity and similar predicted secondary structures, the different hydrodynamic properties and kinetic mechanisms of the two complexes are discussed.

The kinetic mechanism of the SufC ATPase: the cleavage step is accelerated by SufB.

Protein products of the suf operon are involved in iron-sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')-O-N-methylanthraniloyl-ATP (mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC.mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of Pi. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC.mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and -C proteins interact in the processes involved in regulating iron-sulfur transfer.

The plastid of Plasmodium spp.: a target for inhibitors.

Determined efforts are being made to explore the non-photosynthetic plastid organelle of Plasmodium falciparum as a target for drug development. Certain antibiotics that block organellar protein synthesis are already in clinical use as antimalarials. However, all the indications are that these should be used only in combination with conventional antimalarials. The use of antibiotics such as doxycycline and clindamycin may reduce the development of drug resistant parasites and such means to avoid drug resistance should be explored hand-in-hand with drug development. Genomic information predicts that fatty acid type II (FAS II) and isoprenoid biosynthetic pathways are localized to the plastid. However, clinical trials with fosmidomycin (a specific inhibitor of DOXP reductase in the non-mevalonate pathway for isoprenoids) suggest it too should only be used in drug combinations. Prospects for more potent antimalarial compounds have emerged from studies of several of the enzymes involved in the FAS II pathway. Lead antibiotics such as thiolactomycin (an inhibitor of beta-ketoacyl-ACP synthase) and triclosan (a specific inhibitor of enoyl-ACP reductase) have led to structurally similar, active compounds that rapidly kill ring- and trophozoite-stage parasites. The FAS II pathway is of particular interest to the pharma-industry.

Organelle-specific cochaperonins in apicomplexan parasites.

Protein maturation in eukaryotic organelles requires the type I chaperonin system; this comprises chaperonin 60 (Cpn60) and its cochaperonin. We have re-examined and revised the sequence of the nuclear genes specifying organellar cochaperonins in Plasmodium falciparum (Pf). One gene encodes a typical cochaperonin (PfCpn10) whereas the other (encoding PfCpn20) specifies two Cpn10 domains arranged in tandem as in plant chloroplasts. Transfection experiments using fluorescent reporters showed specific localization of PfCpn10 to the mitochondrion and PfCpn20 to the plastid. As P. falciparum also has two Cpn60s, one of which is targeted specifically to the mitochondrion and the other exclusively to the plastid, each organelle has a distinct type I chaperonin system. Comparative sequence analysis extended these findings to several other apicomplexan parasites that have both a mitochondrion and a plastid. Phylogenetic analysis suggests the Cpn10s and Cpn20s of apicomplexans are independently monophyletic. The apicomplexan Cpn10 is phylogenetically related to other mitochondrial versions but a significant relationship between apicomplexan Cpn20s and other cochaperonins was not established.

The plastidic DNA replication enzyme complex of Plasmodium falciparum.

The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.

Parasite plastids: approaching the endgame.

Considerable work still needs to be done to understand more fully the basic processes going on inside the non-photosynthetic plastid organelle of Plasmodium spp., the causative agent of malaria. Following an explosion of genomic and transcriptional information in recent years, research workers are still analysing these data looking for new material relevant to the plastid. Several metabolic and housekeeping functions based on bacterial biochemistry have been elucidated and this has given impetus to finding lead inhibitors based on established anti-microbials. Structural investigations of plastid-associated enzymes identified as potential targets have begun. This review gives a perspective on the research to date and hopes to emphasize that a practical outcome for the clinic should be an important focus of future efforts. Malaria parasites have become resistant to front-line anti-malarials that are widely used and were formerly dependable. This has become a worrying problem in many regions where malaria is endemic. The time lag between hunting for new inhibitors and their application as pharmaceuticals is so long and costly that a steady stream of new ventures has to be undertaken to give a reasonable chance of finding affordable and appropriate anti-malarials for the future. Attempts to find inhibitors of the plastid organelle of the malaria parasite should be intensified in such programmes.

Enzymes for heme biosynthesis are found in both the mitochondrion and plastid of the malaria parasite Plasmodium falciparum.

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway delta-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg2+ (and enhanced by Mn2+) but not by Zn2+. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

The transcriptome: malariologists ride the wave.

The Plasmodium falciparum genome-sequencing project has provided malariologists with vast amounts of new information pertinent to a multitude of cellular processes that previously were only guessed about. In exploring this morass of predicted genes and proteins, there is now a danger of simply re-inventing the cell. Fortunately, new global transcriptional analyses reassure malariologists that they are not dealing with just "any old cell." The informative papers on the plasmodial transcriptome by Le Roch et al. (2003)1 and Bozdech et al. (2003)2 discussed below forge a bridge between the genomics and proteomics of P. falciparum. They are likely to act as a fulcrum upon which much future research will turn: for example, the study of regulation and feed-back loops.

Parasite plastids: maintenance and functions.

Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.

Proteobacteria-like ferrochelatase in the malaria parasite.

A gene encoding the heme biosynthetic enzyme ferrochelatase (FC) was found in the genomic DNA databases of Plasmodium spp. The predicted amino acid sequence of malarial FC is highly conserved and fairly well conserved by comparison with other orthologues. The FC genes of P. falciparum and P. yoelii are transcribed and the mRNAs are processed to encode polypeptides of the expected amino acid sequence. The cloned cDNA for the FC of P. falciparum successfully rescued a FC-null mutant of Escherichia coli, indicating that it encodes an active enzyme. Unlike eukaryotic FCs, the malarial enzyme lacks a characteristic extension at the C-terminus. In addition, the sequence of the malarial FC resembles proteobacterial orthologues rather than eukaryotic enzymes. Strikingly, the malarial FC lacks a bipartite presequence at its N-terminus, unlike delta-aminolevulinic acid dehydratase of the same organism. This suggests an unusual intracellular distribution of heme biosynthetic enzymes, involving multiple subcellular compartments.

The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms.

In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two-dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D-loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.

Progress with parasite plastids.

This review offers a snapshot of our current understanding of the origin, biology, and metabolic significance of the non-photosynthetic plastid organelle found in apicomplexan parasites. These protists are of considerable medical and veterinary importance world-wide, Plasmodium spp., the causative agent of malaria being foremost in terms of human disease. It has been estimated that approximately 8% of the genes currently recognized by the malarial genome sequencing project (now nearing completion) are of bacterial/plastid origin. The bipartite presequences directing the products of these genes back to the plastid have provided fresh evidence that secondary endosymbiosis accounts for this organelle's presence in these parasites. Mounting phylogenetic evidence has strengthened the likelihood that the plastid originated from a red algal cell. Most importantly, we now have a broad understanding of several bacterial metabolic systems confined within the boundaries of the parasite plastid. The primary ones are type II fatty acid biosynthesis and isoprenoid biosynthesis. Some aspects of heme biosynthesis also might take place there. Retention of the plastid's relict genome and its still ill-defined capacity to participate in protein synthesis might be linked to an important house-keeping process, i.e. guarding the type II fatty acid biosynthetic pathway from oxidative damage. Fascinating observations have shown the parasite plastid does not divide by constriction as in typical plants, and that plastid-less parasites fail to thrive after invading a new cell. The modes of plastid DNA replication within the phylum also have provided surprises. Besides indicating the potential of the parasite plastid for therapeutic intervention, this review exposes many gaps remaining in our knowledge of this intriguing organelle. The rapid progress being made shows no sign of slackening.

SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima.

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.

The genome of Plasmodium falciparum encodes an active delta-aminolevulinic acid dehydratase.

The enzyme delta-aminolevulinic acid dehydratase (ALAD) catalyses the second reaction in the heme biosynthetic pathway. It has been suggested previously that the malaria parasite Plasmodium falciparum imports this enzyme from the host cell for de novo heme biosynthesis. However, the parasite's genome encodes an orthologue for ALAD. Here we report molecular cloning of a complete cDNA for the parasite's intrinsic ALAD and show it rescues an ALAD-null mutant of Escherichia coli, indicating that the malarial gene encodes a functional ALAD. The malarial ALAD has a long bipartite extension at its N-terminus, which may function as a plastid targeting signal. The amino acid sequence of the enzyme is related most closely to those of plant/algal chloroplast ALADs, though the malarial version may lack the allosteric Mg2+-binding site, which is conserved among chloroplast ALADs.