Shreya Ray 20091069

Purine Metabolism by Human Malarial Parasite: Plasmodium Falciparum

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Introduction
Plasmodium parasites, including the human malarial parasite Plasmodium falciparum that resides in the Red Blood Cells (erythrocytes) of the patient, cannot synthesize Purines rings de novo. In order to complete their life-cycle, they have to salvage them from the host. The Purine salvage pathway used by P. falciparum includes the synthesis of enzymes for purine salvage and interconversion, as well as altering the host erythrocyte membrane for transport of Purines.

Purine Transport
The erythrocyte pool of purines is not a sufficient purine source. Hence, to meet the purine demand of P. falciparum, transport of extraerythrocytic purines into the infected cell is necessary. Nucleosides and nucleobases may cross the host cell membrane in two ways: 1. High-affinity transport processes mediated by the human Equilibrative Nucleoside Transporter (hENT1) and the human Facilitative Nucleobase Transporter (hFNT1). 2. Nonsaturable, broad-specificity New Permeability Pathways (NPPs) induced by the parasite in the erythrocyte membrane. These include PfNT1, which is a low-affinity transporter that mediates the rapid uptake of adenosine, inosine, hypoxanthine, adenine, guanine, guanosine, and xanthine. In addition to this, adenine, the most rapidly absorbed nucleoside, may have a PfNT1-independent mechanism as well. The P. falciparum genome sequencing project revealed three additional nucleoside transporters: PfNT2, PfNT3, and PfNT4. Once inside the infected erythrocyte, the nucleosides can cross the parasitophorous vacuole membrane via large-diameter, nonselective pores present on this membrane.

The Salvage Pathway for Purine Biosynthesis

Following are the steps in the purine salvage pathway by P.falciparum: 1. Adenosine, Inosine, Hypoxanthine, Guanosine, Guanine, Xanthine and Adenine are be transported across the parasite plasma membrane by PfNT1. Adenine also enters the cell via a second, PfNT1-independent mechanism. 2. Inosine is produced from adenosine by ADA: hADA in the erythrocyte cytoplasm and PfADA in the parasite cytosol. Overall, more Inosine is produced, around twice the amount produced in an uninfected erythrocyte. o P. Falciparum Adenosine Deaminase (Pfada) The enzyme ADA catalyzes the irreversible hydrolytic cleavage of adenosine to produce inosine and ammonia. The hADA and the PfADA essentially have the same Km. However, an important feature of the PfADA is that it exhibits a dual catalytic function: along with adenine it can also act on a 5substituted compound, MTA (methylthioadenosine), that is produced via the polyamine biosynthesis pathway, and converts it to MTI (methylthioinosine.). 3. Inosine is then converted to hypoxanthine by PNP: hPNP in the erythrocyte cytoplas and PfPNP in the parasite cytosol. o P. Falciparum Purine Nucleoside Phosphorylase (Pfpnp) PNP enzymes are responsible for the reversible phosphorolysis of nucleosides into the corresponding nucleobases and ribose groups in the presence of inorganic phosphate. Like PfADA, PfPNP has the ability to act on a 5-substituted compound, MTI, which was produced by PfADA. The abilities of PfADA and PfPNP to utilize methylthiopurines allow the salvage of purines from both the traditional purine nucleoside pathways and the polyamine synthesis pathway. PfPNP can also utilise inosine, guanosine, and deoxyguanosine but not adenosine or xanthosine. 4. PRT activity of PfHGXPRT converts hypoxanthine to IMP, guanine to GMP, and xanthine to XMP. There is some evidence to suggest that the parasite can also convert adenine to AMP by using a parasite-encoded APRT (PfAPRT). o P. Falciparum Phosphoribosyltransferase Enzymes (PfPRTs) Nucleobases can be converted directly to nucleoside monophosphates by the action of PRT enzymes. PRT enzymes catalyze the reversible transfer of a phosphoribosyl group from phosphoribosylpyrophosphate to a nucleobase, resulting in the production of a nucleotide and pyrophosphate. In P. falciparum, PRT activity is catalyzed by a single enzyme, PfHGXPRT. The ability of the enzyme to utilize xanthine as a substrate distinguishes it from its human enzyme counterpart.

A second enzyme PfAPRT is suspected to account for adenine PRT (APRT) activity. But this is still controversial.

Downie et al. Eukaryotic Cell, Aug. 2008, p. 12311237

5. IMP, GMP and XMP, are then converted to guanylate and adenylate nucleotides by the action of several more enzymes. Also, IMP can be converted to XMP by IMP dehydrogenase. XMP can be converted to GMP by GMP synthetase. o Guanylate kinase phosphorylates GMP to form GDP. o To make adenylate nucleotides, IMP is converted to adenylosuccinate by Adenylosuccinate synthetase. Adenylosuccinate lyase then converts adenylosuccinate to AMP. AMP is phosphorylated to ADP by Adenylate kinase. 6. ATP and GTP (or deoxy- ATP and GTP) may now be incorported in RNA (or DNA).

The Salvage Pathway in malaria parasitized erythrocyte (PRBC) observably differs from the unparasitized mature erythrocyte (RBC) in the following ways: 1. PRBC primarily utilize hypoxanthine for synthesis of both adenylates and guanylates 2. PRBC incorporate the base guanine into guanylates at a higher rate than control RBC 3. PRBC do not appear to use adenine effectively due to an overwhelming competition for this base by the whole erythrocyte population 4. Although PRBC cultures show an initial increase in [ATP] this change is interpreted to reflect a generalized RBC response to malaria infection and not a response restricted to PRBC.

Implications for the Future: Chemical Therapies against Malaria

Since the host can obtain both types of bases by either pathway, it may be possible to exploit the parasite's limited capability in nucleotide metabolism. Within the parasite, the metabolism of purines appears to be funneled through hypoxanthine. But we cant exploit this because hypoxanthine is the main purine source found in human serum. Therefore, it seems likely that a block in the purine salvage pathway at the ADA or PNP step could be circumvented by the downstream salvage of hypoxanthine, guanine, or xanthine from the host, all of which are transported into the parasite by PfNT1. The potential of PfADA and PfPNP as therapeutic targets is yet to be validated. The 5-methylthio Coformycins are popular inhibitors of PfADA. Immucillin-H is currently in clinical trials for treating human Tcell malignancies and has been shown to inhibit PfPNP, as well as inhibiting P. Falciparum growth, in vitro. The apparent dependence of the parasite on PfHGXPRT (Pf. hypoxanthine-guaninexanthine PRT) for nucleotide synthesis and the discovery of compounds that can interact preferentially with the parasite enzyme, together with the reported lack of nucleobase phosphoribosylation redundancy in P. falciparum, makes PfHGXPRT a promising target for the development of antimalarial therapies. The reliance of the parasite on PfNT1 makes this protein a worthy target for further investigation. A relatively new approach in inhibiting malarial growth involves the altered nucleoside transporter in the infected cell membrane through which cytotoxic compounds may be selectively targeted into only the infected cell.

Reference
[1] Quashie et al. Malaria Journal 2010, 9:36. Uptake of purines in Plasmodium falciparuminfected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter. [2] Downie et al. Eukaryotic Cell, Aug. 2008, p. 12311237. MINIREVIEWS: Purine Salvage Pathways in the Intraerythrocytic Malaria Parasite Plasmodium falciparum. [3] Webster and Whaun. Prog Clin Biol Res. 1981; 55:557-73. Purine metabolism during continuous erythrocyte culture of human malaria parasites (P. falciparum). [4] Gero and Sullivan. Blood Cells. 1990; 16 (2-3):467-84; discussion 485-98. Purines and pyrimidines in malarial parasites.