Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

Abstract

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (⌬PfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (URO-GEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, ⌬PfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a ⌬PfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than ⌬PfPBGD. More interestingly, ⌬PfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to URO-GEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to URO-GEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.Detailed studies of the metabolic pathways in the malarial parasite hold promise for the identification of new antimalarial drug targets (1, 2). Earlier studies in this laboratory had shown that the malarial parasite synthesizes heme de novo, despite acquiring heme from the host red cell hemoglobin in the intraerythrocytic stage. It has been shown that Plasmodium falciparum contains ␦-aminolevulinate dehydratase (ALAD) 2 of dual origin: one species encoded by the parasite genome (PfALAD) and another imported from the host red cell. Inhibition of ALAD activity in the parasite, the second enzyme of the pathway with a specific inhibitor, succinylacetone, leads to inhibition of heme synthesis and death of the parasite, indicating the potential of the pathway as a drug target (3, 4).Heme biosynthesis from glycine and succinyl-CoA involves eight different steps and the genes for all the enzymes of the pathway, with the exception of uroporphyrinogen III synthase (UROS, hemD) have been located on the parasite genome (5). However, a hemD orthologue has been identified in Toxoplasma gondii and is expected to be targeted to the apicoplast, a chloroplast-like organelle in the parasite (6). Based mostly on bioinformatics-based predictions, some e...

The apicoplast: now you see it, now you don’t

“…An apicoplast pathway to synthesise isopentenyl diphosphate (IPP) – a precursor of isoprenoids for prenylation of proteins, ubiquinone side chains, dolichols and modification of tRNAs (Ralph et al, 2004) – was also identified from the genome data (Jomaa et al, 1999; Guggisberg et al, 2014a; Imlay and Odom, 2014; Armstrong et al, 2015; Imlay et al, 2015). Subsequent data mining revealed that the apicoplast of malaria parasites makes iron sulphur complexes (Seeber, 2002, 2003; Ralph et al, 2004) and cooperates with the mitochondrion in the synthesis of haem (Ralph et al, 2004; Sato et al, 2004; van Dooren et al, 2006; Nagaraj et al, 2008, 2009a,b; Shanmugam et al, 2010). These four anabolic roles for the apicoplast hinted that synthesis of crucial precursors was the essential role of the apicoplast, but the genes did not tell us exactly what was made and how it was essential.…”

Influence of host iron status on Plasmodium falciparum infection

“…Iron is an essential cofactor for the DNA replication enzyme ribonucleotide reductase, and as a result iron is required to fuel this rapid intra-erythrocytic proliferation (Rubin et al, 1993). Iron is also utilized by the parasite for pyrimidine (Krungkrai et al, 1990; Van Dooren et al, 2006) and heme biosynthesis (Sato and Wilson, 2002; Dhanasekaran et al, 2004; Sato et al, 2004; Nagaraj et al, 2008, 2009, 2010, 2013). As with the human host, the malaria parasite must also balance its need for iron against the cytotoxicity of iron.…”

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

“…The first enzyme, δ-aminolevulinate synthase ( Pf ALAS) [8], [9], and the last two enzymes, Protoporphyrinogen IX oxidase ( Pf PPO) and Ferrochelatase ( Pf FC) [10], [11] localize to the mitochondrion. The enzymes that catalyze the intermediate steps: ALA dehydratase ( Pf ALAD) [12], [13], Porphobilinogen deaminase ( Pf PBGD) [9], [14], and Uroporphyrinogen III decarboxylase ( Pf UROD) [15] localize to the apicoplast (a chloroplast relic), whereas, the next enzyme Coproporphyrinogen III oxidase ( Pf CPO) is cytosolic [16]. Figure 1 depicts the pathway.…”