Lipoic acid metabolism of Plasmodium--a suitable drug target

Abstract

α-Lipoic acid (6,8-thioctic acid; LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. In recent years it was shown that biosynthesis and salvage of LA in Plasmodium are necessary for the parasites to complete their complex life cycle. LA salvage requires two lipoic acid protein ligases (LplA1 and LplA2). LplA1 is confined to the mitochondrion while LplA2 is located in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development suggesting a specific function for LplA2 that has yet to be elucidated. LA salvage is essential for the intra-erythrocytic and liver stage development of Plasmodium and thus offers great potential for future drug or vaccine development. LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP) : protein N-octanoyltransferase (LipB) and lipoate synthase (LipA), is exclusively found in the apicoplast of Plasmodium where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis (FAS II) pathway also present in the organelle. LA is the co-factor of the acetyltransferase subunit of the apicoplast located pyruvate dehydrogenase (PDH), which generates acetyl-CoA, feeding into FAS II. LA biosynthesis is not vital for intra-erythrocytic development of Plasmodium, but the deletion of several genes encoding components of FAS II or PDH was detrimental for liver stage development of the parasites indirectly suggesting that the same applies to LA biosynthesis. These data provide strong evidence that LA salvage and biosynthesis are vital for different stages of Plasmodium development and offer potential for drug and vaccine design against malaria.

Fig. (1)

Structure of lipoic acid and the E2-subunit of pyruvate dehydrogenase. Chemical structure of free α-lipoic acid (LA) in its (A) oxidised and (B) its reduced form, dihydrolipoic acid (DHLA). (C) Schematic diagram of the E2-subunit of P. falciparum and human PDH depicting the two lipoyl-domains (LD), the E1/E3 binding domain (E1/E3 BD) and the catalytic domain of the protein. LA is covalently attached to a conserved lysine (K) residue of both LD.

Fig. (2)

Catalytic mechanism of PDH. The reaction mechanism of the three catalytically active subunits of PDH (E1α/E1β forming a heterotetramer; E2 forming a 24- or 60-mer and E3, forming a dimer) is shown schematically. The E1-subunit decarboxylates pyuvate and the acetyl-moiety is covalently attached to the thiamine diphosphate co-factor (TPP) of E1. CO₂ is released during the reaction (1). TPP transfers the acetyl-moiety to the oxidised lipoamide co-factor of E2 (2), which transfers it to CoA to form acetyl-CoA (3), which is released. E3 re-oxidises dihydrolipoamide (4) and NADH + H⁺ is generated (5).

Fig. (3)

Lipoic acid salvage and biosynthesis pathways. α-Lipoic acid is generated and attached to the E2-subunit of KADH or the H-protein of GCS by two mechanisms. In E. coli and eukaryotes (including Plasmodium), LipB transfers the octanoyl-moiety from Oct-ACP directly to the lipoyl-domain of E2 and two sulphurs are subsequently introduced by LipA. In B. subtilis the LipB homologue LipM transfers the ocantoyl-moiety to the H-protein of the bacterial GCS. Octanoylated GCS-H protein is the substrate for the transamidase LipL, which transfers the octanoyl-moiety to the lipoyl-domain of the E2-subunit which is followed by the introduction of the sulphurs via LipA. It is not fully understood if LipA functions before or after the action of LipL. Salvage of α- lipoic acid is achieved through the activity of two proteins (lipoate activating enzyme and lipoyltransferase) in mammals while E. coli (and Plasmodium) only requires the activity of LplA to catalyse the ligation of free α-lipoic acid to the lipoyl-domain of the E2-subunit of KADH or the H-protein of the GCS. In L. monocytogenes, LplA1 catalyses the ligation of α-lipoic acid to the GCS-H-protein before it is transferred by LipL to the E2-subunit of the KADH.

Fig. (4)

Lipoic acid metabolism in Plasmodium. Biosynthesis and ligation of LA to PDH takes place in the apicoplast. Oct-ACP is an intermediate of FAS II and is attached to the E2-subunit of PDH by LipB or LplA2, followed by the insertion of two sulphurs by LipA, generating the PDH-bound lipoyl-moiety. PDH converts pyruvate into acetyl-CoA, which feeds into FAS II. Free LA cannot enter the apicoplast and is exclusively used for the lipoylation of mitochondrial complexes. LA is activated and ligated to the H-protein of the GCS (GCS-H) and the E2-subunit of BCDH and KGDH by LplA1 or LplA2. KGDH converts α-ketoglutarate (KG) into succinyl-CoA as part of the branched TCA metabolism. LA is possibly taken up by the parasite via a pantothenate transporter, located in the parasite plasma membrane.