Abstract
PDC (pyruvate dehydrogenase complex) is a multi-enzyme complex comprising an E1 (pyruvate decarboxylase), an E2 (dihydrolipomide acetyltransferase) and an E3 (dihydrolipoamide dehydrogenase). PDC catalyses the decarboxylation of pyruvate and forms acetyl-CoA and NADH. In the human malaria parasite Plasmodium falciparum, the single PDC is located exclusively in the apicoplast. Plasmodium PDC is essential for parasite survival in the mosquito vector and for late liver stage development in the human host, suggesting its suitability as a target for intervention strategies against malaria. Here, PfaE3 (P. falciparum apicoplast E3) was recombinantly expressed and characterized. Biochemical parameters were comparable with those determined for E3 from other organisms. A homology model for PfaE3 reveals an extra anti-parallel β-strand at the position where human E3BP (E3-binding protein) interacts with E3; a parasite-specific feature that may be exploitable for drug discovery against PDC. To assess the biological role of Pfae3, it was deleted from P. falciparum and although the mutants are viable, they displayed a highly synchronous growth phenotype during intra-erythrocytic development. The mutants also showed changes in the expression of some mitochondrial and antioxidant proteins suggesting that deletion of Pfae3 impacts on the parasite's metabolic function with downstream effects on the parasite's redox homoeostasis and cell cycle.
Highlights
The pyruvate dehydrogenase complex (PDC) belongs to the KADH (α-keto acid dehydrogenases), a family of mega-Dalton multi-enzyme complexes comprising multiple subunits of three different enzymes
As opposed to the previous expression trial reported by McMillan et al [20], which obtained only marginal amounts of recombinant P. falciparum aE3 (Pf aE3), the yield of recombinant Pf aE3 using the pQE30 expression plasmid was 1.5 mg/l of bacterial culture, which allowed for kinetic and structural analyses of the recombinant protein
The three other KADH complexes are mitochondrial [60] and this distribution requires the presence of apicoplast- and mitochondrion-specific dihydrolipoaminde dehydrogenases (E3) that interact with their organelle-specific KADH [20,61]
Summary
The PDC (pyruvate dehydrogenase complex) belongs to the KADH (α-keto acid dehydrogenases), a family of mega-Dalton multi-enzyme complexes comprising multiple subunits of three different enzymes. The data were fitted with a single species model (Figure 2C) and the resultant weight average molecular masses were plotted as a function of Pf aE3 dimer concentration (Figure 2D) yielding an infinite dilution whole-cell weight average molecular mass of 128 kDa, remarkably close to the molecular mass of the Pf aE3 dimer calculated from the amino acid sequence of the recombinantly expressed full-length protein (127 478 kDa) (see above).
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