title: The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases.
abstract: The Plasmodium falciparum genome contains genes encoding three alpha-ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle-specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain alpha-ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N-terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle-specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial alpha-ketoglutarate dehydrogenase and branched chain alpha-ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum.
authors.affiliation: Institute of Biomedical and Life Sciences, Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: McMillan
authors.forename: Luciana M
authors.initials: LM
authors.lastname: Stimmler
authors.forename: Bernardo J
authors.initials: BJ
authors.lastname: Foth
authors.forename: Geoffrey I
authors.initials: GI
authors.lastname: McFadden
authors.forename: Sylke
authors.initials: S
authors.lastname: Müller
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000096:biosynthesis / Q000235:genetics; D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D008058:Dihydrolipoamide Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000469:parasitology; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D066298:In Vitro Techniques; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D018087:Plastids / Q000201:enzymology*; D016053:RNA, Protozoan / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016053:RNA, Protozoan; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D000217:Acyltransferases; C033588:dihydrolipoamide acyltransferase