Heme biosynthetic pathway in apicomplexan parasites

Animal-like heme biosynthetic pathway has been identified in most apicomplexan except for Cryptosporidium and Theileria. Three enzymes normally found in the cytoplasm---5\u27-aminolevulinic acid dehydratase/ALAD, porphobilinogen deaminase/PBGD, and uroporphyrinogen synthase/UroS have been re-localized to the apicoplast and the mitochondrial coproporphyrinogen oxidase/CPO is cytosolic, as seen in yeast. The other step enzymes---aminolevulinic acid synthase/ALAS, protoporphyrinogen oxidase/PPO and ferrochelatase/FC are still located in the mitochondrion and uroporphyrinogen decarboxylase/UroD is cytoplasmic, as is the case with their host counterparts. Phylogenetic analyses indicate the apicoplast-located enzymes (ALAD, PBGD and UroS) are of plastidic origin. However, only ALAD is of cyanobacterial gene ancestry. The mitochondrial located components (ALAS, PPO and FC) along with the cytosolic component (UroD) are of α-proteobacterial origin. The cytosolic CPO is possibly of eukaryotic nuclear origin, which might be relocated to the mitochondrion for efficiency in animals. PPO-targeting herbicides, Acifluorfen-methyl and Oxyfluorfen, are used in pharmacological study, revealing inhibition of this enzyme (IC90 8∼9 μM) leads to the death of intracellular parasites, however the host cell Human foreskin fibroblast does not have significant morphological and metabolic changes under 100μM drug treatment, implying de novo heme biosynthesis could be an effective drug target. Parasite growth inhibition (IC50 ∼2mM) and 14C-labeling heme production assays (IC50 ∼0.3mM) by ALAD inhibitor succinyl acetone further supported the potential of parasite heme pathway as a drug target. Multiple differentially-spliced TgALAD transcripts were discovered in RH strain tachyzoite cDNAs, but only the most abundant of them encodes a functional enzyme, which shares the apicoplast targeting signal with downstream pitrilysin gene. This unique organization is conserved in Plasmodium species. A unique C-terminal extension is required for TgALAD activity, which could serve as parasite-specific drug attacking site. rTgALAD forms an octamer (∼320KDa) with a Km of 188μM at pH 9, dependent on Mg2+ for optimal activity, but retaining ∼30% activity in 25mM EDTA