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Shanmugam D , Wu B , Ramirez U , Jaffe EK , Roos DS
Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential
J Biol Chem. 2010 Jul 16;285(29) :22122-31
PMID: 20442414    PMCID: PMC2903393    URL: https://www.ncbi.nlm.nih.gov/pubmed/20442414
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Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn(2+) and Mg(2+)) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable approximately 320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7-8.5), with a K(m) for aminolevulinic acid of approximately 150 microM and specific activity of approximately 24 micromol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg(2+) but not Zn(2+) and shows two Mg(2+) affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg(2+)-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.
Shanmugam, Dhanasekaran Wu, Bo Ramirez, Ursula Jaffe, Eileen K Roos, David S eng ES003654/ES/NIEHS NIH HHS/ AI28724/AI/NIAID NIH HHS/ R37 AI028724/AI/NIAID NIH HHS/ CA006927/CA/NCI NIH HHS/ R01 ES003654/ES/NIEHS NIH HHS/ AI077577/AI/NIAID NIH HHS/ T32CA00935/CA/NCI NIH HHS/ R56 AI077577/AI/NIAID NIH HHS/ P30 CA006927/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural J Biol Chem. 2010 Jul 16;285(29):22122-31. doi: 10.1074/jbc.M110.107243. Epub 2010 May 4.