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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
Journal of Biological Chemistry. 2010 Jul;285(29) :22122-22131
AbstractApicomplexan 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 (Zn2+ and Mg2+) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable similar to 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 similar to 150 mu M and specific activity of similar to 24 mu mol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg2+ but not Zn2+ and shows two Mg2+ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg2+ -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.
NotesShanmugam, Dhanasekaran Wu, Bo Ramirez, Ursula Jaffe, Eileen K. Roos, David S. National Institutes of Health Grants [AI28724, ES003654, T32CA00935, AI077577, CA006927] This work was supported, in whole or in part, by National Institutes of Health Grants AI28724 (to D.S.R.), ES003654 (to E.K.J.), T32CA00935 (to the Institute for Cancer Research), AI077577 (to E.K.J.), and CA006927 (to Fox Chase Cancer Center). 44 Amer soc biochemistry molecular biology inc; 9650 rockville pike, bethesda, md 20814-3996 usa 622yf