J Biol Chem. 2010 May 4. [Epub ahead of print]
Plastid associated porphobilinogen synthase from toxoplasma gondii: kinetic and structural properties validate therapeutic potential
Shanmugam D, Wu B, Ramirez U, Jaffe EK, Roos DS.
University of Pennsylvania, United States;
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+)), Mg(2+)) and oligomeric states (dimers, hexamers, octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ~320 kDa octamer, and low levels of dimmers -- 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 ~150 muM and specific activity ~24 mumol porphobilinogen/mg protein/hr. 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 offers scope for developing selective inhibitors of the parasite enzyme based on its quarternary structure characteristics.
PMID: 20442414 [PubMed - as supplied by publisher]