title: Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.
abstract: 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.
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Dhanasekaran
authors.initials: D
authors.lastname: Shanmugam
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
authors.forename: Ursula
authors.initials: U
authors.lastname: Ramirez
authors.forename: Eileen K
authors.initials: EK
authors.lastname: Jaffe
authors.forename: David S
authors.initials: DS
authors.lastname: Roos
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016678:Genome / Q000235:genetics; D006418:Heme / Q000096:biosynthesis; D006537:Heptanoates / Q000494:pharmacology; D006801:Humans; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D051379:Mice; D018087:Plastids / Q000187:drug effects / Q000201:enzymology*; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000187:drug effects; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D014122:Toxoplasma / Q000187:drug effects* / Q000201:enzymology*
chemical_list: D000981:Antiprotozoal Agents; D006537:Heptanoates; D007477:Ions; D008670:Metals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006418:Heme; C020804:succinylacetone; D000623:Porphobilinogen Synthase