Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.
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
D
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Wu
Bo
B
Ramirez
Ursula
U
Jaffe
Eileen K
EK
Roos
David S
DS
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*
D000981:Antiprotozoal Agents; D006537:Heptanoates; D007477:Ions; D008670:Metals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006418:Heme; C020804:succinylacetone; D000623:Porphobilinogen Synthase