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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.

SUBMITTER: Shanmugam D

PROVIDER: S-EPMC2903393 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.

Shanmugam Dhanasekaran D Wu Bo B Ramirez Ursula U Jaffe Eileen K EK Roos David S DS

The Journal of biological chemistry 20100504 29

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 ...[more]

PMID: 20442414

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Project description:Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn2+; a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb2+. PBGSs that do not require the soft metal ion Zn2+ at the active site instead are suspected of using the hard metal Mg2+. The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg2+ as an allosteric activator. Mg2+ functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do not use the allosteric Mg2+, there is a spatially equivalent arginine-derived guanidium group. Deprotonation of this residue promotes formation of the hexamer and accounts for the basic arm of the bell-shaped pH vs activity profile of human PBGS. A human inborn error of metabolism known as ALAD porphyria is attributed to PBGS variants that favor the hexameric assembly. The existence of one such variant, F12L, which dramatically stabilizes the human PBGS hexamer, allowed crystal structure determination for the hexamer. Without this crystal structure and octameric PBGS structures containing the allosteric Mg2+, it would have been difficult to decipher the structural basis for PBGS allostery. The requirement for multimer dissociation as an intermediate step in PBGS allostery was established by monitoring subunit disproportionation during the turnover-dependent transition of heteromeric PBGS (comprised of human wild type and F12L) from hexamer to octamer. One outcome of these studies was the definition of the dissociative morpheein model of protein allostery. The phylogenetically variable time scales for PBGS multimer interconversion result in atypical kinetic and biophysical behaviors. These behaviors can serve to identify other proteins that use the morpheein model of protein allostery.

Dataset Information

Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.

Publications

Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.

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