Pseudomonas aeruginosa Contains a Novel Type V Porphobilinogen Synthase with No Required Catalytic Metal Ions †

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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 ϳ320-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 ϳ150 M and specific activity of ϳ24 mol 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.

Section: ⌬C;supporting

confidence: 90%

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii

Shanmugam

Ramirez

et al. 2010

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“…The cognate cluster of aspartic acid residues are present in PBGS crystal structures from the bacterial species P. aeruginosa (PDB codes 1B4K, 1GZG, 2C13, 2C14, 2C15, 2C16, 2C18, 2C19, and 2WOQ) and Chlorobium vibrioforme (PDB codes 1W1Z and 2C1H) and the current eukaryotic TgPBGS structure (PDB code 3OBK). Of the nine P. aeruginosa PBGS structures containing the wild-type sequence in this region, most do not show magnesium at the active site, consistent with kinetic data that suggest that basal P. aeruginosa PBGS activity is metal ion-independent (37). Exceptions include a sulfone-containing inhibitor where the sulfone moiety contributes to active site magnesium binding (PDB code 2C18) and a structure with the inhibitor aleramycin (PDB code 2WOQ), where the modeled magnesium has an unusual pentacoordinate geometry and is not liganded to the inhibitor.…”

Section: Active Site Metal Ions Of Pbgs-supporting

confidence: 78%

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

Jaffe

Shanmugam

Gardberg

et al. 2011

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Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the productbound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N-and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit ␤-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.The myriad forms of cyclic and linear tetrapyrroles, including heme, chlorophyll, siroheme, cobalamine (B 12 ), and the phycobilins, are essential for energy production/utilization in virtually all life forms. Tetrapyrrole biosynthesis pathways are complex and phylogenetically variable, suggesting the potential for species-selective control (1). The universal portion of this pathway consists of only three reactions, the first of which involves biosynthesis of the monopyrrole porphobilinogen from two molecules of 5-aminolevulinic acid (ALA) 3 (Fig. 1), catalyzed by porphobilinogen synthase (PBGS; EC 4.2.1.24; also known as 5-aminolevulinic acid dehydratase or ALAD). The photoreactive and toxic nature of tetrapyrrole biosynthesis intermediates mandates close regulation of this pathway, and various mechanisms are used for control. We recently have described an unusual mechanism of allosteric regulation involving transition between a high activity PBGS octamer and a low activity hexamer (2-4). In plants, an allosteric magnesium ion favors octamer formation by binding at a subunit interface not present in the hexamer (2). The term "morpheeins" has been used to describe oligomers that can disassemble, change shape in the dissociated state, and reassemble to a structurally and functionally distinct oligomer (5). Fig. 2 illustrates the quaternary structure dynamics of plant and mammalian PBGS, using the human structure by way of example. "Hugging" and "detached" dimers (pathway A) are observed as the asymmetric crystallographic units for wild type and mutant forms of human PBGS (PDB codes 1E51 and 1PV8, respectively (2, 6)). The structure of the physiologicall...

“…This is the lowest number of bound, divalent metal ions yet reported for all species of ALAD except for rPfALAD, which is investigated in the present study. Hence, the enzyme from P. aeruginosa has been described as a novel type V ALAD (26,36). rPfALAD resembles the enzyme from P. aeruginosa in most of its properties, except in terms of sensitivity to EDTA and dialysis.…”

Section: Discussionmentioning

confidence: 99%

“…However, the concentration of Mg 2ϩ in the apicoplast is not known. The bacterial enzyme is completely inhibited by EDTA or just by dialysis and the apoenzyme can be reconstituted by Mg 2ϩ (26,36). The crystal structure of ALAD from P. aeruginosa reveals that Mg 2ϩ is bound 14 Å away from the Schiff base-forming nitrogen atom of the active site lysine (16).…”

Section: Discussionmentioning

confidence: 99%

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Dhanasekaran

Chandra

Sagar

et al. 2004

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host ␦-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit M r of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20 -30% upon addition of Mg 2؉ . The enzyme is inhibited by Zn 2؉ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg 2؉ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.Studies in this laboratory had demonstrated that the malaria parasites (Plasmodium falciparum and Plasmodium berghei) are capable of heme biosynthesis de novo, despite acquiring large amounts of heme from the host red cell hemoglobin in the intraerythrocytic stage (1, 2). Inhibition of the de novo heme biosynthetic pathway leads to death of the parasite and the pathway is therefore a drug target (1-3). Studies on the enzymes of the heme-biosynthetic pathway have revealed that the first enzyme, ␦-aminolevulinate synthase, is coded for by the parasite genome and is localized in the parasite mitochondrion (4, 5). Studies from this laboratory have shown that the second enzyme of the pathway, ␦-aminolevulinate dehydratase (ALAD), 1 in the parasite is of host origin and evidence was provided to show that the enzyme is translocated (imported) from the host red cell into the parasite and is functional (2, 3).At the same time genome sequence data available for P. falciparum indicates that the parasite genome codes for ALAD (PfALAD) and other enzymes of the heme-biosynthetic pathway. Sato et al. (6) have shown by phylogenetic amino acid sequence analysis derived from truncated...