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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 product- bound 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 α-sheet, stabilizing a prooctamer 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. [ABSTRACT FROM AUTHOR]

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 (Zn2+ and Mg2+) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ~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 Km for aminolevulinic acid of ~150 μm and specific activity of ~24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg2+ but not Zn2+ and shows two Mg2+ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg2+-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. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Hexokinase gene is dispensable for tachyzoite stage Toxoplasma gondii. • Glycolysis is essential only for tissue cyst formation by T. gondii. • 13C metabolic labelling studies highlight metabolic adaptation. • Toxoplasma gondii can obtain ATP from either glycolysis or oxidative phosphorylation. Abstract Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii , using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii , and validates the versatile nature of central carbon and energy metabolism in this parasite. [ABSTRACT FROM AUTHOR]

Abstract: Perkinsus marinus (Phylum Perkinsozoa) is a protozoan parasite that has devastated natural and farmed oyster populations in the USA, significantly affecting the shellfish industry and the estuarine environment. The other two genera in the phylum, Parvilucifera and Rastrimonas, are parasites of microeukaryotes. The Perkinsozoa occupies a key position at the base of the dinoflagellate branch, close to its divergence from the Apicomplexa, a clade that includes parasitic protista, many harbouring a relic plastid. Thus, as a taxon that has also evolved toward parasitism, the Perkinsozoa has attracted the attention of biologists interested in the evolution of this organelle, both in its ultrastructure and the conservation, loss or transfer of its genes. A review of the recent literature reveals mounting evidence in support of the presence of a relic plastid in P. marinus, including the presence of multimembrane structures, characteristic metabolic pathways and proteins with a bipartite N-terminal extension. Further, these findings raise intriguing questions regarding the potential functions and unique adaptation of the putative plastid and/or plastid genes in the Perkinsozoa. In this review we analyse the above-mentioned evidence and evaluate the potential future directions and expected benefits of addressing such questions. Given the rapidly expanding molecular/genetic resources and methodological toolbox for Perkinsus spp., these organisms should complement the currently established models for investigating plastid evolution within the Chromalveolata. [Copyright &y& Elsevier]

Inflammation is a constant in Non-Alcoholic Fatty Liver Disease (NAFLD), although their relationship is unclear. In a transgenic zebrafish system with chronic systemic overexpression of human IL6 (IL6-OE) we show that inflammation can cause intra-hepatic accumulation of triglycerides. Transcriptomics and proteomics analysis of the IL6-OE liver revealed a deregulation of glycolysis/gluconeogenesis pathway, especially a striking down regulation of the glycolytic enzyme aldolase b. Metabolomics analysis by mass spectrometry showed accumulation of hexose monophosphates and their derivatives, which can act as precursors for triglyceride synthesis. Our results suggest that IL6-driven repression of glycolysis/gluconeogenesis, specifically aldolase b, may be a novel mechanism for fatty liver. This mechanism may be relevant for NAFLD in lean individuals, an emerging class of NAFLD prevalent more in Asian Indian populations. • Chronic IL6 overexpression leads to hepatic steatosis in male zebrafish • The steatotic liver shows transcriptomic signature distinct from diet-induced fatty liver models • Suppression of glycolysis/gluconeogenesis and accumulation of hexose monophosphate observed in the male fatty liver • Down regulation of glycolytic enzyme ALDOB, correlates with NAFLD in patients [ABSTRACT FROM AUTHOR]