The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

Abstract

BackgroundPyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population.Methodology/Principal FindingsWe have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers.ConclusionsWe hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages

“…substrate for the FAS pathways (both type I and II); and c) FAS (45). All of these enzymes are present in T. gondii and could putatively be regulated by an accumulation of acetate following TgACS disruption; PyK is present in two essential isoforms in T. gondii, TgPyKI, likely in the cytosol, and Tg-PyKII, dually localized in the mitochondrion and the apicoplast (46,47), ACCase is also present in two copies: one in the apicoplast to fuel the FASII and one likely in the cytosol potentially for FASI and/or elongases (48). Furthermore, it has also been shown that ACCase can be downregulated when there is the accumulation of the FASII products (49).…”

Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis

“…In many parasites, including Leishmania mexicana , Trypanosoma brucei , Toxoplasma gondii , Plasmodium falciparum and Schistosoma mansoni , PK is highly regulated, indicating that it may play crucial regulatory roles in glycolysis in these parasites [ 15 – 19 ]. PK is considered a compelling therapeutic target for malignant tumours as well as for human pathogens such as apicomplexans [ 20 ]. Nearly all PKs exist as homotetramers [ 18 , 21 ].…”

Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

“…Among the parasitic proteins, L. mexicana pyruvate kinase (LmPyK) is the most thoroughly studied (13 out of 18 entries in the protein data bank), and structural consequences of binding substrates and allosteric regulators have been established from a series of crystal structures [20], [28], [30]. Crystal structures of PyK from three other parasites, T. gondii , Trypanosoma cruzi and P. falciparum (PDBID: 3KHD; unpublished) have also been reported [25], [31]. These studies demonstrated that the structures of PyKs are influenced by the presence or absence of ligands at specific sites in the protein and by the crystallization conditions [25], [30].…”