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The glyoxalase system of the malaria parasite Plasmodium falciparum


    Electrophilic 2-oxoaldehydes are harmful chemicals that are formed in every cell and that need to be metabolized because of their ability to modify nucleophiles in proteins and nucleic acids. Methylglyoxal, for example, can be generated as an unwanted byproduct during glycolysis due to the elimination of phosphate from glyceraldehyde-3-phosphate or dihydroxyacetone-phosphate. Conversion of methylglyoxal and other 2-oxoaldehydes to 2-hydroxycarboxylic acids is catalyzed by the ubiquitous glyoxalase system (Fig. 1) (for a comprehensive review on the system and other glutathione-dependent enzymes see Deponte 2013).


    Fig. 1: The glyoxalase system comprises reduced glutathione (GSH) as a coenzyme as well as the enzymes glyoxalase I and II (GloI and GloII). 2-Oxoaldehydes spontaneously react with GSH to form two diastereomeric hemithioacetals. These are subsequently isomerized to a thioester by GloI. The thioesterase GloII catalyzes the last step of the pathway leading to the regeneration of GSH and the formation of a non-toxic 2-hydroxycarboxylic acid.


    The asexual blood stages of the malaria parasite Plasmodium falciparum divide rapidly (Fig. 2) and rely on a high-power metabolism that is fueled by an excessive glucose consumption. The increased production of harmful methylglyoxal in combination with an inhibition of the glyoxalase system was therefore thought to be detrimental to the parasite (Urscher et al. 2011 and Deponte 2014). However, we recently showed that neither cytosolic GloI nor GloII are essential for blood-stage parasite development and that knockout strains have no significant growth defect (Wezena et al. 2017). Hence, the cytosolic P. falciparum glyoxalases are not suited as drug targets and their physiological relevance remains unknown. An elevated formation of gametocytes after removal of cytosolic GloII suggests that the glyoxalase system might play a role for gametocytogenesis (Wezena et al. 2017).


    Fig. 2, left side: Asexual blood stages of Plasmodium falciparum during the 48 h life cycle. Two ring stages, two early schizonts, three later schizonts, and the release of merozoites are shown clockwise. Right side: Comparison of the domain architectures of homodimeric human GloI and monomeric GloI from P. falciparum. The active sites that are composed of two homologous domains are boxed. Please note that two distinguishable active sites are present in the monomeric enzyme. See Deponte et al. 2007 for details.


    Monomeric GloI from P. falciparum is interesting from an enzymological perspective because the enzyme has two distinguishable active sites in contrast to homodimeric human GloI (Fig. 2). Several years ago, we could show that both active sites of monomeric GloI are not only functional but have different substrate affinities and are allosterically coupled (Fig. 3) (Deponte et al. 2007). As a consequence, the enzyme exists in at least two different states, a high-affinity state and a high-activity state. These states might be a molecular adaptation of the parasite to different host environments and metabolic conditions, whereas the different substrate affinities of the active sites might point to alternative physiological substrates (which might also explain the evolution of monomeric GloI-isoforms in several unrelated organisms).


    Fig. 3: Catalytic model for monomeric GloI from P. falciparum. The enzyme exists in at least two different conformations depending on the substrate concentration. At lower substrate concentrations the high-affinity conformation is found, whereas at higher substrate concentrations substrate binding to one reaction center also stabilizes the high-activity conformation at the other reaction center. See Deponte et al. 2007 for details.


    Inhibition studies by Miriam Urscher revealed that methyl-gerfelin and nanomolar tight binding inhibitors block both active sites of cytosolic P. falciparum GloI. This is in contrast to curcumin, which preferentially interacts with the reaction center formed between the N- and C-terminal domains. Miriam's studies have important implications for the interpretation of previous and future inhibitor screening projects, which are often performed with commercial monomeric glyoxalases. Methyl-gerfelin was also moderately active in cell culture, killing P. falciparum blood stage parasites preferentially at the ring stage (Urscher et al. 2010 and Urscher et al. 2012). More recent inhibition studies in combination with knockout experiments suggest, however, that the detected antimalarial properties of the inhibitors were most likely due to off-target effects (Wezena et al. 2016 and Wezena et al. 2017).


    We also analyzed the catalytic mechanism of cytosolic P. falciparum GloII (Fig. 4), which is a member of the diverse binuclear metallohydrolase family. Our studies revealed that substrate binding (i) is mainly achieved via ionic protein-glutathione interactions and (ii) is a rate-limiting step occurring at a similar rate as product formation. Inhibition studies were in agreement with a Theorell-Chance "hit-and-run" mechanism with the hydroxycarboxylic acid and GSH as first and second product, respectively. A Theorell-Chance mechanism could also explain why the reaction velocity of many glyoxalases is only slightly affected when the metal ion at the reaction center is replaced. In addition, we were lucky to unmask acid-base catalysis and the formation of the hydroxide ion at the metal center for the first time. Our findings resulted in an updated and much more detailed model of GloII catalysis (Urscher and Deponte 2009).

    Fig. 4: Acid-base catalysis and substrate binding of cytosolic GloII from Plasmodium falciparum. The nucleophilic hydroxide ion is formed at the metal center by significantly lowering the pKa value of water. Important substrate-binding residues that interact with the glutathione-moiety are highlighted. See Urscher and Deponte 2009 for details.


    Structure-function analyses of cytosolic P. falciparum GloII also led to the discovery that the enzyme exists in an unexpected monomer-dimer equilibrium, which might play a role under physiological conditions. Moreover, in collaboration with Jude Przyborski, we demonstrated for the first time a plastid localization of a glyoxalase II. In our case the isozyme tGloII was found in the apicoplast of P. falciparum, pointing towards an alternative, yet unidentified, metabolic pathway (Urscher et al. 2010 and Urscher et al. 2011).


    The project was supported by the DFG (grant DE 1431/1).


Further projects


The mitochondrial protein import machinery of parasitic protists

GFP-tagging sheds light on protein translocation


Glutathione-dependent catalysis

Hydroperoxide and thiol-disulfide metabolism - new enzymes, new lessons