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