Download Summary Summary Glutaredoxins (Grx) and Glyoxalases 1 and 2

Summary
Summary
Glutaredoxins (Grx) and Glyoxalases 1 and 2 (Glo1 and Glo2) are ubiquitous glutathionedependent enzymes. During the last few years, my group and I biochemically characterized the novel isoforms ScGrx6, ScGrx7, and ScGrx8 from the yeast Saccharomyces
cerevisiae and PfGlo1, PfcGlo2, and PftGlo2 from the malaria parasite Plasmodium
falciparum. These enzymes turned out to be perfect model proteins for the discovery of
new concepts in glutathione-dependent metabolism with respect to enzyme catalysis,
molecular evolution, and molecular cell biology.
Yeast glutaredoxins
ScGrx6-8 are unique proteins. ScGrx6 and ScGrx7 are not only membrane-anchored, but
are also the first Grx-isoforms targeted to the secretory pathway, namely the cis-Golgi.
Both enzymes are highly suited to study structure-function relationships because they
differ from all monothiol Grx-isoforms investigated so far and rather share features with
canonical dithiol Grx. For example, they are the first active monothiol Grx-isoforms in
the standard enzymatic assay with the model substrate HEDS. Although sequential
kinetic patterns raise questions about the precise mechanism in this assay, ScGrx7 was
clearly shown to reduce the mixed disulfide between glutathione and cysteine via a pingpong mechanism. ScGrx7 and ScGrx6 are furthermore the first Grx forming non-covalently linked dimers, and ScGrx6 is the first monothiol Grx-isoform shown to directly
bind an Fe/S-cluster. The cluster is stabilized by reduced glutathione. Its loss results in
the conversion of tetrameric to dimeric ScGrx6, suggesting a regulatory function. ScGrx8
is a novel dithiol Grx-isoform with a significantly altered substrate binding site and a
Trp14-type cysteine motif at the catalytic center. Deletion of the corresponding gene does
not result in a reduced growth rate under oxidative stress conditions, nor does it enhance
the defects of Δgrx1 and Δgrx2 single or double mutants. ScGrx8 has a low activity and
autoxidation rate and is the first Grx-isoform requiring both cysteine residues for glutathione-dependent catalysis in the HEDS assay. Studies on ScGrx7 and ScGrx8 revealed
that the reductive half-reactions of both enzymes differ significantly and that the standard
catalytic model for Grx is too limited. A refined model for monothiol and dithiol Grxcatalysis explains the differences among all isoforms in vitro and in vivo.
Plasmodium glyoxalases
Malaria parasites possess a cytosolic glyoxalase system and an insular Glo2-isozyme in
the apicoplast. In fact, PftGlo2 is the first plastid glyoxalase discovered so far. Moreover,
PfcGlo2 is the first Glo2-isozyme shown to exist in a rapid monomer-dimer equilibrium.
This feature might have been overlooked for Glo2-isoforms from other organisms and
could be of physiological importance. As far as Glo2-catalysis is concerned, a TheorellChance Bi Bi mechanism was discovered (with a hydroxide ion and the thioester as first
and second substrate, and carboxylic acid and GSH as first and second product, respectively). Glutathione is predominantly bound via two conserved basic residues close to the
C-terminus of the enzyme. The mechanism not only explains acid-base catalysis, but also
other enzyme-features such as unusual kinetics, salt-sensitivity and metal-ion promiscuity.
Evolution of monomeric Glo1-isoforms allowed the formation of a second, modified
active site as revealed for PfGlo1. The latter enzyme has a high- and a low-affinity
binding site for its canonical substrate. In the course of evolution, the low-affinity binding
site might have been optimized for an alternative substrate or a regulator. Both active
sites can adopt two discrete conformations and are allosterically coupled. As a result,
PfGlo1 has an increased affinity at low substrate concentrations and an increased activity
at higher substrate concentrations. This could also be the case for monomeric Glo1isoforms from other organisms with implications for physiology. First inhibitors with
promising activities against both active sites of PfGlo1 have been characterized.
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