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


    Peroxiredoxins (Prx) and glutaredoxins (Grx) are key players in redox metabolism. While Prx reduce hydroperoxides and can also act as chaperones and sensors, Grx catalyze dithiol-disulfide exchange reactions and exert different functions in iron-metabolism (see Deponte 2013 and Deponte 2017 for review). For example, enzymatically active Grx utilize reduced glutathione (GSH) for the reduction of inter- or intramolecular disulfide bonds (Fig. 1).

    Fig. 1: Traditional model of the Grx-dependent reduction of inter- (left side) or intramolecular (right side) disulfides by GSH. Grx have either one or two reactive cysteine residues at the active site and can therefore be classified as monothiol and dithiol glutaredoxins, respectively. Only one cysteine residue is required for the mechanism on the left side. See Eckers et al. 2009 for details.


    Although Prx and Grx are known for a long time, more recent studies revealed that these proteins form mechanistically and funtionally highly heterogeneous groups. Thus, Prx and Grx are great examples of proteins that underwent subtle structural changes throughout evolution resulting in completely different properties and functions. The following questions are addressed:


    - How do the mono- and dithiol isoforms of Prx or Grx differ?


    - What makes Prx and Grx active/inactive in standard enzymatic assays?


    - How do some Prx act as chaperones and Grx bind iron-sulfur clusters?


    - Which features determine the quaternary structure of Prx and Grx?


    - How are Prx- and Grx-dependent reactions exactly catalyzed?


    Regarding the diversity of Prx, we recently established the Prx5-type isoform from Plasmodium falciparum (PfAOP) as a model enzyme for Grx/GSH-dependent peroxiredoxins (Djuika et al. 2013). A mutational follow-up analysis of the enzyme revealed a novel mechanism for balancing substrate turnover and Prx inactivation (Staudacher et al. 2015). This mechanistic model was further supported and strenghtened by stopped-flow kinetic measurements in collaboration with Madia Trujillo and Rafael Radi as well intracellular ratiometric redox measurements with roGFP2-tagged PfAOP in collaboration with Bruce Morgan (Staudacher et al. 2017). In addition, results from a collaboration with Peer Bork's group pointed towards an unusual prokaryotic ancestry and gene fusion event resulting in a dual localization of PfAOP in malaria parasites (Djuika et al. 2015).

    Regarding the diversity of Grx, we previously characterized and compared three novel Grx-isoforms from yeast in collaboration with Vincent Stroobant and Johannes Herrmann's group (Mesecke et al. 2008a, Mesecke et al. 2008b, and Eckers et al. 2009): Grx6 and Grx7 both have a significant enzymatic activity with low molecular weight model substrates in contrast to most other monothiol glutaredoxins. However, the apparently similar proteins have quite different properties considering catalytic turnover, Fe-S cluster binding and subunit oligomerization (Fig. 2).


    Fig. 2: Oligomerization of yeast Grx6 and Grx7 and glutathione-dependent binding of a Fe-S cluster in Grx6. See Mesecke et al. 2008a for details.


    Yeast Grx8 is an unusual dithiol glutaredoxin because it requires both cysteine residues for catalysis and has a rather low activity with model substrates (Eckers et al. 2009). Based on our studies on Grx6-8, we proposed a refined model of Grx catalysis and postulated the existence of two distinct glutathione interaction sites (Fig. 3), which we later confirmed experimentally (Begas et al. 2017). The model is suited to explain the observed phenomena for mono- and dithiol Grx and can be used for the prediction of mechanistic features and physiological functions of Grx-isoforms (Deponte 2013 and Deponte 2017). For example, based on a comparative study on yeast Grx7 and the dithiol Grx from P. falciparum, we showed that Grx can also efficiently convert low-molecular weight disulfide substrates using an alternative mechanism (Begas et al. 2015).

    Fig. 3: Refined mechanistic model for Grx-dependent catalysis. Click on picture to enlarge. See Eckers et al. 2009 for details.


Further projects


The mitochondrial protein import machinery of parasitic protists

GFP-tagging sheds light on protein translocation


Glutathione-dependent catalysis

The glyoxalase system of the malaria parasite Plasmodium falciparum