Changes in the redox state of protein cysteines in response to dynamic alterations of the cellular redox state modulate the activity and structure of proteins. Therefore, several vital cellular processes are redox regulated, including nitric oxide production, apoptosis, immune response, and signaling processes. The aim of this dissertation was to characterize in detail the redox regulation of two target proteins: methionine adenosyltransferase of the malaria parasite Plasmodium falciparum (PfalMAT) and the dye-decolorizing peroxidase 1 of the fungus Mycetinis scorodonius (MscDyP1).PfalMAT is an attractive drug target since its product, S-adenosylmethionine, is the major biochemical methyl group donor and a precursor for crucial downstream pathways such as polyamine metabolism. However, PfalMAT has so far been poorly characterized. This dissertation provides a detailed kinetic analysis, including inhibitor and oligomerization studies to expand the biochemical characterization of this enzyme. Furthermore, the dimeric human homolog hMATIII was characterized here using the same assay system to allow a direct comparison of both enzymes. A second aim of this dissertation was to characterize the interplay of PfalMAT with the plasmodial redoxins thioredoxin-1, glutaredoxin, and plasmoredoxin, as well as to study in detail the impact of the thiol-based post-translational modifications S-glutathionylation and S-nitrosylation on the activity and oligomerization of PfalMAT. In order to identify potential sites of these redox modifications in PfalMAT a homology model was created, which indicated that among the eight cysteines of PfalMAT, Cys52, Cys113, and Cys187 were solvent accessible in the dimeric enzyme. Hence, these cysteines were conservatively mutated via site-directed mutagenesis into serines, and their kinetic and oligomerization behaviors were studied using a spectrophotometric assay and gel filtration experiments, respectively. PfalMAT activity was indeed affected by redox regulatory processes; reduction of cysteines by the reducing agent dithiothreitol and reduced glutathione activated PfalMAT, whereas enzyme inhibition followed oxidative events, i.e. S-glutathionylation and S-nitrosylation. Consequently, the impact of the selected cysteines on these redox regulatory processes was studied, showing that thioredoxin 1 and plasmoredoxin potently activated PfalMAT at subcellular concentrations by specifically changing the oxidation state of Cys52 and Cys113, whereas glutaredoxin did not alter PfalMAT activity. Protein-S-glutathionylation inhibited PfalMAT via a thiol-disulfide exchange between glutathione disulfide and Cys113. Deglutathionylation was mediated by thioredoxin 1 and plasmoredoxin, which restored the activity of PfalMAT at physiological concentrations, suggesting a likely in vivo regulation. Furthermore, PfalMAT was inhibited upon S-nitrosylation of Cys52, Cys113, and Cys187 under NO-stress. These results indicate a coupling of the S-adenosylmethionine status to the cellular redox state of P. falciparum via redox regulation of PfalMAT.The second enzyme on focus of this dissertation, was MscDyP1. This dye-decolorizing peroxidase (DypPrx) is a member of the recently identified eponymous novel class of heme peroxidases, which differ structurally and mechanistically from classic heme peroxidases. Within this dissertation, upon systematic screening and optimization of different crystallization conditions, high resolution protein crystals of MscDyP1 were obtained, that allowed to solve the three-dimensional structure of MscDyP1. This structure revealed interesting features. Two surface-exposed methionine residues, Met302 and Met305, which are not conserved among DypPrx, appeared to play an important structural role, confirmed by site-directed mutagenesis. Furthermore, the single conserved cysteine,Cys360, is connected to the fifth proximal coordination partner of the heme iron, thus contributing to the formation of the active site. Cys360 accessible to small redox-active molecules indicating a redox regulation of MscDyP1 might be possible. Site-directed mutagenesis studies using a recombinant enzmye heteroogously overexpressed in E. coli revealed adverse effect on activity, substrate affinity to hydrogen peroxide. And a higher resistance to suicide inhibition upon high hydrogen peroxide concentrations upon a lack of Cys360, indicating Cys360 participates in the regulation of MscDyP1 via redox processes in vitro. The native enzyme is highly glycosylated, which was shown to protect Cys360 from oxidative effects, indicating redox regulation of MscDyP1 is rather unlikely in vivo.
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