PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...

Indiana University-Purdue University Indianapolis (IUPUI) / Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.

Indiana University-Purdue University Indianapolis (IUPUI) / Oxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC 1.2.3.4 OxOx). In this work, wild-type (WT) Bacillus subtilis OxDC and a series of variants with mutations on conserved residues were characterized to investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided information about the mechanism. The Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen and oxalate. Mutations on the second shell residues in the N-terminal metal binding site affected the enzyme activity properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the previously proposed mechanism in which OxDC and the OxDC/OxOx chimeric variant share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing the interaction between the N-terminal and C-terminal domains.

The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.