Comparison of Fungal Thermophilic and Mesophilic Catalase-Peroxidases for Their Antioxidative Properties.

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

Evaluation of enzymatic stamp removal strategies on handmade (cellulose-based) and machine-made (lignin-containing) papers.

Two different biocleaning techniques for stamp removal from different paper samples (handmade and machine-made) were investigated. Cellulose is the main component of handmade paper, while higher concentration of lignin is present in machine-made paper. Biocleaning methods included the direct application on paper surfaces of the extracellular enzymatic mixture (EEM) extracted from the yeast Sporidiobolus metaroseus and the recombinant protein CthediskatG of Chaetomium thermophilum var. dissitum. The produced microbial enzymes (EEM or CthediskatG) were also combined with agarose hydrogels. The effectiveness of the cleaning ability of the individual methods was determined using different spectrophotometer measurements based on colorimetric analysis and by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). Some tested samples were also subjected to microstructural and chemical analysis using Scanning Electron Microscope-Energy-Dispersive X-ray spectroscopy (SEM-EDX). The analysis showed that the EEM-based approaches were the most suitable, mainly they are less time-consuming and easy to produce, and moreover slight differences were displayed between EEM and CthediskatG during the removal of the stamp by hydrogel-enzyme approaches. Both EEM applications (direct and hydrogel) speed up the stamp removal process from real paper samples. However, for the complete elimination of the stamp smears a quick N,N-dimethylformamide post-treatment is advised too.

Deep Insights into the Specific Evolution of Fungal Hybrid B Heme Peroxidases.

In this study, we focus on a detailed bioinformatics analysis of hyBpox genes, mainly within the genomes of Sclerotiniaceae (Ascomycota, Leotiomycetes), which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium-crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum, although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1-specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae. We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.

Parallel Molecular Evolution of Catalases and Superoxide Dismutases-Focus on Thermophilic Fungal Genomes.

Catalases (CAT) and superoxide dismutases (SOD) represent two main groups of enzymatic antioxidants that are present in almost all aerobic organisms and even in certain anaerobes. They are closely interconnected in the catabolism of reactive oxygen species because one product of SOD reaction (hydrogen peroxide) is the main substrate of CAT reaction finally leading to harmless products (i.e., molecular oxygen and water). It is therefore interesting to compare the molecular evolution of corresponding gene families. We have used a phylogenomic approach to elucidate the evolutionary relationships among these two main enzymatic antioxidants with a focus on the genomes of thermophilic fungi. Distinct gene families coding for CuZnSODs, FeMnSODs, and heme catalases are very abundant in thermophilic Ascomycota. Here, the presented results demonstrate that whereas superoxide dismutase genes remained rather constant during long-term evolution, the total count of heme catalase genes was reduced in thermophilic fungi in comparison with their mesophilic counterparts. We demonstrate here, for the newly discovered ascomycetous genes coding for thermophilic superoxide dismutases and catalases (originating from our sequencing project), the expression patterns of corresponding mRNA transcripts and further analyze translated protein sequences. Our results provide important implications for the physiology of reactive oxygen species metabolism in eukaryotic cells at elevated temperatures.