Hybrid B Heme Peroxidases from Thermophilic and Mesophilic Fungi

Hybrid B heme peroxidases (HyBpox) constitute a distinct family of iron containing oxidoreductases within the peroxidase-catalase superfamily. Recent phylogenetic analysis demonstrated that these metalloenzymes represent evolutionary turning points of the complex history of this protein superfamily. Already over 100 putative sequences are known with interesting motifs at the distal and proximal sides of the catalytic heme center that reveal deviations compared to well known members of the so far described classes. All known HyBpox representatives are spread within the fungal kingdom and the majority seems to be present in various pathogens. Recently, we could demonstrated that hyBpox genes are expressed in phytopathogenic & soil fungi and that their production is induced by various forms of oxidative stress. In this research project we want to investigate the structure-function relationships of three selected representatives: MagHyBpox1 from the pathogenic rice blast fungus Magnaporthe oryzae, CgHyBpox2 is a counterpart from a typical soil fungus Chaetomium globosum and CtheHyBpox is a variant from a thermophilic fungus Chaetomium thermophilum able to grow at 50C. Expression of the corresponding synthetic gene constructs in the Pichia pastoris and protein purification has been shown to work and will be improved. Recombinant proteins will be characterized by mutational analysis and application of a broad set of biophysical/chemical methods including (pre)steady-state kinetics, UV-vis, CD- and EPR spectroscopy, spectroelectrochemistry as well as X-ray crystallography of wild-type and mutant proteins. Based on structure and mechanistic aspects specific inhibitors should be designed based on data derived from high-throughput in silico docking. Potential lead compounds will be tested for future combating of those phytopathogenic fungi.

Peroxidases are essential oxidoreductases responsible for the cleavage of peroxidic bonds in many for the cells potentially harmful and very reactive substances. All known peroxidases can be divided into heme-containing and non-heme representatives. Almost all known heme peroxidases can be classified within four divergent superfamilies. Hybrid B heme peroxidases represent a completely newly discovered unique oxidoreductase subfamily of the previously defined Peroxidase-Catalase superfamily. Their coding sequences are phylogenetically positioned between already well-known Family II (manganese and lignin peroxidases) and Family III (plant secretory peroxidases) members. Within this research project we have investigated selected members of this peculiar heme b peroxidase subfamily from diverse fungi at the level of mRNA and protein macromolecules. First, we have monitored the native expression of several hyBpox genes from filamentous fungi under various physiological conditions. It came out that the corresponding mRNA transcripts are highly induced with various types of oxidative stress. Afterwards, we have chosen 3 typical genes from the genomes of 2 mesophilic and 1 thermophilic filamentous fungi for a heterologous expression in the methylotrophic yeast Pichia pastoris being appropriately codon optimized. After their secretion from donor cells we proceeded with an affinity purification of recombinant peroxidases followed by ion exchange and size exclusion chromatography. Thus, we have obtained highly purified samples that were investigated with divergent biochemical and biophysical methods. By the means of circular dichroism we were able to exactly determine the content of the secondary structure elements within newly discovered heme peroxidases. We could compare them with those peroxidases that already possess known 3D structures. Afterwards, we have investigated the reaction kinetics with up to 12 different electron donors with photometrical and fluorometrical methods and also compared with previously known peroxidases. From a detailed inspection of the protein sequence it could be claimed that hybrid B heme peroxidases contain besides a highly conserved heme domain also additional domains that apparently do not possess any peroxidase activity. We could classified these domains either to carbohydrate-binding modules (CBM classified in the CAZy database) or to stress-dependent domains with water- soluble sugars (WSC). From such analysis one can deduce also the probable physiological function of these unique hybrid peroxidase sugar binding fusion proteins mainly among various phytopathogenic fungi.