Atypical Heme Peroxidase Family

Hybrid type heme peroxidases are yet unknown families of oxidoreductases discovered recently during phylogeny reconstruction. They represent missing links within the peroxidase-catalase superfamily namely between catalase- peroxidases, cytochrome c peroxidases, ascorbate peroxidases and manganese peroxidases. We have chosen four representatives of the hybrid type B family from ascomycete fungi for detailed investigation of their expression as well as for characterization of their biophysical and biochemical properties. All hybrid B peroxidases are secretory proteins with a distinct signal sequence. Two paralog hybrid peroxidase sequences were selected from the rice blast fungus Magnaporthe grisea. One of them appears as a fusion protein containing a peroxidase domain connected with a WSC carbohydrate domain. The third candidate originates from a phytopathogen Sclerotinia sclerotiorum and reveals a quite unusual sequence of the distal catalytic triad not found in any other out of over 5,000 known heme peroxidase sequences. The fourth representative from the soil fungus Chaetomium globosum reveals a moderate variation of the distal catalytic triad essential for the heterolytic cleavage of the peroxidic bond. The investigations will start with a quantitative real-time PCR screening of the expression of mentioned peroxidases under normal and oxidative stress conditions. In parallel also the corresponding enzymatic activities will be monitored with application of specific inhibitors to select among various possible peroxidases activities in crude samples of sac fungi. For the heterologous expression of complete genes simple and advanced levels are suggested. In the simple level synthetic genes for hybrid B peroxidase domains containing a C-terminal his-tag will be produced in E. coli intracellularly. In the advanced level complete RT-PCR products will be expressed in Pichia pastoris or in Aspergillus niger extracellularly. The glycosylation pattern and disulfide bridges formation will be compared between the natural and heterologously expressed forms. Spectroscopical investigations will include UV-vis and resonance Raman spectroscopy as well as electronic circular dichroism spectra. Stability investigations will be performed via differential scanning calorimetry. Redox potential of the Fe3+/Fe2+ couple will be carried out using an optically transparent thin-layer spectroelectrochemical cell. From this measurement valuable information about iron-ligand binding interactions and the electrostatics at the interface between iron and the protein environment can be obtained. Finally, pre-steady state and steady state kinetics can deliver essential informations on the reactivity of these promising heme peroxidases with a variety of potential substrates. The principles of reaction mechanisms will be explained within this attempt. As for other peroxidases the formation of reactive intermediates namely Compound I , Compound II and eventually also Compound III will be followed with stopped flow technique. The effectiveness of Compound II reduction back to the ferric state will be monitored with various electron donors to search for the most probable natural substrate of fungal hybrid peroxidases.

The research project Atypical Heme Peroxidase Family investigated a novel protein family of heme containing peroxidases that are positioned phylogenetically at the border between distinct previously defined structural classes within the large peroxidase-catalase superfamily. Bifunctional catalase-peroxidases, monofunctional ascorbate peroxidases, cytochrome c peroxidases, lignin and manganese peroxidases as well as plant secretory peroxidases are typical representatives of this unique superfamily that are known already for decades. They play essential physiological roles in bacteria, archaea, fungi and plants in detoxifying reactive oxygen species, mainly hydrogen peroxide. H2O2 not only emerges as metabolic by-product but it has also an important signalling and effector function for the cells. There are differences in the reactivity and function of mentioned peroxidases but their active centers are highly conserved. We have intensively searched for evolutionary intermediates between these distinct oxidoreductases and found two hybrid heme peroxidase families that are positioned between the defined families not only phylogenetically but also possessing intermediate enzymatic properties. For our detailed biochemical and biophysical investigations we have chosen the rather atypical hybrid B heme peroxidase family that occurs exclusively among various fungi. It is important to note that several gene paralogs of hyBpox genes are present mainly in genomes of phytopathogenic fungi. This underlines their potential importance for host pathogen interactions, mainly that they could be involved in defence mechanisms of pathogenic fungi against oxidative burst induced in the plant host upon infection. Our detailed sequence analysis revealed that most of hybrid B peroxidases are fusion proteins where the N-terminal peroxidase domain is fused with a C-terminal WSC- sugar binding domain that represents a novel and mostly undiscovered type of fungal sugar-associated domains. We have cloned selected synthetic hyBpox genes and investigated the corresponding recombinant proteins expressed either in E.coli or in P.pastoris to study their structure-function relationships and compare them with typical representatives of heme peroxidases.