Biochemistry of peroxidasins

Members of the peroxidase-cyclooxygenase superfamily catalyse biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. Subfamily 2 of this superfamily is comprised of multidomain oxidoreductases called peroxidasins. Peroxidasins (Pxd) are glycosylated secreted heme peroxidases having in addition leucine-rich repeat domains, C-like immunoglobulin domains as well as a von Willebrand factor C module. These are typical motifs of extracellular proteins that mediate protein-protein interactions. Peroxidasin was initially discovered as a basement membrane constituent and later on was shown to be involved in innate immune reactions, extracellular matrix formation, tissue development, tumor progression as well as oxidative reactions that lead to induction of cell apoptosis. Very recently it was demonstrated that human peroxidasin forms sulfilimine bonds in collagen IV by hypohalous acids thus catalysing a reaction that is important in tissue development and human disease. Despite the upcoming biological importance of this protein family the biochemical knowledge is very poor. Here, based on a comprehensive phylogenetic analysis and comparison with chordata peroxidases as well as successful expression and recombinant production of human peroxidasin 1, we aim at elucidation of structure-function relationships of four peroxidasins at different evolutionary level. In detail the biochemistry of peroxidasin 1 from Caenorhabditis elegans, Pxd from Drosophila melanogaster as well as human peroxidasins 1 & 2 will be investigated. Recombinant production of the four full-size model proteins as well as of truncated forms that lack distinct domains will allow comprehensive biochemical/physical analyses including (i) UV-vis-, fluorescence-, CD-, multi-angle light scattering-, resonance Raman- and EPR spectroscopy, (ii) stopped-flow spectroscopy and polarography, (iii) mass-spectrometry and X-ray crystallography, (iv) site directed mutagenesis, (v) spectroelectrochemistry and (vi) DSC. Those methods will help to elucidate the structure and enzymatic activity of these metalloproteins including (i) oligomeric state and architecture of substrate access channel(s) and heme cavity, (ii) domain interaction and (un)folding pathway(s), (iii) chemistry of the prosthetic group including oxidation and spin-state(s), ligation and posttranslational modification(s), (iv) substrate pattern and specificity, accessibility and binding site of substrate molecules and ligands as well as nature of reaction products, (v) chemistry, reactivity and relevance of enzymatic redox intermediates (ferrous and ferric forms, Compounds I, II and III) and of their redox thermodynamics and (vi) the role of (conserved) active site amino acids in substrate/ligand binding and conversion. It is the clear goal of this project to understand the molecular basis of substrate oxidation and conversion of peroxidasins at all relevant redox sub-steps and to present the first high-resolution structures of these multi-domain oxidoreductases. Moreover, functional and structural comparison of the two invertebrate and the two vertebrate peroxidasins will give new insights into the evolution of this protein family as well as in its proposed roles in innate immunity and/or extracellular matrix formation.