Molecular Evolution of Heme Peroxidases from Cephalochordata

Cephalochordata heme peroxidases represent yet undiscovered ancient evolutionary line leading to well known mammalian thyroid peroxidases that share with them a common ancestor. Together, they exhibit also a very high level of sequence similarity along their entire coding regions with fully conserved active centres for the catalysis. In this project we will investigate the structure-function relationships of these peculiar peroxidases from simplest chordates. We have selected the unknown intracellular peroxidase BbePOX1 from Branchiostoma belcheri and the extracellular counterpart BfPOX2 from Branchiostoma floridae that will be both produced in different cellular expression systems. First, we will unveil the native expression of corresponding lancelet genes under various physiological conditions and different tissues by a very sensitive method, real-time quantitative PCR. In the next stage we will focus on the typical optical spectra of purified recombinant Branchiostoma peroxidases under various conditions and binding of ligands in their active centres. With the mass spectrometry we will also verify whether the typical covalent link formed autocatalytically between the prosthetic heme group and the polypeptide chain, as detected previously in all mammalian heme peroxidases occurs among their lancelet counterparts. Later, we will try to uncover also their typical reaction mechanism with hydrogen peroxide and iodine forming hypoiodous acid that has strong bactericidal effects. The main focus will be set on iodide oxidation as well as iodination of selected molecules which leads to the formation of thyroxine and related animal hormones. After obtaining complex biophysical and biochemical results for both enzymes we want to evaluate whether these peculiar peroxidases can be used as a suitable model system for human thyroid peroxidase that although medically very important is still not sufficiently investigated at the molecular level. With various biophysical methods in solution and in formed protein crystals we will proceed with the determination of a first experimental 3D structure of a peroxidase involved in physiological iodination process. Finally, our results shall significantly contribute to the understanding of how far can these peroxidases with covalently linked heme to protein be involved in the innate immunity of these frequently occurring marine invertebrates.

In this research project, bioinformatic in silico analyses were performed, focusing on genomic data mining, sequence conservation patterns, and robust phylogenetic reconstruction. A robust phylogenetic tree of peroxidases from the entire peroxidase-cyclooxygenase superfamily was constructed by analyzing 620 full-length sequences. This led to the discovery of the particular gene architecture of the peroxidases encoded by the Amphioxus genome, which are phylogenetically positioned between family 1 and family 2 of the peroxidase-cyclooxygenase superfamily. Comparison of the corresponding parts of the genomes revealed a complex evolutionary pattern and showed that putative representatives of the cephalochordate peroxidases are evolutionarily basal to all vertebrate thyroid peroxidases and also to all peroxidasins. The best representative of the cephalochordate peroxidases, the peroxidase from Branchiostoma belcheri, was heterologously expressed in Pichia pastoris and characterized by extensive biophysical and biochemical studies. The same in silco methods were applied to hybrid B heme peroxidases and glutathione peroxidases. In this work, a new hybrid B heme peroxidase was discovered as the only peroxidase in the secretome of the important phytopathogenic fungus Sclerotium cepivorum and was studied at the proteome level, and the construction of a large glutathione peroxidase tree elucidated the evolutionary relationships among all phyla. The molecular phylogeny of peroxygenases, known to be highly versatile biocatalysts, was also reconstructed. These oxidoreductases, which are mainly capable of oxyfunctionalization, constitute the superfamily of peroxidase peroxygenases. Representative reconstruction revealed high diversity but also well-conserved sequence motifs within relatively short protein molecules. Corresponding genes encoding peroxygenases were found in only a few lower eukaryotes. Most peroxygenases originate from the kingdom of fungi and, in particular, clades, which have a very ancient evolutionary origin. There is also a distinct clade formed mainly by phytopathogenic stramenopiles, in which even peroxygenase sequences from amoebozoans can be found. The phylogenetically older peroxygenases are mostly intracellular, but later evolution gave rise to secretory proteins, especially in pathogenic fungi.