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.