FWF - Nonheme Fe(II) - Nonheme Fe(II) hydrolxylases: from structure-function relationship to redesign

Selective oxygenations of non-activated carbon atoms by O2 are a major target in green chemistry, as they are typically inaccessible by abiotic synthetic means. Nature, by contrast has evolved structures that allow the highly selective oxidation of organic molecules by O2, the two most versatile being the P450 heme and the nonheme Fe(II) centers. Despite their unique catalytic potential, and contrary to the P450s, nonheme Fe(II) enzymes are not generally used as a platform for designed oxygenations. Reasons therefore may include their instability outside the cell and complications regarding the establishment of reliable, efficient whole cell screening methods for these systems. These factors limit the applicability of evolutionary methods for enzyme engineering. Rational methods of protein redesign that are rooted in an in depth understanding of the interplay of enzyme structure and function may help to bridge this gap. α-KG dependent nonheme Fe(II) hydroxylases (α-KG-MNH) are an enzyme grouping that transforms cell metabolites via oxidative hydroxylation. They all share a cupin fold and a common metal center organization. Yet, particular exponents of the α-KG-MNHs show high but distinct stereo-, regio-, and substrate-selectivities. In this project the structural basis of this diversity, which is not well understood, will be explored. Therefore exponents of α-KG-MNHs are subjected to a combination of experimental and computational methods. Mutational analysis and kinetic and spectral characterizations are correlated with molecular dynamic studies in order to gain insights into the impact of the protein structure and on particular steps of catalysis. The goal of the study is to introduce new catalytic functions into the protein scaffolds. The applicability of molecular dynamic studies to rationalize and predict catalytic properties in silico based on quantitative descriptors will be assessed. Results will furthermore be put in context with available data of α-KG-MNHs from literature and sequence data, in order to define the structural motifs that bring about stereo-, regio-, and substrate-selectivity in α-KG-MNHs. The overriding aim of the project is to increase the knowledge basis regarding the interplay of protein structure and catalytic properties of α-KG-MNH, in order to facilitate their computer assisted redesign and to expand their catalytic repertoire. Ultimately, this may open new routes for the sustainable biosynthetic production of added-value chemicals from feed stock.