Selective hydroxylation of L-proline and derivatives with proline hydroxylases - An approach to reactivity und mechanism

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Prolinehydroxylases, which belong to the enzyme class of the alpha-ketoglutarate dependent dioxygenases, are an ideal model for investigations in enzyme-catalyzed CH-activation reactions. First, enzymes with different regio- and stereoselectivities are available, so that factors directing regio- and stereoselectivities can be determined more easily. Second, hydroxyprolines and their derivatives are valuable chiral building blocks for the synthesis of pharmaceuticals and other active agents, which are only difficult to access by means of classical organic chemistry. The prolinehydroxylases known so far shall be characterized thoroughly by applying different methodological approaches, which, in combination, allow a deeper understanding of the catalytic activity and the causes of different selectivities. To investigate substrate specificity and catalytic selectivity, the active center of the prolinhydroxylases will be investigated by electron paramagnetic resonance and other spectroscopic methods. In parallel, the catalytic properties of the enzymes will be characterized using an assay with natural and non-natural substrates. By means of mutagenesis variants will be generated and included into the investigation. The aim is to understand the factors determining selectivity and substrate specificity of the prolinehydroxylases to make it possible to modify the catalytic properties of the enzymes in rational approaches. In this way new and biotechnologically usable CH-activating biocatalysts shall be generated.