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

脯氨酸羟化酶属于α -酮戊二酸依赖双加氧酶的一类，是研究酶催化ch活化反应的理想模型。首先，具有不同区域选择性和立体选择性的酶是可用的，因此可以更容易地确定指导区域选择性和立体选择性的因素。其次，羟脯氨酸及其衍生物是合成药物和其他活性物质的重要手性组成部分，仅通过经典有机化学方法难以获得。目前已知的脯氨酸羟化酶应该通过应用不同的方法来彻底地表征，这些方法结合在一起，可以更深入地了解催化活性和不同选择性的原因。为了研究底物特异性和催化选择性，将利用电子顺磁共振和其他波谱方法研究脯氨酸羟化酶的活性中心。同时，酶的催化性能将使用天然和非天然底物的测定来表征。通过诱变，将产生变异并纳入调查。目的是了解决定脯氨酸羟化酶的选择性和底物特异性的因素，从而有可能以合理的方法修饰酶的催化性能。通过这种方式，将产生新的和生物技术上可用的ch活化生物催化剂。

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.