Biosynthesis of Non-Natural Deoxyribonucleotides

非天然脱氧核糖核苷酸是一类重要的化合物，在不同领域具有应用，从细胞生物学到合成生物学再到医学。这些化合物的化学合成是一个困难、耗时和昂贵的过程。生物催化方法已经可用于核糖核苷酸的合成，但不用于脱氧核糖核苷酸。在自然界中，核糖核苷酸还原酶（RNR）催化DNA合成和修复所需的典型核糖核苷酸还原为相应的脱氧核糖核苷酸。本项目的目标是通过评估和扩展这类酶的底物特异性来探索RNR用于非天然脱氧核糖核苷酸的生物合成。RNR的底物特异性在其天然底物方面得到了很好的表征，但对其将非天然核糖核苷酸转化为相应脱氧核糖核苷酸的能力知之甚少。因此，该项目的第一个目标是确定RNR的底物特异性，重点是具有结构不同核碱基的核糖核苷酸。下一步，将应用酶工程将RNR的底物特异性扩展到重要的非天然核糖核苷酸，这些核苷酸不能被天然酶转化。根据特定非天然脱氧核糖核苷酸的所需应用，磷酸化水平可能是至关重要的（例如单磷酸、二磷酸或三磷酸）。所有天然RNR都对核苷二磷酸或核苷三磷酸底物表现出特异性。目的是控制RNR的磷酸特异性，以获得在核苷单磷酸、二磷酸或三磷酸特异性之间进行选择的能力。这将通过酶工程的磷酸盐结合口袋模型RNRs. RNR变体与修改的底物特异性的产生将伴随着结构研究的X射线晶体学的方式实现。通过比较野生型和修饰酶的结构，可以研究导致所需效果的结构变化。这不仅将提供有关这类重要酶的结构-功能关系的有价值的信息，而且还可以实现更有针对性的酶工程策略。该项目将产生两个，一个具有生产重要非天然脱氧核糖核苷酸能力的RNR工具箱，以及RNR的底物特异性如何由其结构定义的知识。这将为RNR在生物学和医学不同领域的广泛应用铺平道路。

Non-natural deoxyribonucleotides are an important group of compounds, with applications in different fields, ranging from cell biology over synthetic biology to medicine. Chemical synthesis of these compounds is a difficult, time-consuming and expensive procedure. Biocatalytical approaches are available for the synthesis of ribonucleotides already, but not for deoxyribonucleotides. In nature, ribonucleotide reductases (RNRs) catalyse the reduction of the canonical ribonucleotides to the corresponding deoxyribonucleotides, required for DNA synthesis and repair. The objective of this project is the exploration of RNRs for the biosynthesis of non-natural deoxyribonucleotides by evaluating and expanding the substrate specificity of this class of enzymes.The substrate specificity of RNRs is well characterized in respect to their natural substrates, but little is known about their ability to convert non-natural ribonucleotides to the corresponding deoxyribonucleotides. Thus, the first target of this project is the determination of the substrate specificity of RNRs with a focus on ribonucleotides with structurally different nucleobases. In the next step, enzyme engineering will be applied to extend the substrate specificity of RNRs to important non-natural ribonucleotides, which can not be converted by the natural enzymes. Depending on the desired application of a specific non-natural deoxyribonucleotide, the phosphorylation level can be crucial (e.g. mono-, di-, or triphosphate). All natural RNRs do exhibit a specificity to either nucleoside diphosphate or nucleoside triphosphate substrates. The objective is to control phosphate specificity of RNRs to gain the ability to select between nucleoside mono-, di-, or triphosphate specificity. This will be realised by means of enzyme engineering of the phosphate binding pocket in model RNRs.The generation of RNR variants with modified substrate specificity will be accompanied by a structural investigation by means of X-ray crystallography. By comparison of the structures of wild-type and modified enzymes, structural changes that lead to the desired effects can be studied. This will not only provide valuable information about the structure-function relationship in this important class of enzymes but also enable more directed enzyme engineering strategies.The project will yield both, a toolbox of RNRs with the capability to produce important non-natural deoxyribonucleotides and the knowledge how the substrate specificity of RNRs is defined by their structure. This will pave the way for a widespread application of RNRs in different fields of biology and medicine.