Biosynthesis of Non-Natural Deoxyribonucleotides

• Applicant: Dr. Christoph Loderer
• Subject Area: Biological Process Engineering
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 445451075

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.

• DFG Programme: Research Grants
• International Connection: Sweden
• Cooperation Partners: Professor Anders Hofer; Professor Daniel Lundin