KRED-Origami: Synthesis and Characterization of Ketoreductase Cascades Assembled on DNA Origami Nanostructures
Final Report Abstract
In this project, concepts of DNA nanotechnology were used for the fabrication and characterization of multi-enzyme systems arranged at the nanometer scale in order to improve the understanding of functional, spatially organized biomolecular networks. To this end, supramolecular DNA origami nanostructures (DON) bearing characteristic arrays of recombinant ketoreductases (KRED) and NAD(P)H-regenerating enzymes (NRE) should be synthesized and characterized. The functionality of the enzyme-DON constructs should be evaluated using quantitative data of their catalytic activity and stereoselectivity by means of a chromatographic assay based on the stereoselective conversion of a CS-symmetrical nitrodiketone substrate. Questions concerned on the one hand methodological aspects, such as the selection and molecular cloning of bioconjugatable KREDs and NREs, the establishment of site-selective DNA ligation methods, and the construction and analysis of origami-based NRE-KRED cascades. Furthermore, overarching questions should be addressed, in particular, whether it is possible to control the overall stereoselectivity of the constructs using cascades carrying both (S)- and (R)- selective KREDs. In addition, supported by theoretical work among others, it should also be evaluated whether design rules for effective multi-enzyme/cascade systems can be identified that contribute to the understanding of the fundamental mechanisms of spatially organized, diffusionbased, multicenter biocatalysts and ultimately can be exploited for practical applications. In this project, on the one hand, refined methodological approaches and tools were successfully developed. In particular, these included efficient linker systems based on genetically encoded binding tags, microbead-based purification strategies for enzyme-decorated DON constructs, highly sensitive analytics based on chiral HPLC coupled with MS/MS detection, and experimental platforms and simulation tools for fluidically controllable biocatalytic reaction environments. The here developed tools and results will help to perform detailed structure-activity relationship studies to investigate the underlying mechanisms of compartmentalized biomolecular cascades and substrate channeling. The nucleic acid-based multienzyme systems used in this project are ideally suited for such basic studies. However, for commercial applications of compartmentalized enzyme cascades, protein-based systems are likely to be advantageous due to their much lower cost.
Publications
- (2017) Orthogonal Surface Tags for Whole-Cell Biocatalysis. Angew Chem Int Ed Engl 56, 2183-2186
Peschke, T., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1002/anie.201609590) - (2017) Self- Immobilizing Fusion Enzymes for Compartmentalized Biocatalysis. ACS Catal 7, 7866-7872
Peschke, T., Skoupi, M., Burgahn, T., Gallus, S., Ahmed, I., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1021/acscatal.7b02230) - (2018) On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes. Angew Chem Int Ed Engl 57, 5539-5543
Maier, M., Radtke, C. P., Hubbuch, J., Niemeyer, C. M., Rabe, K. S.
(See online at https://doi.org/10.1002/anie.201711072) - (2018) Self-Assembling All-Enzyme Hydrogels for Flow Biocatalysis. Angew Chem Int Ed 57, 17028-17032
Peschke, T., Bitterwolf, P., Gallus, S., Hu, Y., Oelschlaeger, C., Willenbacher, N., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1002/anie.201810331) - (2019) Solid-Phase Synthesis and Purification of Protein–DNA Origami Nanostructures. Chem. Eur. J. 25, 3483-3488
Burgahn, T., Garrecht, R., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1002/chem.201805506) - (2019) Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels. Chem Sci 10, 9752-9757
Bitterwolf, P., Gallus, S., Peschke, T., Mittmann, E., Oelschlaeger, C., Willenbacher, N., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1039/c9sc04074a) - (2020) Evaluation of a Microreactor for Flow Biocatalysis by Combined Theory and Experiment. ChemCatChem 12, 2452 – 2460
Burgahn, T., Pietrek, P., Dittmeyer, R., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1002/cctc.202000145) - (2020) Surface Display of Complex Enzymes by In Situ SpyCatcher-SpyTag Interaction. ChemBioChem 21, 2126-2131
Gallus, S., Peschke, T., Paulsen, M., Burgahn, T., Niemeyer, C. M., Rabe, K. S.
(See online at https://doi.org/10.1002/cbic.202000102) - (2021) An Orthogonal Covalent Connector System for the Efficient Assembly of Enzyme Cascades on DNA Nanostructures. Small 17, 2105095
Kröll, S., Rabe, K. S., Niemeyer, C. M.
(See online at https://doi.org/10.1002/smll.202105095) - (2021) Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry. ACS Catal 11, 10695-10704
Ott, F., Rabe, K. S., Niemeyer, C. M., Gygli, G.
(See online at https://doi.org/10.1021/acscatal.1c02076)
