Nano ‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

A biocatalytic cascade consisting of a stereoselective ketoreductase and a cofactor-regenerating enzyme arrayed on DNA nanostructures on dispersed microbeads provides detailed insights into the interplay of nano- and microscale compartmentalization on the coupled reaction-diffusion network. AbstractArtificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot-based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It...

Source: Small - September 21, 2023 Category: Nanotechnology Authors: Sandra Kr öll, Teresa Burgahn, Kersten S. Rabe, Matthias Franzreb, Christof M. Niemeyer Tags: Research Article Source Type: research