RAMA: an Integrative Approach to Identify Hot Spots and Guide the Directed Evolution of High Molecular Weight Enzyme

Directed evolution is a robust method for engineering enzymes by iteratively creating and screening variant libraries. However, the challenge of developing assays for high-throughput screening persists, especially for high molecular weight enzymes, which are constrained by the vast number of possible amino acid sequences. In this study, we introduce an integrative approach named RAMA to reduce the search space in the directed evolution of enzymes. ¹ H- ¹³ C NMR spectra were applied to record the titration of a 90 kDa B-type DNA polymerase Pyrococcus furiosus DNA polymerase ( Pfu pol) with neutralization nanobodies and substrates to recognize ‘hot spots’ involved in its activity. Communal peaks with chemical shift perturbation were analyzed to determine their amino acid types. Following by the molecular docking of Pfu pol-nanobody and Pfu pol-substrate complexes, these hot spots were assigned and used for the directed evolution of the Pfu pol. Saturation mutagenesis of four identified hot spots of Pfu pol were performed to screen polymerase with enhanced catalytical activity. Biochemical and structural analysis of three preferred Pfu pol variants reveal distinct molecular mechanism for their improved performances, indicating a powerful capability of RAMA in the directed evolution of Pfu pol. This proposed approach is also applicable to other high molecular weight enzymes, provided appropriate antibodies are available. Thus, its use will speed up the design and engineer enzymes for many bio-chemical transformations.