NMR-guided rational exploration of co-factors in boosting the Pfu DNA polymerase

With rapid developments of emerging technologies like synthetic biology, the demand for
DNA polymerases with superior activities including higher thermostability and processivity has increased significantly. Thus, rational optimization of the performance of DNA
polymerase is of great interest. Nuclear magnetic resonance (NMR) spectroscopy is a
powerful technique used for studying protein structure and dynamics. It provides the
atomic resolution information of enzymes under their functional solution environment to
reveal the active sites (hot spots) of the enzyme, which could be further used for optimizing the performance of enzymes. In our previous work, we identified hot spot residues
of Pyrococcus furiosus DNA polymerase (Pfu pol). We aim to employ these binding hot
spots to screen for co-factors of Pfu pol, particularly targeting those molecules exhibiting
weak intermolecular interactions. To validate this concept, we first demonstrated the
feasibility of utilizing hot spot residues as screening probes for auxiliary factors by
employing the well-characterized Tween-20 as a model system. Employing these hot
spots as probes, two new co-factors, the heat shock protein TkHSP20 from Thermococcus
Kodakaraensis and the chemical chaperone L-arginine, are identified to interact with Pfu
pol to boost its performance in amplifying long DNA fragments by enhancing the thermal
stability and the processivity of the Pfu pol. This NMR-based approach requires no prior
assignment information of target enzymes, guiding the rational exploration of novel cofactors for Pfu pol. Moreover, our approach is not dependent on structural data or bioinformatics. Therefore, it has significant potential for application in various enzymes to
expedite the progress in enzyme engineering.