Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity

• Composite mutation of the Asp540 and the C-terminal helix improved ligase activity.
• The mutations in the C-terminus of the helix reduced the ΔH value for DNA-binding.
• We developed effective thermostable DNA ligase able to be utilized as a biological tool.

The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.