title: Understanding and Engineering Thermostability in DNA Ligase from  Thermococcus sp. 1519.
abstract: The physical chemical principles underlying enzymatic thermostability are keys to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also an important factor for engineering more thermoresistant enzymes to be used in the industrial setting, such as, for instance, DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, we first address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance thermostability of the enzyme through bioinformatics analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.
authors.forename: Hassan Pezeshgi
authors.initials: HP
authors.lastname: Modarres
authors.forename: Boris D
authors.initials: BD
authors.lastname: Dorokhov
authors.forename: Vladimir O
authors.initials: VO
authors.lastname: Popov
authors.forename: Nikolai V
authors.initials: NV
authors.lastname: Ravin
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Skryabin
authors.forename: Matteo
authors.initials: M
authors.lastname: Dal Peraro
mesh_terms: D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D017433:Protein Structure, Secondary; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D011088:DNA Ligases; D000072481:DNA Ligase ATP