Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9{degrees}N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3[->]P5 phosphoramidate linkages in the presence of 3-amino-2,3-dideoxynucleoside 5-triphosphate (3-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9{degrees}N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9{degrees}N-NRQS) that incorporated 3-NH2-ddNTPs into a 3-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.