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

• Published in: ACS chemical biology 2022-07, Vol.17 (7), p.1924-1936
• Main Authors: Aggarwal, Tushar, Hansen, William A., Hong, Jonathan, Ganguly, Abir, York, Darrin M., Khare, Sagar D., Izgu, Enver Cagri
• Format: Article
• Language: English
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.2c00373

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O–P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N–P linkages using 3′-NH2-ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3′-amino nucleophile in the active site. Further remodeling of the protein–nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNANP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.