Base editing technologies enable the precise conversion of one base pair in genomic DNA into another. The most recent class of base editors, adenine base editors (ABEs), facilitate the efficient modification of A:T base pairs to G⋮C at targeted genomic loci via the deamination of adenine to inosine. ABEs were developed by engineering and evolving a tRNA adenosine deaminase enzyme (TadA) into a single-stranded DNA (ssDNA) editing enzyme. Amechanistic understanding of the contributions of the individual mutations to ssDNA editing efficiency ofABEs remains unknown.To reduce the experimental effort associated with directed evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we employ computational design strategies. All-atom, explicit-solvent, molecular dynamics (MD) simulations of the various TadA-ssDNAcomplexes were performed on GPUs using the CUDA version of PMEMD in AMBER. These simulations help us to explore the structural and functional role played by the initial mutations in the onset of ssDNA modifying activity of the TadA enzyme. Atomistic insights into the system reveal that these early mutations lead to intricate conformational changes in the structure of the protein. These mutations are associated with an enhancement in the binding free energy of the TadA* to ssDNA, which we confirm using umbrella sampling calculations. Our simulations and experimental reversion analyses verify the importance of these mutations in imparting functional promiscuity to the TadA enzyme.We plan on generalizing this engineering approach and aiding the discovery of more genome editors in conjunctionwith canonical directed evolution.