Mechanism of Conformational Selection of tRNA by Bacterial Deaminase TadA.

Base editing is a common mechanism by which organisms expand their genetic repertoire to access new functions. Here, we explore the mechanism of tRNA recognition in the bacterial deaminase TadA, which exclusively recognizes tRNA and converts the wobble base adenosine (A34) to inosine. We quantitatively evaluate the dynamics of tRNA binding by incorporating the fluorescent adenine analogue 2-aminopurine (2-AP) at position 34 in the wobble base of the anticodon loop. Time-resolved fluorescence and anisotropy studies revealed that the recognition process is finely tuned. Mutations in residues directly involved in facilitating deamination, such as E55A and N42A, showed a minimal impact on binding dynamics. In contrast, mutations in the "capping residues", notably R149, unique to prokaryotic TadAs and located 12-15 Å away from the catalytic center, significantly disrupted binding and consequently catalytic activity. The capping residues play a critical role in enabling tRNA recognition, thereby underscoring their importance in enzyme function. Moreover, for effective catalysis, peripheral positively charged residues (R70, R94) that are part of the adjacent subunit in the dimeric assembly are important to splay out the tRNA, assisting in A34 attaining a flipped-out conformation. Perturbations in these extended regions, although 15-20 Å away from the active site, disrupt the binding dynamics and consequently the function, emphasizing the fine regulation of the tRNA recognition process. Analysis reveals that the C-terminal end of the extended helix where R149 is positioned, acts as a selectivity filter imparting exclusivity toward the deamination of tRNA by TadA.