Loop‐Centric Engineering of Terminal Deoxynucleotidyl Transferase for Enhanced Catalysis of 3'‐Modified Nucleotides

ABSTRACT Efficient enzymatic DNA synthesis requires terminal deoxynucleotidyl transferases (TdTs) that can accept reversibly blocked nucleotides, but improving wild‐type enzymes has traditionally depended on labor‐intensive screening. Here, we combine deep learning‐based structure prediction with molecular dynamics simulations to guide loop‐centric engineering of a TdT from Crocodylus porosus . The resulting variant M3 (V253E/L256M/N338R) showed a 26‐fold increase in specific activity toward 3'‐ONH 2 ‐dCTP while maintaining expression and thermostability. Simulations indicated that the mutations reorganized Loop1 dynamics and reproducibly biased the incoming nucleotide toward a more favorable in‐line attack orientation. The key N338R change also improved a second TdT scaffold, supporting N338 as a potentially transferable engineering hotspot. These results provide a practical computational strategy for improving terminal deoxynucleotidyl transferases for incorporation of 3′‐modified nucleotides, reducing reliance on large‐scale screening.