Structural Insights into Single-Stranded DNA Recognition and Modified Substrate Tolerance in an Engineered Terminal Deoxynucleotidyl Transferase

Abstract Terminal deoxynucleotidyl transferase (TdT) is the cornerstone enzyme for de novo enzymatic DNA synthesis (EDS), promising to overcome the length and sustainability limitations of traditional phosphoramidite chemistry. We previously identified a highly active TdT ortholog from Zonotrichia albicollis (ZaTdT) and engineered a variant (R335L/K337G) capable of efficiently incorporating 3’-aminooxy (3’-ONH 2 ) reversible terminators. However, the atomic-level mechanism by which these engineered mutations alter substrate specificity has remained elusive. Here, we present the high-resolution (2.20 Å) crystal structure of the engineered ZaTdT in complex with a single-stranded DNA primer. Structural analysis reveals a conserved catalytic core anchored by a hydrophobic platform (Phe110/Phe257/Trp308) that stabilizes the primer. Crucially, comparative modeling with the homologous murine TdT ternary complex demonstrates that the engineered mutations (corresponding to L190/G192 in the crystal structure) disrupt a rigid salt-bridge network at the active site entrance. This electrostatic remodeling not only reduces local positive charge but also expands the catalytic pocket depth from 3.8 Å to 5.8 Å. This specific spatial expansion provides the structural rationale for the accommodation of bulky 3’-blocking groups, validating our rational design strategy and paving the way for next-generation long-read DNA synthesis.