Noncatalytic surface electrostatic networks tune thermolability in uracil-DNA glycosylase.

Uracil-DNA glycosylases (UDGs) are widely used to prevent carryover contamination in nucleic acid amplification-based diagnostics; however, existing thermolabile UDGs exhibit limited thermal inactivation windows for emerging applications. Here, we combine evolutionary mining, structural analysis, and structure-guided saturation mutagenesis to define non-catalytic determinants that tune UDG thermolability without compromising catalytic function. From 8482 bacterial UDG sequences, we assembled a 24-member diversity panel and identified UDG_7 as a naturally thermolabile scaffold coupling robust low-temperature activity with sharp inactivation near 45 °C. The crystal structure of UDG_7 reveals a canonical family-I α/β fold with a fully conserved active site, closely resembling both mesophilic human and Escherichia coli UDGs and thermolabile cod UDG. These structural insights guided the design of a single-site variant library targeting 48 non-catalytic positions involved in packing and electrostatic networks. Pooled thermal shift assays distinguished a rigid structural core from 16 surface thermolability hotspots. A high-throughput functional screening of 480 single mutants yielded 114 clones with a desirable "on-off-off" profile and, after sequence consolidation, identified 54 unique variants that retained activity at 25 °C but lost activity at 30 to 37.5 °C. Biochemical characterization revealed nine single substitutions, Q51I, T112Y, V144M, D167F, R201F, R201Y, D219M, R221P, and R221D, that markedly lower the melting temperature while preserving near-native activity. Together, these results indicate that UDG_7 thermolability is encoded by a distributed, surface-biased electrostatic network that can be selectively disrupted without perturbing the conserved catalytic core, shifting the functional inactivation boundary downward and supporting robust carryover control under low-temperature amplification constraints.