Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod

The minor groove intercalation loop (leucine-loop) in Uracil-DNA N-glycosylases (UNGs) undergoes significant conformational changes upon substrate interaction. Previous studies have shown that the cold adapted Atlantic cod UNG (cUNG) possesses a tenfold higher catalytic efficiency than human UNG (hUNG). A sequence alignment of the two enzymes revealed amino acid substitutions in and near the leucine-loop of cUNG which may increase the elasticity of the loop and thus explain the observed higher catalytic efficiency of cUNG compared to hUNG. In order to investigate this hypothesis we constructed five single and four multiple mutants of cUNG with cod to human UNG substitutions and characterised them by kinetic experiments at three different temperatures (15, 22 and 37°C) and Differential Scanning Calorimetry (DSC). The results showed that the mutations affected the kcat more than the KM, and mutants with apparent reduced hydrophobic properties (A266T, V267A, A266T/V267A and A266T/ V267A/A274V) showed increased or equivalent kcat to cUNG, while mutants with predicted increased hydrophobicity (A274V, H275Y, L279F, A266T/V267A/A274V/H275Y and A266T/V267A/A274V/H275Y/H250Q) showed reduced kcat compared to cUNG. Thus, mimicking the amino acid composition of the leucine-loop in hUNG reduces the activity of cUNG. The DSC experiments were performed in order to identify a potential increase in the overall stability of the mutants as a result of altered hydrophobicity of the leucine-loop. However, the results showed a slight reduction in the overall stability for mutants with increased kcat and increased stability for mutants with reduced kcat. We suggest these results arise from a need to compensate for a potential increased/reduced elasticity of the leucine-loop which is introduced by the amino acid substitutions. This study confirms the suggested importance of the leucine-loop for efficient catalysis of UNG and shows that it is possible to alter the catalytic capacity of this enzyme by manipulating the amino acid composition of this loop.