Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

Abstract

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

Identification of a prototypical single-stranded uracil DNA glycosylase from Listeria innocua

“…Due to the low catalytic efficiency of Lin SMUG1-like, we determined the kinetic parameters under the conditions that enzyme concentration was in excess of substrate concentration as previously described [34]. The linear increase of k obs over enzyme concentrations precluded acquisition of individual k 2 or K m , but allowed for determination of k 2 / K m (Fig.…”

“…The linear increase of k obs over enzyme concentrations precluded acquisition of individual k 2 or K m , but allowed for determination of k 2 / K m (Fig. S1) [34]. The k 2 / K m values were obtained by ploting k obs against total enzyme concentration by linear regression with equation $k_{\mathit{obs}}=\frac{k_{2}false[Efalse]}{K_m}$.…”

“…The mutual information (MI) analysis was performed as previously described with modifications [34]. Briefly, to encompass the six known families of the UDG superfamily in the analysis, we compiled sequences of the families 2, 3 5 and 6 and added them to the existing set of families 1 and 4 sequences [34].…”

“…Briefly, to encompass the six known families of the UDG superfamily in the analysis, we compiled sequences of the families 2, 3 5 and 6 and added them to the existing set of families 1 and 4 sequences [34]. Subsequently, a multiple sequence alignment was constructed using MEGA6 [37].…”

“…The reference sequence selected was the SMUG1-like glycosylase from Listeria innocua (GenBank accession WP_010991469.1). The parameters were set to those used previously [34]. …”