Unveiling a Single-Metal-Mediated Phosphodiester Bond Cleavage Mechanism for Nucleic Acids: A Multiscale Computational Investigation of a Human DNA Repair Enzyme.

Despite remarkable stability, the phosphodiester bond of nucleic acids is hydrolytically cleaved in critical biological processes. Although this reaction is commonly accepted to take place via a two-metal-assisted mechanism, recent experimental evidence suggests that several enzymes use a single-metal ion, but the precise catalytic mechanism is unknown. In the present work, we employ a multiscale computational approach to decipher the phosphodiester cleavage mechanism for this unique pathway by focusing on the human APE1 repair enzyme, which catalyzes the incision of phosphodiester bonds adjacent to DNA lesions. To resolve ambiguity in the literature regarding the role of the single-metal (Mg(II)) center, several catalytic mechanisms were carefully examined. Our predicted preferred hydrolysis pathway proceeds in two steps via a pentacovalent phosphorane intermediate in the absence of substrate ligation to Mg(II), with a rate-limiting barrier (19.3 kcal/mol) in close agreement with experiment (18.3 kcal/mol). In this mechanism, D210 promotes catalysis by activating water for nucleophilic attack at the 5'-phosphate group with respect to the damaged site. Subsequently, a Mg(II)-bound water triggers leaving group departure by neutralizing the 3'-hydroxyl of the neighboring nucleotide. Consistent with experimental kinetic and mutational data, several other active site residues (N212, Y171, and H309) play multiple roles throughout the reaction to facilitate this challenging chemistry. In addition to revealing previously unknown mechanistic features of the APE1 catalyzed reaction, our work sets the stage for exploring the phosphodiester bond cleavage catalyzed by other single-metal-dependent enzymes, as well as different pharmaceutical and biotechnological applications.