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

Abstract

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.

A structure of substrate-bound Synaptojanin1 provides new insights in its mechanism and the effect of disease mutations

“…This structure thus reveals the interactions with all the important phosphate groups, including the scissile 5-phosphate group. Together with a detailed kinetic analysis of the contribution of these phosphate groups to catalysis, this allows us to propose a refined model for the catalytic mechanism of the 5PPase reaction extending on the previous models based on analogies with the apurinic/apyrimidinic base excision repair endonucleases (40,41,(47)(48)(49), as shown in Figure 6.…”

“…Indeed, structural and sequence comparison revealed similarities in the active site architecture of the 5PPases and APE1, a Mg 2+ dependent enzyme that catalyzes the cleavage of the phosphodiester bond on the 5' side of the abasic site in DNA using a conserved aspartate as catalytic base and a conserved histidine that presumably stabilizes the phosphorane transition state. Moreover, leaving group activation has been proposed to occur via a Mg 2+bound water molecule, although the exact role of the Mg 2+ -ion in the catalytic mechanism of 5PPases has not been fully established (10,38,41). A crystal structure of the S. pombe Synaptojanin (SPSynj) homologue in complex with the inositol-(1,4)-bisphosphate provided a first snapshot of a potential enzyme-product complex (39).…”

Fabrication of highly active phosphatase-like fluorescent cerium-doped carbon dots for in situ monitoring the hydrolysis of phosphate diesters

“…It was believed that the metal ion can activate the phosphate group and a nucleophilic H 2 O molecule, and stabilize the pentacoordinated phosphorus transition state by cooperative effect in metalloenzymes. 17 These mimetic metallohydrolases not only have similar natural enzyme activities and high substrate specicities, but also effectively overcome the disadvantages of natural enzymes, such as low stabilities due to denaturation, sensitivity to the environment, difficult preparation and purication, providing valuable information on the fundamental role of metal ions in promoting the hydrolysis reactions of phosphate ester.…”

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

“…[ 12,13 ] Numerous studies have been performed to understand role of Mg 2+ as natural cofactor. [ 11,14–17 ] But the microscopic origin of the specificity of metal ion cofactor Mg 2+ vis‐à‐vis Ca 2+ in DNA cleavage is unknown. [ 18–20 ]…”

“…performed to understand role of Mg 2+ as natural cofactor. [11,[14][15][16][17] But the microscopic origin of the specificity of metal ion cofactor Mg 2 + vis-à-vis Ca 2+ in DNA cleavage is unknown. [18][19][20] Naturally occurring restriction endonucleases are classified as four types such as type I, II, III and IV, depending on their structure, recognition site, cleavage site etc.…”