The metal dependence of single-metal mediated phosphodiester bond cleavage: a QM/MM study of a multifaceted human enzyme

Abstract

Nucleases catalyze the cleavage of phosphodiester bonds in nucleic acids using a range of metal cofactors. Although it is well accepted that many nucleases rely on two metal ions, the...

Is Metal Stabilization of the Leaving Group Required or Can Lysine Facilitate Phosphodiester Bond Cleavage in Nucleic Acids? A Computational Study of EndoV

“…50 Furthermore, QM calculations on APE1 revealed that indirect metal−substrate ligation is preferred over the direct substrate ligation observed in the crystal structure of the PC (PDB ID: 4IEM, Figure 3a), while QM/MM calculations verified that this metal binding architecture can facilitate the phosphodiester bond hydrolysis in a manner consistent with experimental data. 56,69 A similar water-mediated interaction between Mg 2+ and a nonbridging phosphate oxygen has been detected in the active site of the RC analogue of one-metal-dependent Vvn (PDB ID: 1OUP, Figure 3h). 41 Based on this literature, indirect metal−substrate coordination was investigated for EndoV using MD simulations, in which the octahedral coordination geometry of Mg 2+ was satisfied by D43, D110, and four active site water molecules (Figures S11b, S12b, and Table S2).…”

“…Although no catalytically feasible pathway could be characterized in the present work that involves direct metal coordination to the substrate, an active site configuration involving indirect metal coordination to the substrate has been observed in the endonuclease literature. ,,, Specifically, a crystal structure of a phosphorothioate RC analogue of one-metal-mediated APE1 shows a Mn 2+ -coordinated water within hydrogen-bonding distance to a nonbridging oxygen of the scissile phosphate (PDB ID: 5DG0, Figure b) . Furthermore, QM calculations on APE1 revealed that indirect metal–substrate ligation is preferred over the direct substrate ligation observed in the crystal structure of the PC (PDB ID: 4IEM, Figure a), while QM/MM calculations verified that this metal binding architecture can facilitate the phosphodiester bond hydrolysis in a manner consistent with experimental data. , A similar water-mediated interaction between Mg 2+ and a nonbridging phosphate oxygen has been detected in the active site of the RC analogue of one-metal-dependent Vvn (PDB ID: 1OUP, Figure h) . Based on this literature, indirect metal–substrate coordination was investigated for EndoV using MD simulations, in which the octahedral coordination geometry of Mg 2+ was satisfied by D43, D110, and four active site water molecules (Figures S11b, S12b, and Table S2).…”

“…Although no catalytically feasible pathway could be characterized in the present work that involves direct metal coordination to the substrate, an active site configuration involving indirect metal coordination to the substrate has been observed in the endonuclease literature. 41,50,56,69 Specifically, a crystal structure of a phosphorothioate RC analogue of one-metal-mediated APE1 shows a Mn 2+ -coordinated water within hydrogenbonding distance to a nonbridging oxygen of the scissile phosphate (PDB ID: 5DG0, Figure 3b). 50 Furthermore, QM calculations on APE1 revealed that indirect metal−substrate ligation is preferred over the direct substrate ligation observed in the crystal structure of the PC (PDB ID: 4IEM, Figure 3a), while QM/MM calculations verified that this metal binding architecture can facilitate the phosphodiester bond hydrolysis in a manner consistent with experimental data.…”

“…This choice of methodology was based on previous work that extensively investigated the impact of the method on the description of one-metal-mediated phosphodiester bond cleavage reactions facilitated by enzymes. 69,70 All calculations were performed using the Gaussian 16 program (revision B.01). 97 To identify the preferred catalytic pathway, we compare each computed mechanism to experimental structural, 17 kinetic, 16 and mutational data.…”

“…For example, although the PC analogue of APE1 (PDB ID: 4IEM, Figure a) displays direct metal–substrate ligation, the phosphorothioate RC analogue shows indirect Mg 2+ coordination to the substrate (PDB ID: 5DG0, Figure b), and QM and QM/MM calculations initiated from the crystal structure of the PC suggest that indirect metal–substrate ligation is preferred . Furthermore, while several computational works have characterized the pathway for various two-metal-dependent nucleases, − to the best of our knowledge, the mechanism of one-metal-mediated phosphodiester bond cleavage has only been investigated for APE1. ,, Therefore, the catalytic mechanism of one-metal-dependent EndoV is unclear, with missing atomic-level information leaving unanswered questions surrounding the identity of the residues that activate the water nucleophile, provide TS charge stabilization, and promote leaving group departure, as well as the role of the single metal.…”