Reaction Mechanism of the ε Subunit of E. coli DNA Polymerase III: Insights into Active Site Metal Coordination and Catalytically Significant Residues

Abstract

The 28kDa ε subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which ε carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N–terminal domain containing the exonuclease activity. Both the free–ε and a complex, ε bound to a θ homolog (HOT), were studied. For the ε–HOT complex, Mg2+ or Mn2+ were investigated as the essential divalent metal cofactors, while only Mg2+ was used for free–ε. In all calculations, a water molecule bound to the catalytic metal acts as the nucleophile for the hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed. Subsequently, the nucleophilic attack takes place, followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn2+ is faster than with Mg2+, in agreement with experiment. In addition, the ε–HOT complex shows a slightly lower energy barrier compared to free–ε. In all cases the catalytic metal is observed to be penta–coordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the penta–coordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129 and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA–Pol I, and the DNA polymerase of bacteriophage RB69.

Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease

“…Mutagenesis of the catalytic histidine within the DEDDh exonucleases TREX1, TREX2, and RNase T abolishes exonuclease activity [23, 26–27, 51]. Quantum mechanical/molecular mechanical studies of the catalytic mechanism of the structurally similar epsilon subunit of E. coli DNA polymerase III indicate that a proton is transferred from the nucleophilic water to the catalytic histidine, further supporting a role for the histidine in phosphoryl hydrolysis [25]. …”

Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine

“…The average non-bonded interaction between a particular cytosine derivative and every other residue, ΔE int , is approximated by ΔE int =<ΔE i >, where i represents an individual residue, ΔE i represents the nonbonded interaction (Coulomb or VdW) between residue i and the particular cytosine derivative, and the broken brackets represent averages over the complete production ensemble obtained from the MD simulations. This analysis has been previously employed for QM/MM and MD simulations to study a number of protein systems 21,22,51–54 . The EDA results for all protein residues with mC/hmC/fC/caC are presented in Supplementary Table 2, and specific non-bonded interactions are shown in Supplementary Table 3.…”

Catalytic Mechanism of RNA Backbone Cleavage by Ribonuclease H from Quantum Mechanics/Molecular Mechanics Simulations

“…2,3 Other major unsolved problems concern the proton transfer (PT) processes that take place during the phosphate hydrolysis reaction. 4,5 In many systems, the search is ongoing for the residues that participate in the PT steps involved in activating the water nucleophile or hydroxide ion, and in protonating the leaving group. Only limited experimental information is available concerning the PT steps in the enzymatic reaction.…”