Flap endonucleases (FENs) are nucleic acid hydrolyzing enzymes in charge of excising S'-small DNA and RNA fragments (flaps) protruding from nucleic acid structures during the lagging strand DNA replication or the long-patch base excision repair (LP-BER) processes. In this work we report, for the first time, an atomistic and energetic rendering of the enzymatic catalysis promoted by the human FEN1. After reconstruction of a reactive hFEN/double strand (ds) DNA adduct we employed mixed quantum-classical (QM/MM) metadynamics and umbrella sampling free energy calculations, with the QM part treated with the AM1/d-PhoT Hamiltonian, to perform an extensive characterization of all possible reaction pathways underlying the enzymatic cycle. Our extensive investigation points to a most likely reaction pathway very similar to that recently proposed for ribonuclease H, in which the rate determining step is the nucleophilic attack of a water to the scissile phosphate, which occurs concomitantly with its activation by the pro-Rp oxygen of the nucleobase flanking the scissile phosphate. This step requires a free energy barrier in good agreement with experimental data (Delta G(exp)double dagger = 16.1 kcal/mol vs Delta F-calc double dagger = 16 +/- 2 kcal/mol). Due to the important role of FENs in maintaining nucleic acid fidelity and cell proliferation, a detailed understanding of its enzymatic mechanism has broad interest to elucidate a key enzymatic biological process for preserving genome integrity and has implications for medical and biotechnological applications.

QM/MM MD simulations on the enzymatic pathway of the human flap endonuclease (hFEN1) elucidate common cleavage pathways to RNase H enzymes / J., Sgrignani; Magistrato, Alessandra. - In: ACS CATALYSIS. - ISSN 2155-5435. - 5:6(2015), pp. 3864-3875. [10.1021/acscatal.5b00178]

QM/MM MD simulations on the enzymatic pathway of the human flap endonuclease (hFEN1) elucidate common cleavage pathways to RNase H enzymes

MAGISTRATO, ALESSANDRA
2015-01-01

Abstract

Flap endonucleases (FENs) are nucleic acid hydrolyzing enzymes in charge of excising S'-small DNA and RNA fragments (flaps) protruding from nucleic acid structures during the lagging strand DNA replication or the long-patch base excision repair (LP-BER) processes. In this work we report, for the first time, an atomistic and energetic rendering of the enzymatic catalysis promoted by the human FEN1. After reconstruction of a reactive hFEN/double strand (ds) DNA adduct we employed mixed quantum-classical (QM/MM) metadynamics and umbrella sampling free energy calculations, with the QM part treated with the AM1/d-PhoT Hamiltonian, to perform an extensive characterization of all possible reaction pathways underlying the enzymatic cycle. Our extensive investigation points to a most likely reaction pathway very similar to that recently proposed for ribonuclease H, in which the rate determining step is the nucleophilic attack of a water to the scissile phosphate, which occurs concomitantly with its activation by the pro-Rp oxygen of the nucleobase flanking the scissile phosphate. This step requires a free energy barrier in good agreement with experimental data (Delta G(exp)double dagger = 16.1 kcal/mol vs Delta F-calc double dagger = 16 +/- 2 kcal/mol). Due to the important role of FENs in maintaining nucleic acid fidelity and cell proliferation, a detailed understanding of its enzymatic mechanism has broad interest to elucidate a key enzymatic biological process for preserving genome integrity and has implications for medical and biotechnological applications.
2015
5
6
3864
3875
https://pubs.acs.org/doi/10.1021/acscatal.5b00178
J., Sgrignani; Magistrato, Alessandra
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/32810
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