Atomistic details of the phosphodiester cleavage of ribonuclease H

Abstract

it catalyzes the hydrolysis of phospho-diester linkages of the RNA strand in the DNA:RNAhybrid duplex. RNase H activity is encoded as a part ofthe reverse transcriptase (RT) that converts a retroviralsingle strained RNA genome into double strained DNA.Due to the RNase H activity in HIV reverse transcrip-tase (HIV-RT), it represents a promising target for anti-HIV drug design.In our study we focused on a computational investiga-tion of the hydrolytic mechanism ofhuman RNase HI(PDB Code 2QKK) [1] using a comprehensive QM/MMtheoretical method that is based on DFT/B3LYP calcula-tion of the interactions in the QM region and the inclu-sion of the interactions of the surrounding protein andsolvent water in the MM region as implemented in thesoftware package of NWChem [2].Starting from the X-ray structure of the mutatedenzyme-substrate complex we changed the enzyme intothe active form to reach the reactant state. Furthermore,this structure has been validated by additional dockingstudies. Afterwards, using a series of constrained andrelaxation steps we could model the reaction path, iden-tify the product state and found a stabile intermediatestate along the reaction coordinates (Fig. 1).After a nucleophilic attack of a water molecule on thescissile phosphorous a water-proton is transferred to theO1P oxygen as the first step.In the second, consecutive step the proton is shuttledto the O3’ oxygen and the nucleotide is being cleaved toform the product state. Finally, we performed transitionstate search and energy barrier calculations over thereaction coordinates and identified the rate limiting stepof the reaction.The calculated reaction energy is inexcellent agreement with experimental findings.