The RNase H Domain: Structure, Function and Mechanism

Nowotny, Marcin; Figiel, Małgorzata

doi:10.1007/978-1-4614-7291-9_3

Cited by 1 publication

(1 citation statement)

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“…71 All of the identified enzymes belong to the RNHL superfamily. 70 While the DEDD motif is common among nucleases, 1,72 we identified three different subclasses of this catalytic motif with histidine based on the PDB mining. Two with Glu bound to metal B (RNase H-like, RuvC-like, Figure 6AC) and the other with Glu bound to metal A (RNase T-like, Figure 6B).…”

Section: Role Of Catalytic Histidine and Glutamate In Other Nucleasesmentioning

confidence: 99%

The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H

et al. 2021

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RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present in important drug targets, such as the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs), Escherichia coli (Ec) RNases H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton transfer events are not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2'O atom of the sugar. Additionally, we also describe an alternative proton transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol -1 , encountered in the phosphate cleavage step, matches the slow experimental rate of ∼1-100 min -1 . Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal ion catalytic phosphate cleaving enzymes with a DDE motif.

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