Chemical feasibility of the general acid/base mechanism of glmS ribozyme self‐cleavage

Abstract

In numerous Gram-positive bacteria, the glmS ribozyme or catalytic riboswitch regulates the expression of glucosamine-6-phosphate (GlcN6P) synthase via site-specific cleavage of its sugar-phosphate backbone in response to GlcN6P ligand binding. Biochemical data have suggested a crucial catalytic role for an active site guanine (G40 in Thermoanaerobacter tengcongensis, G33 in Bacillus anthracis). We used hybrid quantum chemical/molecular mechanical (QM/MM) calculations to probe the mechanism where G40 is deprotonated and acts as a general base. The calculations suggest that the deprotonated guanine G40− is sufficiently reactive to overcome the thermodynamic penalty arising from its rare protonation state, and thus is able to activate the A-1(2’-OH) group toward nucleophilic attack on the adjacent backbone. Furthermore, deprotonation of A-1(2’-OH) and nucleophilic attack are predicted to occur as separate steps, where activation of A-1(2’-OH) precedes nucleophilic attack. Conversely, the transition state associated with the rate-determining step corresponds to concurrent nucleophilic attack and protonation of the G1(O5’) leaving group by the ammonium moiety of the GlcN6P cofactor. Overall, our calculations help to explain the crucial roles of G40 (as a general base) and GlcN6P (as a general acid) during glmS ribozyme self-cleavage. In addition, we show that the QM/MM description of the glmS ribozyme self-cleavage reaction is significantly more sensitive to the size of the QM region and the quality of the QM-MM coupling than that of other small ribozymes.

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

“… 1222 , 1237 “Uncorrected” refers here to the fact that no thermodynamic corrections were included for residues that react starting from rare (transient) protonation states (see below). 1238 − 1240 …”

“… 1267 In contrast, the latter mechanism involving G40 – and GlcN6PH + as general base and general acid, respectively, was considered chemically feasible both by the Hammes-Schiffer, Bevilacqua, and co-workers team 1267 as well as by Banas and co-workers based on QM/MM scanning of the reaction path ( Figure 66 ). 1238 Both studies reported the proton transfer from GlcN6PH + to the O5′ leaving group associated with exocyclic cleavage as the rate-determining step of the self-cleavage reaction. 1238 , 1267 This observation suggests that glmS ribozyme cleavage critically depends on the GlcN6P cofactor acting as the general acid.…”

“… 1238 Both studies reported the proton transfer from GlcN6PH + to the O5′ leaving group associated with exocyclic cleavage as the rate-determining step of the self-cleavage reaction. 1238 , 1267 This observation suggests that glmS ribozyme cleavage critically depends on the GlcN6P cofactor acting as the general acid.…”

Mechanistic insights into the phosphoryl transfer reaction in cyclin-dependent kinase 2: A QM/MM study

“…Among the limitations that this last study revealed is the small QM region used (only 48 atoms), which did not include most of the residues coordinating the Mg 2+ cofactor, this due to the costly sampling at a DFT level. Nonetheless, it is known that this simplification could lead to overpolarization of the QM fragments, and specially in this case the coordinating metal, affecting the energetics of the system [28]. Furthermore, the free energy barrier is dependent on the selection of the reaction coordinate, which they showed can vary considerably depending on how the combination of the reactive distances is made.…”

Understanding in-line probing experiments by modeling cleavage of nonreactive RNA nucleotides

“…Computational techniques are an established tool for the investigation of structural and dynamical properties of RNA at an atomistic level (Schlick 2010;Cheatham and Case 2013;Šponer et al 2013) and could in principle allow for an investigation and interpretation of reactivity patterns in RNA. In particular, quantum mechanical/molecular mechanical (QM/MM, Warshel and Levitt 1976) approaches, where only the reactive portion of the system is described at the QM level, have been used to characterize cleavage reactions within catalytic RNA systems (Banáš et al 2008;Nam et al 2008b,a;Lee et al 2008;Mlýnský et al 2011;Rosta et al 2011;Xu et al 2012;Gu et al 2013;Ganguly et al 2014;Mlýnský et al 2015;Dubecký et al 2015;Zhang et al 2015;Thaplyal et al 2015;Casalino et al 2016). Single point QM/MM calculations evaluate potential energy surfaces neglecting entropic contributions.…”