Unraveling the Critical Role Played by _Ado762′OH in the Post-Transfer Editing by Archaeal Threonyl-tRNA Synthetase

Abstract

Archaeal threonyl-tRNA synthetase (ThrRS) possesses an editing active site wherein tRNA that has been misaminoacylated with serine (i.e., Ser-tRNA) is hydrolytically cleaved to serine and tRNA. It has been suggested that the free ribose sugar hydroxyl of Ado76 of the tRNA (2'OH) is the mechanistic base, promoting hydrolysis by orienting a nucleophilic water near the scissile Ser-tRNA ester bond. We have performed a computational study, involving molecular dynamics (MD) and hybrid ONIOM quantum mechanics/molecular mechanics (QM/MM) methods, considering all possible editing mechanisms to gain an understanding of the role played by 2'OH group. More specifically, a range of concerted or stepwise mechanisms involving four-, six-, or eight-membered transition structures (total of seven mechanisms) were considered. In addition, these seven mechanisms were fully optimized using three different DFT functionals, namely, B3LYP, M06-2X, and M06-HF. The M06-HF functional gave the most feasible energy barriers followed by the M06-2X functional. The most favorable mechanism proceeds stepwise through two six-membered ring transition states in which the2'OH group participates, overall, as a shuttle for the proton transfer from the nucleophilic HO to the bridging oxygen (3'O) of the substrate. More specifically, in the first step, which has a barrier of 25.9 kcal/mol, the 2'-OH group accepts a proton from the attacking nucleophilic water while concomitantly transferring its proton onto the substrates C-O center. Then, in the second step, which also proceeds with a barrier of 25.9 kcal/mol, the 2'-OH group transfers its proton on the adjacent3'-oxygen, cleaving the scissile C-O3' bond, while concomitantly accepting a proton from the previously formed C-OH group.

Recent Progresses in Ab Initio Electronic Structure Calculation toward Understandings of Functional Mechanisms of Biological Macromolecular Systems

“…We further suggested that the ribozymal mechanism that we discovered is common in the editing reaction of various aaRS systems beyond the classes ( Table 1) [63]. In fact, Thermus thermophilus IleRS (class Ia) [77], Pyrococcus abyssi ThrRS (class IIa) [72,74,75,78], and Enterococcus faecalis ProRS (class IIa) [71] showed the similar binding mode of the nucleophilic water in the catalytic site. Furthermore, Kumar et al also suggested that the editing reaction of the complex of prolyl-tRNA synthetase (ProRS) and alanyl-tRNA Pro exhibited a similar mechanism, in which the 2 0 -OH group of A76 of tRNA Pro was involved in the substrate binding and the activation of the nucleophilic water [71].…”

Mechanistic insights into the chemistry of compound I formation in heme peroxidases: quantum chemical investigations of cytochromecperoxidase

“…2,17,18 Indeed, water-assisted mechanism is well-known approach in facilitating the reaction in other fundamental enzymes. 19,20 Cytochrome c peroxidase (Ccp1) served for decades as a benchmark biochemical model for understanding the chemical and structural properties of other peroxidases. 6 Its principal function is to eliminate the toxic H 2 O 2 from the cell with the assistance of its redox partner cytochrome c (Cc).…”

“…2,17,18 Indeed, water-assisted mechanism is well-known approach in facilitating the reaction in other fundamental enzymes. 19,20…”