Elucidation of Enzymatic Catalysis Mechanism of Methyltransferase Trm5 by Combined Molecular Dynamics Simulation and Quantum Chemical Calculations

<jats:p>Trm5 is an S-adenosyl-methionine (SAM)-dependent methyltransferase introducing the essential m1G37 modification to the tRNAs of Archaea and Eukarya. Despite extensive crystallographic, mutational, and kinetic characterization, its dynamic motions that support catalysis remain poorly understood. In the present work, we combine molecular dynamics (MD) simulations with quantum-mechanical (QM) calculations based on density functional theory to investigate the Trm5 dynamics in the reaction mechanism. First, the MD simulations establish that while the active site experiences considerable conformational flexibility, no general base residue is positioned near the N1 atom of G37 to conduct the necessary incipient 2 deprotonation. The following evaluation, by QM calculations, of possible deprotonation pathways through extended water bridges shows that a proton transfer to the bulk solution is the most feasible alternative. Thus, the catalytic function of the enzyme during this first chemical step is to increase the acidity of G37 and to stabilize the resulting anionic form of the nucleobase, through interaction with the nearby arginine R145. Subsequent MD simulations of the enzyme-substrate-SAM complex of the deprotonated G37, using purpose-optimized forcefield parameters, reveal a markedly different active-site conformation. This rearrangement suggests that, in the crystal structure of Trm5–tRNA–SAM complex, G37 is trapped in its deprotonated state, half-way through the mechanism. Finally, QM calculations of the methyl transfer step reveal activation energy barrier consistent with the experimentally measured value. Overall, this study demonstrates that computational modeling of the enzyme dynamics during catalysis provides mechanistic insights that cannot be obtained from experimental measurements or structural analysis alone.</jats:p>