Abstract
Mg2+ is required for the catalytic activity of TrmD, a bacteria-specific methyltransferase that is made up of a protein topological knot-fold, to synthesize methylated m1G37-tRNA to support life. However, neither the location of Mg2+ in the structure of TrmD nor its role in the catalytic mechanism is known. Using molecular dynamics (MD) simulations, we identify a plausible Mg2+ binding pocket within the active site of the enzyme, wherein the ion is coordinated by two aspartates and a glutamate. In this position, Mg2+ additionally interacts with the carboxylate of a methyl donor cofactor S-adenosylmethionine (SAM). The computational results are validated by experimental mutation studies, which demonstrate the importance of the Mg2+-binding residues for the catalytic activity. The presence of Mg2+ in the binding pocket induces SAM to adopt a unique bent shape required for the methyl transfer activity and causes a structural reorganization of the active site. Quantum mechanical calculations show that the methyl transfer is energetically feasible only when Mg2+ is bound in the position revealed by the MD simulations, demonstrating that its function is to align the active site residues within the topological knot-fold in a geometry optimal for catalysis. The obtained insights provide the opportunity for developing a strategy of antibacterial drug discovery based on targeting of Mg2+-binding to TrmD.
Highlights
Methyl transfer reactions play vital roles in a variety of physiological processes
This is in contrast to, for instance, phosphoryl transfer reactions catalyzed by kinases, which strictly require divalent metal ions to orient the γ-phosphoryl group of ATP “in-line” with respect to the substrate to create the correct geometry for catalysis.[5]
We describe a combined molecular dynamics (MD) simulation and quantum mechanics (QM) investigations aimed at elucidating the Mg2+-dependent catalysis by TrmD
Summary
Methyl transfer reactions play vital roles in a variety of physiological processes. They most often employ the methyl donor S-adenosyl-methionine (SAM) for methyl transfer. Even in the rare cases, wherein metal ions are required, their role is usually limited to stabilizing substrates and enhancing selectivity,[1−4] but are not directly involved in the actual breaking and formation of chemical bonds This is in contrast to, for instance, phosphoryl transfer reactions catalyzed by kinases, which strictly require divalent metal ions to orient the γ-phosphoryl group of ATP “in-line” with respect to the substrate to create the correct geometry for catalysis.[5]. Our current computational studies, validated by mutational experiments, uncover a new site for Mg2+ binding within the active site, but away from G37, which was missed previously These results demonstrate that a primary role of the ion is to optimize the conformations of the active site and of SAM for methyl transfer
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