Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis.

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. While both TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3' position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor, TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinities to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed "half-of-the-sites" reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel to those between the two classes of aminoacyl-tRNA synthetases, which use distinct active-site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.

The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N1 of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg2+ is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg2+ is not involved in substrate binding, but in promoting methyl transfer. Based on the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg2+ is to help to increase the nucleophilicity of N1 of G37 and stabilize the negative developing charge on O6 during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg2+ contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial post-transcriptional modifications to tRNA.

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N 1 position of G37 directly 39 to the anticodon. While the biological role of m 1 G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O 6 , and for E185 as a general base to accept the proton from G37-N 1 , are suggested based on the mutational analysis.

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N 1 of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.