Stereochemical mechanisms of tRNA methyltransferases

Abstract

Methylation of tRNA on the four canonical bases adds structural complexity to the molecule, and improves decoding specificity and efficiency. While many tRNA methylases are known, detailed insight into the catalytic mechanism is only available in a few cases. Of interest among all tRNA methylases is the structural basis for nucleotide selection, by which the specificity is limited to a single site, or broadened to multiple sites. General themes in catalysis include the basis for rate acceleration at highly diverse nucleophilic centers for methyl transfer, using S-adenosylmethionine as a cofactor. Studies of tRNA methylases have also yielded insights into molecular evolution, particularly in the case of enzymes that recognize distinct structures to perform identical reactions at the same target nucleotide.

Figure 1

Mechanism of E. coli tRNA (m⁵U54) methyltransferase (RUMT, TrmA). Attack of a conserved enzyme cysteine sulfydryl group (Cys324) on C6 leads first to accommodation of the negative charge on O4, allowing for nucleophilic attack by the C5 carbon on the methyl group of SAM, with the sulfonium ion serving as electron sink to produce the SAH product. Base abstraction of a proton from C5 (by Glu358) then leads to elimination of the covalent adduct and regeneration of active enzyme.

Figure 2

Proposed mechanism for T. thermophilus TrmFO-catalyzed formation of ribothymidine at position 54 of tRNA. Nucleophilic attack on C6 generates in turn a nucleophilic C5 position that attacks the methylene group of 5,10-methylenetetrahydrofolate (MTHF). The covalent intermediate (upper right) is resolved by base abstraction of a proton at C5, analogous to the TrmA mechanism depicted in Figure 1A, but producing a methylene group at C5. The methylene is reduced to a methyl group by a redox chain involving both FAD and NADP⁺ cofactors.

Figure 3

Proposed mechanism for methylation at exocyclic adenine N6 by the DNA methylase M.TaqI – a potential model for tRNA methylases operating at adenine N6, guanine N2, and cytosine N4. Attack on the SAM methyl group is depicted as proceeding via attack by nitrogen in its sp² hybridization state, because proton removal prior to nucleophilic attack is highly unfavorable. Proton abstraction from the positively charged quaternary nitrogen, by contrast, is relatively facile. See text for details.

Figure 4

Proposed mechanism for M. jannaschii Trm5. Proton abstraction during docking of G37 in the active site (upper left) yields deprotonated G37 as visualized in the crystal structure (upper right). Nucleophilic attack on SAM by deprotonated N1 then leads to product formation.