Poster abstracts
Poster number 30 submitted by Aiswarya Krishnamohan
Mechanistic features of the bifunctional tRNA methyltransferase, Trm10
Aiswarya Krishnamohan (Ohio State Biochemistry Program, Dept. of Chemistry and Biochemistry), Jane Jackman (Ohio State Biochemistry Program, Dept. of Chemistry and Biochemistry)
Abstract:
The tRNA m1G9 methyltransferase (Trm10) family enzymes methylate the 9th nucleotide position of multiple tRNAs using S-adenosyl methionine (SAM) as the methyl group donor. Although the founding member of the Trm10 family, Saccharomyces cerevisiae Trm10 (ScTrm10), is an obligate m1G9 methyltransferase, some homologs of Trm10 have been shown to also methylate adenosine (A9) to form m1A9, an expansion of substrate specificity not observed in other characterized methyltransferases. Moreover, although Trm10 has been classified as a member of the SpoU-TrmD (SPOUT) family of methyltransferases, it does not share key mechanistic features associated with members of this enzyme family, including the characteristic dimeric structure that is essential for forming the active site in most SPOUT enzymes. These observations suggest that Trm10 enzymes exhibit a distinct active site and therefore a novel mechanism of catalysis compared to other SPOUT members. To address these questions about the mechanism and the unique substrate specificity of Trm10 enzymes, a detailed biochemical and kinetic analysis of the m1G9 catalyzing ScTrm10 and a m1A9/m1G9 catalyzing bifunctional Thermococcus kodakarensis Trm10 (TkTrm10) was performed, providing evidence for an atypical tRNA methyltransferase mechanism that characterizes this enzyme family.
Using a structure of Trm10 solved in the absence of tRNA as a guide, we investigated the catalytic roles of residues proposed to interact with the SAM methyl donor, a putative general base, and a proposed tRNA binding surface. To study the monomeric form of Trm10 binding to tRNA we used chemical and nuclease footprinting to identify elements on the tRNA that is recognized by Trm10. While our data support the crystallographically observed SAM binding site, we provide direct evidence contradicting the identification of the proposed general base and tRNA binding surface. These new and unexpected findings will enable future efforts to fully understand the mechanism of catalysis and substrate recognition by Trm10 so that its complex effects on biology can be understood.
Keywords: tRNA modification, tRNA methylation, Enzyme mechanism