Poster abstracts
Poster number 73 submitted by Aiswarya Krishnamohan
Studying the catalytic mechanism of the m1G9/m1A9 methyltransferase (Trm10) family enzymes
Aiswarya Krishnamohan (Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Chemistry and Biochemistry, The Ohio State University)
Abstract:
The tRNA m1G9 methyltransferase (Trm10) methylates the guanosine residue at the 9th position (G9) of some tRNAs using S-adenosyl methionine (SAM) as the methyl group donor. Trm10 was originally discovered in Saccharomyces cerevisiae but is nearly ubiquitous in Archaea and Eukarya, and has been classified based on limited sequence similarity as a member of the SpoU-TrmD (SPOUT) family of methyltransferases. Intriguingly, some Trm10 orthologs also methylate A9 residues, representing an expansion of nucleotide substrate specificity not observed in any other methyltransferase family, and which is expected to require different catalytic groups based on the very different pKa values of the N1 atom of guanosine vs. adenosine nucleotides. Although a crystal structure of fungal Trm10 was recently determined, a mechanism of action that explains the dual specificity of these enzymes has not yet been elucidated. Thus, this mechanism is of interest as Trm10 enzymes likely use a novel mechanism to carry out N-1 methylation compared to other well-studied methyltransferases. Moreover, a complete understanding of the biological significance of Trm10 family enzymes also remains elusive, as deletion of the Trm10 gene in Saccharomyces cerevisiae does not result in an obvious phenotype, but mutations in a human homolog, TRMT10A, were recently implicated in numerous abnormalities including defects in glucose metabolism and neurological dysfunction. This direct correlation to human health has also elevated the importance of studying this unique group of enzymes. Here we use site-directed mutagenesis to mutate conserved residues that are implicated in Trm10 catalytic activity based on their positions in the crystal structure and utilize kinetic assays to evaluate their resulting activities. Although the SAM-binding site predicted by the crystal structure is supported by this analysis, the evidence argues against the proposed involvement of a conserved aspartate as a general base in the reaction. Analysis of additional Trm10 variants is underway with the ultimate goal of elucidating the complete catalytic mechanism of this unusual methyltransferase enzyme family.
Keywords: tRNA, methyltransferase, Trm10