Abstract
The tRNA methyltransferase Trm10, conserved throughout Eukarya and Archaea, catalyzes N1-methylation of purine residues at position 9 using S-adenosyl methionine as the methyl donor. The Trm10 family exhibits diverse target nucleotide specificity, with some homologs that are obligate m1G9 or m1A9-specific enzymes, while others are bifunctional enzymes catalyzing both m1G9 and m1A9. This variability is particularly intriguing given different chemical properties of the target N1 atom of guanine and adenine. Here we performed an extensive kinetic and mutational analysis of the m1G9 and m1A9-catalyzing Trm10 from Thermococcus kodakarensis to gain insight into the active site that facilitates this unique bifunctionality. These results suggest that the rate-determining step for catalysis likely involves a conformational change to correctly position the substrate tRNA in the active site. In this model, kinetic preferences for certain tRNA can be explained by variations in the overall stability of the folded substrate tRNA, consistent with tRNA-specific differences in metal ion dependence. Together, these results provide new insight into the substrate recognition, active site and catalytic mechanism of m1G/m1A catalyzing bifunctional enzymes.
Highlights
TRNAs undergo extensive post-transcriptional modifications, with the resulting nucleotide diversity affecting the biological and structural properties of these RNAs
Trm10, the tRNA m1 R9 MTase (R = G or A) that catalyzes N1-methylation at position 9 of tRNAs in Eukarya and Archaea, is an atypical member of the SPOUT family that acts in monomeric form [6,7,8,9]
Wild-type T. kodakarensis Trm10 (TkTrm10) was cloned into AVA421, a previously described plasmid for heterologous expression of N-terminal His6 -tagged protein in E. coli, from T. kodakarensis genomic DNA using ligation-independent cloning [6,20]
Summary
TRNAs undergo extensive post-transcriptional modifications, with the resulting nucleotide diversity affecting the biological and structural properties of these RNAs. Almost all the methyltransferases (MTases) that catalyze these modifications in tRNAs belong to two major structural classes (i) Class. The tRNA m1 R9 MTase (R = G or A) that catalyzes N1-methylation at position 9 of tRNAs in Eukarya and Archaea, is an atypical member of the SPOUT family that acts in monomeric form [6,7,8,9]. Loss of m1 A9 methylation in mammalian mitochondrial tRNALys (catalyzed by a human mitochondrial Trm homolog hTRMT10C) results in acute misfolding, forming an extended hairpin loop making the tRNA unusable in translation [10]. Mutations in two different human paralogs (hTRMT10A and hTRMT10C) have been implicated in multiple disease states [11,12,13,14,15].
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