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• W2806263203 abstract "tRNA molecules get heavily modified post-transcriptionally. The N-1 methylation of purines at position 9 of eukaryal and archaeal tRNA is catalyzed by the SPOUT methyltranferase Trm10. Remarkably, while certain Trm10 orthologs are specific for either guanosine or adenosine, others show a dual specificity. Structural and functional studies have been performed on guanosine- and adenosine-specific enzymes. Here we report the structure and biochemical analysis of the dual-specificity enzyme from Thermococcus kodakaraensis (TkTrm10). We report the first crystal structure of a construct of this enzyme, consisting of the N-terminal domain and the catalytic SPOUT domain. Moreover, crystal structures of the SPOUT domain, either in the apo form or bound to S-adenosyl-l-methionine or S-adenosyl-l-homocysteine reveal the conformational plasticity of two active site loops upon substrate binding. Kinetic analysis shows that TkTrm10 has a high affinity for its tRNA substrates, while the enzyme on its own has a very low methyltransferase activity. Mutation of either of two active site aspartate residues (Asp206 and Asp245) to Asn or Ala results in only modest effects on the N-1 methylation reaction, with a small shift toward a preference for m1G formation over m1A formation. Only a double D206A/D245A mutation severely impairs activity. These results are in line with the recent finding that the single active-site aspartate was dispensable for activity in the guanosine-specific Trm10 from yeast, and suggest that also dual-specificity Trm10 orthologs use a noncanonical tRNA methyltransferase mechanism without residues acting as general base catalysts." @default.
• W2806263203 created "2018-06-13" @default.
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• W2806263203 date "2018-05-30" @default.
• W2806263203 modified "2023-09-27" @default.
• W2806263203 title "Structural and biochemical analysis of the dual-specificity Trm10 enzyme from <i>Thermococcus kodakaraensis</i> prompts reconsideration of its catalytic mechanism" @default.
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• W2806263203 doi "https://doi.org/10.1261/rna.064345.117" @default.
• W2806263203 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6049504" @default.
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