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Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism

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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 m 1G formation over m 1A 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.

Tijdschrift: RNA (New York, N.Y.)
ISSN: 1355-8382
Issue: 8
Volume: 24
Pagina's: 1080-1092
Jaar van publicatie:2018
Trefwoorden:Dual specificity, Methyl transferase, SPOUT, TRNA modification, Substrate Specificity/genetics, Adenosine/chemistry, S-Adenosylmethionine/metabolism, Models, Molecular, Crystallography, X-Ray, tRNA Methyltransferases/genetics, Catalytic Domain/physiology, RNA Processing, Post-Transcriptional/physiology, Molecular Docking Simulation, Catalysis, Guanosine/chemistry, Binding Sites, S-Adenosylhomocysteine/metabolism, Thermococcus/enzymology
BOF-keylabel:ja
BOF-publication weight:2
CSS-citation score:1
Auteurs:Regional
Authors from:Higher Education
Toegankelijkheid:Open