1. Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism
- Author
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Wim Versées, Lina Wauters, Ranjan Kumar Singh, Dany Van Elder, André Feller, Martine Roovers, Louis Droogmans, Cell Biochemistry, Vrije Universiteit Brussel, Structural Biology Brussels, Faculty of Sciences and Bioengineering Sciences, and Department of Bio-engineering Sciences
- Subjects
Catalytic Domain/physiology ,Models, Molecular ,0301 basic medicine ,S-Adenosylmethionine ,Adenosine ,PROTEIN ,METHYLTRANSFERASE TRM10 ,Crystallography, X-Ray ,Substrate Specificity ,chemistry.chemical_compound ,Catalytic Domain ,CRYSTAL-STRUCTURE ,Thermococcus/enzymology ,RNA Processing, Post-Transcriptional ,methyl transferase ,23S RIBOSOMAL-RNA ,chemistry.chemical_classification ,tRNA Methyltransferases ,RNA Processing, Post-Transcriptional/physiology ,Guanosine ,biology ,Substrate Specificity/genetics ,RNA MODIFICATION ENZYME ,S-Adenosylmethionine/metabolism ,S-Adenosylhomocysteine ,Molecular Docking Simulation ,Thermococcus ,INSIGHTS ,MITOCHONDRIAL RNASE P ,ESCHERICHIA-COLI ,Transfer RNA ,RIBOSOMAL-RNA ,Life Sciences & Biomedicine ,Biochemistry & Molecular Biology ,TRNA modification ,Stereochemistry ,Article ,Catalysis ,03 medical and health sciences ,YEAST ,Binding site ,S-Adenosylhomocysteine/metabolism ,Molecular Biology ,Adenosine/chemistry ,Science & Technology ,Binding Sites ,dual specificity ,IDENTIFICATION ,Guanosine/chemistry ,TRNA Methyltransferase ,RECOGNITION ,Active site ,biology.organism_classification ,tRNA Methyltransferases/genetics ,SPOUT ,GENE ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,METHYLTRANSFERASE ,tRNA modification - 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.
- Published
- 2018