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

TitleStructural and biochemical analysis of the dual-specificity Trm10 enzyme from prompts reconsideration of its catalytic mechanism.
Publication TypeJournal Article
Year of Publication2018
AuthorsSingh, R. Kumar, A. Feller, M. Roovers, D. Van Elder, L. Wauters, L. Droogmans, and W. Versées
JournalRNA
Volume24
Issue8
Pagination1080-1092
Date Published2018 08
ISSN1469-9001
KeywordsAdenosine, Binding Sites, Catalysis, Catalytic Domain, Crystallography, X-Ray, Guanosine, Models, Molecular, Molecular Docking Simulation, RNA Processing, Post-Transcriptional, S-Adenosylhomocysteine, S-Adenosylmethionine, Substrate Specificity, Thermococcus, tRNA Methyltransferases
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 (Trm10). 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 -adenosyl-l-methionine or -adenosyl-l-homocysteine reveal the conformational plasticity of two active site loops upon substrate binding. Kinetic analysis shows that Trm10 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 mG formation over mA 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.

DOI10.1261/rna.064345.117
Alternate JournalRNA
PubMed ID29848639
PubMed Central IDPMC6049504
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