6ems | pdb_00006ems
Crystal Structure of dual specific Trm10 construct from Thermococcus kodakaraensis.
Structural highlights
Function[TRM10_THEKO] Catalyzes the S-adenosyl-L-methionine-dependent formation of either N(1)-methyladenine or N(1)-methylguanine at position 9 (m1A9 or m1G9) in tRNA.[1] Publication Abstract from PubMedtRNA molecules get heavily modified posttranscriptionally. 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 orthologues 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 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(1)G formation over m(1)A 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 orthologues use a non-canonical tRNA methyltransferase mechanism without residues acting as general base catalysts. Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism.,Singh RK, Feller A, Roovers M, Van Elder D, Wauters L, Droogmans L, Versees W RNA. 2018 May 30. pii: rna.064345.117. doi: 10.1261/rna.064345.117. PMID:29848639[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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