6EMV

Crystal Structure of dual specific Trm10 construct from Thermococcus kodakaraensis.

Summary for 6EMV

• Entry DOI: 10.2210/pdb6emv/pdb
• Descriptor: tRNA (guanine(9)-/adenine(9)-N1)-methyltransferase, S-ADENOSYL-L-HOMOCYSTEINE (3 entities in total)
• Functional Keywords: trm10, dual specificity enzymes, spout, mtases, rna binding protein
• Biological source: Thermococcus kodakarensis (Pyrococcus kodakaraensis)
• Total number of polymer chains: 3
• Total formula weight: 68421.03

Authors

Singh, R.K.,Versees, W. (deposition date: 2017-10-03, release date: 2018-06-13, Last modification date: 2024-05-01)

Primary citation

Singh, R.K.,Feller, A.,Roovers, M.,Van Elder, D.,Wauters, L.,Droogmans, L.,Versees, W.
Structural and biochemical analysis of the dual-specificity Trm10 enzyme fromThermococcus kodakaraensisprompts reconsideration of its catalytic mechanism.
RNA, 24:1080-1092, 2018

PubMed 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.

PubMed: 29848639
DOI: 10.1261/rna.064345.117

Experimental method

X-RAY DIFFRACTION (2.90001435892 Å)