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Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism
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 specif...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Cold Spring Harbor Laboratory Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049504/ https://www.ncbi.nlm.nih.gov/pubmed/29848639 http://dx.doi.org/10.1261/rna.064345.117 |
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author | Singh, Ranjan Kumar Feller, André Roovers, Martine Van Elder, Dany Wauters, Lina Droogmans, Louis Versées, Wim |
author_facet | Singh, Ranjan Kumar Feller, André Roovers, Martine Van Elder, Dany Wauters, Lina Droogmans, Louis Versées, Wim |
author_sort | Singh, Ranjan Kumar |
collection | PubMed |
description | 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 ((Tk)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 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 (Tk)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 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 orthologs use a noncanonical tRNA methyltransferase mechanism without residues acting as general base catalysts. |
format | Online Article Text |
id | pubmed-6049504 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-60495042019-08-01 Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism Singh, Ranjan Kumar Feller, André Roovers, Martine Van Elder, Dany Wauters, Lina Droogmans, Louis Versées, Wim RNA Article 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 ((Tk)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 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 (Tk)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 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 orthologs use a noncanonical tRNA methyltransferase mechanism without residues acting as general base catalysts. Cold Spring Harbor Laboratory Press 2018-08 /pmc/articles/PMC6049504/ /pubmed/29848639 http://dx.doi.org/10.1261/rna.064345.117 Text en © 2018 Singh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Article Singh, Ranjan Kumar Feller, André Roovers, Martine Van Elder, Dany Wauters, Lina Droogmans, Louis Versées, Wim Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
title | Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
title_full | Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
title_fullStr | Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
title_full_unstemmed | Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
title_short | Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
title_sort | structural and biochemical analysis of the dual-specificity trm10 enzyme from thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049504/ https://www.ncbi.nlm.nih.gov/pubmed/29848639 http://dx.doi.org/10.1261/rna.064345.117 |
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