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An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basi...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973310/ https://www.ncbi.nlm.nih.gov/pubmed/24371284 http://dx.doi.org/10.1093/nar/gkt1331 |
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author | Friedt, Jenna Leavens, Fern M. V. Mercier, Evan Wieden, Hans-Joachim Kothe, Ute |
author_facet | Friedt, Jenna Leavens, Fern M. V. Mercier, Evan Wieden, Hans-Joachim Kothe, Ute |
author_sort | Friedt, Jenna |
collection | PubMed |
description | Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism. |
format | Online Article Text |
id | pubmed-3973310 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-39733102014-04-04 An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation Friedt, Jenna Leavens, Fern M. V. Mercier, Evan Wieden, Hans-Joachim Kothe, Ute Nucleic Acids Res Nucleic Acid Enzymes Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism. Oxford University Press 2014-04 2013-12-26 /pmc/articles/PMC3973310/ /pubmed/24371284 http://dx.doi.org/10.1093/nar/gkt1331 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Nucleic Acid Enzymes Friedt, Jenna Leavens, Fern M. V. Mercier, Evan Wieden, Hans-Joachim Kothe, Ute An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation |
title | An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation |
title_full | An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation |
title_fullStr | An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation |
title_full_unstemmed | An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation |
title_short | An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation |
title_sort | arginine-aspartate network in the active site of bacterial trub is critical for catalyzing pseudouridine formation |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973310/ https://www.ncbi.nlm.nih.gov/pubmed/24371284 http://dx.doi.org/10.1093/nar/gkt1331 |
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