An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

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dc.contributor.authorFriedt, Jenna
dc.contributor.authorLeavens, Fern M. V.
dc.contributor.authorMercier, Evan
dc.contributor.authorWieden, Hans-Joachim
dc.contributor.authorWieden-Kothe, Ute
dc.date.accessioned2017-03-17T22:44:24Z
dc.date.available2017-03-17T22:44:24Z
dc.date.issued2014
dc.descriptionSherpa Romeo green journal. Permission to archive final published versionen_US
dc.description.abstractPseudouridine 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 finetuning 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.en_US
dc.description.peer-reviewYesen_US
dc.identifier.citationFriedt, J., Leavens, F.M.V., Mercier, E., Wieden, J., & Kothe, U. (2014). An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation. Nucleic Acids Research, 42(6), 3857-3870. doi:10.1093/nar/gkt1331en_US
dc.identifier.urihttps://hdl.handle.net/10133/4805
dc.language.isoen_USen_US
dc.publisherOxford University Pressen_US
dc.publisher.departmentDepartment of Chemistry and Biochemistryen_US
dc.publisher.facultyArts and Scienceen_US
dc.publisher.institutionMax Planck Institute for Biophysical Chemistryen_US
dc.publisher.institutionUniversity of Lethbridgeen_US
dc.subjectArginineen_US
dc.subjectAspartateen_US
dc.subjectCatalysisen_US
dc.subjectPseudouridineen_US
dc.subjectTruBen_US
dc.subjecttRNAen_US
dc.subjectTransfer RNAen_US
dc.titleAn arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formationen_US
dc.typeArticleen_US
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Wieden, Hans-Joachim
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