Show simple item record Friedt, Jenna Leavens, Fern M. V. Mercier, Evan Wieden, Hans-Joachim Wieden-Kothe, Ute 2017-03-17T22:44:24Z 2017-03-17T22:44:24Z 2014
dc.identifier.citation Friedt, 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/gkt1331 en_US
dc.description Sherpa Romeo green journal. Permission to archive final published version en_US
dc.description.abstract 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 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.language.iso en_US en_US
dc.publisher Oxford University Press en_US
dc.subject Arginine en_US
dc.subject Aspartate en_US
dc.subject Catalysis en_US
dc.subject Pseudouridine en_US
dc.subject TruB en_US
dc.subject tRNA en_US
dc.subject Transfer RNA en_US
dc.title An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation en_US
dc.type Article en_US
dc.publisher.faculty Arts and Science en_US
dc.publisher.department Department of Chemistry and Biochemistry en_US
dc.description.peer-review Yes en_US
dc.publisher.institution Max Planck Institute for Biophysical Chemistry en_US
dc.publisher.institution University of Lethbridge en_US

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