An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation
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Date
2014
Authors
Friedt, Jenna
Leavens, Fern M. V.
Mercier, Evan
Wieden, Hans-Joachim
Wieden-Kothe, Ute
Journal Title
Journal ISSN
Volume Title
Publisher
Oxford University Press
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.
Description
Sherpa Romeo green journal. Permission to archive final published version
Keywords
Arginine , Aspartate , Catalysis , Pseudouridine , TruB , tRNA , Transfer RNA
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