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

Article abstract of DOI:10.1093/nar/gkt1331

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. The Author(s) 2013. Published by Oxford University Press.

Full text of DOI:10.1093/nar/gkt1331

Published online 26 December 2013
Nucleic Acids Research, 2014, Vol. 42, No. 6 3857–3870
doi:10.1093/nar/gkt1331
An arginine-aspartate network in the active site of
bacterial TruB is critical for catalyzing
pseudouridine formation
Jenna Friedt, Fern M. V. Leavens, Evan Mercier, Hans-Joachim Wieden and Ute Kothe*
Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of
Lethbridge, Lethbridge AB T1K 3M4, Canada
Received October 29, 2013; Revised November 27, 2013; Accepted November 30, 2013
conversion of uridines to pseudouridines (1,2). Similar to
DNA glycosylases, these enzymes catalyze the breakage of
a glycosidic bond, albeit not by hydrolysis. Instead, the
uracil base is reoriented and subsequently reattached to
the ribose through an unusual C5–C1’ glycosidic bond.
Based on this reaction mechanism including breakage of
the glycosidic bond and formation of a new C-C bond,
pseudouridine synthases are sometimes also considered
RNA lyases. Through numerous structural studies, it
has become clear that all pseudouridine synthases share
a common catalytic domain despite significant heterogen-
eity in primary structure (3–10). Therefore, it has been
suggested that all pseudouridine synthases share the
same catalytic mechanism (11).
After more than a decade of research, the details of
the catalytic mechanism of pseudouridine formation still
remain under debate with three different mechanisms
being proposed. The only universally conserved residue
in the active site of all pseudouridine synthases is an as-
partate residue that is essential for catalysis (12–20). This
aspartate may contribute to catalysis either by forming a
covalent bond to C1’ of the ribose (13), to C6 of the uracil
base (21) or by abstracting a proton from C2’ of the ribose
(22). Currently the two mechanisms are favored where the
aspartate acts on the ribose rather than the uracil (22).
Hardly anything is known about the contribution of
other residues to the formation of pseudouridine. Within
the active site, there are two additional residues that are
highly conserved, a basic and an aromatic residue (2). The
basic residue in the active site of pseudouridine synthases
is either an arginine or a lysine that forms a salt bridge
interaction with the catalytic aspartate. It may be the role
of the basic residue to render the aspartate more nucleo-
philic, to position the aspartate for catalysis or to serve as
a source of protons (2). Additionally, an aromatic residue
in the active site is found to stack against the target uracil
base likely stabilizing the conformation of the base within
the active site (23). All pseudouridine synthases use a
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 cataly-
sis 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, biochem-
ical and kinetic studies reveal that this residue is not
critical for substrate binding but influences cataly-
sis significantly as replacement of D90 with glutam-
ate or asparagine reduces the catalytic rate 30- and
50-fold, respectively. In agreement with molecu-
lar 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, nega-
tively charged residues similar to D90 are found in a
number of pseudouridine synthases, suggesting
that this might be a general mechanism.
INTRODUCTION
Pseudouridine synthases are tRNA-uridine uracil mutases
(E.C. 5.4.99) that catalyze the most common modifica-
tion in functional RNA, namely the posttranscriptional
*To whom correspondence should be addressed. Tel: +1 403 332 5274; Fax: +1 403 329 2057; Email: ute.kothe@uleth.ca
Present address:
Evan Mercier, Max Planck Institute for Biophysical Chemistry, Department of Physical Biochemistry, Gottingen, Germany.
ß The Author(s) 2013. Published by Oxford University Press.
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

3858 Nucleic Acids Research, 2014, Vol. 42, No. 6
tyrosine for this role, except for TruD where this residue is
instead a phenylalanine (7–9). The tyrosine has also been
proposed to act as a general base abstracting a proton
from C5 to complete the isomerization process (23).
One of the best characterized pseudouridine synthases
that often serves as a model for the other enzymes is the bac-
terial pseudouridine synthase TruB. This enzyme modifies
U55 in the TÉC arm of all elongator tRNAs (24). Although
not the first pseudouridine synthase to be identified, TruB
was the first to be crystallized in the presence of RNA
providing significant insight into the architecture of the
active site of pseudouridine synthases (5). Subsequent struc-
tures of TruB, including its apo form, revealed an induced-
fit mechanism of substrate binding involving the movement
of TruB’s thumb loop, insert 1 segment, as well as its
C-terminal PUA domain (found in pseudouridine synthases
and archaeosine-transglycosidases) (25–27). The RNA also
undergoes some conformational changes, as three consecu-
tive bases including the target uridine are flipped out of the
T loop into the active site of TruB. In addition, a number of
biochemical studies on TruB have been reported including a
detailed analysis of its substrate specificity (28), the import-
ance of the catalytic aspartate residue (14,25), the role of the
conserved tyrosine residue in the active site (23), investiga-
tions on the pH profile (29) and determination of catalysis
as the rate-limiting step in TruB’s kinetic mechanism (30).
The bacterial TruB enzyme is a homologue of the
archaeal and eukaryotic Cbf5 protein (31) that acts as the
pseudouridine synthase in H/ACA small (nucleolar)
ribonucleoproteins (32).
In this study, we have investigated the function of the
basic residue in the active site (R181 in TruB) as well as
the role of a nearby second-shell residue (D90 in insert 1 of
TruB) using a combination of site-directed mutagenesis,
biochemistry, biophysics and computational methods.
Our results underline the importance of both residues
for catalysis of pseudouridine formation, but not for
tRNA interaction. In addition, we identify the electro-
static properties of these residues as their most important
characteristic, which—together with molecular dynamic
simulations—suggests that the catalytic aspartate, the
conserved basic residue and the second-shell D90 residue
form an electrostatic interaction network that is critical
for catalysis of pseudouridylation.
site-directed mutagenesis (Stratagene) to encode the
amino acid substitutions D90E, D90N, D90A, R181K,
R181M and R181A. TruB wild type and variants were
expressed and purified as previously described (30) using
affinity and size-exclusion chromatography. Final protein
concentration was determined using A280 (molar extinc-
tion coefficient of 20 985 Mꢀ1 cmꢀ1) and by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) comparison. A260 and urea-PAGE analysis
showed that protein preparations were not detectably
contaminated with nucleic acid, and final protein purity
was >95% as judged by SDS-PAGE.
[3H]-labeled tRNA preparation
E. coli [3H]-tRNAPhe was prepared as described (30). In
brief, template DNA for tRNAPhe was amplified from the
pCF0 plasmid (33) and used for in vitro transcription of
tRNAPhe including 100 mM [C5-3H]-UTP. The [3H]-tRNA
was purified with a Nucleobond PC100 column (Macherey
& Nagel). Scintillation counting and absorbance measure-
ments at 260 nm were used to quantify the tRNA concen-
tration and specific activity.
Tritium release assay
[3H]-tRNA was refolded in 1ꢁ TAKEM4 buffer by
incubation at 65ꢂC for 5 min followed by cooling to
room temperature for 10 min. TruB was diluted in 1ꢁ
TAKEM4 buffer and pre-warmed at 37ꢂC before adding
the folded tRNA and incubating at 37ꢂC. Samples were
taken from the reaction at various time points and the
amount of released tritium corresponding to the amount
of pseudouridine formation was determined as
described (30). To determine the apparent rate of catalysis
(kapp), single-turnover experiments were analyzed by one-
exponential fitting:
Y ¼ Ymax+Amp ꢁ expðꢀkapp ꢁ tÞ
2AP-tRNA preparation
2AP-tRNA was prepared by annealing two halves of the
tRNA and ligating to give full-length tRNA. The follow-
ing 30-half was purchased from Dharmacon and contained
a 2AP label at position 57 (full-length numbering): 50-AA
AUCCCCGUGUCCUUGGUUCG-2AP-UUCCGAGU
CCGCGCACCA-30. The 50-half was generated by
assembly and amplification of template DNA followed
by in vitro transcription. First, the template was assembled
by polymerase chain reaction using the 50-half sense
primer (50-GCGTAATACGACTCACTATAGCGCGG
ATAGCTCAGTCG-30) and the 50-half antisense primer
(50-TCAATCCCCTGCTCTACCGACTGAGCTATCC
G-30); second, the product of this assembly was further
amplified using the sense T7 promoter primer (50-GCGT
AATACGACTCACTATAG-30) and a methylated reverse
MATERIALS AND METHODS
Buffers and reagents
TAKEM4 buffer: 50 mM Tris–HCl, pH 7.5, 70 mM
NH4Cl, 30 mM KCl, 1 mM EDTA, 4 mM MgCl2.
[C5-3H]-UTP for in vitro transcriptions was purchased
from Moravek. T4 RNA ligase was purchased from
New England Biolabs. All other enzymes and chemicals
were obtained from Fermentas (Fisher Scientific).
2-aminopurine (2AP)-labeled 30-half tRNA was purchased
from Dharmacon (Thermo Fisher Scientific).
primer
(50-mUmCAATCCCCTGCTCTACCGAC-30).
The 50-half tRNA template was then used in 2 ml in vitro
transcription reactions using the same procedure as
outlined for [3H]-tRNA, but including 3 mM nonradio-
active UTP. Following ethanol precipitation, the RNA
Protein expression and purification
The Escherichia coli TruB coding region in the pET28a-
EcTruB plasmid (30) was mutated using QuikChangeÕ

Nucleic Acids Research, 2014, Vol. 42, No. 6 3859
was purified using the BioRad Model 491 Prep Cell con-
tinuous electrophoresis purification system. The RNA
(1.44 ml) was loaded on a 5-cm 12% urea-PAGE (28 mm
diameter) and a constant voltage of 250 V was applied while
eluting with 1ꢁ Tris/Borate/EDTA (TBE) buffer at a flow
rate of 0.75 ml/min at the bottom of the gel. Peak fractions
(A254) were analyzed by 12% urea-PAGE, pooled and
ethanol-precipitated in the presence of 0.05 mg/ml
glycogen. To generate full-length 2AP-tRNA, 15 mM 50-
half tRNA was annealed in 1.5ꢁ excess to 2AP-30-half
tRNA in 1ꢁ T4 RNA ligase buffer (New England
Biolabs) by heating at 95ꢂC for 3 min, 65ꢂC for 10 min
and then slowly cooling from 65 to 20ꢂC over 45 min. T4
RNA ligase (0.02 U/ml) was added to the mixture followed
by incubation at 37ꢂC for 4 h. Ligated tRNA was purified
by urea-PAGE, ultraviolet-shadowing and electroelution
(EluTrap electroelution system, Whatman).
place the thumb-loop structure (residues 124–152) (26).
Therefore, to simulate TruB-apo, first a homology
model of E. coli TruB-apo was generated using the
Swiss-Model server (34,35) and the Thermotoga maritima
TruB-apo crystal structure [PDBID: 1ZE1 (27)] as a
template such that the ordered thumb-loop could be
added to the crystal structure of E. coli TruB-apo
(PDBID: 1R3F). To do this, the T. maritima TruB struc-
ture was superimposed with the crystal structure of E. coli
TruB-apo with respect to the catalytic domain. The coord-
inates of residues 124–152 corresponding to the thumb-
loop of T. maritima TruB were added to the coordinates
of E. coli TruB-apo.
To generate a starting structure for TruB-bound, the
crystal structure for E. coli TruB bound to a short RNA
[PDBID: 1K8W (5)] and the TruB-apo model were used.
Since the crystal structure for TruB-bound was missing
amino acids at both the N- and C- termini, E. coli
TruB-bound was superimposed with TruB-apo with
respect to residue 10, and the coordinates of residues
8–9 from TruB-apo were added to the coordinates of
TruB-bound. The structures were then superimposed
with respect to residues 310–311, and the coordinates of
residues 312–314 from TruB-apo were added to the coord-
Fluorescence spectroscopy and stopped-flow experiments
2AP-tRNA (0.05–0.2 mM) diluted in 1ꢁ TAKEM4 buffer
was excited at 325 nm, titrated with TruB and the emis-
sion was measured from 340 to 400 nm using a Quanta
Master 60 fluorescence spectrometer (Photon Technology
International). After the first addition of TruB, the sample
was incubated at room temperature for 30 min in the dark
to allow for pseudouridine formation to occur. Similarly,
TruB was titrated into buffer, and the measured fluores-
cence intensity was subtracted from the titration with
2AP-tRNA. Relative fluorescence changes at 364 nm
were plotted against protein concentration. The data
were fitted with a hyperbolic function to determine the
KD for TruB binding to 2AP-tRNA:
˚
inates of TruB-bound. Water molecules within 10 A of the
protein as observed in the crystal structure (PDBID:
1K8W) were also included in the TruB-bound model.
This was not done for the TruB-apo model because
there were no water molecules identified in the crystal
structure. The model for the TruB D90N simulation was
generated from the TruB-bound model, modifying D90 to
N90 with the Mutator plugin in Visual Molecular
Dynamics (VMD) (36). Hydrogen atoms were added to
each model using the Psfgen plugin in VMD.
Y ¼ Bmax ꢁ ½proteinꢃ=ðKD+½proteinꢃÞ
All models were solvated using the solvate package in
˚
VMD to create a water box that extended at least 10 A
Pre–steady-state fluorescence experiments were per-
formed using a KinTek SF-2004 stopped-flow apparatus.
A final concentration of 0.1 mM 2AP-tRNA was rapidly
mixed with 3 mM TruB and excited at 325 nm. Emission
was monitored at wavelengths >350 nm. For TruB wild
type, the time courses were fitted with a 3-exponential
function:
around the protein in each direction. The models were
then minimized in five stages using a 0.5 fs time step, in
which the protein molecules were fixed for the first 10 000
steps, followed by fixing the water molecules for the next
10 000 steps, repetition of the first two stages and releasing
all constraints for 100 000 steps or until the energy of the
system stabilized. The final minimized structures were
ionized and neutralized using the autoionize package in
VMD. Since wet-lab work with TruB was conducted
using TAKEM4 buffer, the structures were ionized and
subsequently neutralized by placing 100 mM K+,
F ¼ Fmax+Amp1 ꢁ expðꢀkapp1 ꢁ tÞ+Amp2
ꢁ expðꢀkapp2 ꢁ tÞ+Amp3 ꢁ expðꢀkapp3 ꢁ tÞ
where Fmax is the fluorescence end-level; Amp is the
respective amplitude of fluorescence change for each
phase; t is time; and kapp indicates the apparent rate of
the respective phase. Experiments with the TruB variants
were best fit with a 2-exponential function:
100 mM Clꢀ and 3 mM Mg2+ no closer than 5 A to each
˚
other or to the protein. The models were further
minimized for 100 000 steps at 300 K without any atom
constraints. The final model for TruB-apo contained 4811
protein atoms, 45 321 water atoms, 37 K+ ions, 30 Clꢀ
ions and 1 Mg2+ ion (50 200 atoms total). The final
model for TruB-bound contained 4811 protein atoms,
44 634 water atoms, 37 K+ ions, 30 Clꢀ ions and 1 Mg2+
ion (49 513 atoms total). The final model for TruB D90N
contained 4813 protein atoms, 44 826 water atoms, 36 K+
ions, 30 Clꢀ ions and 5 Mg2+ ions (49 710 atoms total).
Before starting the simulations, the models were
equilibrated at 310 and 360 K for 150 ps with a step
size of 0.5 fs at constant pressure (1 atm) and
F ¼Fmax+Amp1 ꢁ expðꢀkapp1 ꢁ tÞ+Amp2
ꢁ expðꢀkapp2 ꢁ tÞ
TruB molecular dynamics simulations
TruB wild type was simulated in two different conform-
ations as observed in the presence (bound) and absence
(apo) of RNA. In crystal structures of E. coli TruB in the
absence of RNA, insufficient resolution was obtained to

3860 Nucleic Acids Research, 2014, Vol. 42, No. 6
Figure 1. Conserved residues in the active site of the bacterial pseudouridine synthase TruB. (A) Sequence alignment of TruB from different bacteria
(E. coli, Serratia marcescens, Yersinia enterocolitica, Vibrio cholera, Haemophilus influenzae), Saccharomyces cerevisiae Pus4 and Pyrococcus furiosus
Cbf5. The conserved active site residues D48 and R181 as well as the conserved second-shell residue D90 in insert 1 (E. coli numbering) are indicated
by asterisks. (B) TruB sequence conservation mapped onto the E. coli TruB apo structure (PDBID: 1R3F) and represented by a color gradient of red
to white to blue, indicating 0–100% conserved, respectively (a colour version of this figures is available online). The conservation of each residue was
determined from a multiple sequence alignment of 100 bacterial TruB sequences. Stick models of the conserved residues D48, D90 and R181 are
˚
˚
shown in yellow. D48–R181 C-C distance: 4.5 A, D90–R181 C-C distance: 3.8 A. (C) Close-up view of TruB active site (cyan), including 22-mer T-
loop RNA (dark blue). Stick models show proximity of D48 (green) and R181 (blue) of TruB to U55 of RNA [red, 5-fluoro-6-hydroxy-pseudouracil
(5FhÉ55) in this structure], as well as proximity of D90 of TruB (purple) in insert 1 (also purple) to C56 of RNA (yellow). D48–R181 C-C distance:
˚
˚
4.0 A, D90-R181 C-C distance: 5.7 A. Image was generated in VMD using PDBID: 1K8W.
variable volume (NPT ensemble) while invoking periodic
boundary conditions. A Nose-Hoover Langevin piston
RESULTS
´
To identify and analyze interactions between active site
residues of TruB, a multiple sequence alignment of 100
bacterial TruB sequences was used to determine highly
conserved residues that were then mapped onto the
E. coli TruB structure (Figure 1). As known before, the
active site and the RNA-interaction face of the protein
are highly conserved including three known active site
residues, i.e. catalytic aspartate 48, tyrosine 76 that is
thought to help stabilize the conformation of the
flipped-out RNA bases after binding (23) and arginine
181 that interacts with D48 (5,25,26,39). Although R181
has been proposed to be a catalytic residue of pseudourid-
ine synthases based on its conservation and location in the
active site, its function has not been tested before (to the
best of our knowledge). In addition, residues L200 and
R202 are universally conserved and located on the
b-sheet behind the active site. Notably, the only other in-
variant residue close to the active site is aspartate 90, a
second-shell residue located in insert 1. As reported previ-
ously, this D90 residue contacts the strictly required C56
of the RNA substrate (26–28).
was used for pressure control, and Langevin dy-
namics were used to maintain a constant temperature.
Production phase simulations were performed at 310 K
using the final atomic velocities from the equilibration at
310 K and the atomic coordinates from the equilibration
at 360 K. The particle mesh Ewald method was used to
calculate full-system electrostatics. Simulations were
generated in NAMD2 with CHARMM27 parameters
(37,38) for 40 ns using periodic boundary conditions,
NPT ensemble conditions with a Nose-Hoover Langevin
´
piston for pressure control and Langevin dynamics for
temperature control. The simulations were analyzed
using scripts written in house, evoked in VMD. Data
from the trajectories was sampled every 2 ps. Root-
mean-square deviation (RMSD) was calculated using the
backbone atoms of all residues and all residues excluding
the thumb-loop (residues 124–152). Root-mean-square
fluctuation (RMSF) was calculated for the backbone
atoms of each residue excluding the first 5 ns when the
simulation was still stabilizing. Active site interactions
were analyzed by measuring the distance between the
terminal carbon atom of the carboxylate side chain
group of aspartate and the terminal carbon of the
guanidinium side chain group of arginine.
Here, we aimed to elucidate the exact role of the highly
conserved active site residues R181 and D90 for the
function of E. coli TruB. Toward this goal, site-directed
mutagenesis was used to substitute D90 (with glutamate,

Nucleic Acids Research, 2014, Vol. 42, No. 6 3861
Table 1. Dissociation constants determined from fluorescence titra-
tions (Figure 2)
TruB
KD, mM
Wild type
D90E
D90N
0.7 0.1
0.4 0.2
0.4 0.1
0.4 0.2
0.7 0.1
0.5 0.2
D90A
R181K
R181M
R181A
2
1
KD values are stated with their standard deviation derived from hyper-
bolic fitting of the titration curves (Figure 2).
Figure 2. Binding of 2AP-tRNA by TruB variants. 2AP-tRNA
(50–200 nM) was titrated with increasing concentrations of TruB, and
the relative fluorescence change of 2AP at 364 nm was recorded. Three
representative titrations are shown for TruB wild type (closed circles,
dashed line), TruB D90E (closed squares) and TruB R181K (open
affinity for unmodified substrate tRNA is determined.
Since TruB binds substrate and product tRNA with
similar affinities (14,30), the determined dissociation con-
stants should be comparable even for heterogeneous
populations of substrate and product tRNA. Hence, our
results show that substitutions of R181 or D90 in the
active site of TruB do not strongly affect tRNA binding.
squares). Fitting a hyperbolic function to each titration curve
provided the dissociation constants (KD) summarized in Table 1.
asparagine or alanine) and R181 (with lysine, methionine
or alanine). The substitutions were chosen to disrupt the
charge and alter the size of the original residue at each
position. These TruB variants were over-expressed
and purified by affinity and size-exclusion chromatog-
raphy resulting in >95% purity with no detectable RNA
contamination.
Effects of amino acid substitutions on catalysis of
pseudouridine formation
Next, the catalytic activity of each TruB variant was
assessed in tritium release assays using [C5-3H]-labeled
tRNA. Upon formation of the new C5-C1’ glycosidic
bond in pseudouridine, the tritium is released and can be
quantified by extraction and scintillation counting (41).
Initially, multiple-turnover conditions were used by
incubating 20 nM of TruB with 950 nM tRNA.
Surprisingly, hardly any pseudouridine formation was
detected within 60 min for all D90 and R181 variants,
while TruB wild type rapidly converted nearly all substrate
into product within the first 5 min (data not shown). When
the enzyme concentration was increased to 200 nM,
complete conversion of substrate to product was seen
for TruB wild type in <1 min, whereas only TruB D90E
and D90N showed significant product formation under
these conditions (Figure 3A). TruB D90A was able to
form pseudouridine, but only reached 30% product for-
mation after 120 min. When incubating for 30 h,
pseudouridylation could be detected with TruB R181K,
but not TruB R181M and R181A (Figure 3B). In conclu-
sion, substitutions of residue R181 and D90 lead to severe
reductions in the ability of TruB to form pseudouridines,
and the yields in multiple-turnover reactions are too low
to conduct Michaelis–Menten titrations.
To detect residual activity of the TruB R181M and
TruB R181A variants, as well as to determine the rates
for catalysis by all TruB variants, single-turnover experi-
ments were performed, again using 950 nM tritium-labeled
tRNA, but an excess of TruB enzyme (3 mM). Under these
conditions of high enzyme concentration, product forma-
tion was observed for all D90 and R181 TruB variants
(Figure 4A and B), albeit at much slower rates than for
TruB wild type. In fact, TruB R181M and R181A
required incubation with tRNA for up to 30 h before
tRNA affinity of TruB D90 and R181 variants
First, the ability of the TruB variants to bind tRNA was
tested by fluorescence titrations using a novel assay based
on tRNAPhe containing a 2AP in position 57; at this
position, TruB has been reported to accept any nucleobase
(28). 2AP is a fluorescent adenine analog that is quenched
when involved in base-stacking interactions and is often
used to study RNA folding (40). We hypothesize that 2AP
at position 57 will undergo a fluorescence change upon
binding to TruB when nucleotides 55–57 of the tRNA
are flipped into the active site of the enzyme. The 2AP-
tRNA was generated by ligating an in vitro transcribed
50 half of the tRNA to a chemically synthesized 30 half
containing the 2AP label (for details see ‘Materials and
Methods’ section). 2AP-tRNA was titrated with TruB
wild type, D90 and R181 variants, and the relative fluor-
escence change at 364 nm was recorded (Figure 2).
A hyperbolic function was fitted to the data yielding the
dissociation constant (KD) (Table 1). For TruB wild-type,
a KD of 0.7
0.1 mM was recorded, which is in excellent
agreement with previous absorbance-based measurements
(30). Therefore, we conclude that the 2AP label does not
significantly affect tRNA binding and is useful for TruB–
tRNA interaction studies. The TruB D90 variants showed
no decrease in tRNA affinity compared with the wild type,
and the same is true for TruB R181K and R181M. Only
TruB R181A displayed a 3-fold increase in the dissoci-
ation constant compared with the wild type enzyme
(Table 1). During the incubation period before fluores-
cence measurements, active TruB variants will modify
the tRNA, and consequently the affinity for product
tRNA is measured. For inactive TruB variants, the

3862 Nucleic Acids Research, 2014, Vol. 42, No. 6
Figure 3. Pseudouridine formation by TruB variants under multiple-turnover conditions. Tritium-labeled tRNAPhe (0.95 mM) was incubated with
TruB (0.2 mM) at 37ꢂC, and pseudouridine formation was quantified at selected time points with the help of a tritium release assay. (A) Two-hour
time courses with TruB wild type (closed circles), TruB D90E (closed squares), TruB D90N (closed triangles) and TruB D90A (closed diamonds).
(B) Thirty-hour time courses with TruB wild type (closed circles) and TruB R181K (open squares) to confirm detectable activity (no enzyme control,
open circles).
Figure 4. Pseudouridine formation by TruB variants under single-turnover conditions. The percentage of pseudouridine formed was determined
during incubations of 0.95 mM titrium-labeled tRNAPhe with an excess of 3 mM TruB at 37ꢂC. (A) Analysis of TruB variants with substitutions of
D90: TruB wild type (closed circles), TruB D90E (closed squares), TruB D90N (closed triangles) and TruB D90A (closed diamonds). (B) Effect of
substitutions of R181: TruB wild type (closed circles), TruB R181K (open squares), TruB R181M (open triangles) and TruB R181A (open
diamonds). (C) Long time courses with TruB R181M (open triangles) and TruB R181A (open diamonds). No enzyme control, open circles.
(A–C) A single-exponential equation was fitted to the data to determine the rate of catalysis, kapp (smooth lines, see Table 2).
pseudouridylation activity could be reliably detected
(Figure 4C). The single-turnover time courses were fit
with single-exponential functions to obtain the rate of
pseudouridine formation (Table 2). Compared with the
single-turnover rate of TruB wild type (30), a drastic re-
duction in catalytic rate was seen for each variant. The
D90 variants were generally more active than the R181
variants, showing a reduction in catalytic rate of 30 -,
50- and 500-fold for substitutions D90E, D90N and
D90A, respectively. TruB R181K displayed a 2500-fold
reduction in catalytic activity compared with wild type,
and TruB R181M and R181A were the most impaired
at pseudouridine formation with a reduction in catalytic
rate of >20 000-fold. Importantly, the tritium release assay
is able to detect pseudouridine formation while the tRNA
is still in the active site of TruB (the enzyme is denatured
and the tritium released during quenching of the reaction).
Also, the single-turnover assays are independent of
product release as each enzyme modifies only one sub-
strate tRNA. Therefore, the decrease in rate is due to

Nucleic Acids Research, 2014, Vol. 42, No. 6 3863
slow catalysis (or an earlier step in the kinetic mechanism),
but cannot be explained by slow product release alone.
increase has a relatively small amplitude rendering it dif-
ficult to determine precise apparent rates (82 61 sꢀ1 for
wild type), but qualitatively there is no difference for kapp1
among the different TruB variants analyzed. The second
fluorescence increase has a larger amplitude and takes
place with a rate of 4.2 0.5 sꢀ1 for the wild type
enzyme. This apparent rate is similar to a previously
determined overall rate of tRNA binding to TruB based
on absorbance changes (30). As these earlier studies
showed that this rate is independent of the TruB concen-
tration, it was concluded that tRNA binding to TruB
takes place in at least two steps. With the 2AP reporter
group, this second phase has the largest amplitude and
likely represents base-flipping into the active site of
TruB. As the 2AP label is highly sensitive to its environ-
ment, it might be that the first rapid fluorescence increase
detected here, which has a comparably small amplitude,
represents the initial bimolecular encounter of tRNA and
TruB. The D90 and R181 variants display kapp2 values
that are only 2-fold lower than observed for TruB wild
type (see Table 3 for all kapp2 values), suggesting that the
substitutions do not significantly influence the kinetics of
tRNA binding and the subsequent base-flipping into
TruB’s active site. This is in agreement with comparable
affinities of these proteins to tRNA (Table 1). Hence, the
observed strong impairments in pseudouridylation are not
a result of deficiencies in tRNA binding and likely not of
base-flipping, but stem directly from effects on the
chemical steps in TruB’s mechanism.
Kinetics of tRNA binding to TruB variants
To convert uridine to pseudouridine, a number of steps
have to take place including binding of tRNA, flipping of
the target uridine and the two subsequent bases into
TruB’s active site, breaking of the N1–C1’ glycosidic
bond, rotation or flipping of the detached uracil base, for-
mation of a new C5–C1’ glycosidic bond and release of the
modified tRNA. To narrow down the step in catalysis in
which the TruB variants were impaired, rapid-kinetic
stopped-flow analysis was performed using tRNAPhe con-
taining a 2AP in position 57. To observe the interaction of
tRNA with TruB wild type and variants in real time,
0.1 mM 2AP-tRNA was rapidly mixed with an excess of
TruB (3 mM). When 2AP-tRNA interacted with TruB wild
type, a two-phase rapid increase in fluorescence was
detected followed by a slower decrease in fluorescence
(Figure 5). In contrast, when mixing 2AP-tRNA with
TruB D90 or TruB R181 variants, only the biphasic fluor-
escence increase was observed without a subsequent fluor-
escence decrease. Fitting of the time courses with three- or
two-exponential functions yielded the apparent rates for
each fluorescence change. The first rapid fluorescence
Table 2. Apparent rates of pseudouridine formation by TruB variants
The apparent rate of the fluorescence decrease observed
uniquely with TruB wild type was 0.26 0.02sꢀ1 and
similar to the previously determined rate of catalysis for
TruB [0.5 0.2 sꢀ1 (30)]. Presumably, this fluorescence
decrease reflects release of the tRNA, which is rate-
limited by catalysis for TruB wild type (30). As the TruB
D90 and TruB R181 variants are extremely slow in pseudo-
uridine formation (Figure 4), it seems that the tRNA stays
bound to the protein within this observation time (<1 min).
Longer stopped-flow time courses with the TruB D90 and
TruB R181 variants did not allow reliable determination of
the rate of fluorescence decrease as this took place on a
similar timescale as photobleaching. In summary, TruB
D90 and TruB R181 variants interact with tRNA on a
TruB
kapp, sꢀ1
Fold decrease
Wild type
D90E
D90N
0.5 0.2
0.017 0.003
0.010 0.003
0.0010 0.0002
0.0002 0.0001
< 3 ꢁ 10ꢀ5
30
50
500
2500
>20 000
>20 000
D90A
R181K
R181M
R181A
<3 ꢁ 10ꢀ5
Apparent rates, kapp, were determined by single-exponential fitting of
time courses recorded under single-turnover conditions (Figure 4). The
fold decrease in catalytic rate is given relative to the rate constant of
pseudouridine formation by TruB wild type as previously determined
(30). kapp represents average rate from duplicate data standard
deviation.
Figure 5. Kinetics of tRNA interaction with TruB observed by 2AP fluorescence. tRNA (0.1 mM) was rapidly mixed in a stopped-flow apparatus
with TruB (3 mM) wild type (gray), TruB D90 variants (A) and R181 variants (B). Each time course was fitted with a 3-exponential function for TruB
wild type (smooth black line), or a 2-exponential function for TruB D90E and R181K (smooth white lines), D90N and R181M (dashed white lines)
and D90A and R181A (dotted white lines). Apparent rates for the strong fluorescence increase (kapp2) derived from the fitting are summarized in
Table 3; other apparent rates are described in the results.

3864 Nucleic Acids Research, 2014, Vol. 42, No. 6
Table 3. Apparent rates of tRNA binding to TruB wild type and
variants
domain had the highest RMSF values, as these elements
have been shown to undergo conformational changes on
RNA binding (26). Interestingly, the flexibility of insert 1
differed between the three MD simulations with high
RMSF values for the TruB-bound conformation, but
2-fold lower values in the TruB-apo and the TruB D90N
simulations—even though the latter one is built on the
TruB-bound conformation. As D90 is located in insert
1, these findings suggest that the presence of an asparagine
residue at position 90 in some way restricts the motion of
this structural element.
TruB
k
app2, sꢀ1
Wild type
D90E
D90N
4.2 0.5
1.5 0.7
1.9 0.4
2.0 0.4
1.9 0.5
D90A
R181K
R181M
R181A
3
4
1
1
Apparent rates (kapp2
) for the strong fluorescent increase were
Specifically, changes in the interaction patterns between
D48, R181 and D90 were analyzed to determine potential
roles of these residues for catalysis and to better under-
stand the impact of the D90N substitution. These three
residues may interact through hydrogen bonds and/or
electrostatic interactions. To best characterize these inter-
actions in a single measurement, the distance between the
carboxylate carbon atom of the aspartate side chains and
the guanidinium carbon atom of the arginine side chain
was measured for D48–R181 and D90–R181 throughout
each simulation (Figure 7). Interestingly, the D48–R181
interaction was not as stable as predicted based on the
analysis of TruB crystal structures (5,25–27). In particular
in both TruB wild type MD simulations, there were sig-
nificant fluctuations in the distance between D48 and
R181. However, in the TruB D90N simulation, these
determined by 3-exponential fitting of stopped-flow time courses for
TruB wild type and 2-exponential fitting for the TruB variants
(Figure 5). kapp2 represents the average apparent rate from at least
five time courses standard deviation.
similar timescale as TruB wild type, but do not release
tRNA rapidly due to their impairment in catalysis.
Molecular dynamics simulations of TruB
As D90 is not in direct contact with the substrate tRNA,
the strong effects of substitutions of this residue on cataly-
sis of pseudouridine formation are not immediately
apparent. To shed more light on the intramolecular inter-
actions and functional dynamics in the active site of TruB,
we conducted a series of molecular dynamics (MD) simu-
lations of TruB wild type as well as TruB D90N. To
account for the different structural states of TruB, TruB
wild type was modeled in two functionally different con-
formations, its apo conformation as observed in crystal
structures lacking RNA [PDBID: 1R3F (26)], as well as
its conformation when bound to tRNA [PDBID: 1K8W
(5)]. To avoid complications of simulating a protein–RNA
complex, the RNA was removed from the latter structure
(see ‘Materials and Methods’ section for details of model
building). In the following, the two simulations of TruB
wild type are named ‘TruB-apo’ and ‘TruB-bound’.
Furthermore, a model for TruB D90N was generated
based on the TruB-bound model by substituting D90 for
asparagine, as this conformation is more likely to repre-
sent an active conformation. All models were solvated and
ionized with the addition of K+, Clꢀ and Mg2+ ions and
simulated for 40 ns at 310 K.
˚
two residues approached each other closely (<5 A) for
most of the interaction time; this interaction appeared
stable from 15 to 40 ns of the simulation. In contrast to
the D48–R181 interaction, we observed a short distance
˚
(<5.5 A) between D90 and R181 for the majority of time
in all three MD simulations. The TruB-bound simulation
was the only one where some fluctuations of the
D90–R181 distance were observed in the first 10 ns and
the last 5 ns of simulation. For the wild-type simulations,
the D90–R181 distance was in general shorter than the
D48–R181 distance as evident in the histograms in
Figure 7. For the TruB D90N simulation, however, the
D48–R181 distance was generally shorter than the
N90–R181 distance. This is particularly evident during
the last 25 ns of the TruB D90N simulation (Figure 7C).
In summary, the MD simulations reveal a stable
D90–R181 interaction that was not described based on
crystal structures (5,26,27) and a D48–R181 interaction
that is less stable with the exception of the TruB D90N
simulation.
As a first analysis of the MD simulations, the RMSD
of the backbone atoms were calculated for all residues of
TruB wild type (Figure 6A) and TruB D90N (Figure 6C),
as well as for all residues excluding the thumb-loop
residues 124–152 (shown only for TruB D90N,
Figure 6C). After 5 ns of TruB wild type simulations, the
DISCUSSION
˚
˚
RMSD values stabilized at ꢄ3.5 A and at ꢄ3 A when the
thumb-loop residues were omitted from the calculations
(data not shown). Similarly, the RMSD values from the
In the present study, we have analyzed by biochemical,
biophysical and computational means the detailed role
of residues R181 and D90 for the function of the pseudo-
uridine synthase TruB. Our data suggest that the active
site residue R181 and in particular its charge is critical for
catalysis of efficient pseudouridine formation without
influencing substrate binding. Interestingly, residue D90
is also important for catalysis although it is not in dir-
ect contact with the target uridine and is located in the
second-shell surrounding the active site. Again, the charge
˚
TruB D90N simulations stabilized at ꢄ3 A when omitting
the highly mobile thumb loop (Figure 6C). Based on these
findings, further analyses were performed omitting the
first 5 ns of the simulations. Next, RMSF values were
calculated for all TruB residues from 5 to 40 ns (Figure
6B and D) to identify the most flexible regions of TruB
during the simulations. As expected, insert 1 (residues 85–
100), the thumb loop, helix a6 and the C-terminal PUA

Nucleic Acids Research, 2014, Vol. 42, No. 6 3865
Figure 6. Global fluctuations of TruB during MD simulations. TruB wild type (in the absence of RNA, panels (A and B) was simulated at 310 K in
the apo and bound conformation based on available crystal structures with and without RNA, respectively (for details see ‘Materials and Methods’
section). TruB D90N (C and D) was simulated in the bound conformation only. (A) RMSD of the backbone atoms of all residues for TruB wild type
simulations (apo conformation, gray; bound conformation, black). (B) RMSF values for simulation of TruB wild type from 5–40 ns, with insert 1
(asterisk) and thumb-loop (double asterisk) residues indicated by dashed boxes (apo conformation, gray; bound conformation, black). (C) TruB
D90N simulation. RMSD of the backbone atoms of all residues (black) and for backbone atoms of all residues excluding thumb-loop residues
124–152 (gray). (D) RMSF values for TruB D90N residues from 5 to 40 ns of simulation; insert 1 (asterisk) and thumb loop (double asterisk).
of this residue seems to be its most important character-
istic, and substitutions of this residue do not affect the
interaction of TruB with substrate tRNA. MD simula-
tions of TruB wild type and TruB D90N suggest that
the three residues, D48, R181 and D90, form an electro-
static interaction network.
formation, and therefore this residue can be classified as a
catalytic residue. For TruB specifically, the lysine substi-
tution might result in significantly less activity compared
with TruB wild type (with arginine in position 181) either
because lysine has fewer potential hydrogen-bond donors
or because it is shorter. In this respect, it is noteworthy
that we observed in the MD simulations that R181
often interacted simultaneously with both D48 and D90
(Figure 8), which might be facilitated by the guanidinium
group forming multiple interactions. Also, visual inspec-
tion of substituting R181 with lysine in the crystal struc-
ture of TruB in the bound conformation revealed that
lysine should still be long enough to effectively interact
with either D48 or D90. As expected for a catalytic
residue and similar to the catalytic aspartate (14,30),
R181 does not contribute significantly to substrate
binding by TruB, presumably since the majority of
contacts between TruB and tRNA are formed outside
the active site. In conclusion, a basic residue in position
181 is essential for catalysis of pseudouridine formation,
and in TruB an arginine might be strictly required as it is
able to simultaneously interact with D48 and other
residues such as D90.
Residue R181 of TruB is a catalytic residue
Although the conserved basic residue in the active site,
R181 in TruB, has been discussed in a number of publi-
cations describing pseudouridine synthases (5,25), no bio-
chemical studies testing the exact role of this residue have
been reported. Here, we unambiguously demonstrate the
importance of R181 in bacterial TruB for catalysis of
pseudouridine formation. Even a conservative substitu-
tion of this residue with lysine leads to a 2500-fold reduc-
tion in the rate of pseudouridylation while substitutions
with methionine or alanine almost abolish activity. This is
particularly remarkable considering that lysine is found in
the corresponding position in enzymes from the RsuA,
TruD and Pus10 families (3,8,42). A basic residue in this
position is evidently essential for catalyzing pseudouridine

3866 Nucleic Acids Research, 2014, Vol. 42, No. 6
Figure 7. Active site residue interactions during MD simulations. Every 2 ps of simulation time, distances were measured between the guanidinium
carbon of R181 and the carboxylate carbon of D48 and D90. The D48–R181 interaction is depicted in gray while the D90–R181 interaction is shown
in black. Both time-resolved changes (left) as well as histograms of C-C distances (right) are reported. (A) TruB wild type in the apo conformation.
(B) TruB wild type in the bound conformation. (C) TruB D90N in the bound conformation.
Residue D90 in insert 1 is critical for pseudouridine
binding determinant. As D90 and C56 are located on
formation by TruB
the surface of the TruB–tRNA complex, we suspect that
We initially predicted the importance of residue D90 in
insert 1 of TruB based on its remarkably high conserva-
tion and proximity to the active site. Since D90 has been
seen in crystal structures to interact with C56 in the tRNA
(26,27), it was expected that D90 substitutions would have
a greater effect on tRNA binding than on catalysis.
However, our results show that all TruB D90 variants
had a similar affinity for tRNA as TruB wild type,
indicating that D90 is not critical for substrate binding
and that the hydrogen bond to C56 is not a major
this hydrogen bond can be replaced by interactions with
the surrounding water. Moreover, C56 can also be
recognized by residues in the thumb loop (27), and this
interaction may also compensate for substitutions of D90.
Surprisingly though, all three substitutions of D90
severely impaired pseudouridine formation, with even
the most active variant (TruB D90E) displaying a 30-
fold decrease in catalytic rate. As the effect of replacing
aspartate 90 with asparagine was even larger than
substituting with glutamate, it seems that the negative

Nucleic Acids Research, 2014, Vol. 42, No. 6 3867
TruB, R181 is in a bent orientation pointing away from
D90 (Figure 1B). This difference might be explained by
the absence of RNA in the MD simulations, as the RNA
may influence the orientation of nearby residues, in par-
ticular of R181. However, superimposing the RNA from
crystal structures onto representative frames from the MD
simulations reveals that a close network of interactions
between D48, R181 and D90 is compatible with RNA
binding and does not result in steric clashes (Figure 8).
It is therefore feasible that these three residues adopt
multiple conformations before and during the interaction
with tRNA and that one of these conformations is a close
network between D48, R181 and D90 as observed in the
MD simulations.
The MD simulations presented here strongly support
the suggestion that the proximity of the negatively
charged D90 residue influences the nearby R181—
independent of the exact orientation of these residues.
This is particularly evident in the simulations of TruB
D90N where this interaction is reduced to the formation
of a hydrogen bond instead of an electrostatic interaction.
In this case, the interaction between R181 and D48 seems
to become even stronger as the two residues are in close
proximity without major fluctuations for 25 ns of the
simulation. Therefore, changing D90 to asparagine appar-
ently stabilized the interaction between R181 and D48, but
did not drastically alter the interaction between R181 and
N90. The only other notable difference in the TruB D90N
simulations, compared with simulations of TruB wild type
in the bound conformation, was the reduced mobility of
insert 1 containing the D90N substitutions (Figure 6D).
The exact molecular nature of this stabilization of insert 1
is unclear. These observations offer two explanations for
the 50-fold reduced catalytic activity of TruB D90N
compared with wild type. It might be that flexibility of
insert 1 is important for catalysis; however, this is difficult
to reconcile as movements of this structural element
are typically associated with RNA binding or release.
Alternatively, and maybe more likely, catalysis may be
impaired in the TruB D90N variant because of the
strengthened interaction between the catalytic aspartate
48 and arginine 181.
Figure 8. Selected conformation of TruB active site residues during
MD simulations. Representative active site conformations where
R181 (blue) was found in close proximity to both D48 (green) and
D90 within insert 1 (purple) are shown. RNA from the TruB-RNA
crystal structure (PDBID 1ZL3) was modeled into the simulation
frames (red/yellow/light purple). (A) Snapshot at 9.1 ns of the TruB-
˚
bound simulation. D48–R181 C-C distance: 3.7 A, D90–R181 C-C
˚
distance: 3.9 A. (B) Snapshot at 15.6 ns of the TruB D90N simulations.
˚
˚
D48–R181 C-C distance: 3.6 A, N90-R181 C-C distance: 4.6 A. In
neither frame do the interactions between the active site residues
clash with the crystal structure position of the RNA substrate.
charge of this residue is most important for TruB’s
activity. This highlights a role of this residue for catalysis
rather than tRNA binding as the hydrogen bond of
residue 90 to C56 of the tRNA should be retained in
the TruB D90N variant. The critical importance of the
negative charge at position 90 suggests that electrostatic
interactions of this residue contribute to pseudouridine
formation. Both the TruB apo crystal structure (26) as
well as our MD simulations reveal that R181 is a potential
electrostatic interaction partner of D90 and the only posi-
tively charged group in the vicinity of D90. The potential
role of the electrostatic interactions of D90 for catalysis of
pseudouridine formation will be discussed in more detail
below.
Dynamics of active site interactions in TruB
Here, we also report the first MD simulations of TruB in
two different conformations (i.e. the apo conformation
and the RNA-bound conformation) although the RNA
was omitted from the simulations for technical reasons.
It is noteworthy that our data confirm the presence of
several flexible regions in TruB; namely, insert 1, the
thumb loop, helix 6 and the PUA domain as evident in
the RMSF values (Figure 6B and D). The flexible nature
of these structural elements likely contributes to the rela-
Model for the role of R181 and D90 in pseudouridine
formation by TruB
Based on the findings from biochemical and computa-
tional studies, we propose that a finely balanced inter-
action network of residues D48, R181 and D90 is
required for efficient catalysis of pseudouridylation by
TruB. As evident from the TruB R181M and R181A
variants, catalysis is almost completely abolished if D48
is not able to interact with R181. On the other hand, if the
interaction between R181 and D48 is too stable, as was
seen in the TruB D90N MD simulation, the rate of cataly-
sis is also reduced. We propose that R181 must interact
with D48, but the strength of the interaction needs to be
modulated by additional contacts such as to D90. For
example, the contact between R181 and D90 may be
important for disrupting the interaction of R181 with
D48 to make the catalytic aspartate 48 residue available
˚
tively high RMSD values (ꢄ3 A) observed throughout our
simulations (Figure 6A and C) (43,44). Since the RMSDs
(excluding the thumb loop for TruB D90N) do not display
any significant drifts after the first 5 ns of simulation, we
consider the simulations to be stable allowing us to
analyze interactions in TruB’s active site.
The MD simulations of TruB revealed that R181 was
capable of forming a salt bridge with D90 (Figure 8) in
addition to the previously described interaction with D48
(5,25,26,39). Interestingly, such an interaction between
R181 and D90 is also visible in the apo TruB crystal struc-
ture (Figure 1A), while in all other crystal structures of

3868 Nucleic Acids Research, 2014, Vol. 42, No. 6
for nucleophilic attack of the target uridine. The D90
residue could thereby sequester the positively charged
R181 residue while D48 is participating in the chemical
reaction. This would mitigate a highly disfavored uncom-
pensated positive charge on R181 in the protein’s interior.
Previously, it was hypothesized that the thumb loop could
partially fulfill this role (25), but it seems likely that the
negatively charged D90 residue contributes to interacting
with R181 while D48 is engaged in the chemical reaction.
Lastly, we asked whether the contribution of an electro-
static network to catalysis could be a common principle
in pseudouridine synthases. The catalytic role of a
basic residue for pseudouridine formation seems to be
conserved in all pseudouridine synthases, as they all
contain either an arginine or a lysine in the corresponding
position. In contrast, D90 is 100% conserved in insert 1 in
all bacterial TruBs, but insert 1 is unique to TruB.
Therefore, we searched for similarly conserved, negatively
charged residues in other pseudouridine synthases that are
in the spatial vicinity of the conserved basic residue. We
identified an aspartate in position 178 of E. coli RluA that
is 100% conserved in all aligned RluA sequences and in
close proximity to RluA’s catalytic arginine (Figure 9A).
This residue is located in a unique insert of the RluA
protein compared with other pseudouridine synthases.
Also, in RluF, we found a highly conserved aspartate resi-
due, D109, found in 80% of the sequences, that could
interact with the catalytic arginine 190 (Figure 9B).
Lastly, there is a negatively charged residue at position
243 in 73 of 100 analyzed TruA sequences that could po-
tentially interact with the conserved arginine 205 on
changing its orientation (Figure 9C). However, we could
not find such a conserved negatively charged residue
in Cbf5 or in Nop10 in H/ACA small (nucleolar)
ribonucleoproteins. Also, the nearest phosphate in the
˚
H/ACA RNA is 13 A away from the active site arginine
Figure 9. Conserved negatively charged residues in the active site of
bacterial pseudouridine synthases. Sequence conservation mapped onto
crystal structures of E. coli pseudouridine synthases (A) RluA (PDBID:
2I82), (B) RluF (PDBID: 3DH3) and (C) TruA (PDBID: 2NQP), each
bound to RNA. The conservation of each residue was determined from
a multiple sequence alignment of 100 bacterial sequences of the protein
and is represented by a colour gradient of red to white to blue,
indicating 0–100% conserved, respectively (a colour version of this
figures is available online). Stick models are shown in yellow of
conserved residues for the catalytic aspartate and arginine (D48 and
R181 in TruB) as well as a highly conserved negatively charged residue
in close proximity. (A) The alignment of RluA showed D178 to be
100% conserved, which has a C-C distance to R165 of 4.0 A. (B)
D109 in RluF is 77% conserved (16% P, 4% E) with a C-C distance
˚
to R190 of 3.6 A. (C) E243 in TruA was found to be 62% conserved
˚
(11% D, 9% G) and has a C-C distance to R205 of 7.9 A in accordance
with its orientation pointing away from R205.
such that it is unlikely to fulfill the same role as D90 in
TruB. Furthermore, there seems to be no conserved acidic
residue in TruD, which is the most divergent pseudourid-
ine synthase. Interestingly, the conserved basic residue
(K21) in TruD is located in a different structural element
than in other pseudouridine synthases. This further
supports the suggestion that residues in nonequivalent
positions, such as the different second-shell acidic
residues identified here, can fulfill the same functional
role (7–9). In conclusion, it seems highly likely that the
proposed network of the catalytic aspartate with a basic
residue and another acidic residue plays a role in catalysis
in several pseudouridine synthases in addition to TruB.
In summary, we have proven the catalytic role of the
conserved basic residue in the active site of pseudouridine
synthases. While in the different pseudouridine syn-
thases this residue can be either an arginine or a lysine
interacting with the catalytic aspartate residue, the exact
type of residue is highly conserved for each enzyme family.
Substitutions of an arginine for lysine result in a 2500-fold
reduced activity in the case of TruB, corresponding to an
increase of activation energy from 77.8 kJ molꢀ1 to 97.5 kJ
molꢀ1 for this reaction. This discovery indicates that the
interactions in the active sites of pseudouridine synthases
are finely balanced to optimize activity. For TruB, we
˚
have identified D90 in insert 1 as a critical residue for effi-
cient pseudouridine formation. Rather than contributing
significantly to substrate binding, D90 seems to form an
electrostatic interaction network with D48 and R181
thereby facilitating catalysis. Similar conserved, negatively
charged second-shell residues have been identified in
RluA, RluF and TruA. These findings shed new light
onto the complexity of pseudouridine formation. The
challenge remains to identify the exact catalytic mechan-
ism of pseudouridine synthases;
a
comprehensive

Nucleic Acids Research, 2014, Vol. 42, No. 6 3869
11. McDonald,M.K., Miracco,E.J., Chen,J., Xie,Y. and Mueller,E.G.
(2010) The handling of the mechanistic probe 5-fluorouridine by
the pseudouridine synthase TruA and its consistency with the
handling of the same probe by the pseudouridine synthases TruB
and RluA. Biochemistry, 50, 426–436.
description thereof will have to include the contribution
of the catalytic basic residue and the second-shell acidic
residue identified here.
12. Raychaudhuri,S., Niu,L., Conrad,J., Lane,B.G. and Ofengand,J.
(1999) Functional effect of deletion and mutation of the
Escherichia coli ribosomal RNA and tRNA pseudouridine
synthase RluA. J. Biol. Chem., 274, 18880–18886.
ACKNOWLEDGEMENTS
The authors thank Adrian Ferre-D’Amare, Juli Feigon,
´ ´
13. Huang,L., Pookanjanatavip,M., Gu,X. and Santi,D.V. (1998) A
conserved aspartate of tRNA pseudouridine synthase is essential
for activity and a probable nucleophilic catalyst. Biochemistry, 37,
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Identification and site of action of the remaining four
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Marc Roussel and Steven Mosimann for critical discus-
sion of this research. Furthermore, we are grateful to
Laura Keffer-Wilkes for initial nitrocellulose filtration
assays and preparation of tritium-labeled tRNA. Lastly,
we appreciate the advice by Daniel Lafontaine on tRNA
ligation.
FUNDING
16. Conrad,J., Niu,L., Rudd,K., Lane,B.G. and Ofengand,J. (1999)
16S ribosomal RNA pseudouridine synthase RsuA of Escherichia
coli: deletion, mutation of the conserved Asp102 residue, and
sequence comparison among all other pseudouridine synthases.
RNA, 5, 751–763.
17. Kaya,Y. and Ofengand,J. (2003) A novel unanticipated type of
pseudouridine synthase with homologs in bacteria, archaea, and
eukarya. RNA, 9, 711–721.
18. Zebarjadian,Y., King,T., Fournier,M.J., Clarke,L. and Carbon,J.
(1999) Point mutations in yeast CBF5 can abolish in vivo
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synthases. Arch. Biochem. Biophys., 489, 15–19.
20. Behm-Ansmant,I., Urban,A., Ma,X., Yu,Y.T., Motorin,Y. and
Branlant,C. (2003) The Saccharomyces cerevisiae U2
snRNA:pseudouridine-synthase Pus7p is a novel multisite-
multisubstrate RNA:Psi-synthase also acting on tRNAs. RNA, 9,
1371–1382.
Natural Science and Research Council of Canada
[NSERC Discovery Grant 341996-2009 to U.K. and
Discovery Grant 326979-2011 to H.-J. W.]; the Canada
Foundation for Innovation (CFI Leaders Opportunity
Fund, Project Number 13152, U.K.); the University of
Lethbridge [University of Lethbridge Research Fund
No. 13724, U.K.]; an NSERC CGS-M and an Alberta
Innovates—Technology Futures Graduate Student
Scholarship (to J.F.). Supercomputer time was provided
by the WestGrid/Compute Canada Resource Allocation
Committee. Funding for open access charge: The publica-
tion charges will be covered by research grants to Ute
Kothe (NSERC Discovery Grant) and Hans-Joachim
Wieden (AITF Strategic Chair).
Conflict of interest statement. None declared.
21. Kammen,H.O., Marvel,C.C., Hardy,L. and Penhoet,E.E. (1988)
Purification, structure, and properties of Escherichia coli tRNA
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fluorouridine by the action of the pseudouridine synthase TruB
disfavor one mechanism and suggest another. J. Am. Chem. Soc.,
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