Carbohydrate-conjugated 4-(1,3,2-dithiarsolan-2-yl)aniline as a cytotoxic agent against colorectal cancer

Article abstract of DOI:10.1039/C8RA07860B

Arsenic trioxide (As₂O₃) has been approved for the treatment of acute promyelocytic leukemia (APL); however, its use in the treatment of solid tumors is limited due to its pharmacokinetic properties. Organic arsenic compounds provide better options for pharmaceutical optimization. p-Aminophenyl arsenoxide (p-APAO), an organic arsenic compound, was found to interact with the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) fusion protein in a similar manner to arsenic trioxide. Analogs of p-APAO such as 4-(1,3,2-dithiarsolan-2-yl)aniline (p-APDTAs) were recently found to show improved cytotoxicity toward several solid tumor cell lines with lower toxicity to normal cells. Here, we synthesized a carbohydrate-conjugated 4-(1,3,2-dithiarsolan-2-yl)aniline (p-APDTAs) and showed that it exhibited reduced cytotoxicity to normal cells, suggesting a feasible approach to improve the therapeutic index of arsenic-containing compounds as chemotherapeutic agents.

Full text of DOI:10.1039/C8RA07860B

Designing seryl-tRNA synthetase for improved serylation
of selenocysteine tRNAs
1
,†
1
1
1,2
Xian Fu , Ana Crnkoviꢀc
, Anastasia Sevostyanova
and Dieter S o€ ll
1
2
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA
Department of Chemistry, Yale University, New Haven, CT, USA
Correspondence
D. S o€ ll, Department of Molecular Biophysics
Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead,
Sec
seryl-tRNA synthetase (SerRS) produces Ser-tRNA , which is subsequently
and Biochemistry, Yale University, New
Haven, CT 06511, USA
Sec
converted by selenocysteine synthase to Sec-tRNA . Escherichia coli SerRS
Sec
Fax: +1 203 432 6202
serylates tRNA poorly; this may hinder efficient production of designer
selenoproteins in vivo. Guided by structural modelling and selection for chlo-
Tel: +1 203 432 6200
E-mail: dieter.soll@yale.edu
ramphenicol acetyltransferase activity, we evolved three SerRS variants cap-
Sec
†
Present address
able of improved Ser-tRNA
increased kcat/KM values compared to wild-type SerRS using synthetic
synthesis. They display 10-, 8-, and 4-fold
BGI-Shenzhen, Shenzhen 518083, China
Sec
tRNA
species as substrates. The enzyme variants also facilitate in vivo
Xian Fu and Ana Crnkovi ꢀc contributed
read-through of a UAG codon in the position of the critical serine146 of chlo-
ramphenicol acetyltransferase. These results indicate that the naturally
evolved SerRS is capable of further evolution for increased recognition of a
specific tRNA isoacceptor.
equally to this work.
(Received 5 September 2018, revised 28
September 2018, accepted 3 October 2018,
available online 24 October 2018)
Keywords: protein engineering; selenoproteins; seryl-tRNA synthetase;
synthetic biology; tRNA
doi:10.1002/1873-3468.13271
Edited by Michael Ibba
Selenocysteine (Sec), genetically encoded by UGA, is
the only naturally occurring amino acid that lacks a
cognate aminoacyl-tRNA synthetase. Instead, seryl-
Secondary structures and altered nucleotides (com-
UTuX
Sec
pared to tRNA ) of tRNA
UTu1
SecUX
, tRNA
and
are presented in Fig. 1. Significantly, the
tRNA
three base pairs (green) in the centre of tRNA
Sec
Sec
tRNA synthetase (SerRS) acylates tRNA with serine
(Ser); subsequently, the selenocysteine synthase SelA
transforms the RNA-bound Ser to Sec. The resulting
Sec
Sec-tRNA is delivered to the ribosome by the dedi-
are
identity elements for SelB recognition and reject EF-
Tu. Replacing these base pairs with G7:C66, C/U49:
G65 and A/C50:U64 allows these tRNAs to be recog-
nized by EF-Tu. Sec insertion by these variant tRNAs
cated elongation factor SelB, which in the presence of
a specific mRNA structure aids accurate recoding of
UGA for Sec insertion into proteins [1]. Thus, E. coli
may be feasible for any codon virtually [5].
Escherichia coli SerRS charges five tRNA
Ser
isoac-
ceptors with equal efficiency [6]. However, compared
Sec
tRNA evolved to optimally interact with SerRS,
SelA and SelB, and to reject the general elongation
factor EF-Tu. Yet, strategies for in vivo synthesis of
Ser
Sec
to the tRNA
times more poorly (as judged by kcat/K ) [8]. This is
species, tRNA
[7] is serylated 100
M
Sec
selenoproteins in E. coli rely on tRNA variants that
plausible; given that there is only one selenoprotein in
E. coli [9], efficient genome translation requires
interact with EF-Tu and SelA. For this reason, a
Sec
Sec
Ser
number of tRNA
variants were designed [2–4].
very much less Sec-tRNA
than Ser-tRNA
(for
Abbreviations
aaRS, aminoacyl-tRNA synthetase; CAT, chloramphenicol acetyltransferase; NTD, N-terminal tRNA binding domain; SelA, selenocysteine
synthase; SerRS, seryl-tRNA synthetase.
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies
3759

Sec
Upgrading E. coli SerRS for tRNA
serylation
X. Fu et al.
A
C
A
C
A
A
C
C
C
G
C
C
C
G C
G C
G
G C
G C
G
G
G C
G C
G C
G C
A
U
G C
G C
G C
G C
A
A
U
U
A
A
U
U
A
A
U
U
G C
G C
G C
A
U
U
A
A
U
U
A
A
U
U
A
A
A
U
U
U
U
U
A
C
U
C
U
C
U
C
C G
U
U
A
G C
U
U
A
G C
U
U
A
CCC
GGG
G
UGUCC
GCA GG
G
GUCCC
CA GGG
G
GUUCC
UCA GG
U
U
U
G
C
U
U
G
C
U
U
G
C
U
U
G
G
G
CUA UC U
GA UA GA
G
G
CUCUGC
GA GGC G
G
G
CUGCC G
GA A GGC
G
G
CUCUGC
GA GGC G
C
U
C
C
C
U
G
G
G
A
A
U
U
G
C
G
A
A
G
U
G
G
U
G
U
G
C
U
C
C G
G U
C
C
U
G
U
C
G
C
C
U
G
U
C
U
C
C
G
G
C
G
G
G
A
C
G
C
G
C G
A
G
C
A
C
G
C
G
U
C
C
U
G
U
G C
G C
C
G
G
G
U
C
C
A
G
G
G
G
G
A
U
C
G
G
G C
C
C
G C
C
C G
A
C G
C
C
A
A
U
U
A
U
G
G A
A
U
G
A
A
C
A
C
A
C
A
C
C
C
A
C
C
U
A
U
A
U
A
U
A
U
A
C
A
C
A
U
C
U
C
A
U
tRNASec
tRNAUTuX
tRNASecUX
tRNAUTu1
Sec
Fig. 1. Wild-type and synthetic selenocysteine tRNAs. Identity nucleotide determinants of wild-type Escherichia coli tRNA
are shaded
blue, and the variable arm, whose length and orientation are specifically recognized by SerRS is highlighted in yellow. Nucleotides that
Sec
govern the tertiary fold of tRNA
are given in pink. Base pairs acting as EF-Tu antideterminants are shown in green. tRNA anticodon
sequences are in light blue. Synthetic selenocysteine tRNAs (origin referenced in the text) are given in black. Mutations introduced into
Sec
UTuX
SecUX
UTu1
tRNA
and tRNA
are indicated in red. tRNA
is too dissimilar to E. coli tRNA
to highlight nucleotide differences.
translation of >78 000 codons). In the light of our
synthetic biology efforts to develop an efficient EF-Tu-
dependent translation system for UAG directed, site-
specific Sec incorporation into proteins [10], we
cat gene where an amber stop-codon was introduced at
position 146 [15]. The cassette and pACYC184 backbone
were amplified using primers listed in Table S1 and ligated
using In-Fusion cloning. pXF113, pXF114 and pXF115
UTuX
SecUX
Pyl
contained genes from tRNA
, tRNA
and tRNA
decided to design a variant SerRS enzyme endowed
Sec
respectively. Plasmids pXF113 containing coding sequences
UTu1
with improved serylation of the tRNA
UTuX
variants
[4].
UTu1
SecUX
for tRNA
, SupD and SelC(amb) were created by
tRNA
[3], tRNA
[10]), and tRNA
Gibson Assembly using sets of primer listed in Table S1.
Sec
We based our E. coli SerRS mutagenesis strategy on
the examination of Thermus thermophilus SerRSꢀ
The gene for E. coli tRNA
with an amber anticodon
Ser
Sec
(selC(amb)) was amplified using a previously published
template [2]. To generate pBAD-SerRS plasmids, oligo-
nucleotides EcSerSf and EcSerSr (Table S1) were used to
amplify the SerRS gene. The product was then digested with
NcoI and KpnI and inserted into a modified pKTS vector
containing araC gene and PBAD promoter. SerRS variants
containing S55A mutation or a GGGGS linker were con-
structed by In-Fusion cloning, using primer sets listed in
Table S1. Plasmids constitutively expressing tRNAs and
wild-type or SerRS variants were created by inserting a
lpp-tRNA-rrnC cassette into pBAD-SerRS plasmids by
In-Fusion cloning (Table S1). pTrc99A-SerRS plasmids
were generated by In-Fusion using the primer sets listed in
Table S1. To generate pTrc99A-SerRS.V20-A55S, the
plasmid was mutagenized using primers A55S.F and
A55S.R (Table S1). For protein isolation purposes, SerRS
variants were cloned into pET15 vector (Novagen) using
Gibson Assembly (NEB). The open reading frames of
E. coli SerRS genes and the pET15 backbone were ampli-
tRNA
[11] and Homo sapiens SerRSꢀtRNA
[12]
co-crystal structures, corresponding library construc-
tion and variant selection by an in vivo chlorampheni-
col acetyltransferase (CAT) assay [13]. This approach
led to the discovery of three desired SerRS variants.
Materials and methods
Reagents and strains
Pfu Ultra II DNA polymerase was obtained from Agilent
Technologies (Santa Clara, CA, USA); restriction enzymes,
T4 DNA ligase, and the Gibson Assembly mix were from
New England Biolabs (Ipswich, MA, USA). In-Fusion clon-
ing kit was from Clontech (Mountain View, CA, USA). The
thermosensitive E. coli strain KL235 [14] was obtained from
the Coli Genetic Stock Center (Yale University).
Sec
fied using primer pairs listed in Table S1. Artificial tRNA
UTuX
Plasmid construction
SecUX
UTu1
genes (tRNA
, tRNA
, tRNA
, [3,4,10]) and
Ser
For in vivo serylation assays plasmids pXF113, pXF114
and pXF114 were created by inserting a lpp-tRNA-rrnC
cassette into a modified pACYC184 backbone containing a
wild-type tRNA CGA were amplified using primer pairs
listed in Table S1 and cloned using Gibson Assembly (NEB)
into a modified pUC18 vector (T7 promotor adjacent to the
3
760
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies

Sec
X. Fu et al.
Upgrading E. coli SerRS for tRNA
serylation
ꢁ
1
ꢁ1
BamHI restriction site). Construction of pUC19 with wild-
Sec
type E. coli tRNA was described in [16].
extinction coefficients (68 760 M ꢀcm
for wild-type
and SerRS v.20 and v.22, and 71 740 Mꢁ1ꢀcmꢁ1 for
SerRS v.8).
Construction of Escherichia coli knock-out strains
Transcript preparation and purification
Strains of DH10B DselAB::zeo and WL30153 were used to
test serylation of different tRNAs in vivo and for selection
purposes. DH10B DselAB::zeo was created by k-Red
recombination [17] using primers EM7-zeo FW and EM7-
zeo RV (Table S1) to amplify the EM7 – zeocin cassette.
Escherichia coli strain WL30153 (MC4100 DselAB) was
described [18].
Ser
pUC18 plasmids containing genes for tRNA
and
Sec
tRNA variants were transformed into E. coli DH5a cells,
grown overnight and isolated using Plasmid Maxi kit
(Qiagen, Gaithersburg, MD, USA). Following BstNI diges-
0
tion to generate appropriate 3 -CCA ends, tRNAs were
generated by in vitro transcription using recombinantly pro-
duced T7 RNA polymerase [19]. Transcripts were purified
on a HiTrap DEAE FF column (GE Healthcare), as
described [20]. Prior use, tRNAs were heated to 92 °C for
3 min and slowly cooled to room temperature in the pres-
Library creation and chloramphenicol-resistance
selection
ence of 5 mM MgCl
tration was estimated according to its absorbance at
60 nm and the percentage of active transcript was
2
to allow renaturation. tRNA concen-
Four fragments of serS gene were amplified by primers
listed in Table S1 to generate SerRS coding sequences that
are fully randomized at given positions (residues K86, A87,
D90, S160 and D228 and T229) by overlap PCR. The mix-
ture of pBAD-SerRS plasmids expressing the wild-type and
the S55A variant with 1:1 ratio was used as the template to
amplify serS gene fragments. Inserts were then digested
with NcoI and NdeI and ligated into pRSF-based vector,
under the control of a PBAD promoter. The SerRS library
2
obtained from the plateau charging (see below).
In vitro aminoacylation assays
All aminoacylation reactions were performed at 37 °C,
essentially as reported [21], and contained 100 mM Tris-Cl
8
transformation resulted with 8.5 9 10 clones, oversam-
8
pling the theoretical diversity of 3.5 9 10 . For selection
(pH 7.5), 10 mM KCl, 10 mM MgCl
2
, 1 mM DTT, 10 mM
ꢁ1
ATP and 0.4 mgꢀmL bovine serum albumin, 0.05 U ther-
mostable inorganic pyrophosphatase (NEB) and 125 lM
purposes, the library was co-transformed with pXF113 into
14
DH10B DselAB::zeo, and plated onto LB agar supple-
[ C]serine (Perkin Elmer, Waltham, MA, USA). Aliquots
of the reaction were removed at given intervals, spotted
onto 3 MM filter papers and immersed in 10% trichloroa-
ꢁ
1
mented with 55 mgꢀL Cm.
cetic acid to precipitate aminoacyl-tRNA. For the K
UTuX
M
and
Protein purification
k
cat determination in case of tRNA
, tRNA concentra-
tion was varied over the range of ~0.1–5.6 lM (for SerRS
Escherichia coli BL21(DE3) cells harbouring pET15b-
serS plasmids were grown to OD600 0.4 at 37 °C, after
which 0.3 mM isopropyl b-D-1-thiogalactopyranoside
V8 and V20) and ~0.1–8.6 lM (for wt and SerRS V22).
SecUX
In case of tRNA
, tRNA concentration was varied
UTu1
over the range of ~0.6–11.1 lM and in case of tRNA
over the range of ~0.2–12 lM for all SerRS enzymes. To
determine the plateau of any selenocysteine tRNA seryla-
tion, 2 lM wt SerRS was incubated with of 2 lM transcript
for 30 min.
(
IPTG) was added. Following induction, cells were
grown for 3 hr, pelleted and resuspended in buffer A
0.1 M Tris-Cl pH 7.4, 0.1 M NaCl, 5 mM 2-mercap-
(
toethanol, 0.3 mM PMSF). Cells were then lysed by
ultrasonic treatment and soluble proteins collected by
centrifugation at 12 000 g. Wild-type SerRS and SerRS
variants were then purified by two rounds of anionic
exchange, first on HiTrap DEAE FF Sepharose (GE
Healthcare, Chicago, IL, USA) and then HiTrap Q (GE
Healthcare). Purification was executed in a linear gradi-
ent of salt using buffers A and B (0.1 M Tris-Cl pH 7.4,
Complementation assay
A culture of E. coli strain KL235 (in which lack of growth
at elevated temperatures is a result of a thermosensitive
SerRS [14]) was grown to early exponential phase at 30 °C.
The cells were made electrocompetent and were trans-
formed with plasmids pTrc99A, in which the genes for
wild-type and SerRS variants were placed under the control
of trc promoter. As a negative control, a batch of cells were
transformed with pTrc99A lacking the insert. Individual
clones of KL235 containing desired plasmids were grown
overnight, diluted to OD600=1 and spotted in serial
1
M
NaCl, 5 mM 2-mercaptoethanol). Eluted proteins
were concentrated and stored at ꢁ80 °C in buffer S
0.1 M Tris-Cl pH 7.4, 0.15 M NaCl, 50% glycerol,
mM 2-mercaptoethanol). Prior to the aminoacylation
(
5
assay, proteins were diluted in buffer D (50 mM Tris-Cl
pH 8.0, 1 mM 1,4-dithiothreitol (DTT)) and their con-
centration was measured according to the theoretical
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies
3761

Sec
Upgrading E. coli SerRS for tRNA
serylation
X. Fu et al.
Ser
minimal set of recognition parameters in tRNA and
dilutions onto LB plates containing 0.05 and 0.1 mM IPTG.
Sec
Colonies were then grown at 30, 37 and 42 °C for 20 h.
tRNA [8,28,29], the NTD of TtSerRS also appears
Ser
to interact with the T loop of tRNA (Fig. 2, [11]).
Sec
Sec
Model of the Escherichia coli SerRSꢀtRNA
Escherichia coli SerRS was comparatively modelled by
Similarly, T loop of tRNA
NTD in the human SerRSꢀtRNA
Given the difference in nucleotide composition of TwC
is also in proximity of
Sec
complex [12].
Ser
Sec
Phyre2 (Protein Homology/analogY Recognition Engine
arms of tRNA and tRNA , we hypothesized that
certain residues in NTD might account for different
mode of recognition of these tRNAs. Most closely
[
22]). Superposition of the model structure of E. coli SerRS
Sec
onto SerRSꢀtRNA complex was achieved by UCSF Chi-
mera [23].
Ser
placed to tRNA , charged residues E84, E85, and
R88 of T. thermophilus SerRS are likely to form direct
interactions with the tRNA (Fig. 2). Thus, we decided
to randomize the corresponding residues of EcSerRS
Results
(K86, A87, and D90) to generate a SerRS variant cap-
able of improved serylation of tRNA
Designing a SerRS variant library
Sec
.
Sec
Ser
SerRS recognizes tRNA
and tRNA
in a similar
In addition to NTD residues, we wanted to simulta-
neously target the amino acids located in the catalytic
domain of the enzyme. Compared to T. thermophilus,
E. coli SerRS has a much longer motif 1 loop (resi-
dues R221-N231) [30]. This exposed motif 1 loop and
S160 of EcSerRS are in proximity to the base of the
acceptor arm according to our structural model
(Fig. 2). Nucleotides in the tRNA that are likely to
form interactions with SerRS occupy positions 65 and
manner. The two tRNA species share a set of minor
identity elements (discriminator base G73, C72, and
acceptor stem pairs G2:C71, A3:U70, Fig. 1) [24,25].
However, the most important element is the long vari-
able arm, which is recognized by SerRS in a sequence-
independent manner; the length and relative orienta-
tion have the greatest effect. The binding of the vari-
able arm (of both tRNA species) is accompanied by
the movement of the N-terminal tRNA binding
Ser
Sec
Ser
66; they differ in tRNA and tRNA (tRNA has
Sec
C65 and G66 while tRNA contains G65 and A66).
domain (NTD) [11,12].
Another idiosyncratic feature of tRNA
Sec
is the
G19:U20:C56 base triplet that defines its tertiary
Thus, to examine the role of catalytic domain residues
in differential recognition of these two tRNA species,
we targeted D228 and T229 in the motif 1 loop and
S160.
fold [12,26] (Fig. 1). Interestingly, U20 protrudes in
Sec
the human SerRSꢀtRNA
structure and is solvent
accessible [12,26], unlike the corresponding C20 in
Ser
TttRNA that is buried in the tRNA core [11]. The
Read-through of UAG codons in chloramphenicol
acetyltransferase
highly conserved S61 residue presumably allows SerRS
Ser
Sec
to distinguish between tRNA and tRNA by inter-
acting specifically with nucleotide determinant in posi-
tion 20 [12]. Both S61 (S55 in E. coli SerRS) and U20
UTuX
The synthetic selenocysteine tRNAs tRNA
SecUX
,
(Fig. 1) charged with Ser
UTu1
tRNA
and tRNA
Sec
are present in EcSerRS and EctRNA
Sec
respectively.
SecUX
are recognized by EF-Tu, as shown by minor mis-
incorporation of Ser instead of Sec into reporter pro-
teins [2,3,10]. Therefore, the capacity of SerRS to
Furthermore, all tRNA
UTu1
derivatives (tRNA ,
, Fig. 1) retain this uridine.
UTuX
tRNA
and tRNA
Sec
Sec
However, our attempts to improve tRNA serylation
via S55 mutagenesis, or by increasing NTD flexibility
aminoacylate these tRNA variants can be monitored
in vivo via stop-codon read-through in a selA deficient
strain.
Using the CAT with a TAG stop-codon at position
in order to accommodate the longer acceptor-TwC
Sec
arm of tRNA
Information for detailed explanations).
were unsuccessful (see Supporting
UTuX
SecUX
112 as reporter, tRNA
production of the full-length CAT protein at moderate
and tRNA
facilitated
Next, we decided to simultaneously target multiple
Ser
ꢁ1
ꢁ1
residues that, in the T. thermophilus SerRSꢀtRNA
Cm levels (50 mgꢀL and 25 mgꢀL for the respective
tRNAs, Fig. S1). Since a number of different amino
acids in position 112 lead to CAT activity, we switched
complex, are in close contact (Fig. 2) [11]. We decided
Sec
Ser
to use this structure because tRNA
and tRNA
share some common features responsible for their
recognition by SerRS [25], and because TtSerRS and
EcSerRS are more closely related than HsSerRS
and EcSerRS [27]. While the discriminator base G73
and the orientation of the variable arm constitute the
to a cat gene TAG variant (position 146) that required
UTuX
Ser for activity [31]. Ser-tRNA
facilitated UAG
ꢁ
SecUX
1
suppression that allowed growth at ≤ 20 mgꢀL , while
no growth was observed with tRNA
(Fig. S2).
Both types of assay were conducted in DH10B DselAB::
3
762
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies

Sec
X. Fu et al.
Upgrading E. coli SerRS for tRNA
serylation
E85
E84
R88
CTD
Motif-1 loop
Ser
Sec
Fig. 2. Key interactions between SerRS and tRNA
as a guide for SerRS engineering. (left) Cartoon representation of SerRSꢀtRNA
productive complex. SerRS is given in two shades of pink, for each monomer of the homodimer. The N-terminal tRNA binding domain
NTD), C-terminal catalytic domain (CTD) and the active site (star) are indicated. Cognate tRNA binds across the dimer. Acceptor, TwC,
Sec
(
variable, anticodon and D arms of tRNA
are shown in red, orange, yellow, green and blue respectively. (right) Magnified view of the two
Ser
regions targeted for mutagenesis. (up) In the crystal structure of T. thermophilus SerRSꢀtRNA
complex (PDB ID: 1SER) NTD residues
E84, E85 and R88 are in the vicinity and/or form contacts with the TwC arm. These residues correspond to K86, A87 and D90 of
Escherichia coli SerRS. (down) Compared to T. thermophilus SerRS (light pink), E. coli SerRS (purple) has a longer motif 1 loop, which may
interact with the base of the acceptor arm.
ꢁ
1
zeo in which there was no observable near-cognate sup-
pression (Fig. S1).
grew on an LB plate supplemented with 55 mgꢀL Cm
UTuX
when tRNA
nies appeared when tRNA
was expressed, while only a few colo-
Pyl
was provided. Repeated
testing of 22 positives showed that four SerRS variants
Selection of desirable variants (a) recognition of
UTuX
tRNA
confer Cm resistance up to 65 mgꢀLꢁ in a tRNA
1
UTuX
-
dependent manner (Fig. S4). After recloning only vari-
ants V8, V20 and V22 showed reproducible improvement
in CAT read-through assay. Relevant SerRS sequences
of these three variants are shown in Fig. 3A. All three
variants possess different mutations, except for D228. In
all cases, D228 is converted to a positively charged K or
A SerRS library containing fully randomized positions
8
4, 85, 88, 160, 228 and 229 was created using both wt
and SerRS-S55A coding sequences as templates. SerRS
coding sequence was placed under the control of the
PBAD promoter to regulate its expression and reduce
its toxicity on cell growth. After confirming the library
quality, library plasmids were re-transformed into the
DH10B DselAB::zeo strain harbouring the selection
R, implicating the importance of this motif 1 residue in
UTuX
the interaction with tRNA
. Furthermore, S55 is
mutated to Ala in SerRS V20. However, this sequence
heterogeneity is not accidental. After mutating Ala55 to
wild-type serine, SerRS V20 loses its ability to efficiently
plasmid (pXF113 that carries the cat-146TAG and
UTuX
tRNA
genes). The number of apparent cat rever-
Pyl
tants was determined with pXF115 carrying tRNA
UTuX
charge tRNA
and other selenocysteine tRNAs
[
15] (see above).
To identify SerRS variants with improved activity
(Fig. S5). Thus, the S55A mutation is advantageous
when accompanied by other specific mutations.
UTuX
towards tRNA
tated cell growth at 55 mgꢀLꢁ Cm. About 100 colonies
, we selected those clones that facili-
Sequencing of the entire plasmids that encode the vari-
ants revealed a partial deletion of the araC gene. As
1
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies
3763

Sec
Upgrading E. coli SerRS for tRNA
serylation
X. Fu et al.
A
B
Cm/(mg·L–1
)
45
60
0
45
NTD
Motif 1
Empty plasmid
wt SerRS
Position
8
6 87 90 160 228 229
Variant
wt SerRS
K
A
Y
T
L
D
F
V
T
S
T
D
K
R
R
T
M
N
N
SerRS V8
SerRS V8
SerRS V20
SerRS V22
C
K
A
SerRS V20
N
P
SerRS V22
IPTG/mM
0.05
0.1
0.5
0.05
0.1
0.5
0.05
0.05
tRNAUTuX
tRNAPyl
UTuX
Fig. 3. SerRS variants improve tRNA
-mediated amber stop-codon suppression. (A) Three most proficient SerRS variants contain
different mutations, except for D228; this negatively charged motif-1 residue is converted into a basic (K or R) amino acid in all variants
(
highlighted). Locations of the residues are indicated (NTD or motif 1). In addition to the mutations listed here, SerRS V20 also contains a
UTuX
S55A mutation. (B) Comparison of in vivo acylation activity between wt and mutant SerRS for tRNA
UTuX
DselAB::zeo expressing tRNA
UTuX
tRNA
. Escherichia coli cells DH10B
ꢁ1
and SerRS variants were grown on LB plates with different concentration of Cm (0, 45, 60 mgꢀL ).
was under the control of lpp promoter; a CAT gene with a TAG codon at position 146 was on the same plasmid. SerRS
UTuX
expression was controlled by the trc promoter on the second plasmid. The read-through facilitated by the variants is tRNA
Pyl
as no resistance is conferred in the presence of tRNA
- dependent,
.
mutations in the AraC protein may give rise to various
induction/repression phenotypes [32] we did not want to
take a guess on the SerRS variant expression levels
(Table 1). SerRS V22 provides a more moderate
improvement (about 4-times), yet this is not sufficient to
allow higher suppression levels in vivo (Fig. 3B). Interest-
ingly, the major contribution to improved activity is the
consequence of increased turnover (8- to 19-increased kcat
for V20 and V8 respectively), rather than increased affin-
(Fig. S4 and discussion therein). To unambiguously
demonstrate that increased Cm resistance correlates with
the activity of evolved SerRS variants, and not caused by
unintended mutations in the vector, we recloned the
SerRS coding sequences under the control of a trc pro-
moter into another low-copy plasmid (Fig. 3B).
ity (Table 1). Taken together, SerRS variants V8 and
UTuX
V20 aminoacylate tRNA
enzyme under in vitro and in vivo conditions.
better than the wild-type
The CAT-146TAG read-through assay by the SerRS
variants placed under the trc inducible promoter
revealed that SerRS V8 and V20 outperform wt SerRS
and allow growth at higher Cm levels (Fig. 3B). How-
ever, high Cm resistance (45 and 60 mgꢀLꢁ ) could only
be achieved if expression of the variants was induced
with 0.05 and 0.1 mM IPTG, presumably allowing only
modest level of expression (Fig. 3B). Higher IPTG con-
centrations led to decreased cell viability suggesting that
SerRS overexpression is toxic to the cell [33]. This may
explain why AraC deletion variants accumulated during
selection of the SerRS library. Our results indicate that
Selection of desirable variants (b) recognition of
SecUX
UTu1
tRNA
and tRNA
1
Sec
As the different synthetic tRNA species displayed dif-
ferences in selenoprotein formation, we tested the sub-
SecUX
UTu1
and tRNA
strate properties of tRNA
(Fig. 1)
UTuX
Table 1. Steady-state kinetic parameters for tRNA
UTuX
type SerRS and evolved variants. The tRNA
for wild-
concentrations
were corrected for plateau charging (48%). Enzyme concentrations
were: 650 nM wild-type SerRS, 187 nM SerRS V8, 92 nM SerRS
V20, 224 nM SerRS V22.
the efficiency of CAT stop-codon suppression is related
UTuX
to increased levels of Ser-tRNA
.
k
cat/K
M
Relative
To examine the biochemical properties of the evolved
ꢁ1
(minꢁ1ꢀlMꢁ1
Enzyme
K
M
(lM)
k
cat (min
)
)
improvement
UTuX
variants with tRNA
activity in vitro (Table 1). Using an established protocol
29] and similar active enzyme concentrations (deter-
mined by active site titration), SerRS V8 and V20 serylate
as substrate, we tested their
wt
1.3
2.4
1.2
7.2
0.075
1.4
0.057
0.58
0.48
0.21
1
V8
10
[
V20
V22
0.56
1.5
8.4
3.6
UTuX
tRNA
10- and 8-times better than wild-type SerRS
3
764
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies

Sec
X. Fu et al.
Upgrading E. coli SerRS for tRNA
serylation
Ser
[4,10,29] with our SerRS variants in vitro and in vivo. In
enzyme-kinetic assays (described above), we determined
SecUX
the steady-state parameters. The values for tRNA
Ser-tRNA sufficient for active cell growth, we deter-
mined whether the variant enzymes could restore
growth of E. coli strain KL235 that carries a tempera-
ture-sensitive SerRS [1]. This strain contains a genomic
reveal significant serylation improvement by SerRS V8,
while the kinetic parameters of SerRS V20 were only
modestly improved (Table 2). As was the case with
copy of serS15, encoding
a thermolabile SerRS
enzyme; as a result the host strain is unable to grow at
42 °C. The SerRS variants were induced with low
amounts of IPTG (0.05 and 0.1 mM) for 20 h at
42 °C. Robust growth was detected for KL235 cells
expressing either wild-type SerRS or variant V20 (Fig-
ure 5). Toxicity of SerRS overexpression was more
apparent in case of SerRS V22 that can replace SerS15
only when expressed at a very low level (0.05 mM
IPTG). Similarly, SerRS V8, which can (albeit
pretty inefficiently) rescue growth when induced with
0.05 mM IPTG, loses this capacity altogether when
0.1 mM IPTG is added. This indicates that SerRS V20
is able to supply sufficient serylated tRNA species for
vigorous growth.
UTuX
tRNA
, the improvement of SerRS V8 activity arises
from a kcat increase (Tables 1, 2). This is plausible as
UTuX
SecUX
both tRNA
both sequence and structure. Interestingly, SerRS V8 and
and tRNA
are highly similar in
UTu1
V20 aminoacylate the more dissimilar tRNA
and 6-times better than wild-type SerRS. Although
[10] 11-
UTu1
tRNA
the tRNA
, as allo-tRNA [15], differs significantly from
UTuX
species used in SerRS V8 and V20 evolu-
tion, both of these enzyme variants have higher affinity
UTu1
towards tRNA
UTuX
than the wild-type SerRS. As with
SecUX
tRNA
and tRNA
, both variants have signifi-
cantly higher turnover numbers (~3–6-times for SerRS
V20 and V8, respectively, Table 2).
The CAT-146TAG read-through assay with
Additional information on the serylation properties of
SerRS came from a serine-specific CAT-146TAG read-
through assay. In order to decode the 146 UAG codon,
SecUX
UTu1
tRNA
and tRNA
in vitro data. Both SerRS V8 and V20 confer signifi-
corroborated the above
Ser
cantly higher Cm resistance compared to wt SerRS
SecUX
we used the natural E. coli tRNA suppressor SupD
(
Fig. 4). In case of tRNA
, both SerRS variants
[34]. The suppression efficiencies mediated by the SerRS
V8ꢀSupD and V20ꢀSupD pairs were comparable, and
even higher than in case of wt SerRS. SupD allowed
ꢁ1
allow growth at 20 mgꢀL Cm, while the wt enzyme
does not. Using the much stronger UAG suppressor
UTu1
ꢁ1
tRNA
[10], SerRS V8 and V20 allow robust
moderate to good growth at 300 mgꢀL Cm (Fig. S6).
ꢁ1
growth at Cm 400 mgꢀL . Wild-type SerRS can also
facilitate growth at these Cm levels albeit modestly,
and only if expressed at a very low level (0.05 mM
IPTG, Fig. 4). Thus, in case of all three selenocysteine
tRNAs, the evolved SerRS variants allow growth at
higher Cm concentrations but also operate in a larger
dynamic range of expression.
However, in vitro aminoacylation reveals somewhat
reduced affinity of SerRS V8 and V20 for SupD; both of
these enzymes have about 2-times higher K , while k is
M
cat
2–3-times lower, compared to the wt SerRS (Table S2).
This suggests that certain mutations improving seleno-
cysteine tRNA recognition may have adverse effects on
Ser
tRNA aminoacylation by SerRS variants (Figure 5).
SerRS variant V20 supports Escherichia coli
growth in the absence of other active SerRS
enzymes
Discussion
Ser
Escherichia coli SerRS catalyzes serylation of tRNA
Sec
and tRNA with a 100-fold difference in kcat/KM [8].
To examine if the mutations, that gave rise to our
SerRS variants, influenced their ability to provide
One of the requirements for effective SelA recognition,
Sec
the 8-bp long acceptor stem of tRNA , negatively
SecUX
UTu1
SecUX
Table 2. Steady-state kinetic parameters for tRNA
UTu1
and tRNA
for wild-type SerRS and variants V8 and V20. The tRNA
SecUX
and
tRNA
concentrations were corrected for plateau charging (39% and 55% respectively). For tRNA
UTu1
concentrations were: 500 nM wild-type SerRS, 187 nM SerRS V8, 92 nM SerRS V20; for tRNA
wild-type SerRS, 34 nM SerRS V8, 34 nM SerRS V20, 224 nM SerRS V22.
serylation the enzyme
the enzyme concentrations were: 150 nM
ꢁ1
(minꢁ1ꢀlMꢁ1
Enzyme
K
M
(lM)
k
cat (min
)
k
cat/K
M
)
Relative improvement
tRNA
tRNA
SecUX
wt
3.7
4.9
3.7
5.7
3.1
2.7
0.14
1
0.038
0.21
0.071
0.86
9.3
1
5.5
1.9
1
V8
V20
wt
0.26
4.9
UTu1
tRNA
V8
29
13
11
5.7
V20
4.9
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies
3765

Sec
Upgrading E. coli SerRS for tRNA
serylation
X. Fu et al.
IPTG/mM
wt SerRS
0.05
0.1
UTu1
SerRS V8
tRNA
SerRS V20
wt SerRS
SerRS V8
SecUX
tRNA
SerRS V20
OD600
10–1 10–2 10–3 10–4 10–5 10–6 10–1 10–2 10–3 10–4 10–5 10–6
UTuX
SecUX
UTu1
Fig. 4. In addition to tRNA
UTu1
, SerRS V8 and V20 improve tRNA
and tRNA
ꢁ1
-mediated TAG codon suppression. WL30153 cells
SecUX
expressing tRNA
and SerRS variants were grown on LB plates with 400 mgꢀL Cm. The same strain expressing tRNA
and SerRS
ꢁ1
variants was grown on LB plates with 20 mgꢀL Cm; both tRNAs were under the control of lpp promoter, and the cat gene with TAG at
codon position 146 was on the same plasmid. SerRS expression was controlled by the trc promoter on the second plasmid.
IPTG/mM
wt SerRS
0.05
0.1
SerRS V8
SerRS V20
SerRS V22
no insert
30 °C
wt SerRS
Fig. 5. SerRS V20 rescues the
thermolabile SerRS at nonpermissive
temperature. Plasmids pTrc99A containing
wt or SerRS variants were transformed
into Escherichia coli strain KL235 [1]; as a
negative control, pTrc99A without an
insert was also used. Plates were
SerRS V8
SerRS V20
SerRS V22
42 °C
no insert
removed from the incubator after 20 h at
3
0 °C (upper panels) and 42 °C (lower
10–1 10–2 10–3 10–4 10–5 10–6
10–1 10–2 10–3 10–4 10–5 10–6
OD600
panels).
UTuX
influences recognition by SerRS, as demonstrated by
Sec
the improved activity of tRNA variants lacking the
times better aminoacylated than tRNA
(Tables 1,
has about 9-times lower
kcat/KM value than SupD (Tables 2, S2). The improved
UTu1
2). However, even tRNA
5A:67A bp [8]. These differences are also reflected by
the extent of CAT synthesis in vivo, where the con-
Sec
serylation of the synthetic tRNA species by the
designed SerRS V8 and V20 variants (Tables 1, 2)
ferred resistance matches relative serylation levels of
Sec
the SupD tRNA suppressor and the synthetic tRNA
proves that SerRS can be adapted to better recognize
Sec
species (see above). Enzyme-kinetic analyses of these
UTuX
atypical tRNA
these variants are inferior in the SupD tRNA acyla-
structures. Compared to wt SerRS,
Ser
tRNAs with wild-type SerRS reveal tRNA
SecUX
marginally better substrate than tRNA
to be a
(kcat/KM is
Sec
tion (4–5 fold), but superior for synthetic tRNA spe-
cies (up to 11-fold).
UTu1
1.5-time higher, Tables 1, 2), and that tRNA
is 15-
3
766
FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies

Sec
X. Fu et al.
Upgrading E. coli SerRS for tRNA
serylation
Given the central role of aminoacyl-tRNA syn-
5 Br o€ cker MJ, Ho JM, Church GM, S o€ ll D and
thetases in protein synthesis, the last three decades saw
much work directed toward ‘improvement’ of these
enzymes. This effort led to tRNA synthetases endowed
with an altered or expanded substrate spectrum for
either tRNAs [35] or amino acids [36], or unnatural
amino acid substrates for synthetic biology applications
O’Donoghue P (2014) Recoding the genetic code with
selenocysteine. Angew Chem Int Ed Engl 53, 319–323.
Roy KL and S o€ ll D (1970) Purification of five serine
transfer ribonucleic acid species from Escherichia coli
and their acylation by homologous and heterologous
seryl transfer ribonucleic acid synthetases. J Biol Chem
6
2
45, 1394–1400.
[37]. In this work, we addressed a different challenge, to
7
8
Sch o€ n A, B o€ ck A, Ott G, Sprinzl M and S o€ ll D (1989)
The selenocysteine-inserting opal suppressor serine
tRNA from E. coli is highly unusual in structure and
modification. Nucleic Acids Res 17, 7159–7165.
Baron C and B o€ ck A (1991) The length of the
aminoacyl-acceptor stem of the selenocysteine-specific
design variants of a normal cellular tRNA synthetase
that aminoacylates multiple cognate isoacceptors. While
Ser
wt E. coli SerRS serylates its cognate tRNA species
Sec
00-fold better than its tRNA isoacceptor [8], we cre-
1
ated an enzyme variant with improved serylation prop-
Sec
erties only for tRNA
species; yet this enzyme fully
Sec
tRNA of Escherichia coli is the determinant for
Ser
maintains its essential role in providing Ser-tRNA
for cellular protein synthesis. This is another illustration
of the great plasticity of the translation machinery.
binding to elongation factors SELB or Tu. J Biol Chem
2
66, 20375–20379.
9
B o€ ck A & Thanbichler M (2004) Selenocysteine. EcoSal
Plus, https://doi.org/10.1128/ecosalplus.1123.1126.1121.
1
121.
0 Mukai T, Sevostyanova A, Suzuki T, Fu X and S o€ ll D
2018) A facile method for producing selenocysteine-
containing proteins. Angew Chem Int Ed Engl 57, 7215–
219.
Acknowledgments
1
1
1
(
The authors are grateful to Takahito Mukai, Li-Tao
Guo, and Yuchen Liu for enlightened discussions and
Anusha Manglik for help with experiments. This work
was supported by the National Institute of General
Medical Sciences (R35GM122560 to DS), and the
Division of Chemical Sciences, Geosciences and Bio-
sciences, Office of Basic Energy Sciences of the
Department of Energy (DE-FG02-98ER20311 to DS)
for the genetic experiments.
7
1 Biou V, Yaremchuk A, Tukalo M and Cusack S (1994)
ꢁ
The 2.9 A crystal structure of T. thermophilus seryl-
Ser
tRNA synthetase complexed with tRNA . Science
2
63, 1404–1410.
2 Wang C, Guo Y, Tian Q, Jia Q, Gao Y, Zhang Q,
Sec
Zhou C and Xie W (2015) SerRS-tRNA complex
structures reveal mechanism of the first step in
selenocysteine biosynthesis. Nucleic Acids Res 43,
1
0534–10545.
Author contributions
1
3 Liu DR and Schultz PG (1999) Progress toward the
evolution of an organism with an expanded genetic
code. Proc Natl Acad Sci USA 96, 4780–4785.
14 Low B, Gates F, Goldstein T and S o€ ll D (1971)
Isolation and partial characterization of temperature-
sensitive Escherichia coli mutants with altered leucyl-
and seryl-transfer ribonucleic acid synthetases. J
Bacteriol 108, 742–750.
15 Mukai T, Vargas-Rodriguez O, Englert M, Tripp HJ,
Ivanova NN, Rubin EM, Kyrpides NC and S o€ ll D
(2017) Transfer RNAs with novel cloverleaf structures.
Nucleic Acids Res 45, 2776–2785.
XF, AC and DS designed the experiments and wrote
the manuscript. XF, AC and AS executed the experi-
ments. All the authors read and edited the manuscript.
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FEBS Letters 592 (2018) 3759–3768 ª 2018 Federation of European Biochemical Societies