Catalytic acetalization of glycerol to biofuel additives over NiO and Co3O4 supported oxide catalysts: experimental results and theoretical calculations

Article abstract of DOI:10.1016/j.mcat.2020.111186

A series of NiO and Co₃O₄ supported oxide catalysts was synthesized and investigated in the acetalization of glycerol with butyraldehyde to biofuels production. The catalysts were characterized by XRD, N₂ sorption, FTIR and Raman spectroscopy, SEM-EDS and HRTEM techniques. The influence of the catalysts mass, glycerol to butyraldehyde molar ratios, reaction temperature and presence of solvent were deeply investigated. The dispersion of Ni and Co oxides gave distinct effects on the catalytic performances of the solids with AlTi and CeMn being actives in all conditions tested. The mechanistic aspect of the reaction was investigated by density functional theory (DFT). Among the catalysts, the high acidity of the Co/CeMn and Ni/CeMn promoted the acetalization of glycerol more efficiently to produce 4-methanol-2-propyl-1,3-dioxolane, in spite of the glycerol oligomers formation. The correlation of the structure, acidity, morphology and texture of the solids with the catalytic performance was illustrated for solventless acetalization of glycerol with butyraldehyde reaction. The theoretical calculations by DFT studies revealed a more favorable route to 1,3-dioxolane production.

Full text of DOI:10.1016/j.mcat.2020.111186

Article
Dimer–Dimer Interaction of the Bacterial
Selenocysteine Synthase SelA Promotes
Functional Active-Site Formation and
Catalytic Specificity
Yuzuru Itoh1, 2, 3, †, Markus J. Bröcker4, †, Shun-ichi Sekine1, 2, 5
Dieter Söll4, 6 and Shigeyuki Yokoyama1, 2, 7
,
1 - Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
2 - RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
3 - Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
4 - Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA
5 - Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi,
Yokohama 230-0045, Japan
6 - Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA
7 - RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
Correspondence to Dieter Söll and Shigeyuki Yokoyama: D. Söll is to be contacted at: Department of Molecular
Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA; S. Yokoyama, RIKEN Structural
Biology Laboratory, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan. yokoyama@riken.jp
http://dx.doi.org/10.1016/j.jmb.2014.01.003
Edited by M. Guss
Abstract
The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized
on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec
,
formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-
phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a
molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA
complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers,
with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site
is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site
formation remained elusive. In the present study, we examined the quaternary interactions and
demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined
the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that
prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and
confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the
conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I
pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary
structures.
© 2014 The Authors. Published by Elsevier Ltd. All rights reserved.
tRNASec-ligated serine (Ser-tRNASec) generated by
Introduction
seryl-tRNA synthetaseis converted to tRNASec-ligated
Selenocysteine (Sec) is known as the 21st amino
acid incorporated translationally into proteins and is
synthesized on its specific tRNA (tRNASec) [1]. The
Sec (Sec-tRNASec), where selenophosphate,
synthesized by selenophosphate synthetase, is
used as a selenium donor [2]. In Eukaryotes and
0022-2836/$ - see front matter © 2014 The Authors. Published by Elsevier Ltd. All rights reserved. J. Mol. Biol. (2014) 426, 1723–1735

1724
Dimer–Dimer Interaction of SelA
Fig. 1. The enzymatic reaction. Schematic views of the enzymatic reactions of SelA, SepSecS, and cystathionine
β-lyase (CBL). These enzymes catalyze α position deprotonation, followed by β position elimination, to generate
tRNA-ligated or non-esterified 2-aminoacrylate intermediates, conjugated with PLP. The substrate of CBL is cystathionine,
and the enzyme–substrate interaction is responsible for the α-carboxyl group [8] (c) while SepSecS interacts with the
phosphate group of its substrate Sep-tRNASec [9]. However, Ser-tRNASec, the substrate of SelA, has neither a carboxyl
nor a phosphate group. Furthermore, the leaving group in the SelA-catalyzed reaction is a hydroxide ion, which is less
suitable as compared to the phosphate and the deprotonated homocysteine eliminated in the SepSecS- and
CBL-catalyzed reactions, respectively.
Archaea, Ser-tRNASec is phosphorylated by
dimer–dimer interface, the significance of the forma-
tion of an (α2)5 SelA structure in the generation of
functional catalytic sites remained unclear.
O-phosphoseryl-tRNASec kinase (PSTK) to gen-
erate O-phosphoseryl-tRNASec (Sep-tRNASec
)
[3], followed by Sep-to-Sec conversion catalyzed by
SepSecS [4,5]. On the other hand, in Bacteria, the
selenocysteine synthase SelA directly converts
Ser-tRNASec to Sec-tRNASec, without a previous
phosphorylation step [6]. Both SelA and SepSecS
are pyridoxal 5′-phosphate (PLP)-dependent
enzymes.
SelA is a homodecameric enzyme with an overall
molecular mass of about 500 kDa. We previously
determined the crystal structures of SelA with and
without tRNASec [7]. The SelA decamer is organized
as a pentamer of intimate dimers, and it binds 10
tRNASec molecules. Residues from each of the four
SelA subunits of two intimate dimers are involved in
processing one Ser-tRNASec molecule, indicating
that the decameric quaternary structure is essential
to bind Ser-tRNASec [7]. We proposed the SelA
catalytic mechanism based on the co-crystal struc-
ture with thiosulfate, an analog of selenophosphate.
While the tRNA-binding pocket is formed at the
The enzymes SelA and SepSecS belong to the
largest PLP-dependent enzyme group, the fold-type-I
superfamily, and catalyze the replacement of the
amino acid β position (β replacement) in an α,β
elimination reaction proceeding via a 2-aminoacrylate
intermediate and a subsequent addition step (Fig. 1)
[6]. Although β replacement is a common reaction,
there are two major difficulties with the Sec synthesis
step. First, the precursor, Ser-tRNASec, lacks the
α-carboxyl group, which is used for the substrate–
enzyme interaction in all fold-type-I PLP-dependent
enzymes that work with α-amino acids [10]. Second,
the leaving group in the β elimination step is a
hydroxide ion from the Ser moiety (Fig. 1) [6]. In
general, a hydroxide ion is not a suitable leaving
group, and no other fold-type-I members can eliminate
it from the β position of an α-amino acid. In Eukarya/
Archaea, these difficulties are overcome with the
assistance of PSTK. PSTK phosphorylates the Ser
moiety to generate Sep-tRNASec [3], which is the

Dimer–Dimer Interaction of SelA
1725
Fig. 2. Structure of SelA. (a) Overall view of the crystal structure of decameric SelA-FL. PLPs are represented as sphere
models. The N-terminal domain, the N-linker, the core domain, and the C-terminal domain of subunit A are colored
cornflower blue, magenta, blue, and medium-purple, respectively, while those of subunit B are colored green-yellow,
yellow, green, and aquamarine, respectively. Subunits C–J are colored gray. (b and c) Close-up views of the interactions at
interfaces I (b) and II (c) between intimate dimers. Interactions between subunits A and J are shown. The colors of the
ribbon diagrams are the same as in (a). (d and e) Overall views of the dimeric SelA mutants, dSelA-FL (d) and dSelA-ΔN
(e). The ribbon diagrams are colored as in (a). dSelA-FL lacks PLP.
substrate of SepSecS (Fig. 1). The β elimination is
thus facilitated because the phosphate is an excellent
leaving group. Furthermore, the phosphate group is
predominantly responsible for the substrate binding to
SepSecS [9]. In contrast, the bacterial system differs
strikingly in that the single enzyme SelA directly
Table 1. In vivo mutational analyses of E. coli SelA
SelA
mutant
Amino acid change
E. coli numbering
A. aeolicus
Numbering
Function of residue
PLP occupancy
(Mut10a– Mut10d) (%)
Activity
in vivo
1
2
3
4
5
6
7a
7b
8
ΔGln233–Thr236
ΔGln233–Gly234
Arg185Ala
Thr202Tyr
Thr203Tyr
His207Ala
Asp210Ala
Asp210Arg
ΔThr203–Asn204
Thr202–Arg205 = 2 × Ala
Lys295Ala
Lys295Ala, Asp294Lys
Lys295Ala, Leu296Lys
Lys295Ala, Leu297Lys
Glu222–Val225
Glu222, Gly223
Arg174
Thr191
Thr192
Lys196
Asp199
Asp199
Thr192, Asn193
Thr191–Lys194
Lys285
Lys285, Asp284
Lys285, Leu286
Lys285, Leu287
Decamerization interface I
Decamerization interface I
Decamerization interface II/III
Decamerization interface II/III
Decamerization interface II/III
Decamerization interface II/III
Decamerization interface II/III
Decamerization interface II/III
Decamerization interface II/III
Decamerization interface II/III
Covalent PLP ligand
−
−
+++
−
−
+++
+++
−
−
9
−
10a
10b
10c
10d
n.d.
25
37
−
Covalent PLP ligand
Covalent PLP ligand
covalent PLP ligand
−
−
30
−
+++, fully active; +, significantly reduced activity; −, inactive; n.d., below detection limit.
Mutations introduced in E. coli SelA, numbering of the corresponding residues for A. aeolicus SelA, and functions of the residues as
determined from the structures and catalytic activities of the corresponding E. coli SelA variants in vivo are indicated. PLP occupancy for
SelA variants 10a–10d is indicated. All SelA variants lacking catalytic activity in vivo were purified and tested for solubility to exclude the
possibility that the activity loss was due to inclusion body formation.

1726
Dimer–Dimer Interaction of SelA
Fig. 3. Catalytic site comparison. (a–c) Catalytic site comparison between the decameric and dimeric SelAs. The
β-sheet β6•β7 and α7 in the catalytic site also participate in the pentamerizing interaction in the decameric SelA-ΔN (a). On
the other hand, dSelA-FL (b) and dSelA-ΔN (c) lack the β6•β7 sheet, and α7 changes its orientation; therefore, the catalytic
site structure is disrupted. dSelA-FL also lacks PLP. Thiosulfate ions are bound in different sites among (a)–(c). Possible
hydrogen bonds or ionic interactions are shown as broken lines. (d) Close-up view of the TS4- and TS5-binding sites of
dSelA-ΔN. The 2Fo − Fc electron density map at the 1.0 σ level is shown. (e–h) Catalytic sites of SepSecS (PDB ID: 2Z67
[11]) (e), SepCysS (PDB ID: 2E7J [12]) (f), CBL (PDB ID: 1CL1 [12]) (g), and MJ0158 (PDB ID: 2AEV [13]) (h). They lack
the β6•β7 counterpart, and the orientations of their α7 counterparts are closer to those of α7 in depentamerized SelA
mutants. The phosphate/sulfate ions at the catalytic site are shown. The SepSecS structure 2Z67 is from the archaeon
Methanococcus maripaludis. The focused Arg and Lys residues are labeled according to their numbering in human/M.
maripaludis SepSecS.
converts Ser-tRNASec to Sec-tRNASec, without phos-
Results and Discussion
phorylation. Our previous study suggested a putative
binding site for the 3′-terminal A76 of tRNASec, and the
The dimer–dimer interface in the pentamer-of-
dimers structure of SelA
binding site could compensate for the lack of the
α-carboxyl group [7]. However, the mechanism that
facilitates the hydroxyl-group elimination has not been
elucidated.
A. aeolicus SelA is a homodecamer in which the
10 subunits, designated here as subunits A to J, form
a “pentamer of dimers” (Fig. 2a). The “intimate
dimers” A•B to I•J have the largest intersubunit
interfaces (e.g., A•B interface, 4067 Å2) on which
the enzyme active sites are built. Therefore, the
intimate dimer is the basal building block of SelA. In
the pentamer-of-dimers quaternary structure, inti-
mate dimers I•J and A•B, for example, interact with
each other through subunits J and A. The dimer–
dimer interface encompasses 1140 Å2 and charac-
teristically constitutes the overall decameric arrange-
ment of SelA. The dimer–dimer interface may be
divided into three parts. The first part (part I, 438 Å2)
has internal 2-fold symmetry and is composed of two
sets of residues from Asn218 to Phe224, constituting
strand β11 within the core domain (Fig. 2b). Strand
In this study, we have determined the structural
basis for productive SelA active-site formation, which
requires the cyclization of SelA dimers to a full
decamer. Analyses of the structures of Aquifex
aeolicus SelA and dimeric SelA variants revealed
that the active-site conformations of SelA intimate
dimers differ significantly from those of decameric
SelA. Structural rearrangements upon decameriza-
tion generate 10 catalytically functional active sites
that properly accommodate and position Ser-tRNASec
for Sec formation. Based on a comparison of SelA with
phylogenetically related enzymes, we describe a
refined mechanism for the direct hydroxyl-group
elimination facilitated by SelA and propose an
evolutionary hypothesis for the emergence of the
decameric SelA architecture.

Dimer–Dimer Interaction of SelA
1727
Fig. 4. Proposed scheme of the SelA-catalyzed reaction. (a) Docking model of the Ser-ligated terminal nucleotide (A76)
of tRNASec and selenophosphate (Se-P). A76 and the Ser moiety are modeled based on the mutational analyses, and their
carbon atoms are colored salmon and spring green, respectively. Arg86 is located in the vicinity of Lys285 and Ser.
Phe224 from subunit J and Asn218 from subunit A form the A76-binding pocket. The Ser moiety is attached covalently to
PLP. Se-P occupies the TS1-binding site. (b) The reaction scheme of the protonation of the β-hydroxyl group by Lys285,
which enables the elimination of the leaving group (a water molecule). Arg86 may provide the basic environment to
strengthen the Lys285 protonation activity. (c) The interaction interface between the core and C-terminal domains of SelA.
The N-linker (magenta) and the SelA-specific insertions I and IV (cyan) participate in the interaction between the core and
C-terminal domains. The other regions are colored as in Fig. 2a. β1 in the N-linker forms a β-sheet with β18 in the
C-terminal domain. Arg86 in the N-linker participates in the catalytic site.
β11 from subunit J, for example, and the correspond-
ing strand from subunit A form an antiparallel β-sheet
between intimate dimers I•J and A•B. The other parts,
II and III (307 Å2 each), are symmetrically related
to each other. Parts II/III between subunits J and
A comprise Arg163, Leu166, Glu168, and Arg174
(η4-loop-β6-loop-β7) from subunit J and Glu188,
Thr191, Thr192, Asn193, Lys196, and Asp199
(β8-loop-β9-α8) from subunit A and vice versa
(Fig. 2c).
We introduced mutations into the dimer–dimer
interface by using Escherichia coli SelA (Table 1).
The amino acid residues in the dimer–dimer inter-
face are conserved between the A. aeolicus and
E. coli SelAs, although the conservation in part I
is relatively low (Table 1). The Mut1 and Mut2
mutations were designed to impair the antiparallel
β11•β11 interaction in part I, by deleting residues
222–225 and 222–223, respectively (residue num-
bering according to A. aeolicus SelA). The enzymes
bearing the Mut1 and Mut2 mutations were catalyt-
ically inactive in vivo (Table 1), indicating that the
integrity of strand β11 and the formation of the
resultant antiparallel β-sheet are critical for the
function of SelA. Mutations targeting parts II/III
were also examined (Table 1). Here, the Ala
mutations of individual residues in parts II/III exhib-
ited no significant effects on the SelA activity (Mut3,
Mut6, and Mut7a). In contrast, the Thr191Tyr,
Thr192Tyr, and Asp199Arg mutations (Mut4, Mut5,
and Mut7b), which were designed to cause steric
clashes, inactivated E. coli SelA in vivo. Similarly, the
deletion of Thr192–Asn193 (Mut8) or the replacement
of four residues, Thr191–Lys194, with two Ala residues
(Mut9) rendered E. coli SelA inactive in vivo. These
results suggested that the integrity of parts II/III is also
important for the SelA activity.
Crystal structure of dimeric SelA
The interface between the intimate dimers is much
smaller than that between the subunits within the
intimate dimer, suggesting weaker interactions
between the dimer units. In fact, a quadruple
mutation (Tyr220Pro-Asp199Arg-Thr191Tyr-
Thr192Tyr) abolished the pentamerization and
resulted in an inactive “dimerized” or “depentamer-
ized” enzyme [7]. (In the following, we refer to the
depentamerized enzyme as dimeric SelA.) Impor-
tantly, dimeric SelA is stable and can be purified for
crystallization. In this study, we crystallized the
full-length and N-terminally truncated (ΔN, deletion
of residues 1–61) dimeric SelA proteins and
determined their structures at 3.35 and 2.40 Å
resolutions, respectively (Fig. 2d and e). The lysine
residues of the dimeric full-length SelA (dSelA-FL)
were methylated for crystallization improvement,
while the dimeric SelA-ΔN (dSelA-ΔN) was crystal-
lized without Lys methylation. The overall structures
of dSelA-FL and dSelA-ΔN are similar to each other
and to those of the intimate dimers of decameric
SelA-FL and SelA-ΔN, respectively. Each subunit
of dSelA-FL consists of the N-terminal domain
(residues 1–66), the N-linker (residues 67–89), the

1728
Dimer–Dimer Interaction of SelA
Fig. 5. Structure comparison with other fold-type-I PLP-dependent enzymes. (a–d) Overall crystal structures of
SepSecS (PDB ID: 2Z67 [8]) (a), SepCysS (PDB ID: 2E7J [11]) (b), cystathionine β-lyase (CBL) (PDB ID: 1CL1 [12]) (c),
and MJ0158 (PDB ID: 2AEV [13]) (d). The N-terminal domains, the N-linkers, the core domains, and the C-terminal
domains of subunits A and B are colored as in Fig. 2a. The N-terminal regions of SepCysS and MJ0158 correspond to the
SelA N-linker. Like the SelA N-linker, these regions form β-sheets with their C-terminal domains. On the other hand, the N
regions of the homotetrameric enzymes SepSecS and CBL participate in the interactions between the two intimate dimers,
and thus, they occupy different positions from that of the SelA N-linker. The intimate dimers C•D of SepSecS and CBL are
colored gray in the top panels and are not shown in the bottom panels. (e–h) Comparison of the C-terminal domain
orientation with SelA. The A subunits of SepSecS (e), SepCysS (f), CBL (g), and MJ0158 (h) were superposed on the SelA
subunit A, by fitting their core domains. The core and C-terminal domains of SelA are colored blue and medium-purple,
respectively, as in Fig. 2a, while those of the other PLP-dependent enzymes are colored light blue and yellow, respectively.
The major helices α12 and α14, located in the core and C-terminal domains, respectively, are connected by the short
helices η7 and α13 in SelA. In contrast, the α12 and α14 counterparts are directly connected in SepSecS and form a
continuous single helix in SepCysS and CBL. On the other hand, MJ0158 has the α13 counterparts, and the orientation of
its C-terminal domain is somewhat closer to that of the SelA C-terminal domain.
core domain (residues 90–338), and the C-terminal
domain (residues 339–452) (Fig. 2d). The β11 strand
in part I of the dimer–dimer interface is disordered in
the dSelAs.
the intimate dimer unit of SelA. PLP is covalently
bound to Lys285, as a Schiff base. Surprisingly, the
catalytic site structures of dSelA-FL and dSelA-ΔN
differ distinctly from that of the SelA decamer
(Fig. 3a–c). The dimeric SelAs lack a SelA-specific
β-sheet (β6•β7), and α7 has a distinct orientation.
These different catalytic site conformations of the
dimeric mutants resemble those of other fold-type-I
PLP enzymes (Fig. 3b, c, and e–h). The position of
Catalytic site of dimeric SelA
The catalytic site is located at the subunit interface,
involving the core domains and the N-linker, within

Dimer–Dimer Interaction of SelA
1729
PLP in the dSelA-ΔN catalytic site is the same as
that in the SelA decamer, while dSelA-FL lacked
PLP in its catalytic site. PLP may have been
removed during the Lys methylation of dSelA-FL,
whereas the decameric SelA-FL and SelA-ΔN
retained PLP during the methylation [7]. This
indicated that the depentamerization increased the
accessibility of Lys285, which probably resulted in
the replacement of PLP by a methyl group, although
the methyl group was not visible in the structure due
to the high mobility of the Lys285 side chain.
The thiosulfate-binding sites also differ from those of
the decameric SelA-ΔN (Fig. 3a–c). The decameric
SelA-ΔN binds three thiosulfate ions (TS1–TS3) in
each catalytic site. TS1 may mimic the substrate
selenophosphate, and TS2 and TS3 may mimic the
phosphate groups of the nucleotides A76 and C75 of
the tRNASec CCA terminus [7]. In contrast, dSelA-ΔN
binds two thiosulfate ions, TS4 and TS5. TS4 may
correspond to TS1, but TS5 does not correspond to
any of TS1–TS3. TS1 interacts with Arg86A (subunit
A residue) and Arg312B and Arg315B (subunit B
residues), whereas TS4 interacts with Ser172A,
Arg174A, Arg116B, and Arg315B (Fig. 3d). The
distance between TS1 and TS4 is about 3.5 Å. TS5
interacts with Arg312B and Arg315B. dSelA-FL also
has two thiosulfate ions, TS5 and TS6. TS5 is the
same as that in dSelA-ΔN, whereas TS6 occupies the
position of the PLP phosphate group. The difference
between the thiosulfate-binding sites of dSelA-ΔN and
dSelA-FL may be caused by the lack of PLP in the
catalytic sites of dSelA-FL.
enzyme to eliminate a hydroxyl group. SelA has a
putative binding pocket for the A76 adenine ring
(Fig. 4a) [7] and can therefore accommodate the Ser
moiety of Ser-tRNASec. This binding pocket is
critically important because Ser-tRNASec lacks the
α-carboxyl group, which is used for the substrate–
enzyme interaction in all of the fold-type-I super-
family PLP-dependent enzymes working on α-amino
acids [10].
To examine the mechanism of hydroxyl-group
protonation, we compared SelA with other PLP-
dependent enzymes. SelA is the only fold-type-I
enzyme that eliminates the hydroxyl group from the
substrate amino acid (or amino acid moiety).
Therefore, we compared SelA with cystathionine
β-synthase (CBS), a fold-type-II PLP-dependent
enzyme that catalyzes a β replacement reaction.
CBS eliminates the hydroxyl group from serine to
generate 2-aminoacrylate and then adds homo-
cysteine to synthesize cystathionine (Supplemen-
tary Fig. 1a). The β elimination step is the same as
that in SelA catalysis (Fig. 1). A crystallographic
study revealed that Tyr227 of CBS is responsible
for the protonation of the serine hydroxyl group [14].
Interestingly, cysteine synthase (CysS), the close
homolog of CBS, lacks the corresponding Tyr residue
[15]. CysS catalyzes the β replacement reaction that
eliminates an acetyl group from O-acetylserine, and it
adds hydrogen sulfide to generate cysteine (Supple-
mentary Fig. 1b). In this reaction, the leaving group is
acetate, and thus, hydroxyl-group protonation is
unnecessary.
In decameric SelA, the β6•β7 sheet is stabilized
by the pentamerizing interactions. The main chain
NH and carbonyl moieties of Glu168A in β6 interact
with Asn193J, and the Arg174A side chain forms a
salt bridge with Asp199J (Fig. 3a). Since Arg174A
also participates in α7, the α7 orientation is
dependent on the Arg174A position, which is fixed
by the Arg174A•Asp199J salt bridge. Although the
β6•β7 sheet has no polar interactions with the
thiosulfate ions, Gly170A in the β6–β7 loop is close
to TS1 and forms a van der Waals interaction (Fig. 3a).
Therefore, the β6•β7 sheet contributes to TS1 binding
and is important for catalysis.
These results indicated that the formation of the
catalytic site structure is dependent on the pentamer-
ization of intimate dimers and that the dimeric mutant
cannot properly bind its substrates. The activity loss
by the mutations introduced to the pentamerization
interfaces (Table 1) should result from both the
prevention of decamer-dependent tRNASec binding
and the disruption of the catalytic core structure.
On the other hand, SelA lacks residues such as Tyr,
His, and Cys that could serve as proton donors in the
vicinity of the Ser-moiety-binding site, except for
Lys285, which forms a Schiff base with PLP in the
waiting state. Some enzymes in the sugar aminotrans-
ferase subfamily catalyze the elimination of the
α-hydrogen and the β-hydroxyl group. Here, the α
position indicates the position of the amino group
conjugated with PLP during catalysis. The enzymes
GDP-6-deoxy-α-D-lyxo-hexopyranos-4-ulose dehydra-
tase (ColD) and CDP-6-deoxy-α-D-xylo-hexopyrano-
s-4-ulose dehydratase (E1) employ a His residue,
which is conserved at the position of the PLP-coordi-
nating Lys285 of SelA, to catalyze the α,β elimination
step (Supplementary Fig. 1c and d) [16,17]. In contrast,
5-amino-3-hydroxybenzoate (AHBA) synthase, anoth-
er enzyme that eliminates the α-hydrogen and the
β-hydroxyl group (Supplementary Fig. 1e), contains
a conserved Lys residue, which is considered to
protonate the β-hydroxyl group [18]. Here, the gener-
ation of a benzene ring in the product is likely to
promote the α,β elimination reaction.
How does SelA eliminate the hydroxyl group
from the Ser moiety?
These findings suggested that Lys285 in SelA can
act as proton donor for the β-hydroxyl group (Fig. 4b).
As enzymes such as ColD and E1 utilize His, instead
of Lys, to strengthen the protonation activity and
AHBA generates a benzene ring to promote the
In general, proper binding and hydroxyl-group
protonation of the substrate are essential for an

1730
Dimer–Dimer Interaction of SelA
Fig. 6. Structure-based phylogenetic tree of the fold-type-I PLP-dependent enzymes. SelA and the crystal structures of
the fold-type-I PLP enzymes, deposited in the Protein Data Bank, were analyzed. The enzyme names are colored based
on their catalysis type: the enzymes that catalyze aminotransfer (AT), α-carboxyl group replacement, α-hydrogen
replacement, reverse aldol reaction, α,β elimination, β replacement (type 1), β replacement (type 2), α,γ elimination, and γ
replacement are colored dark blue, blue, sky blue, blue-violet, magenta, red, orange, spring green, and green,
respectively. The enzymes with unknown functions are colored gray. The structure-based phylogenetic analysis revealed
that SelA, SepSecS, and SepCysS [20] belong to distinct taxa of the fold-type-I PLP-dependent enzymes. SelA is likely to
form an independent group related to the cystathionine γ-synthase (CGS) family. SepCysS is close to the cysteine
desulfurase and kynureninase families, while SepSecS also forms another independent group close to the sugar
aminotransferase (AT) family.
protonation and elimination of the β-hydroxyl group,
an additional auxiliary mechanism to support proton-
ation and β-hydroxyl elimination might be plausible for
SelA as well. Such a mechanism would involve the
Arg86 residue (Fig. 4a and b). Our in vivo mutational
assay revealed that Arg86 is mandatory for activity [7].
Arg86 is located in the vicinity of Lys285 (Fig. 4a), and
the positive charge of Arg86 may reinforce the
protonation activity of Lys285. Arg86 is located in
the N-linker between the N and core domains. The
β-strand β1 in the N-linker forms a β-sheet with β18
from the C-terminal domain (Fig. 4c). Although the
corresponding β-sheet is present in many fold-type-I
PLP-dependent enzymes, the C-terminal domain

Dimer–Dimer Interaction of SelA
1731
Fig. 7. Possible steps of SelA (a) and SepSecS (b) evolution. The dimeric SelA ancestor accommodated the tRNASec
acceptor arm within the cleft between the core and C-terminal domains. The ancestor interacted with the substrate
Sep-tRNASec by binding its Sep phosphate group at the selenophosphate (Se-P)-binding site. Therefore, the Sep-to-Sec
reaction and the tRNA discrimination were dependent on the Sep phosphate group. Tetramerization created the
A76-binding pocket, and decamerization stabilized the interactions among intimate dimers. Finally, the acquired
N-terminal domain discriminated tRNASec from tRNASer, by interacting with the unique D arm of tRNASec. The A76-binding
pocket and the tRNASec discrimination enabled Ser-to-Sec conversion, and thus, SelA became independent of the Sep
phosphate group. On the other hand, the dimeric SepSecS interacted with the extra arm side of the tRNASec acceptor arm,
since it lacks the cleft between the core and C-terminal domains. Tetramerization stabilized the tRNASec binding by
interacting with its T and extra arms, without recognizing the unique D arm structure. Therefore, SepSecS is unable to
become independent of the Sep phosphate group of Sep-tRNASec
.
orientation in SelA differs from those of the other
homologs (Fig. 5). The unique C-terminal domain
orientation in SelA results in the specific positioning of
the N-linker that allows participation in the catalytic
site. This structural feature is the reason why the
N-linker can provide Arg86 in the vicinity of Lys285.
Here again, the characteristic tertiary and quaternary
structures of SelA are essential for its unique catalytic
activity to eliminate the hydroxyl group.
SepSecS also possesses an Arg residue (Arg75
in human SepSecS) that occupies the space
corresponding to SelA Arg86 [9]. However, Arg75 is
located in the core domain of SepSecS and is not an
alignment-based counterpart of SelA Arg86, which is
in the N-linker. In fact, Arg75 forms an intersubunit
interaction with Lys-bound PLP; that is, Arg75B
interacts with PLP-Lys284A (Fig. 3e). In contrast, the
Arg86 and PLP-Lys285 interaction is an intrasubunit
one (Figs. 3a and 4a). Moreover, the PLP-ligated
Lys284 in SepSecS cannot protonate the β-hydroxyl
group of the Ser moiety; SepSecS cannot bind
Ser-tRNASec because the phosphate group of the
Sep moiety is essential for the substrate Sep-tRNASec
binding.
Why did two different pathways for Sec forma-
tion evolve?
Considerable conservation of the translation ma-
chinery components exists among the three domains
of life. However, the pathways of aminoacyl-tRNA
formation evolved much more independently. For
instance, the synthesis of Asn-tRNA or Gln-tRNA
involves tRNA-dependent and tRNA-independent
amino acid biosynthesis routes [19]. In the case of
Sec, nature also designed distinct routes based on the
different enzyme structures operating in Bacteria
versus Archaea/Eukaryotes. Our phylogenetic analy-
sis (Fig. 6) revealed that SelA is related to the
cystathionine γ-synthase (CGS) family members
catalyzing the β elimination of cysteine derivatives or
the γ elimination and γ replacement of homocysteine
products. Thus, the ancestor of SelA may have been
an enzyme involved in sulfur metabolism. The protein

1732
Dimer–Dimer Interaction of SelA
Table 2. Data collection and refinement statistics.
dSelA-FL4KA (SeMet)
dSelA-ΔN (SeMet)
Lys methylation
Data collection
Beam line
Wavelength (Å)
Space group
Cell parameters
a (Å)
+
−
SPring-8 BL41XU
0.97915
I422
SPring-8 BL41XU
0.97915
P21
144.1
144.1
273.4
59.2
81.3
95.5
b (Å)
c (Å)
β (°)
92.2
Resolution (Å)
Unique reflections
Completeness (%)
Redundancy
50.0–3.35 (3.47–3.35)a
21,096 (2066)a
50.0–2.40 (2.49–2.40)a
35,457 (3520)a
99.6 (100.0)a
3.8 (3.8)a
99.7 (100.0)a
a
6.0 (6.1)
Rsym
0.102 (0.712)a
17.2 (2.75)a
0.082 (0.561)a
15.9 (2.40)a
b
I/σ(I)
Structure refinement
Working-set reflections
Test-set reflections
Resolution (Å)
Number of SelA subunits
Number of protein atoms
Number of PLP
19,970
1062
50.0–3.35
2 (one dimer)
7035
33,593
1770
50.0–2.40
2 (one dimer)
6018
0
2
Number of ions
4 (thiosulfate)
0
0.194/0.250
5 (thiosulfate)
153
0.180/0.230
Number of solvent molecules
c
Rwork/Rfree
Average B-factor (Å2)
Overall
Protein
PLPs
Ion
Solvent
RMSD bond lengths (Å)
RMSD bond angles (°)
144.3
144.2
—
162.4
—
0.009
1.27
64.4
64.6
49.8
79.0
56.7
0.008
1.12
a
The statistics in the highest-resolution shell are given in parentheses.
Rsym = ∑hkl∑i[|Ii(hkl) − 〈I(hkl)〉|]/∑hkl∑i[Ii(hkl)], where Ii(hkl) is the intensity of the ith measurement of hkl and 〈I(hkl)〉 is the average
b
value of Ii(hkl) for all ith measurements.
c
Rwork,free = ∑hkl(||Fo| − k|Fc||)/∑hkl(|Fo|), where Rwork and Rfree are calculated using the working-set and test-set reflections (5% of the
total reflections), respectively.
closest to SelA in the phylogenetic tree is the archaeal
MJ0158, a homodimeric protein of unknown function
that does not interact with tRNASec (Fig. 6) [13].
Interestingly, the orientation of the C-terminal domain
of MJ0158 is similar to that of the SelA C-terminal
domain (Fig. 5d and h), suggesting that the change in
the domain orientation occurred before SelA acquired
its function. Although the N-linker of MJ0158 occupies
the similar space to that of SelA, MJ0158 has a Ser
residue at the corresponding position of SelA Arg86,
which may reflect its functional difference from SelA.
Since the binding of the A76-Ser moiety of Ser-
tRNA is impossible without the decameric assembly
(the putative A76-binding site is formed at the dimer–
dimer interface) [7], it is plausible that a dimeric SelA
ancestor depended on the phosphate group and
simply converted Sep to Sec without tRNASec
discrimination, as in the case of SepSecS (Fig. 7).
In fact, the extant SelA can produce Sec-tRNASec
from Ser-tRNASec and from Sep-tRNASec [5],
confirming that the Sec synthesis by SelA does not
rely on the phosphate group of the substrate
aminoacyl moiety. Like SepSecS, the SelA ancestor
may have used its selenophosphate-binding site to
bind the Sep phosphate moiety in order to facilitate
the elimination of the phosphate group from Sep. In
this case, either PSTK or an enzyme with a similar
function must have existed for the phosphorylation
of Ser and the discrimination of tRNASec from tRNASer
.
Alternatively, the ability of SepSecS to convert Ser to
Sec was lost when PSTK evolved for more efficient
Sec production.
Evolving decamerization
In a subsequent evolutionary step, the collabora-
tion of the two SelA dimers could have occurred to

Dimer–Dimer Interaction of SelA
1733
Rosetta 2(DE3) (Stratagene) was transformed with the
expression plasmids, and the native and selenomethionine
(SeMet)-substituted proteins were overexpressed and purified
as previously described [21]. In order to improve the
crystallization efficiency, we methylated the lysine residues of
dSelA-FL4KA with formaldehyde and a dimethylamine–borane
complex, as previously described [22]. Prior to methylation, the
protein was diluted with 20 mM Hepes–NaOH buffer (pH 7.5),
containing 0.8 M NaCl and 10 mM 2-mercaptoethanol.
generate the A76-binding pocket at the dimer–dimer
interface, thereby improving the efficiency and
specificity of substrate binding. Nonetheless, in
such a tetramer, only two of the four catalytic sites
would have been functional (the “half-of-the-sites”
stage). The relative orientations between the dimers
could then be stabilized by acquiring decameric ring
closure involving five dimers. Simultaneously, the full
usage of all 10 catalytic sites was achieved (Fig. 7a).
Interestingly, the non-productive catalytic site con-
formation of the dimeric mutant dSelA-ΔN resembles
those of the other fold-type-I members (Fig. 3b, c,
and e–h) and therefore appears to be more primeval.
The loop region before α7 interacts with PLP in
dSelA-ΔN (Fig. 3b and c), while it forms a β-sheet
(β6•β7) in the SelA decamer (Fig. 3a). Hence, the
catalytic site may have evolved by taking advantage of
the decameric quaternary structure. The N-terminal
domain was then acquired to increase its affinity for
tRNASec and was further utilized for the discrimination
Crystallization and X-ray diffraction data collection
The SelA proteins were crystallized at 20 °C by the
sitting-drop vapor diffusion method, by mixing 0.75 μl of the
sample with 0.75 μl of reservoir solutions. Prior to crystal-
lization, dSelA-FL4KA (SeMet substitution, Lys methylation)
and dSelA-ΔN (SeMet substitution, without methylation)
were dialyzed against 20 mM Tris–HCl buffer (pH 7.5),
containing 200 mM NaCl and 10 mM 2-mercaptoethanol,
and were concentrated to 9.6 and 7.8 mg/ml, respectively.
Crystal Screen 2, Natrix (Hampton Research), Wizard I,
and Wizard II (Emerald Bio-Science) screening kits
were used for the initial screening of the crystallization
conditions. The optimized reservoir solutions were
100 mM trisodium citrate–HCl buffer (pH 5.6), containing
580–590 mM trisodium citrate, 100 mM L-serine, and
100 mM Na2S2O3 for dSelA-FL4KA, as well as 100 mM
sodium 4-morpholineethanesulfonate HCl buffer (pH 6.5),
containing 2.5% polyethylene glycol 4000, 150 mM
Na2S2O3, and 70 mM NaNO3 for dSelA-ΔN.
The crystals thus obtained were transferred into cryo-
protective solutions composed of 100 mM trisodium
citrate–HCl buffer (pH 5.6), containing 1.3 M trisodium citrate,
75 mM L-serine, and 75 mM Na2S2O3 for dSelA-FL4KA, as well
as 100 mM sodium 4-morpholineethanesulfonate HCl buffer
(pH 6.5), containing 30% dimethyl sulfoxide, 10% polyethylene
glycol 4000, 50 mM Na2S2O3, and 70 mM NaNO3 for
dSelA-ΔN, and were flash-cooled in a cryo-N2 stream. X-ray
diffraction data were collected at 90 K by using synchrotron
radiation at the BL41XU of SPring-8 (Harima, Japan). The data
were processed with the HKL2000 program [23]. The statistics
of the diffraction data collection are listed in Table 2.
of tRNASec from tRNASer
.
Conclusion
The natural decameric arrangement of five SelA
dimers allows the formation of 10 fully occupied and
catalytically functional active sites. Since SelA dec-
amerization is driven by dimer–dimer interactions,
disruptive mutations at the interaction interfaces solely
yield SelA dimers. In striking contrast to decameric
SelA, these dimers are catalytically deficient and
exhibit a distinctly distorted active-site formation that
prevents productive tRNASec coordination. In fact, the
dimeric SelA conformationally resembles similar
related PLP enzymes, such as the tetrameric SepSecS.
The evolution of the SelA decamer may have been
driven by enhanced catalytic efficiency obtained from
two sources: First, the decamer allows for an active-site
conformation that is optimal for direct, tRNA-dependent
Ser-to-Sec conversion. Second, the 10-subunit archi-
tecture of SelA generates an N-terminal domain with the
Structure determination and refinement
ability to discriminate tRNASec
.
The structures of the dimeric SelAs, dSelA-FL4KA and
dSelA-ΔN, were solved by the molecular replacement
method, using the Phaser program [24]. The search model
was the SelA-ΔN structure. The Coot program [25] was
used for manual fitting of the models to the electron density
map. The structures were refined against the diffraction
data by using the CNS [26] and PHENIX [27] programs,
with iterative cycles of positional and temperature factor
refinements (Table 2).
Materials and Methods
Protein preparation
The genes encoding the full-length and ΔN SelA genes
from A. aeolicus were cloned into the vectors pET25b and
pET22b (Novagen), respectively. The full-length SelA used
for crystallization was a quadruple mutant, with the non-
conserved Lys residues in the loop regions of the N-terminal
domain replaced by Ala residues, Lys19Ala-Lys21Ala-
Lys46Ala-Lys48Ala (SelA-FL4KA). The dimerizing quadruple
mutation, Tyr220Pro-Asp199Arg-Thr191Tyr-Thr192Tyr, was
introduced into SelA-FL4KA and SelA-ΔN by QuikChange
site-directed mutagenesis (Stratagene). The E. coli strain
Production and purification of E. coli SelA variants
Mutations were introduced by site-directed mutagenesis
(QuikChange®; Agilent) to E. coli K12 selA, from the ASKA
E. coli K12 ORF library (GFP-minus), cloned in the pC24N
vector [28]. E. coli BL21 cells were transformed with the
expression plasmids. For protein overproduction, the cells

1734
Dimer–Dimer Interaction of SelA
were grown at 25 °C in LB selective medium, supplemented
with 50 μM isopropyl β-D-1-thiogalactopyranoside. Cells
sedimented from overnight cultures were resuspended in
lysis buffer (50 mM KH2PO4, pH 8, 300 mM NaCl, 15%
glycerol, 10 mM imidazole) and disrupted by sonication.
After centrifugation (50,000g, 45 min, 4 °C), the clarified
lysates were subjected to immobilized metal affinity chro-
matography, using Ni-NTA affinity resin (Qiagen) on
gravity-flow columns. The immobilized 6× His-SelA fusion
proteins were washed with lysis buffer supplemented with
40 mM imidazole, and SelA was eluted with 240 mM imidazole
in lysis buffer. The excess imidazole was removed by dialysis
(50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 15% glycerol), and
the purified SelA variants were analyzed by SDS-PAGE and
stored at −80 °C. The amounts of protein-bound PLP in the
SelA variants were determined by UV/vis spectroscopy at
420 nm and in comparison to a PLP standard curve (0–2 mM).
Accession numbers
The coordinates and structure factors have been depos-
ited in the Protein Data Bank (PDB IDs: 3WCN and 3WCO).
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jmb.2014.01.003.
Acknowledgments
We thank the staff members of SPring-8 BL41XU
and the Photon Factory beam lines for assistance
with our data collection. We thank T. Imada, K. Ake,
and T. Nakayama for assistance with the manuscript
preparation and P. O'Donoghue and M. Simonovic
(University of Illinois, Chicago) for critical discus-
sions. We are grateful to the National BioResource
Project in Japan for providing the ASKA E. coli
K12 ORF library. Y.I. was supported by Research
Fellowships from the Japan Society for the Promotion
of Science. M.J.B. holds a Feodor Lynen Postdoctoral
Fellowship of the Alexander von Humboldt Founda-
tion (Bonn, Germany). This work was supported in
part by Japan Society for the Promotion of Science
Grants-in-Aid for Scientific Research (A) to S.Y. and
(C) to S.S. This work was also supported in part by the
Targeted Proteins Research Program, and the
Platform for Drug Discovery, Informatics, and Struc-
tural Life Science from the Ministry of Education,
Culture, Sports, Science, and Technology (to S.Y.).
D.S. gratefully acknowledges support from the Divi-
sion of Chemical Sciences, Geosciences, and Bio-
sciences, Office of Basic Energy Sciences of the U.S.
Department of Energy (DE-FG02-98ER20311; for
funding the genetic experiments), from the National
Institute of General Medical Sciences (GM22854),
and from the Defense Advanced Research Projects
Agency (contracts N66001-12-C-4020 and
N66001-12-C-4211).
E. coli SelA in vivo activity assays
For in vivo SelA assays, a marker-less E. coli selA deletion
strain was used. The initial selA deletion strain JS1 was
constructed by the replacement of the selA gene with a
kanamycin cassette in E. coli strain BW25113, as previously
described [29], and the genomic insertion of T7 polymerase
by P1 transduction [4]. The kanamycin resistance marker of
E. coli strain JS1 was then excised by FLP recombinase-
mediated homologous recombination between the FRT sites
flanking the kanamycin resistance cassette, upon transfor-
mation of the JS1 strain with the plasmid pCP20 [30], resulting
in the marker-less E. coli ΔselA strain JS2. E. coli ΔselA was
then transformed with the plasmids encoding the E. coli SelA
variants and grown on LB selective medium. Overnight
cultures of these clones were plated on selective LB agar
plates supplemented with 10 μM isopropyl β-D-1-thiogalacto-
pyranoside, 1 μM Na2MoO4, 1 μM Na2SeO3, and 50 mM
sodium formate, as described previously [4], and grown
anaerobically at 37 °C overnight. For the oxygen-sensitive
benzyl viologen assay, the plates were transferred to an
anaerobic chamber (90% N2, 5% H2, 5% CO2) and overlaid
with top agar containing 1 mg/ml benzyl viologen, 250 mM
sodium formate, and 25 mM KH2PO4/K2HPO4 (pH 7.0).
Wild-type SelA served as a positive control. The benzyl
viologen assay allows the colorimetric monitoring of SelA
activity by the formation of purple-colored reduced benzyl
viologen. Benzyl viologen reduction is catalyzed by FDHH,
which requires Sec insertion for catalytic activity. The Sec
formation, finally, is dependent on the SelA activity.
Received 8 November 2013;
Received in revised form 9 January 2014;
Accepted 12 January 2014
Available online 20 January 2014
Keywords:
fold-type-I PLP-dependent enzyme;
selenium metabolism;
Structure-based phylogenetic tree
The STAMP program [31] in the MultiSeq module of VMD
[32] was used to superpose and calculate the structural
similarity, QH, among the coordinates of SelA-ΔN and the other
fold-type-I PLP-dependent enzymes obtained from the Protein
Data Bank. The enzymes with N50% amino acid sequence
identity were removed, and thus, the core domains of 187
coordinates, including the coordinates of both chains A and B
of the heterodimeric glycine decarboxylase (PDB ID: 1WYT),
were superposed. The other 185 enzymes were homooligo-
meric ones. The structural similarity QH was calculated [33],
and the neighbor-joining tree, based on the QH values, was
drawn using the SplitsTree program [34].
tRNASec
;
pentamer of dimers;
21st amino acid
This is an open-access article distributed under the terms
of the Creative Commons Attribution-NonCommercial-
ShareAlike License, which permits non-commercial use,
distribution, and reproduction in any medium, provided the
original author and source are credited.
Y.I. and M.J.B. contributed equally to this work.

Dimer–Dimer Interaction of SelA
1735
[16] Cook PD, Thoden JB, Holden HM. The structure of GDP-4-
keto-6-deoxy-D-mannose-3-dehydratase: a unique coenzyme
B6-dependent enzyme. Protein Sci 2006;15:2093–106.
[17] Chen XM, Ploux O, Liu HW. Biosynthesis of 3,6-
dideoxyhexoses: in vivo and in vitro evidence for protein–
protein interaction between CDP-6-deoxy-L-threo-D-glycero-4-
hexulose 3-dehydrase (E1) and its reductase (E3). Biochemistry
1996;35:16412–20.
[18] Eads JC, Beeby M, Scapin G, Yu TW, Floss HG. Crystal
structure of 3-amino-5-hydroxybenzoic acid (AHBA) synthase.
Biochemistry 1999;38:9840–9.
[19] Ambrogelly A, Palioura S, Soll D. Natural expansion of the
genetic code. Nat Chem Biol 2007;3:29–35.
Abbreviations used:
AHBA, 5-amino-3-hydroxybenzoate; CBS, cystathionine
β-synthase; CysS, cysteine synthase; PLP, pyridoxal
5′-phosphate; PSTK, O-phosphoseryl-tRNASec
kinase; SeMet, selenomethionine.
References
[1] Böck A, Forchhammer K, Heider J, Leinfelder W, Sawers G,
Veprek B, et al. Selenocysteine: the 21st amino acid. Mol
Microbiol 1991;5:515–20.
[2] Ehrenreich A, Forchhammer K, Tormay P, Veprek B, Böck A.
Selenoprotein synthesis in E. coli. Purification and charac-
terisation of the enzyme catalysing selenium activation. Eur J
Biochem 1992;206:767–73.
[3] Carlson BA, Xu XM, Kryukov GV, Rao M, Berry MJ,
Gladyshev VN, et al. Identification and characterization of
phosphoseryl-tRNA[Ser]Sec kinase. Proc Natl Acad Sci U S A
2004;101:12848–53.
[4] Yuan J, Palioura S, Salazar JC, Su D, O'Donoghue P, Hohn
MJ, et al. RNA-dependent conversion of phosphoserine
forms selenocysteine in eukaryotes and archaea. Proc Natl
Acad Sci U S A 2006;103:18923–7.
[5] Xu XM, Carlson BA, Mix H, Zhang Y, Saira K, Glass RS, et al.
Biosynthesis of selenocysteine on its tRNA in eukaryotes.
PLoS Biol 2007;5:e4.
[6] Forchhammer K, Böck A. Selenocysteine synthase from
Escherichia coli. Analysis of the reaction sequence. J Biol
Chem 1991;266:6324–8.
[7] Itoh Y, Bröcker MJ, Sekine S, Hammond G, Suetsugu S, Söll D,
et al. Decameric SelA•tRNASec ring structure reveals mechanism
of bacterial selenocysteine formation. Science 2013;340:75–8.
[8] Clausen T, Huber R, Laber B, Pohlenz HD, Messerschmidt
A. Crystal structure of the pyridoxal-5′-phosphate dependent
cystathionine beta-lyase from Escherichia coli at 1.83 Å. J
Mol Biol 1996;262:202–24.
[9] Palioura S, Sherrer RL, Steitz TA, Söll D, Simonovic M. The
human SepSecS-tRNASec complex reveals the mechanism
of selenocysteine formation. Science 2009;325:321–5.
[10] Eliot AC, Kirsch JF. Pyridoxal phosphate enzymes: mecha-
nistic, structural, and evolutionary considerations. Annu Rev
Biochem 2004;73:383–415.
[11] Araiso Y, Sherrer RL, Ishitani R, Ho JM, Söll D, Nureki O.
Structure of a tRNA-dependent kinase essential for selenocys-
teine decoding. Proc Natl Acad Sci U S A 2009;106:16215–20.
[12] Fukunaga R, Yokoyama S. Structural insights into the second
step of RNA-dependent cysteine biosynthesis in archaea:
crystal structure of Sep-tRNA:Cys-tRNA synthase from
Archaeoglobus fulgidus. J Mol Biol 2007;370:128–41.
[13] Kaiser JT, Gromadski K, Rother M, Engelhardt H, Rodnina
MV, Wahl MC. Structural and functional investigation of a
putative archaeal selenocysteine synthase. Biochemistry
2005;44:13315–27.
[20] Sauerwald A, Zhu W, Major TA, Roy H, Palioura S, Jahn D,
et al. RNA-dependent cysteine biosynthesis in archaea.
Science 2005;307:1969–72.
[21] Itoh Y, Sekine S, Yokoyama S. Crystallization and preliminary
X-ray crystallographic analysis of Aquifex aeolicus SelA, a
bacterial selenocysteine synthase. Acta Crystallogr Sect F
Struct Biol Cryst Commun 2012;68:1128–33.
[22] Walter TS, Meier C, Assenberg R, Au KF, Ren J, Verma A,
et al. Lysine methylation as a routine rescue strategy for
protein crystallization. Structure 2006;14:1617–22.
[23] Otwinowski Z, Minor W. Processing of X-ray diffraction
data collected in oscillation mode. Methods Enzymol
1997;276:307–26.
[24] McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD,
Storoni LC, Read RJ. Phaser crystallographic software. J
Appl Crystallogr 2007;40:658–74.
[25] Emsley P, Cowtan K. Coot: model-building tools for
molecular graphics. Acta Crystallogr Sect D Biol Crystallogr
2004;60:2126–32.
[26] Adams PD, Pannu NS, Read RJ, Brunger AT. Cross-
validated maximum likelihood enhances crystallographic
simulated annealing refinement. Proc Natl Acad Sci U S A
1997;94:5018–23.
[27] Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW,
Echols N, et al. PHENIX: a comprehensive Python-based
system for macromolecular structure solution. Acta Crystal-
logr Sect D Biol Crystallogr 2010;66:213–21.
[28] Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T,
Inamoto E, Toyonaga H, et al. Complete set of ORF clones
of Escherichia coli ASKA library (a complete set of E. coli K-
12 ORF archive): unique resources for biological research.
DNA Res 2005;12:291–9.
[29] Datsenko KA, Wanner BL. One-step inactivation of chromo-
somal genes in Escherichia coli K-12 using PCR products.
Proc Natl Acad Sci U S A 2000;97:6640–5.
[30] Yuan J, Hohn MJ, Sherrer RL, Palioura S, Su D, Söll D. A
tRNA-dependent cysteine biosynthesis enzyme recognizes
the selenocysteine-specific tRNA in Escherichia coli. FEBS
Lett 2010;584:2857–61.
[31] Roberts E, Eargle J, Wright D, Luthey-Schulten Z. MultiSeq:
unifying sequence and structure data for evolutionary analysis.
BMC Bioinformatics 2006;7:382.
[14] Koutmos M, Kabil O, Smith JL, Banerjee R. Structural basis
for substrate activation and regulation by cystathionine beta-
synthase (CBS) domains in cystathionine β-synthase. Proc
Natl Acad Sci U S A 2010;107:20958–63.
[15] Burkhard P, Tai CH, Ristroph CM, Cook PF, Jansonius JN.
Ligand binding induces a large conformational change in O-
acetylserine sulfhydrylase from Salmonella typhimurium. J
Mol Biol 1999;291:941–53.
[32] Humphrey W, Dalke A, Schulten K. VMD: visual molecular
dynamics. J Mol Graphics 1996;14:27–8.
[33] O'Donoghue P, Luthey-Schulten Z. Evolutionary profiles
derived from the QR factorization of multiple structural
alignments gives an economy of information. J Mol Biol
2005;346:875–94.
[34] Huson DH, Bryant D. Application of phylogenetic networks in
evolutionary studies. Mol Biol Evol 2006;23:254–67.