Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from a hyperthermophilic archaeon

Article abstract of DOI:10.1016/j.bbrc.2019.01.141

The hypothetical OCC_00372 protein from Thermococcus litoralis is a member of the ProR superfamily from hyperthermophilic archaea and exhibits unique bifunctional proline racemase/hydroxyproline 2-epimerase activity. However, the molecular mechanism of th

Full text of DOI:10.1016/j.bbrc.2019.01.141

Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications
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Crystal structure of substrate-bound bifunctional proline racemase/
hydroxyproline epimerase from a hyperthermophilic archaeon
,
,
b
,
c
,
, b
Yasunori Watanabe a b, Seiya Watanabe a
*, Yoshika Itoh b, Yasuo Watanabe a
a Department of Bioscience, Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan
b Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan
c Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The hypothetical OCC_00372 protein from Thermococcus litoralis is a member of the ProR superfamily
from hyperthermophilic archaea and exhibits unique bifunctional proline racemase/hydroxyproline 2-
epimerase activity. However, the molecular mechanism of the broad substrate specificity and extreme
thermostability of this enzyme (TlProR) remains unclear. Here we determined the crystal structure of
TlProR at 2.7 Å resolution. Of note, a substrate proline molecule, derived from expression host Escherichia
coli cells, was tightly bound in the active site of TlProR. The substrate bound structure and mutational
analyses suggested that Trp241 is involved in hydroxyproline recognition by making a hydrogen bond
between the indole group of Trp241 and the hydroxyl group of hydroxyproline. Additionally, Tyr171 may
contribute to the thermostability by making hydrogen bonds between the hydroxyl group of Tyr171 and
catalytic residues. Our structural and functional analyses provide a structural basis for understanding the
molecular mechanism of substrate specificity and thermostability of ProR superfamily proteins.
© 2019 Elsevier Inc. All rights reserved.
Received 23 January 2019
Accepted 31 January 2019
Available online xxx
Keywords:
Proline racemase
Hydroxyproline 2-epimerase
Hyperthermophilic archaeon
Thermococcus litoralis
Crystal structure
1. Introduction
Alternatively, direct biosynthesis from free L-proline to L-Hyp,
including T4LHyp and T3LHyp, has been identified in a few bacteria
and fungi that produce peptide antibiotics [12]. The catabolism of L-
Hyp by (micro)organisms has recently been investigated at the
molecular level. Among them, hydroxyproline 2-epimerase (EC
5.1.1.8; HypE) is responsible for the first step of the T4LHyp meta-
bolic pathway from bacteria and catalyzes the isomerization of
Proline racemase (EC 5.1.1.4; ProR) is a member of the pyridoxal
5’-phosphate-independent racemase family (COG3938), and cata-
lyzes the deprotonation/reprotonation of the chiral carbon (Ca) of
proline, resulting in the stereo inversion of chiral centers [1e3]. Its
catalysis is based on the same 1,1-proton transfer mechanism using
two general acidic/basic cysteine residues located on opposite faces
of the active site. In specific clostridium bacteria [4,5] and Trypa-
T4LHyp to cis-4-hydroxy-
being -ketoglutarate [13,14]. On the other hand, in mammals,
bacteria, and archaea, T3LHyp is converted to
1-pyrroline-2-
carboxylate by T3LHyp dehydratase (EC 4.2.1.77; T3LHypD), with
the final product being -proline [15e17]. HypE and T3LHypD belong
D-proline (C4DHyp), with the final product
a
nosoma species [6e10], ProR is involved in the utilization of
L-
D
proline with the production of 5-aminopentanoate (so-called
“Stickland fermentation”), and the mechanisms underlying the
escape of parasites from host immunity as a B-cell mitogen,
respectively.
L
to the COG3938 protein superfamily, and the former possesses two
equivalent cysteine residues with ProR at the active sites, con-
forming to their homologous reactions [15,18]. On the other hand,
the substrate specificities of ProR and HypE are very strict.
Although the ProR-like protein was only originally considered to
be present in mesophilic eukaryotes, bacteria, and fungi, we
recently showed that the hypothetical OCC_00372 and OCC_00387
proteins from the hyperthermophilic archaea Thermococcus litoralis
functioned as ProR (TlProR) and T3LHypD, respectively [17,19]. The
L
-Hydroxyproline (L-Hyp) has been found in specific proteins
including collagen, the cell wall of plants, and some peptide
antibiotics. In mammalian and plant systems, -proline residues are
post-translationally hydroxylated to trans-4-hydroxy- -proline
(T4LHyp) and/or trans-3-hydroxy- -proline (T3LHyp) [11].
L
L
L
former is of interest because not only
T3LHyp are significant substrates; to the best of our knowledge, this
L-proline, but alsoT4LHyp and
* Corresponding author. Department of Bioscience, Graduate School of Agricul-
ture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan.
E-mail address: irab@agr.ehime-u.ac.jp (S. Watanabe).
https://doi.org/10.1016/j.bbrc.2019.01.141
0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Watanabe et al., Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from
a hyperthermophilic archaeon, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.01.141

2
Y. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
is the first example of a bifunctional ProR/HypE. Although a site-
directed mutagenic analysis revealed several important amino
acid residues for the recognition of hydroxyproline (more hydro-
philic and bulkier than proline), it was unsuccessful for completely
eliminating HypE activity similar to a natural ProR enzyme [19]. To
gain mechanistic insights into the unique substrate specificity and
extreme thermostability, we determined the crystal structure of
TlProR. Intriguingly, a proline molecule derived from E. coli cells
was tightly bound in the active site of TlProR during purification
and crystallization. Structure-based mutational analyses showed
that Trp241 in the substrate binding site is responsible for substrate
specificity for hydroxyproline and that Tyr171 might contribute to
the thermostability by making hydrogen bonds to the side chains of
catalytic residues. The crystal structure of TlProR bound with pro-
line gives a basis for understanding the molecular mechanism of
substrate specificity and thermostability.
mixing equal volumes (0.5
TlProR in Buffer C) and reservoir solution, and equilibrated against
70 L of reservoir solution. TlProR crystals were obtained using
m
L) of protein solution (23 mg mLꢀ1
m
reservoir solution consisting of 0.1 M HEPES pH 7.0 and 30% (v/v)
Jeffamine ED-2001 pH 7.0. Because the concentration of Jeffamine
ED-2001 in the reservoir solution was sufficient as a cryoprotectant,
the crystal of TlProR was directly mounted on a nylon loop and then
flash-cooled and maintained in a stream of nitrogen gas at 100 K
during data collection. X-ray diffraction data were collected on a
MAR225HE charge-coupled device detector using beamline
BL38B1, SPring-8 (Hyogo, Japan) at a wavelength of 1.00 Å. Data
processing was performed by HKL-2000 [24]. The structure of
TlProR was determined by the molecular replacement method with
PHASER [25] in CCP4 suite [26], for which the crystal structure of
Trypanosoma cruzi proline racemase (PDB code, 1W61) was used as
a search model. Further model building was performed manually
with COOT [27], and crystallographic refinement with CNS [28] and
PHENIX [29]. Detailed data collection and processing statistics are
shown in Table 1.
2. Materials and methods
2.1. Protein expression and purification
2.3. Spectrophotometric enzyme assay
The TlProR gene from T. litoralis DSM5473 (OCC_00372) was
previously cloned into pETDuet-1 (Novagen), a plasmid vector that
encodes the addition of an N-terminal (His)6 tag on the proteins
expressed [19]. Mutations for the described amino-acid sub-
stitutions were introduced by PCR-based site-directed mutagen-
An
action mixture contained 0.05 mM 2,6-dichloroindophenol
(Cl2Ind) and purified L-ProDH (20 g) in 50 mM Tris-HCl buffer
(pH 8.0). The -proline oxidation reaction was initiated by the
addition of 1 mM TlProR (100 L) or 20 mM Tris-HCl buffer (pH 8.0)
containing 150 mM NaCl (100 L) with a final reaction volume of
L-proline oxidation assay was performed as follows. The re-
m
L
esis. To construct E. coli expression plasmids for
L-proline
m
m
dehydrogenase (L-ProDH) from Aeropyrum pernix [20,21], the gene
encoding the enzyme (APE_1267.1) was amplified by PCR from the
A. pernix genome, digested using BamHI and KpnI, and cloned into
pETDuet-1 using the same sites. To construct E. coli expression
1 mL, and then the absorbance at 600 nm was measured at 50 ꢁC
using a Shimadzu UV-1800 spectrophotometer (Shimadzu GLC Ltd.,
Tokyo, Japan).
plasmids for D-proline dehydrogenase (D-ProDH) from Pyrobac-
Racemase/epimerase activity of TlProR toward
L-proline,
ulum islandicum [22], the synthetic gene optimized with E. coli
codon usage was obtained from Eurofins Genomics, digested using
BamHI and XhoI, and cloned into pETDuet-1 using the same sites.
All of the constructs were sequenced to confirm their identities.
Target proteins with 14 additional residues (MGSSHHHHHHSQDL)
at their N-terminus, were expressed in E. coli strain BL21-
CodonPlus(DE3)-RIL cells (Novagen) cultured in Luria-Bertani (LB)
medium containing ampicillin (100 mg Lꢀ1) and chloramphenicol
(30 mg Lꢀ1) at 37 ꢁC. When the optical density of the culture at
T4LHyp, or T3LHyp was assayed at 50 ꢁC by monitoring the
reduction rate of an artificial electron acceptor in the coupling
system with dehydrogenase for proline or hydroxyproline as
described previously [19]. To measure the activity toward L-proline,
Table 1
Data collection and refinement statistics. Values in parentheses are for the highest
resolution shell.
600 nm reached ~0.8, isopropyl-b-D-thiogalactopyranoside (IPTG)
Data collection
was added to a final concentration of 0.1 mM to induce protein
expression. After the addition of IPTG, the cultures were grown at
20 ꢁC for a further 20 h in order to induce the expression of the
target proteins. The cells were harvested, resuspended in Buffer A
(50 mM sodium phosphate pH 8.0, 500 mM NaCl, and 20 mM
imidazole), disrupted by sonication, and then centrifuged to pellet
the insoluble debris. The supernatant was loaded onto a Ni-NTA
Superflow column (Qiagen) equilibrated with lysis buffer. After
washing the column with Buffer A, target proteins were eluted
using Buffer B (50 mM sodium phosphate pH 8.0, 100 mM NaCl, and
250 mM imidazole). Expression and purification of C4DHyp dehy-
drogenase (D-HypDH) was carried out as described previously [23].
L-ProDH, D-ProDH, and D-HypDH were then dialyzed against
Buffer C (20 mM Tris-HCl pH 8.0 containing 150 mM NaCl). TlProR
was further purified by size-exclusion chromatography using a
HiLoad 16/600 Superdex 200-pg column (GE Healthcare) eluted
with Buffer C. The main single-peak fractions were collected and
concentrated by ultrafiltration with Amicon Ultra-15 (Millipore).
Space group
a, b, c (Å)
Wavelength (Å)
Resolution range (Å)
Rmerge
P6322
125.38, 125.38, 140.63
1.00000
50.0e2.70 (2.75e2.70)
0.180 (1.941)
0.185 (1.993)
0.734
25.4 (2.0)
100.0 (100.0)
20.1 (19.7)
Rmeas
CC1/2 high resolution shell
I/
s(I)
Completeness (%)
Redundancy
Refinement
Resolution (Å)
No. reflections
R/Rfree
No. atoms
Protein
50.0e2.7
18,509
0.245/0.296
2562
8
29
Ligand (proline)
Water
B-factors (Å2)
Protein
58.2
58.8
47.6
Ligand (proline)
Water
2.2. Crystallization and X-ray crystallography
R.m.s. deviations
Bond lengths (Å)
Bond angles (ꢁ)
Crystallization screening was performed at 20 ꢁC using the
sitting-drop vapor-diffusion method. Each drop was formed by
0.009
1.0
Please cite this article as: Y. Watanabe et al., Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from
a hyperthermophilic archaeon, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.01.141

Y. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
10 mM
L-proline was incubated with 0.05 mM Cl2Ind, purified D-
ProDH (20
mg), and 50 nM TlProR in 1 mL of 50 mM Tris-HCl buffer
(pH 8.0). To measure the activity toward T4LHyp, 10 mM T4LHyp
was incubated with 0.25 mM p-iodonitrotetrazolium violet,
0.06 mM phenazine methosulfate, purified D-HypDH (20 mg), and
50 nM TlProR in 1 mL of 50 mM Tris-HCl buffer (pH 8.0). To measure
the activity toward T3LHyp, 10 mM T3LHyp was incubated with
0.05 mM Cl2Ind, purified D-ProDH (20 mg), and 250 nM TlProR in
1 mL of 50 mM Tris-HCl buffer (pH 8.0).
2.4. Amino acid sequence alignment
Amino acid sequences of TlProR and its homologs were analyzed
using the Clustal Omega [30] and ESPript 3.0 [31].
3. Results and discussion
3.1. Crystal structure of TlProR
In order to elucidate the substrate recognition mechanism of
TlProR, we attempted to determine the crystal structure of TlProR.
TlProR was expressed in E. coli cells, purified using (His)6-tag af-
finity chromatography and size-exclusion chromatography. We
obtained the TlProR crystals belonging to the space group P6322.
The crystal structure of TlProR was determined using the molecular
replacement method using the crystal structure of T. cruzi proline
racemase (PDB code, 1W61) as a search model and subsequently
refined to 2.7 Å resolution against the data set from a crystal of
TlProR (Table 1). The crystallographic asymmetric unit contains one
TlProR molecule, corresponding to a Matthews coefficient of
4.2 Å3Daꢀ1 and an estimated solvent content of 70.6%. The subunit
forms the
a/
b
fold comprised of four
a-helices and three antipar-
allel -sheets formed by 19
b
b-strands. (Fig. 1A). To the best of our
knowledge, this is the first structure of a bifunctional ProR/HypE
from archaea. Similar to other proline racemase superfamily pro-
teins [1,32], TlProR forms a symmetric homodimer. The C-terminal
b
19 strand contributes to dimer formation by forming an inter-
molecular antiparallel -sheet (Fig. 1A), and the gel-filtration
b
analysis indicated that TlProR exists as a dimer with a calculated
MW of 76 kDa in solution (Fig. 1B). Structural comparison between
TlProR and other ProR superfamily proteins; T. cruzi ProR (PDB
code, 1W61) and Pseudomonas protegens HypE (PDB code, 4J9X)
[32] shows the r.m.s. differences of 1.4e1.7 Å, indicating that the
structure of TlProR is similar to those of other ProR superfamily
proteins (Fig. 1C).
The dimer interface of TlProR covers 1929.1 Å2 (14% of the total
surface area of one subunit) and is more extensive than those of
T. cruzi ProR (1468.3 Å2, 10% of the total surface area of one subunit)
and P. protegens HypE (1161.9 Å2, 9% of the total surface area of one
subunit). The broad dimer interface area may contribute to the
extreme thermostability of TlProR.
Fig. 1. Crystal structure of TlProR. (A) Overall structure of a TlProR dimer with one
subunit in pink and another subunit in light brown. (B) Gel-filtration elution profile of
TlProR showing that TlProR exists as a dimer in solution. The void volume (>2000 kDa)
and molecular weight markers are indicated. (C) Superposition of TlProR (pink), T. cruzi
ProR (cyan) and P. protegens HypE (magenta). (For interpretation of the references to
colour in this figure legend, the reader is referred to the Web version of this article.)
electron acceptor, Cl2Ind. L-ProDH catalyzes the oxidation of
L-
3.2. A proline molecule is tightly bound to recombinant TlProR
proline to
D
1-pyrroline-5-carboxylate with reduction of Cl2Ind [21].
The assay is based on spectrophotometrically monitoring the
decrease of Cl2Ind that absorbs at 600 nm. In the assay, purified L-
ProDH and TlProR are incubated with Cl2Ind, and oxidation of L-
Surprisingly, clear electron density was observed in the active
site in the center of the TlProR molecule, although no substrate was
added during purification and crystallization (Fig. 2A). We assumed
proline bound to TlProR by L-ProDH will lead to reduction of Cl2Ind
i.e. decrease in the absorbance at 600 nm. As shown in Fig. 2C, in the
presence of TlProR, absorbance at 600 nm was efficiently decreased
in a time-dependent manner as compared with the negative control
without TlProR (Fig. 2C). These results indicated that the proline
molecule derived from E. coli cells is tightly bound to the purified
TlProR during purification and crystallization, and that TlProR co-
crystallized with the proline molecule.
that an L-proline derived from E. coli cells is bound to the active site
of TlProR in the crystal. The proline molecule fitted well into the
electron density map and was completely enclosed by TlProR, sug-
gesting that TlProR forms a substrate-binding closed conformation
(Fig. 2A and B). The Ca atom of proline is in close contact with the SH
groups of catalytic cysteine residues (Cys88 and Cys251). To confirm
that the proline molecule exists in purified TlProR, we performed
the L-proline oxidation assay using purified L-ProDH and an artificial
Please cite this article as: Y. Watanabe et al., Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from
a hyperthermophilic archaeon, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.01.141

4
Y. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. The proline molecule bound to TlProR. (A) Electron density map of the bound proline molecule. The simulated annealing Fo-Fc difference Fourier map was calculated by
omitting the proline molecule, and is shown with blue meshes countered at 3.0 . Distances from the C atom of the bound proline to the sulfur atoms of Cys88 and Cys251 are
indicated. (B) Cutaway representation of TlProR at the level of the active site. The bound proline molecule is shown as a stick model. (C) 100 M TlProR or buffer (indicated as
“without TlProR”) was mixed with 0.05 mM Cl2Ind and purified L-ProDH (20
g), and then the absorbance at 600 nm was monitored at 50 ꢁC for 240 s. (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of this article.)
s
a
m
m
3.3. Substrate recognition mechanism of TlProR
Cys226 in P. protegens HypE are close to the hydroxyl oxygen atom
of the bound T4LHyp (Fig. 3C). These observations suggested that
these residues are responsible for the substrate specificity. Actually,
a previous study showed that L221H and W241F mutations in
TlProR resulted in a shift in substrate specificity toward proline and
4-hydroxyproline, respectively [19]. In the TlProR structure, the
indole nitrogen atom of Trp241 is relatively close to the C-4 atom of
the bound proline, suggesting that Trp241 makes a hydrogen bond
with the hydroxyl oxygen atom of 4-hydroxyproline and thus
contributes to the substrate specificity toward 4-hydroxyproline
(Fig. 3A). On the other hand, the hydroxyl oxygen atom of Tyr171
in TlProR is relatively close to the C-3 atom of the bound proline,
suggesting that Tyr171 contributes to the substrate specificity to-
ward 3-hydroxyproline (Fig. 3A).
The bound proline molecule is entirely buried in the mostly
hydrophobic active site of TlProR, as shown in Fig. 3A. In the active
site, the side chain carboxylic oxygen atom of Asp247 is within the
distance that allows the formation of hydrogen bonds with the
pyrrolidine nitrogen atom of the bound proline, and the imidazolyl
nitrogen atom of His90 and the hydroxyl oxygen atom of Thr253
are within the distance that allows the formation of hydrogen
bonds with the carboxylate group of the bound proline. The hy-
drophobic pyrrolidine ring is in contact with hydrophobic side
chains of Phe62, Leu85, Tyr171, Leu221, and Trp241 in the active site
(Fig. 3A). These interactions of TlProR and the substrate are partially
similar to those of ProR superfamily proteins (e.g. T. cruzi ProR and
P. protegens HypE; Fig. 3B and C). Structural comparison among
these enzymes showed that Trp241 in TlProR is substituted to
Phe290 in T. cruzi ProR and that Leu221 and Trp241 in TlProR are
substituted to His208 and Cys226 in P. protegens HypE, respectively.
The side chain of Phe290 in T. cruzi ProR is in contact with the
hydrophobic pyrrole ring of the bound inhibitor pyrrole-2-
carboxylic acid (PYC) (Fig. 3B). The side chains of His208 and
3.4. Mutational analyses of the substrate binding site of TlProR
To further investigate the roles of Tyr171 and Trp241 in the
substrate specificity of TlProR, we introduced mutations to Tyr171
and Trp241, and measured the activities for
L-proline, T4LHyp and
T3LHyp (Fig. 4A and B). The W241F mutation slightly increased the
Fig. 3. Structural comparison. Close-up view of the substrate binding sites of (A) TlProR, (B) T. cruzi ProR and (C) P. protegens HypE. Distances between the indole nitrogen atom of
Trp241 in TlProR and the C-4 atom of the bound proline and between the hydroxyl oxygen atom of Tyr171 in TlProR and the C-3 atom of the bound proline are indicated.
Please cite this article as: Y. Watanabe et al., Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from
a hyperthermophilic archaeon, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.01.141

Y. Watanabe et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Accession number
The coordinates and structural factors of TlProR have been
deposited under the Protein Data Bank accession code: 6J7C.
Conflicts of interest
The authors declare no conflicts of interest associated with this
manuscript.
Acknowledgments
We would like to thank the staff at beamline BL38B1 of SPring-8,
Japan for data collection support. This work was supported by JSPS
KAKENHI Grant 16K07297 (to S.W.) and the Sumitomo Foundation
(Basic Science Research Projects) (to S.W.).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.01.141.
References
Fig. 4. Site-directed mutagenesis of Tyr171 and Trp241 in TlProR. (A) Purified TlProR
mutants were analyzed by SDS-PAGE followed by CBB staining. The arrowhead in-
dicates purified TlProR. (B) Measurement of racemase/epimerase activities of TlProR
mutants toward L-proline (left panel), T4LHyp (center panel), and T3LHyp (right panel)
by spectrophotometric enzyme assay (see details in Materials and methods). The ac-
tivity of wild-type TlProR was set to 100%. Values are mean SEM (n ¼ 3). (C) Close
view of Tyr171 in TlProR. Broken lines designate possible hydrogen bonds.
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epimerase activities with T4LHyp and T3LHyp (Fig. 4B), consistent
with the previous mutational analyses [19]. On the other hand, the
W241C mutation abolished both the racemase and epimerase ac-
tivities, suggesting that the bulky aromatic residue at the Trp241
position in TlProR is essential for both the racemase and epimerase
activities of TlProR. However, P. protegens HypE has a conserved
cysteine residue (Cys226) at the Trp241 position in TlProR (Fig. 3C
and Fig. S1). A proline residue next to Cys226 in P. protegens HypE is
also conserved in the HypE subfamily and, therefore, might
contribute to the proper formation of the substrate binding site of
P. protegens HypE. Unexpectedly, the Y171F mutation also abolished
both the racemase and epimerase activities (Fig. 4B), although
Tyr171 in TlProR is substituted to phenylalanine in the ProR sub-
family and HypE subfamily (Fig. S1). The hydroxyl oxygen atom of
Tyr171 is within the distance that allows the formation of hydrogen
bonds with the hydroxyl oxygen atom of Thr253 and the sulfur
atom of catalytic Cys251 (Fig. 4C). Given that Tyr171 is conserved in
the bifunctional ProR/HypE subfamily from hyperthermophilic
archaea but not in the ProR subfamily and HypE subfamily (Fig. S1),
Tyr171 in TlProR may contribute to the thermostability, rather than
substrate specificity, by the formation of hydrogen bonds with
catalytic residues. However, Tyr171 is also conserved in the
T3LHypD subfamily (Fig. S1), implying that the tyrosine residue in
T3LHypD has a possible role not only in thermostability, but also in
substrate recognition for T3LHyp.
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Please cite this article as: Y. Watanabe et al., Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from
a hyperthermophilic archaeon, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.01.141

6
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Please cite this article as: Y. Watanabe et al., Crystal structure of substrate-bound bifunctional proline racemase/hydroxyproline epimerase from
a hyperthermophilic archaeon, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.01.141