Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Article abstract of DOI:10.1016/j.bbrc.2014.03.030

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 A. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

Full text of DOI:10.1016/j.bbrc.2014.03.030

Biochemical and Biophysical Research Communications 446 (2014) 911–915
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Structural basis for high substrate-binding affinity and enantioselectivity
of 3-quinuclidinone reductase AtQR
a
a
b,c
a
c
Feng Hou , Takuya Miyakawa , Michihiko Kataoka , Daijiro Takeshita , Shoko Kumashiro ,
d
b,c
a
c,e
a,
⇑
Atsuko Uzura , Nobuyuki Urano , Koji Nagata , Sakayu Shimizu , Masaru Tanokura
a
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531, Japan
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
Research & Development Center, Nagase & Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241, Japan
b
c
d
e
Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 March 2014
Available online 15 March 2014
(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantiose-
lectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-
dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and
showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reduc-
tase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three
NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit
Keywords:
Crystal structure
Short-chain dehydrogenases/reductases
Quinuclidinone
Agrobacterium tumefaciens
of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the a7 helix. This
helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity.
AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydro-
phobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue
on the
a7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a
direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation
and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indis-
pensable residue for the activity.
Ó 2014 Elsevier Inc. All rights reserved.
1
. Introduction
Alzheimer’s disease, the most common form of dementia, is a
synthesize (R)-3-quinuclidinol by the enantioselective reduction
of 3-quinuclidinone. The enzyme was first isolated from Rhodotor-
ula rubra (RrQR). RrQR requires NADPH as a cofactor and converts
3-quinuclidinone to (R)-3-quinuclidinol with a >99.9% enantio-
meric excess [10]. Recently, a novel 3-quinuclidinone reductase
was isolated from Agrobacterium tumefaciens (AtQR), which also
produces the (R)-3-quinuclidinol with a >99.9% enantiomeric ex-
cess. Based on the amino acid sequence identity (43% identical
to RrQR), AtQR belongs to the short-chain dehydrogenases/reduc-
tases (SDR) family like RrQR. However, AtQR requires only NADH
as a cofactor. In addition, AtQR shows a 100 times higher sub-
strate-binding affinity than RrQR [11], which indicates that AtQR
is a more practical enzyme for application in the biosynthesis of
(R)-3-quinuclidinol.
The structural analysis of AtQR would help us to elucidate the
structural basis for its high substrate affinity and enantioselectivi-
ty, and contribute to the exploration on the general understanding
of higher substrate affinity and enantioselectivity in enzymes.
Here, we report the crystal structure of AtQR and its structural
progressive degenerative illness characterized by continuing loss
of memory, impaired cognition, and change in and loss of person-
ality. Talsaclidine (WAL 2014 FU) is a nonselective muscarinic ace-
tylcholine receptor agonist that acts as a full agonist of the M
subtype, and as a partial agonist of the M and M subtypes [1–
]. The compound is intended for muscarinic substitution therapy
1
2
3
3
in patients with Alzheimer’s disease. Talsaclidine appears to be a
promising new candidate because even high doses cause only sal-
ivation as a side effect in healthy volunteers [4].
(
R)-3-quinuclidinol is used as a chiral building block applicable
to the synthesis of various pharmaceuticals including talsaclidine
5–9]. Chiral compounds are chemically synthesized by several
chemical reactions, whereas 3-quinuclidinone reductase can easily
[
⇑
Corresponding author. Fax: +81 3 5841 8023.
E-mail address: amtanok@mail.ecc.u-tokyo.ac.jp (M. Tanokura).
http://dx.doi.org/10.1016/j.bbrc.2014.03.030
006-291X/Ó 2014 Elsevier Inc. All rights reserved.
0

9
12
F. Hou et al. / Biochemical and Biophysical Research Communications 446 (2014) 911–915
difference from RrQR. Structural and mutational analyses were
conducted to explain the high substrate-binding affinity and
enantioselectivity.
Table 1
Data collection and refinement statistics.
AtQR
Data collection
Space group
P2
1
2
. Materials and methods
Cell dimensions
a, b, c (Å)
62.0, 126.4, 62.0
90.0, 110.5, 90.0
4
2.1. Protein expression, purification and crystallization
a
, b, c (°)
Molecules/asymmetric unit
The recombinant AtQR was overexpressed as a fusion protein
Wavelength (Å)
1.0000
20.0–1.72 (1.76–1.72)
4.3 (16.0)
21.7 (5.1)
97.9 (80.7)
3.6 (1.8)
a
with an N-terminal His
6
tag in Eschrichia coli Rosetta(DE3) (Nova-
Resolution (Å)
a,b
R
sym
gen) and purified by sequential chromatographies with Ni-NTA
superflow (QIAGEN), DEAE Sepharose (GE Healthcare) and Hiload
6/60 superdex 200 prep grade (GE Healthcare) as previously de-
<I/
r
(I)>a
a
Completeness
Redundancy
2
a
scribed [12].
Refinement
Resolution (Å)
19.8–1.72
15.0/18.3
257
c
R
work/Rfree (%)
2.2. X-ray diffraction data collection and structure determination
No. Protein residues in monomers
No. non-protein atoms
Ligand
Solvent
For the crystallization of AtQR with its cofactor, 2 mM NADH
132
545
20
was added to the protein solution. AtQR was crystallized under a
reservoir solution condition of 0.2 M ammonium acetate, 0.1 M
HEPES pH 8.5 and 24% (w/v) PEG3350 [12]. The X-ray diffraction
data were collected on the BL-5A beamline at the Photon Factory
Others
Mean B-factor (Å )
2
14.9
Root mean square deviations
Bond lengths (Å)
Bond angles (Å)
0.021
2.025
(
Tsukuba, Japan) using an ADSC Quantum 315r CCD detector. The
diffraction data were indexed, integrated and scaled using XDS
13]. The crystal belonged to the primitive monoclinic space group
P2 , with unit-cell parameters of a = 62.0 Å, b = 126.4 Å, c = 62.0 Å
and b = 110.5°.
Ramachandran plot
Most favorable (%)
Allowed (%)
[
97.2
1.9
0.9
1
Disallowed (%)
a
Values in parentheses are for the highest resolution shell.
b
2.3. Structure determination and refinement
R
sym
=
R
hkl [(R
i
|I
i
– hIi|)/
i i i
R |I |], where I is the ith intensity measurement of
reflection hkl, including symmetry-related reflections, and ‹I› is its average.
c
The Rfree factor was calculated using 5% of the reflections omitted from the
We chose meso-2,3-butanediol dehydrogenase from Klebsiella
refinement.
pneumonia (PDB: 1GEG [14]) as a molecular replacement search
model, because it also belongs to the SDR family and its sequence
is the most similar to AtQR (43% identical) among the proteins
whose structures are available. Molecular replacement was carried
out using Phaser [15]. The initial model of AtQR was refined using
the programs REFMAC5 and Coot [16,17]. The refinement statistics
are summarized in Table 1.
decrease in absorbance. One unit of AtQR activity is calculated as
the amount which oxidizes 1 lmol of NADH in 1 min.
3. Results and discussion
3.1. Overall structure of AtQR
2.4. Computational analysis
AtQR was overexpressed, purified and crystallized in the pres-
Molecular graphics were depicted using the program PyMOL
http://www.pymol.org). The solvent-accessible surface areas were
ence of the cofactor NADH. The crystal structure of AtQR was
determined at 1.72 Å resolution and refined to a crystallographic
R-factor of 0.150 and R-free of 0.183 (Table 1). Four protomers in
an asymmetric unit formed a tetramer (Fig. 1A), in which only
three protomers (chain A, B and C) formed complexes with NADH
and the fourth protomer (chain D) showed an NADH-free structure.
(
calculated using AREAIMOL [18]. Hydrogen bonds with the NADH
+
were calculated and depicted using LIGPLOT [19].
2
.5. Site-directed mutagenesis
The root-mean-square deviations (r.m.s.d.) of chain A with chain B
The expression plasmids for the mutants of AtQR were prepared
and chain C are 0.199 and 0.275 Å for the Ca atoms, respectively.
using site-directed mutagenesis kit (Agilent Technologies). PCR
was carried out with the expression plasmid for the wild-type
AtQR, pET-28a(+)-AtQR, as template. Digestion with the restriction
enzyme DpnI was carried out at 37 °C for 1 h to remove the parent
DNA, and the mutations were confirmed by DNA sequencing.
The close r.m.s.d. values indicate that these protomers do not adopt
large conformation changes while bound to NADH. In order to ex-
plain the structural feature of the NADH-bound form, we chose the
2
chain C protomer because of its lowest overall B-factor (13.0 Å )
among the three protomers (Table 2). As most other SDR family
proteins, the protomer consists of two domains (Fig. 1B), a large
domain and a small domain. The large domain is comprised of a
typical Rossmann fold with a seven-stranded parallel b-sheet
2.6. Enzyme activity measurements
Reductase activity was determined at 37 °C by measuring
the decrease rate of NADH. The standard assay solution (2.5 ml)
(b1–b7), which is flanked by seven
four 310-helices ( 1– 4) on both sides. The small domain contains
two helices ( 7 and 8) and protrudes from the globular body of
a-helices (a1–a6 and a9) and
g g
contains 618 M 3-quinuclidinone-HCl, 320 M NADH, 200 mM
l
l
a
a
potassium phosphate buffer (pH 7.0) and the appropriate amount
of the purified enzyme. After incubation for 2 min, the reaction
was started by adding the enzyme. The absorbance at 340 nm
was measured at 37 °C. The activity can be calculated from the
the large domain. An NADH molecule was clearly observed in the
cleft of the large domain (Fig. 1B and C). Meanwhile, the NADH-
free protomer (chain D) can also be superimposed on chain A with
an r.m.s.d. of 0.474 Å for the Ca atoms. Chain D shows almost the

F. Hou et al. / Biochemical and Biophysical Research Communications 446 (2014) 911–915
913
Fig. 1. (A) Quaternary structure of AtQR. The assembly of AtQR is a homotetramer, in which each protomer of AtQR is colored in green (chain A), cyan (chain B), magenta
chain C) or yellow (chain D). (B) Ribbon diagram of AtQR protomer bound to NADH (chain C). The ribbon is shown from blue (N-terminus) to red (C-terminus). NADH is
(
shown as a stick model. (C) 2F
o
ꢀ F
c
electron density map of NADH is shown by the blue mesh that is contoured at 1.0r.
3
.2. NADH-binding mechanism and NADH-induced conformational
Table 2
changes
NADH-dependent 3-quinuclidinone reductase average B-
factor.
NADH was located in a deep cleft of the large domain and bound
B-factor (Å2)
at the C-terminal end of the b-sheet. The adenosine moiety of
NADH was bound to a pocket formed by Gly16, Leu41, Val62,
Asp63, Val64, Thr65, Ala91, Val93 and Val113 (Fig. 2A). The ribose
ring of the adenosine moiety formed direct and water-mediated
hydrogen bonds with the side-chain atoms of Asp40 and Lys19,
respectively (Fig. 2A). In particular, Asp40 occupied the space for
Chain A
Chain B
Chain C
Residues 190–200 in chain C
Residues 190–200 in chain D
14.6
14.3
13.0
15.9
45.8
0
the additional phosphate moiety at the 2 -position of the ribose
ring of the adenosine moiety. The residue is speculated to be the
critical to the NADH selectivity of AtQR. The D40A mutant showed
decreased enzymatic activity, which indicates that Asp40 plays an
important role in binding to NADH (Table 3). The nicotinamide ring
same backbone but with a slight shift of the a7 helix. The signifi-
cance of this helix and the influence from the shift will be dis-
cussed later.
The homotetramer of AtQR is stabilized by three protein–protein
interfaces (A–B, A–C, and A–D). The A–B interface consists of the two
Table 3
NADH-dependent 3-quinuclidinone reductase activity.
long helices: a9 and b7 and several loops. The buried area of the A–B
2
interface is 3476 Å per dimer, which is equivalent to 15.7% of the
Enzyme
Activity
total surface area of the protomer. The A–D interface consists of
Wild type
D40A
100%
ND
2
a5 and
a6 with a buried surface area of 3103 Å per dimer (13.6%
of the total surface area). In addition to these major interprotomer
R196A
E197A
Y216V
76%
ND
31%
contacts, protomers A and C also make minor contacts with a buried
2
surface area of 148 Å (0.7% of the total surface area). These three
interfaces are highly conserved among the SDR family proteins.
ND, not detected.
+
Fig. 2. (A) Schematic diagram for the non-covalent interactions between AtQR and NADH. The interactions were identified with the program LIGPLOT [18]. (B) Superposition
of structures between the ligand-free form and NADH-bound form. Chains C (NADH-bound) and D (ligand-free) are colored in yellow and purple-blue, respectively. (C)
Interactions between NADH and the residues on the a7 helix. Residues interacting with NADH are represented by a stick model. Water molecule is shown by a sphere model.

9
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F. Hou et al. / Biochemical and Biophysical Research Communications 446 (2014) 911–915
contacted Ile21, Thr141, Ala142, Ser143, Phe186, Gly187 and
Met193 through hydrophobic interactions, and the amide group
formed hydrogen bonds with Val189 and Thr191 (Fig. 2A). The ri-
bose ring connecting to the nicotinamide ring formed hydrogen
bonds with the side-chain atoms of the residues Asn90, Tyr156
and Lys160 and hydrophobic interactions with Thr141 and Gly92
structure formation [20,21]. Secondly, the a7 helix in the small do-
main of AtQR is not conserved in RrQR (Fig. 3A). Thirdly, the en-
trances of substrate-binding pockets are different between AtQR
and RrQR (Fig. 3B). In the structure of AtQR, the additional
occupies the space corresponding to the entrance of RrQR. How-
ever, the loop between the 2-helix and 6-helix of AtQR is short-
a7 helix
g
a
(Fig. 2A). The pyrophosphate moiety interacted with the main-
er than that of RrQR, which enable the substrate to reach its active
chain atoms of Ile21, Thr191 and Arg196 (Fig. 2A).
site.
Superimposition of chain C and chain D reveals that a small
shift occurred on the
average B-factors suggest that NADH binding also contributes to
the stabilization of the 7 helix (residue 190–200) (Table 2). Some
residues around the 7 helix interacted with NADH and contrib-
uted to the stabilization of the helix. Thr191 formed a hydrogen
bond with the pyrophosphate moiety of NADH as mentioned above
a
7 helix upon NADH binding (Fig. 2B). The
3.4. Structural insight into high substrate-binding affinity
and enantioselectivity
a
a
The catalytic mechanism of the SDR family enzymes has been
well examined [22–24]. A catalytic triad of Ser-Tyr-Lys residues
is conserved in the SDR family enzymes. The tyrosine residue func-
tions as a catalytic base and abstracts a proton from the substrate.
The serine residue plays a role in the stabilization of the bound
substrate, and the lysine residue indirectly interacts with the hy-
(
Fig. 2A and C). Another residue Arg196 also interacted with the
pyrophosphate moiety (Fig. 2C). R196A caused decreased activity,
which indicates that the stabilization of the 7 helix contributes
a
0
to the catalytic activity of AtQR (Table 3). In addition, the nicotin-
amide ring formed a hydrogen bond with the side chain of Gln194
through a water molecule. The conformational change upon NADH
binding reveals that NADH not only acts as a hydride donor but
droxyl of the tyrosine residue via the 2 -hydroxyl of the nicotin-
amide ribose of the cofactor and lowers its pKa value. In the
active site of AtQR, Ser143, Tyr156 and Lys160 are spatially con-
served as the catalytic triad residues (Fig. 3C). In addition, the
asparagine residue, which is important for the stabilization of the
water molecule in the proton relay system [25], is also conserved
as Asn114 in AtQR. AtQR also possesses two distinctive hydropho-
bic residues, Leu144 and Phe188, around the active site. The corre-
sponding residues of RrQR (Ile167 and Phe212) were concluded to
be a critical structural basis in relation to the hydrophobic wall for
substrate recognition with direct hydrophobic interactions, which
leads to the (R)-specific reaction. Therefore, high enantioselective
reduction could be defined by this common structural basis of
also contributes to the stabilization of the
.3. Comparison of overall structures with RrQR
Fig. 3A shows the superimposition of the AtQR and RrQR struc-
a7 helix.
3
tures [20,21]. Although the overall structures of both enzymes are
a
primarily identical (r.m.s.d. 4.198 for 231 C atoms), there are also
three apparent differences. Firstly, AtQR does not possess the N-
terminal region of RrQR, which contributes to the quaternary
Fig. 3. (A) Superposition of AtQR and RrQR structures. Protein structures are shown by ribbon diagrams colored in pink for AtQR and aquamarine for RrQR. Circles show the
different structural elements between the two enzymes. (B) Substrate entrances of AtQR (green) and RrQR (magentas). The entrances are indicated by arrows. (C) Active sites
of AtQR (left) and RrQR (right). Catalytic-triad residues are colored in yellow. Residues forming the hydrophobic wall are highlighted by the stick-and-sphere model colored in
blue. In the structure of AtQR (left), Glu197 is colored in slate. (D) Interactions between Tyr216 and
molecule is shown by a sphere model. Hydrogen bonds are shown as dashed lines.
a7 helix. Residues involved in the interactions are shown as sticks. Water

F. Hou et al. / Biochemical and Biophysical Research Communications 446 (2014) 911–915
915
the 3-quinuclidinone reductases. In addition, AtQR also has a dis-
tinctive residue Glu197 that orients towards the substrate-binding
site (Fig. 3C). The E197A mutant showed no activity, which indi-
cates that Glu197 is related to the reductase activity as a non-cat-
alytic residue (Table 3). It is speculated that the Glu197 improves
the binding affinity of 3-quinuclidinone by forming a salt bond
with its tertiary amine. The binding could also induce 3-quinuclid-
inone to a certain orientation which is favorable for proton trans-
[4] W.S. Adamus, J.P. Leonard, W. Tröger, Phase I clinical trials with WAL, a new
muscarinic agonist for the treatment of Alzheimer’s disease, Life Sci. 56 (1995)
(
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[
[
5] M. Takeuchi, R. Naito, M. Hayakawa, Y. Okamoto, Y. Yonetoku, K. Ikeda, Y.
Isomura, Novel quinuclidine derivatives and medicinal composition thereof,
European patent EP 0801067 (1996).
6] V.A. Alabaster, Discovery & development of selective M
clinical use, Life Sci. 60 (1997) 1053–1060.
3
antagonists for
[7] J.S. Ward, L. Merritt, D.O. Calligaro, F.P. Bymaster, H.E. Shannon, C.H. Mitch, C.
Whitesitt, D. Brunsting, M.J. Sheardown, P.H. Olesen, M.D. Swedberg, L.
Jeppesen, P. Sauerberg, 1,2,5-Thiadiazole analogues of aceclidine as potent
fer. Additionally, Glu197 belongs to the
a7 helix and is not
M
1
muscarinic agonists, J. Med. Chem. 41 (1998) 379–392.
conserved in RrQR. Therefore, Glu197 may be the key residue for
enhancing the substrate-binding affinity. Tyr216 interacts with
[8] A. Leusch, W. Tröger, A. Greischel, W. Roth, Pharmacokinetics of the M -agonist
1
talsaclidine in mouse, rat, rabbit and monkey, and extrapolation to man,
Xenobiotica 30 (2000) 797–813.
Glu201 of the
a7 helix and also forms a hydrogen bond with
[
9] T. Ishihara, H. Kakuta, H. Moritani, T. Ugawa, I. Yanagisawa, Synthesis and
biological evaluation of quinuclidine derivatives incorporating phenothiazine
moieties as squalene synthase inhibitors, Chem. Pharm. Bull. (Tokyo) 52 (2004)
Glu197 through a water molecule (Fig. 3D). The Y216V mutation
causes decreased activity, which supports that the orientation of
Glu197 is important for the substrate binding (Table 3).
1204–1209.
[
10] A. Uzura, F. Nomoto, A. Sakoda, Y. Nishimoto, M. Kataoka, S. Shimizu,
Stereoselective synthesis of (R)-3-quinuclidinol through asymmetric
reduction of 3-quinuclidinone with 3-quinuclidinone reductase of
Rhodotorula rubra, Appl. Microbiol. Biotechnol. 83 (2009) 617–626.
3.5. Conclusion
[
[
11] S. Shimizu, M. Kataoka, F. Nomoto, A. Uzura, Alcohol dehydrogenase, gene
encoding the same and method for producing optically active (R)-3-
quinuclidinol using the same, Japanese Patent 2008-212144 (2008).
12] F. Hou, T. Miyakawa, D. Takeshita, M. Kataoka, A. Uzura, K. Nagata, S. Shimizu,
M. Tanokura, Expression, purification, crystallization and X-ray analysis of 3-
quinuclidinone reductase from Agrobacterium tumefaciens, Acta Crystallogr, F
Struct. Biol. Cryst. Commun. 68 (2012) 1237–1239.
We provide the crystal structure of the 3-quinoclidinone reduc-
tase AtQR in both the NADH-bound state and NADH-free state.
Structural comparison between the two states revealed that the
co-factor NADH not only acts as a proton donor, but also contrib-
utes to the stabilization of the
a7 helix that is not conserved in
[
13] K. W., Automatic processing of rotation diffraction data from crystals of
initially unknown symmetry and cell constants, J. Appl. Crystallogr. 26 (1993)
RrQR. Although the catalytic triad of the SDR family and the hydro-
phobic wall are well conserved in both AtQR and RrQR, a critical
795–800.
residue, Glu197, located at the additional
a
7 helix in AtQR, is found
[14] M. Otagiri, G. Kurisu, S. Ui, Y. Takusagawa, M. Ohkuma, T. Kudo, M. Kusunoki,
Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with
NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of
chiral substrate recognition mechanisms, J. Biochem. 129 (2001) 205–208.
[15] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J.
Read, Phaser crystallographic software, J. Appl. Crystallogr. 40 (2007) 658–674.
near the active site and confirmed to be activity-related. Therefore,
the Glu197 is considered to be the residue that is critical to the
high activity. Based on these findings, we conclude that the stabil-
ization of the
a7 helix contributes to the high activity by orienting
[
16] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of macromolecular
a critical residue Glu197 to the active site.
structures by the maximum-likelihood method, Acta Crystallogr
Crystallogr 53 (1997) 240–255.
D Biol
3.6. Accession numbers
[17] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta
Crystallogr. D Biol. Crystallogr. 60 (2004) 2126–2132.
[
18] B. Lee, F.M. Richards, The interpretation of protein structures: estimation of
static accessibility, J. Mol. Biol. 55 (1971) 379–400.
The atomic coordinate and structure factor of AtQR have been
deposited to Protein Data Bank (PDB) with accession code 3WDS.
[19] R.A. Laskowski, M.B. Swindells, LigPlot+: multiple ligand-protein interaction
diagrams for drug discovery, J. Chem. Inf. Model. 51 (2011) 2778–2786.
[
20] D. Takeshita, M. Kataoka, T. Miyakawa, K. Miyazono, S. Kumashiro, T. Nagai, N.
Urano, A. Uzura, K. Nagata, S. Shimizu, M. Tanokura, Structure basis of
stereospecific reduction by quinuclidinone reductase, AMB Express 4 (2014) 6.
21] D. Takeshita, M. Kataoka, T. Miyakawa, K. Miyazono, A. Uzura, K. Nagata, S.
Shimizu, M. Tanokura, Crystallization and preliminary X-ray analysis of the
NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra, Acta
Crystallogr. F Struct. Biol. Cryst. Commun. 65 (2009) 645–647.
Acknowledgments
[
We would like to thank the beamline staff at Photon Factory.
The synchrotron radiation experiments were performed at BL5A
in the Photon Factory, Tsukuba, Japan (Proposal No. 2008S2-001).
This research is supported by the Targeted Proteins Research Pro-
gram (TPRP) of the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT), Japan.
[
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