Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase

Article abstract of DOI:10.1039/c4cc01752h

Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs' stereoselectivity. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01752h

Volume 50 Number 58 25 July 2014 Pages 7729–7906
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Yan Xu, Rey-Ting Guo et al.
Unconserved substrate-binding sites direct the stereoselectivity of
medium-chain alcohol dehydrogenase

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COMMUNICATION
Unconserved substrate-binding sites direct the
stereoselectivity of medium-chain alcohol
dehydrogenase†
Cite this: Chem. Commun., 2014,
0, 7770
5
Received 8th March 2014,
Accepted 30th April 2014
a
a
a
a
b
Shanshan Wang,‡ Yao Nie,‡ Yan Xu,* Rongzhen Zhang, Tzu-Ping Ko,
c
c
c
d
Chun-Hsiang Huang, Hsiu-Chien Chan, Rey-Ting Guo* and Rong Xiao
DOI: 10.1039/c4cc01752h
www.rsc.org/chemcomm
Structure-guided design of substrate-binding pocket inversed the
stereoselectivity of an NADH-dependent medium-chain alcohol
dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming
amino acids, especially the unconserved residues as hotspots, play critical
roles in directing MDRs’ stereoselectivity.
Optically active alcohols play a rapidly growing role as building
blocks for the synthesis of pharmaceuticals and fine chemicals.
Among them, enantiopure aryl alcohols serve as valuable inter-
mediates for preparation of antidepressants, anti-asthmatics,
cholesterol-lowering agents, adrenergic receptor agonists, and
Scheme 1 Asymmetric reduction of aryl ketones (1a–8a) to the corres-
ponding alcohols (1b–8b) by RCR using NADH as a hydrogen donor.
1
NK1 antagonists, etc. Attractive access to the alcohols involves
alcohol dehydrogenase (ADH)-mediated asymmetric reduction ketones to corresponding alcohols with excellent stereo-
6
of ketones with unparalleled chemo-, regio-, and stereoselectiv- selectivity in Prelog configuration (Scheme 1).
2
ities under benign conditions. Concerning the stereochemical
To date, only human ADH aa has the opposite stereoselectivity in
patterns of hydride transfer from NAD(P)H to ketones, ADHs structure-solved stereoselective MDRs. Thermoanaerobacter
perform either the Prelog or anti-Prelog stereoselectivity. In brockii ADH (TbADH) has a substrate-size-induced reversal of
nature, very few ADHs follow the anti-Prelog’s rule, particularly stereoselectivity (Table S1, ESI†). Moreover, the same stereo-
7
3
8
4
medium-chain ADHs (MDRs). Furthermore, the limited anti- selective enzymes bear a variety of stereopreferences towards
5
Prelog ADHs require more expensive NADPH as a cofactor.
different substrates. Thus, it was proposed that the diversity of
Therefore, anti-Prelog NADH-dependent ADHs are extremely in MDRs’ stereoselectivity would be dominated by the primary
demand. Previously, NADH-dependent (R)-specific carbonyl structure and the cavity shape in active sites of enzymes.
reductase (RCR) from Candida parapsilosis, an MDR member,
was found to catalyze asymmetric reduction of prochiral aryl controlled reactions. By contrast, stereoselectivity switch of MDRs is
rare. So far, only the NADPH-dependent ADH from T. ethanolicus was
Recent research studies have focused on the design of stereo-
9
reported to alter stereoselectivity by substituting Ala for I86 and C295, or
Thr for S39. Although it is known that MDRs’ stereoselectivity depends
a
10
School of Biotechnology and Key Laboratory of Industrial Biotechnology,
Ministry of Education, Jiangnan University, Wuxi 214122, China.
E-mail: yxu@jiangnan.edu.cn; Fax: +86-510-85864112; Tel: +86-510-85918201
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of
Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
E-mail: guo_rt@tib.cas.cn; Fax: +86-022-84861926; Tel: +86-022-84861999
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway,
NJ 08854, USA
on the relative sizes of two substituents neighboring the carbonyl group,
a puzzling question remains on the molecular basis of enzyme
stereorecognition and substrate orientation. Therefore, engineering
MDRs performing anti-Prelog stereoselectivity is still a major challenge.
The structural details and the stereoselective mechanism of RCR are yet
b
c
d
unclear, although RCR shares 31.5% sequence identity with ADH-‘A’
11
(
PDB codes: 2XAA and 3JV7). Herein, we solved the complex structures
†
Electronic supplementary information (ESI) available: Experimental section, supple-
of RCR with a cofactor and a substrate/product (Table S2, ESI†), and
performed the rational design of a Prelog NADH-dependent ADH for
inversion of stereoselectivity towards aryl ketones.
mentary tables and figures, and crystal structures deposited in the PDB under the
accession codes: 3WLE, 3WLF and 3WNQ. See DOI: 10.1039/c4cc01752h
‡
These authors contributed equally to this work.
7770 | Chem. Commun., 2014, 50, 7770--7772
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The holo-RCR with NAD revealed as a homo-tetramer, and
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+
At present, only one structure of anti-Prelog MDR, human
each subunit was composed of a cofactor-binding domain and a ADH aa, has been determined, in which the T48 and A93 are
+
7,13
catalytic domain. Between these two domains lay a NAD molecule. key residues contributing to the anti-Prelog stereoselectivity.
+
The NAD binding induced a significant conformational change Compared to isoenzyme aa, S46 in RCR with the smaller side
from an open form to a closed form of the enzyme (Fig. S1–S3, ESI†), chain than T48 lines the large pocket. Interestingly, although
12
which resembles other MDRs. In the binary complex with (R)-1b, G91 in RCR (corresponding to A93 in isoenzyme aa F93 in
the molecule was bound in the hydrophobic cavity formed by HLADH) has no side chain, the bulky side chain of W286 as an
residues L55, W116, L119, D154 and F285. Most importantly, the important residue like F93 in HLADH forms the small pocket,
OG of S46 was in hydrogen-bonding distance to the O1 of (R)-1b. The in agreement with the same Prelog stereoselectivity of HLADH
N of W286 and the OG1 of T158 interacted with the O2 of (R)-1b by (Fig. S6, ESI†). In addition, RCR had more bulky side chains at
hydrogen bonds, which determined its orientation along with the F285 and W286, located at the entrance of and inside of the
hydrophobic interactions (Fig. S4, ESI†). The RCR_H49A mutant small pocket, respectively, than those of other MDRs, such as
complex with 1a further confirmed its binding orientation in the I-F in HLADH, L-V in SsADH, I-V in PaADH and CbADH (Fig. S7,
substrate pocket.
ESI†). So we considered that the F285 and W286 might play
Based on the solved holo-RCR, the structure model of a ternary critical roles. It is hypothesized that non-conservative substitu-
+
complex with NAD and 1a displayed a hydrophobic substrate- tions with Ala at the positions would greatly enlarge the small
binding pocket, matching the observation that this enzyme was pocket and affect the enantiomeric specificity of RCR.
6
adapted to accept aryl ketones. The substrate-binding cavity can be
Three variants of F285A, W286A and F285A/W286A were
divided into a large and a small pocket (Fig. 1A). The large pocket is constructed (Table S3, ESI†) and examined in asymmetric
composed of S46, H49, V50, L55, C57, H65, W116, L119 and L262, reduction of aryl ketones (1a–8a). As expected, the variants
while the small pocket is formed by C44, D154, T158, F285 and indeed changed the stereoselectivity towards the substrates. It
W286. The architecture of pockets allows the phenyl ring of 1a to fit is likely that stepwise enlarged small pocket from the wide type
into the large pocket, and the hydroxymethyl group to enter the (WT) to the double-mutant F285A/W286A led to a stepwise
small pocket. Consequently, the orientation of 1a ensures that change in stereoselectivity and stereopreference towards 2a
NADH delivers its pro-R hydride to the re-face of 1a to produce and 4a–8a. Exceptionally, all three variants completely inversed the
(R)-1b, following the Prelog’s rule (Fig. S5, ESI†).
enantioselectivity towards 3a to the (S)-product with 499.9% ee.
Although every MDR has its unique substrate-binding pocket, F285A/W286A catalyzed all substrates with the opposite enantio-
and possesses versatile stereoselectivity and function, one question selectivity, exhibiting high enantioselectivity towards 3a and good
is how the amino acid composition determines the pocket shape enantioselectivity towards 1a, 4a and 6a. W286A also successfully
and controls the stereoselectivity and stereopreference. Combined inverted the enantioselectivity towards 2a–4a and 6a, and F285A
with other solved stereoselective MDR structures, we are interested lost most of the original enantioselectivity and contributed to
in the pocket-forming amino acids (Fig. S6, ESI†). The residues double-mutant F285A/W286A with the inverted enantioselectivity
equivalent to S46, H49, V50 and H65 lining the large pocket of RCR (Table 1). The ee values of the desired enantiomer products might
are relatively conserved in MDRs, with H49 and H65 nearly not be all ideal for practical utility, whereas the landmark results
invariant. However, the positions at L55, C57, W116, L119 and will serve as a starting point for further protein engineering of RCR
L262 are varied. Moreover, in the small pocket, the C44, D154 and and other related enzymes.
T158 are relatively conserved, while at the F285 and W286 are
To understand the molecular basis of the altered stereoselectivity
optional (Fig. 1B). These varied residues might lead to the different and stereopreference, we performed automated docking experi-
substrate-binding pockets and stereopreferences of MDRs.
ments. Models of variants with the substrate 1a bound to the active
sites showed that a reasonable docking conformation in F285A
yielded the (R)-enantiomer, while two opposite conformations in
W286A produced (R)- and (S)-enantiomers (re-face orientation still
had slightly higher affinity than that of si-face), which was qualita-
tively consistent with the maintained enantioselectivity of F285A and
a
Table 1 Enantioselectivity for RCR WT and variants towards aryl ketones
Substrate
WT
F285A
W286A
77.3 (R)
35.1 (S)
499.9 (S)
57.4 (R)
23.3 (S)
52.4 (R)
8.1 (R)
F285A/W286A
1a
2a
3a
4a
5a
499.9 (R)
499.9 (R)
499.9 (R)
97.3 (S)
73.8 (S)
499.9 (S)
80.6 (S)
499.9 (R)
59.4 (R)
499.9 (S)
35.1 (S)
24.4 (S)
71.9 (S)
48.9 (S)
71.7 (S)
73.3 (S)
47.5 (S)
499.9 (S)
85.6 (R)
15.3 (R)
76.1 (R)
63.6 (R)
21.6 (R)
Fig. 1 The substrate-binding pocket of RCR. (A) Surface of the large pocket of
the phenyl-binding site (blue) and the small pocket of the hydroxymethyl-
+
6a
binding site (green). NAD (yellow) and 1a (cyan) are shown as thick sticks. (B)
7
8
a
a
The pocket-forming residues are shown as thin sticks. The varied residues
499.9 (S)
64.6 (S)
(
magentas and orange) and the conserved residues (blue and green) in the
a
large and the small pocket. The zinc ion (gray) is shown as a sphere.
Determination of enantiomeric excess by HPLC analysis (Fig. S8–S15, ESI).
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7770--7772 | 7771

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difference in pocket shape and substrate orientation, and thus direct
the diversity of reaction stereoselectivity and stereopreference. It is
probable that MDRs, even members with unknown structure, can be
engineered by varying these sites to create more stereocomplemen-
tary features for production of desired optically active alcohols for
the synthesis of pharmaceuticals and fine chemicals.
This work was financially supported by the National
Key Basic Research and Development Program of China
(2011CB710800), the National Hi-Tech Research and Develop-
ment Program of China (2011AA02A209, 2011AA02A210), the
National Natural Science Foundation of China (21336009,
21376107), the Program of Introducing Talents of Discipline
to Universities (111-2-06), the High-End Foreign Experts
Recruitment Program (GDW20133200113), and the Project
Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions. We thank the National
Synchrotron Radiation Research Center of Taiwan for beam-
Fig. 2 Comparison of docking the substrates 1a (A), 3a (B) and 4a (C) in
the active sites of (a) F285A (blue), (b) W286A (orange and green, the time allocation and data-collection assistance.
affinity of orange conformation higher than that of the green conforma-
tion) and (c) F285A/W286A (cyan) with WT (magentas).
Notes and references
1
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1
2
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3
4
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5
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9
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1
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7792–7798.
7772 | Chem. Commun., 2014, 50, 7770--7772
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