Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones

Article abstract of DOI:10.1039/c0ob00938e

The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potential. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes.

Full text of DOI:10.1039/c0ob00938e

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PAPER
Novel anti-Prelog stereospecific carbonyl reductases from Candida
parapsilosis for asymmetric reduction of prochiral ketones†
Yao Nie,a,b Rong Xiao,a Yan Xu*b,c and Gaetano T. Montelione*a
Received 27th October 2010, Accepted 21st February 2011
DOI: 10.1039/c0ob00938e
The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies
for the replacement of chemical routes due to its environmentally benign reaction conditions and
unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl
reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene
cluster identified in Candida parapsilosis that consists of four open reading frames including three
putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh).
These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities
for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the
coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel
anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone
derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards
ethyl 4-chloro-3-oxobutyrate, a b-ketoester with important pharmaceutical potential. Our study also
showed that genomic mining is a powerful tool for the discovery of new enzymes.
yeasts,5 plants,6 and tissues from several mammalian species.7
Introduction
However, despite the diversity of stereospecific oxidoreductases,8
their range of applications remains modest. This situation may
be attributed to several perceived limitations including the stere-
ospecificity and availability of the enzymes. In addition, research
into the molecular mechanisms of oxidoreductases is still in its
infancy. So far, most enzymes catalyzing asymmetric reductions
generally follow the Prelog’s rule in terms of stereochemical
outcomes,9 while enzymes with anti-Prelog stereospecificity, such
as Pseudomonas sp. ADH and Lactobacillus kefir ADH, are
still limited among the carbonyl reductases classified to four
stereochemical patterns concerning hydride transfer from reduced
cofactor (NAD(P)H) to ketone.10 In particular, the precise mech-
anism of enzymatic anti-Prelog stereo-preference in asymmetric
reduction is not yet fully understood.
Although most oxidoreductases possessing anti-Prelog selec-
tivity have been identified in bacteria, such as Geotrichum sp.,11
Lactobacillus brevis,12 Lactobacillus kefir,13 and Pseudomonas
sp.,14 Candida yeast species are attractive as sources of highly-
stereospecific oxidoreductases.15 In a previous study, an NADPH-
dependent alcohol dehydrogenase of anti-Prelog type has been
discovered from C. parapsilosis that catalyzes asymmetric reduc-
tion of 2-hydroxyacetophenone into (S)-1-phenyl-1,2-ethanediol
(PED),16,17 a versatile chiral building block for the synthesis of
pharmaceuticals, agrochemicals, and liquid crystals. PED is also
a precursor for the production of chiral biphosphines and a chiral
initiator for stereoselective polymerization.18
The NAD(P)H-dependent carbonyl reductases catalyze reduction
of a variety of endogenous and xenobiotic carbonyl compounds,
including biologically and pharmacologically active substrates.1
There is considerable interest in the use of carbonyl reduc-
tases in the pharmaceutical and fine chemicals industries for
the production of chiral alcohols, important building blocks
for the synthesis of chirally pure pharmaceutical agents.2 For
the production of chiral auxiliaries from their corresponding
prochiral ketones, carbonyl reductases have inherent advantages
over chemo-catalysts in terms of their highly chemo-, enantio-,
and regioselectivities. These features make stereospecific carbonyl
reductases very interesting from both scientific and industrial
perspectives.3
Stereospecific carbonyl reductases are ubiquitous in nature and
have been characterized from diverse sources including bacteria,4
aCenter for Advanced Biotechnology and Medicine, Department of Molec-
ular Biology and Biochemistry, Rutgers University, Piscataway, New Jer-
sey 08854, USA. E-mail: guy@cabm.rutgers.edu; Fax: +1-732-235-5633;
Tel: +1-732-235-5321
bSchool of Biotechnology and Key laboratory of Industrial Biotechnology,
Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China
cState Key Laboratory of Food Science and Technology, Jiangnan University,
Wuxi, Jiangsu 214122, China. E-mail: yxu@jiangnan.edu.cn; Fax: +86-
510-85918201; Tel: +86-510-85864112
† Electronic supplementary information (ESI) available: Experimental
details including docking settings and analytical methods for chiral alcohol
products, and 1H-NMR spectrum of 1-phenyl-1,2-ethanediol. See DOI:
10.1039/c0ob00938e
As a result of recent advances in genomics, proteomics,
and bioinformatics, the area of biocatalysis has developed
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significantly, and the availability of genome sequences from large
numbers of microorganisms allows scientists to discover novel
enzymes with potential applications.19 The genome sequence of
C. parapsilosis has been completed recently by the Wellcome
Trust Sanger Institute Pathogen Genomics group (http://www.
sanger.ac.uk/sequencing/Candida/parapsilosis/), which pro-
vided us with an opportunity to look into the genome of C.
parapsilosis in extensive detail. We have identified three open read-
ing frames (ORFs) in the 960-kb contig005802 of C. parapsilosis
coding for putative stereospecific carbonyl reductase genes scr1,
scr2, and scr3, respectively. These ORFs have been cloned and
expressed, and the encoded proteins were purified to homogeneity
and their functions were confirmed as stereospecific carbonyl
reductases (SCR1, SCR2, and SCR3), with anti-Prelog selec-
tivity that converts 2-hydroxyacetophenone to (S)-PED. These
oxidoreductases have unique specificities that are useful for fine
biochemical synthesis.
Fig. 1 Map of contig005802 of Candida parapsilosis genome including
the four open reading frames, scr1, scr2, scr3, and cpadh.
the C. parapsilosis genome. The scr1, scr2, and scr3 genes comprise
846, 840, and 840 bp, encoding polypeptides of 281, 279, and
279 amino acid residues with the calculated molecular masses of
30 061, 29 993, and 30 097 Da, respectively, and thus there are
no introns found in these ORFs. Multiple sequence alignment of
these four ORFs (Fig. 2) revealed high sequence identity between
CPADH and SCR1 (68%), SCR2 (88%), and SCR3 (84%). From
the amino acid sequence and secondary structure prediction,
the three putative enzymes exhibit a classic a/b Rossmann-fold
structure, the cofactor-binding motif, Gly43-X-X-X-Gly47-X-Gly49
in SCR1 and Gly41-X-X-X-Gly45-X-Gly47 in SCR2 and SCR3, and
the catalytic triad, Ser174-Tyr189-Lys193 in SCR1 and Ser172-Tyr187
Lys191 in SCR2 and SCR3.20
-
Results and discussion
To gain insights into the proposed mechanism of cofactor
binding, structure models of SCRs were obtained by homology
modeling based on the X-ray structure of CPADH (PDB code:
3ctm). Because of the high degree of sequence identity, it can be
expected that these four proteins including CPADH and SCRs
are very similar in overall conformation, except for some loop
regions due to amino-acid sequence difference. Thus, the cofactor-
binding domain is expected to fold into the classic Rossmann-fold
structure, a seven-strand parallel b-sheet flanked on both sides
Identification of putative stereospecific carbonyl
reductases-encoding genes
Bioinformatic analysis based on sequence-similarity with
CPADH, a known stereospecific alcohol dehydrogenase,17 in the
C. parapsilosis genome revealed additional homologous ORFs,
named here as scr1, scr2, and scr3 coding for putative stereospecific
carbonyl reductases (SCRs). As shown in Fig. 1, these three ORFs,
as well as the cpadh gene, are located in the 960-kb contig005802 of
Fig. 2 Amino acid sequence alignment of CPADH (GenBank accession number DQ675534), SCR1 (GenBank accession number FJ939565), SCR2
(GenBank accession number FJ939563), and SCR3 (GenBank accession number FJ939564) from C. parapsilosis. Gaps in the aligned sequences are
indicated by dashes. Identical amino acid residues are enclosed in boxes.
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by a-helices.21 Based on the highly conserved cofactor-binding
domain, putative enzyme-cofactor docking was performed with
NADPH and NADH, respectively (Fig. 3). In both lowest-energy
conformational ensembles, structure comparison with different
cofactors suggested that the steric conformation in the cofactor-
binding domain is more favorable to NADPH.
binding phosphate group in NADPH is replaced by weak basic
residue His,24 indicating that the cofactor dependence of the
putative carbonyl reductases might be not rigidly restricted to
NADPH and these enzymes still show somewhat low activity with
NADH. These highly-conserved characteristic sequence motifs
suggested that the SCRs belong to the cP2 subfamily of the
classical SDR superfamily, one of the three NADPH-dependent
subfamilies.24
Cloning, expression, and purification of SCRs
The sequence and structure information involving the overall
protein structure analysis and the conserved SDR structural
characteristics revealed that the identified genes and cpadh show
the absence of introns and the encoded proteins are all not
glycoproteins. We then attempted to express these genes in the
Escherichia coli system. From the nucleotide sequence of ORF, the
scr1, scr2, and scr3 were amplified by PCR from genomic DNA
of C. parapsilosis CCTCC M203011, and the PCR products were
inserted into pET21c vector by ligation-independent cloning to
construct the recombinant plasmids. These three plasmids, pET21-
SCR1, pET21-SCR2, and pET21-SCR3, were then transformed
into expression host E. coli BL21(DE3) pMgK cells, and recombi-
nant SCR1, SCR2, and SCR3 were produced in E. coli as fusion
proteins containing a C-terminal His6 tag. All three recombinant
enzymes were expressed at very high levels. Of them, SCR1 and
SCR3 were expressed as soluble forms at yields of 50 mg L-1 broth
and 46 mg L-1 broth, respectively, while SCR2 has relatively low
solubility with a yield of 5 mg L-1 broth (Fig. 4).
Fig. 3 Stereoview of the overall model structure of enzyme–cofactor
docking with NADPH (a) and NADH (b), respectively, illustrated by
the PyMOL program.
Additionally the positively charged residue His (His70 in SCR1,
His68 in SCR2 and SCR3) is expected to play a role in the affinity
of the enzyme for NADPH, rather than NADH, by forming a salt-
bridge with the phosphate moiety at the 2¢-position of AMP.22
According to the catalytic properties and primary structure
information, stereospecific oxidoreductases including alcohol de-
hydrogenases and carbonyl reductases are mainly classified into
three different groups: the zinc-dependent alcohol dehydrogenase,
the short-chain dehydrogenase/reductase (SDR), and the aldo-
keto reductase (AKR).8 All four proteins share sequence motifs
characteristic of the SDR superfamily, including the cofactor-
binding motif Gly-x-x-x-Gly-x-Gly (x denotes any amino acid)
and the catalytic triad of Ser-Tyr-Lys, and further the extended
tetrad of Asn-Ser-Tyr-Lys observed in the majority of SDRs.23 In
addition, the SCRs also have the conserved sequence motifs of
secondary structure elements and key positions for assignment
of coenzyme specificity of the cP2 subfamily in classic SDRs,
except that the conserved basic residue Lys/Arg responsible for
Fig. 4 Analysis of the overexpression of SCR1, SCR2, and SCR3. The
proteins were separated on a 12% SDS-polyacrylamide gel and stained
with Coomassie Brilliant Blue G-250. Lane 1, total protein for SCR1;
Lane 2, soluble fraction for SCR1; Lane 3, total protein for SCR2; Lane 4,
soluble fraction for SCR2; Lane 5, total protein for SCR3; Lane 6, soluble
fraction for SCR3; Lane 7, molecular mass standard.
The three recombinant enzymes were purified to homogeneity
as judged by Coomassie Brilliant Blue staining of SDS-PAGE
(Fig. 5) by Ni affinity purification followed by gel filtration
chromatography. The relative molecular masses of the SCR1 and
SCR3 were estimated to be 124.6 kDa and 123.4 kDa by analytical
gel filtration and static light scattering using the same low salt
buffer,25,26 but SCR2 was detected as aggregated form. Since the
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Fig. 5 SDS-PAGE analysis of purified enzymes. The purified proteins
were resolved by SDS-PAGE on a 12% polyacrylamide gel and stained
with Coomassie Brilliant Blue G-250. Lane 1, molecular mass standard;
Lane 2, purified SCR1; Lane 3, purified SCR2; Lane 4, purified SCR3.
Fig. 6 pH dependence of SCR1, SCR2, and SCR3 catalyzing 2-hydrox-
yacetophenone reduction. The enzyme activities of SCR1 (ꢀ), SCR2 (᭡),
and SCR3 ( ) were measured in 0.1 M acetate buffer (pH 4.0 to 6.0) or
᭹
0.1 M sodium phosphate buffer (pH 6.0 to 8.0) or 0.1 M Tris-HCl buffer
(pH 8.0 to 8.5) with 2-hydroxyacetophenone as the substrate and NADPH
as the cofactor. Maximal enzyme activity observed was set as 100% relative
activity for each enzyme.
relative molecular mass of the monomer of the recombinant
enzymes should be around 30 kDa based on their amino acid
composition, these results suggested that both SCR1 and SCR3
have tetrameric structures.
Table 1 Activities and kinetic parameters for reduction of 2-
hydroxyacetophenone (2-HAP) by SCR1, SCR2, SCR3, and CPADH,
using NADPH as cofactor
Catalytic properties of recombinant SCRs
Because SCR1, SCR2, and SCR3 showed high homology
to CPADH, which exhibits catalytic activity towards 2-
hydroxyacetophenone,17 the enzymatic activities of these three
SCRs were investigated for reduction of 2-hydroxyacetophenone.
Under the assay conditions, SCR1 gave the highest specific activity
of 5.16 U mg-1, and SCR3 had catalytic activity of 4.23 U mg-1,
while SCR2 had a lower specific activity of 1.55 U mg-1.
In addition, corresponding to the results from the cofactor-
binding model and secondary structure prediction and analysis,
the SCRs all displayed catalytic activity with NADPH as the
coenzyme, but very low activities with NADH, indicating that
these three enzymes, like CPADH,17 are all NADPH-dependent
oxidoreductases.
Since environmental pH value can have an influence on the
stereochemistry of enzymatic reactions,27 the effect of the reaction
pH on the activities of SCRs catalyzing 2-hydroxyacetophenone
reduction was also investigated. All three enzymes exhibited
the highest activity at pHs ranging from 5.0 to 6.0 (Fig. 6).
Subsequently, the enzymes were evaluated under their individ-
ual optimal pH, and apparent kinetic parameters were further
measured by double reciprocal Lineweaver–Burk plots at vari-
ous 2-hydroxyacetophenone concentrations with fixed NADPH
concentrations. As shown in Table 1, for reduction of 2-
hydroxyacetophenone, these three enzymes exhibited different
kinetic parameters.
Specific activity /
pHa U mg-1
V
max/mmol
Enzyme
Km /mM min-1 mg-1
K
cat/s-1
SCR1
SCR2
SCR3
5.0
5.5
6.0
5.16
1.55
4.23
2.70
9.83
4.81
4.68
5.83
42.0
8.83
32.3
18.3
21.04
4.41
16.20
9.18
CPADH 4.5
a Activity assay was carried out at the optimum pH for each enzyme
carbonyl reductases towards 2-hydroxyacetophenone. Of them,
however, SCR2 is not as efficient as the other two enzymes, cor-
responding to its lower activity. Like the homologous anti-Prelog
specific alcohol dehydrogenase, CPADH, these data demonstrate
that the analogous SCR1, SCR2, and SCR3 enzymes are anti-
Prelog-type stereospecific carbonyl reductases.17,28
Oxidoreductases perform a wide variety of asymmetric re-
ductions, differing in stereospecificity and substrate specificity,
and have been used for producing optically active alcohols
from various prochiral ketones, ketoacids, and ketoesters. The
SCRs catalyze (S)-specific reduction of 2-hydroxyacetophenone,
an anti-Prelog type reaction.28 Therefore, these new enzymes
complement the stereospecific oxidoreductases described to date
for catalysis of the reduction of prochiral carbonyl compounds
to the corresponding optically pure alcohols with anti-Prelog
stereopreference.
In addition, the finding of stereospecific carbonyl re-
ductases capable of catalyzing (S)-specific reduction of 2-
hydroxyacetophenone from the same host would give us a
profound knowledge of the reaction mechanism of C. parapsilosis
whole-cell mediated stereoinversion, which involves two steps, the
oxidation step of (R)-PED to the intermediate (2-hydroxyaceto-
phenone) and the reduction step of the intermediate to (S)-PED.16
The deracemization of racemic PED to the (S)-enantiomer by
Stereoselectivity to prochiral carbonyl group
Using 2-hydroxyacetophenone as the substrate, optically pure
1-phenyl-1,2-ethanediol (PED) as (S)-enantiomer (>99% e.e.)
was produced by each of SCR1, SCR2, and SCR3, respectively
(Fig. 7). These three enzymes are capable of catalyzing asymmetric
reduction of prochiral carbonyl compounds and are all (S)-specific
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alcohol oxidase and a single carbonyl reductase (Scheme 1).17,29
In addition, the contribution of newly discovered SCRs to the
whole-cell mediated PED stereoinversion can be further proved
by comparing the kinetic parameters such as the maximum initial
reaction rates and Michaelis constants between the two steps
involved in the one-pot redox reaction, in which the maximum
initial reaction rate of reduction is almost 10 times higher than
the oxidation reaction.30 Thus, the reduction step catalyzed by
CPADH and SCRs would drive the whole reaction process to the
product formation and the oxidation step is the restrictive step in
the sequential two-step reaction.
Scheme 1 One-pot concurrent tandem stereoinversion of 1-phenyl-1,2-
ethanediol by Candida parapsilosis involving CPADH and SCRs.
Substrate specificity
Since the three new enzymes showed distinct (S)-specific carbonyl-
reducing activity, we then further examined the substrate speci-
ficity of these enzymes towards various carbonyl compounds
including aryl ketones, aliphatic ketones, a- and b-ketoesters. As
shown in Table 2, on the one hand, the enzymes all exhibited
higher catalytic activity towards ketoesters than towards alkyl
and aromatic ketones, while compared with the other two en-
zymes, SCR2 generally showed lower activities towards keto sub-
strates, corresponding to the observation in activity and kinetics
evaluation of these enzymes catalyzing 2-hydroxyacetophenone
reduction; on the other hand, the enzymes exhibited diversity
in specificity towards aryl ketones and ketoesters, respectively.
For both substituted acetophenone and 2-hydroxyacetophenone
derivatives, bearing chloro or methyl at various positions of the
phenyl ring, the ortho substituted derivatives were poor substrates
for these enzymes, indicating that the substitution at ortho position
might have a steric effect on the hydrogen attack from electron
donator NADPH to the carbonyl group and therefore significantly
influence the reactivity of the enzymes. However, SCR3 was more
specific to p-Cl-2-hydroxyacetophenone, while SCR1 had higher
activity towards m-Cl-2-hydroxyacetophenone. Concerning the
aryl ketones, despite its low activities towards the substrates, SCR2
was more active towards acetophenone and its derivatives than
towards 2-hydroxyacetophenone, which would be a trend observed
different from SCR1 and SCR3. For ketoesters, the enzymes exhib-
ited high activity towards those with small groups, but compared
with SCR3, SCR1 was more active towards bulky ketoesters with
Fig. 7 Asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to
1-phenyl-1,2-ethanediol (PED) enantiomer by SCR1, SCR2, and SCR3,
respectively. (a) Standard sample of (R)-PED. (b) Standard sample of
(S)-PED. (c) SCR1 catalyzed asymmetric reduction of 2-HAP. (d) SCR2
catalyzed asymmetric reduction of 2-HAP. (e) SCR3 catalyzed asymmetric
reduction of 2-HAP.
C. parapsilosis whole cells should be considered as a one-pot
concurrent tandem process involving at least four stereoselective
alcohol dehydrogenases and carbonyl reductases, not just a single
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Table 2 Substrate specificity and stereoselectivity of SCR1, SCR2, and SCR3 towards various carbonyl compounds
Substrate
R1
Relative activitya/%
R2
SCR1
SCR2
SCR3
Product configuration
Aryl ketones
1
2
3
4
5
6
7
8
H
H
CH3
1.27
2.84
0.60
14.61
2.96
—
6.31
10.90
1.96
—
1.65
0.05
1.63
1.88
0.88
2.02
—
3.36
0.78
1.74
2.21
2.97
—
0.32
0.16
—
—
0.42
0.57
0.51
1.21
1.68
3.87
2.57
3.08
0.19
5.91
18.49
—
2.08
0.81
0.93
—
0.12
2.50
7.29
0.36
2.33
6.54
—
R
S
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
CH3
CH3
CH3
CH3
CH3
2¢-Cl
S
3¢-Cl
S
S
4¢-Cl
4¢-OCH3
H
—
R
R
R
—
R
R
R
R
R
R
—
H
H
H
2¢-CH3
3¢-CH3
4¢-CH3
2¢-Cl
3¢-Cl
4¢-Br
9
10
11
12
13
14
15
16
17
CH3
CH3
4¢-OCH3
Aliphatic ketones
—
18
19
20
21
22
23
CH3
CH3
CH3
CH3
CH3
CH2CH3
a-Ketoesters
CH2CH3
0.93
0.65
0.86
0.15
1.39
0.37
—
—
0.83
0.52
—
—
0.91
0.10
0.40
0.28
0.52
0.82
—
—
R
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
CH(CH3)2
R
R
—
24
25
26
27
CH3
Phenyl
CH3
Phenyl
b-Ketoesters
CH3
CH3
CH2CH3
CH2CH3
67.12
32.93
65.32
19.15
18.92
5.76
19.89
2.78
74.12
3.82
80.72
0.19
R
S
R
S
28
29
30
31
32
33
34
35
36
CF3
CH3
CH2CH3
4-Fluorophenyl
CH3
CH2CH3
Chloromethyl
Phenyl
CH2CH3
CH3
CH3
29.51
29.31
9.44
14.12
45.43
8.79
100.00
7.01
4.44
15.24
9.88
7.82
5.62
8.17
7.16
17.58
—
11.86
4.96
5.05
S
R
R
S
R
R
S
S
S
CH3
3.89
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
21.31
20.85
53.20
0.22
3,4-Dimethoxyphenyl
—
0.47
a The enzyme activities of SCRs towards various substrates and the configuration of produced alcohols were measured as described in the text. Maximal
enzyme activity observed was set as 100% relative activity for the enzymes towards various substrates.
phenyl ring and generally showed higher activities towards b-
ketoesters. For stereoselective reduction of carbonyl compounds
with different chemical structures, these three enzymes followed
the anti-Prelog rule for their behaviors in catalyzing asymmetric
reduction of 2-hydroxyacetophenone, although chiral alcohol
enantiomers were produced in different absolute configurations,
affording aryl diols, phenylhydroxyl esters, ethyl 4-trifluoro-3-
hydroxybutyrate, and ethyl 4-chloro-3-hydroxybutyrate in (S)-
configuration, whereas (R)-antipode for others due to the switch
in CIP priority.
Among the tested b-ketoesters, additionally, SCR1 produced
the highest activity towards ethyl 4-chloro-3-oxobutyrate, an
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important chiral building block. The enzyme–cofactor–substrate
docking model, as shown in Fig. 8, revealed that, in the lowest-
energy conformational ensemble, the substrate-binding domain is
favorable to this kind of substrate, and the carbonyl carbon is in
a position proximal to the C4 atom of the nicotinamide ring of
NADPH. These results suggested that, for enzymatic reactions,
the enzyme structure generally has a significant influence on
the enzyme activity and affinity towards substrates. The steric
conformation of the active site in the catalytic domain determines
the substrate recognition, binding, and orientation in the enzyme.31
other chemicals used in this work were of analytical grade and
commercially available.
Cloning and expression of genes encoding stereospecific reductases
The genes encoding SCR1, SCR2, and SCR3 were amplified
by polymerase chain reaction from C. parapsilosis genomic
DNA. PCR-amplified DNA products were purified by QI-
Aquick PCR Purification Kit (QIAGEN, USA) and inserted
into pET21c expression vector (Novagen, USA) by ligation-
independent cloning (LIC) using In-Fusion PCR Cloning Kit
(Clontech, USA) for construction of recombinant plasmids. The
infusion reaction mixtures were used to transform E. coli XL-10
gold cells. The plasmids isolated from these transformants were
verified by DNA sequence analysis using BigDye Terminator cycle
sequencing kit and an ABI PRISM 310 Genetic Analyzer (Applied
Biosystems, USA). The plasmids with the correct inserts, pET21-
SCR1, pET21-SCR2, and pET21-SCR3, were transformed into
E. coli BL21(DE3) pMgK competent cells for the production of
SCRs. These plasmids provide SCRs with a six-His tag fused at
the C-terminus.
E. coli BL21 (DE3) pMgK transformants were cultivated at
◦
37 C in Luria broth (LB) medium in the presence of ampicillin
(100 mg mL-1) and kanamycin (50 mg mL-1). When the optical
density of the culture at 600 nm reached 0.6, the temperature
was changed to 17 ◦C and isopropyl-b-D-thiogalactopyranoside
(IPTG) was added to the culture to give a final concentration
of 1 mM for induction of gene expression. After an additional
incubation of 20 h at 17 ◦C, cells harvested by centrifugation were
disrupted by sonication, and expressions of the recombinant pro-
teins were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). For expression of SCR2, expression
conditions involving a lower concentration of IPTG (0.1 mM) and
a shorter induction time (12 h) were adopted, besides the above
standard expression conditions.
Fig. 8 Stereoview of the model structure of SCR1–NADPH–ethyl
4-chloro-3-oxobutyrate (COBE) docking, illustrated by the PyMOL
program.
It is worth noting that, as shown above, SCR1 catalyzes the
reduction of a broad spectrum of ketones including aryl ketones,
aliphatic ketones, and a- and b-ketoesters, and shows a partic-
ular substrate specificity towards ethyl 4-chloro-3-oxobutyrate, a
precursor for the synthesis of ethyl 4-chloro-3-hydroxybutyrate
which is an important pharmaceutical intermediate.32 Therefore,
the newly discovered stereospecific carbonyl reductases should be
useful enzymes with potential applications.
Purification of recombinant enzymes
Experimental
The cells were suspended in binding buffer (20 mM Tris-HCl,
pH7.5, 0.3 M NaCl, 40 mM imidazole, 1 ¥ protease inhibitors,
1 mM Tris (2-carboxyethyl) phosphine (TCEP)) and disrupted
on ice by sonication. The supernatant of the cell lysate was
Materials
C. parapsilosis strain CCTCC M203011 was obtained from the
China Center for Type Culture Collection (CCTCC, Wuhan,
China). E. coli XL-10 gold cells were used for gene cloning
and plasmid preparation, and E. coli BL21 (DE3) pMgK
competent cells, a rare codon-enhanced strain, were used for
gene expression. High-fidelity PCR kit including DNA poly-
merase was purchased from FINNZYMES (Finland). The
plasmid pET21c was obtained from Novagen (USA). (R)-
and (S)-1-phenyl-1,2-ethanediol, all the aliphatic ketones and
ketone esters, aryl ketones including acetophenone and its
derivatives, 2-hydroxyacetophenone, propiophenone, butyrophe-
none, valerophenone, hexanophenone, and coenzymes including
NAD(P)H and NAD(P)+ were purchased from Sigma-Aldrich
(USA). All other 2-hydroxyacetophenone derivatives including
o-Cl-2-hydroxyacetophenone, m-Cl-2-hydroxyacetophenone, p-
Cl-2-hydroxyacetophenone, and p-CH3O-2-hydroxyacetophenone
were prepared using the method described by Itsuno.33 All
◦
collected by centrifugation at 26 000 ¥ g for 40 min at 4 C and
purified by an AKTAxpress system using HisTrap HP affinity
column followed by Superdex 75 gel filtration column (GE
Healthcare, USA), and the purified fractions were exchanged
into low salt buffer (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl,
0.02% NaN3, 5 mM D,L-dithiothreitol).25,26 The final recombinant
enzymes were purified with an apparent homogeneity on sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
with 12% polyacrylamide gels. Their molecular masses were mea-
sured by matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometry (Applied Biosystems, USA),
and their oligomerization states were determined by analytical
gel filtration using Agilent 1200 series HPLC system followed
by static light scattering (Wyatt Technology, USA). These final
preparations of purified SCRs were used in all of the experiments
in this study.
4076 | Org. Biomol. Chem., 2011, 9, 4070–4078
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Enzyme assays
with AutoDock Vina 1.0.34 A docking algorithm that took account
of ligand flexibility but kept the protein rigid was employed.
Docking runs were carried out using the standard parameters of
the program for interactive growing and subsequent scoring (for
settings of grid box dimensions and centers see the ESI†).
Carbonyl reductase activity was measured by a continuous spec-
trophotometric assay using 2-hydroxyacetophenone as a substrate.
One unit of enzyme activity was defined as the amount of enzyme
catalyzing the oxidation of 1 mmol NAD(P)H per min under the as-
say conditions. The standard assay mixture for the enzyme activity
comprised of 0.1 M potassium phosphate buffer (pH 6.5), 0.3 mM
NAD(P)H, 0.7 mM 2-hydroxyacetophenone and the appropriate
enzyme in a total volume of 100 mL. The decrease in the amount
of the coenzyme was measured spectrophotometrically at 340 nm
(extinction coefficient [e] = 6.22 mM-1 cm-1). Protein concentration
was determined using Bradford reagents (Bio-Rad) with bovine
serum albumin as a standard. The pH dependence of the enzyme
activity was determined over a pH range of 4.0 to 8.5 using the
following buffers: 0.1 M acetate (pH 4.0 to 6.0), 0.1 M sodium
phosphate (pH 6.0 to 8.0) and 0.1 M Tris-HCl (pH 8.0 to 8.5).
The substrate specificity of the SCRs was investigated under the
same conditions as described above. Various carbonyl compounds
including aryl ketones, aliphatic ketones, a- and b-ketoesters were
used as the substrates with the cofactor of NADPH.
Nucleotide sequence accession number
The nucleotide sequence for the stereospecific carbonyl reductase
genes scr1, scr2, and scr3 have been deposited in the GenBank
database under accession numbers FJ939565, FJ939563, and
FJ939564, respectively.
Conclusion
In this work, we described a new gene cluster of enantioselective
oxidoreductases with unusual stereospecificity in C. parapsilosis.
We confirmed that these genes encode three unique stereospecific
carbonyl reductases through cloning, expression, purification,
and characterization of the corresponding gene products. We
verified the enantiomer configuration of the enzymatic products
from asymmetric reduction of prochiral carbonyl groups using
multiple substrates. SCR1, SCR2, and SCR3, like CPADH, all
exhibit a novel anti-Prelog stereospecificity in reducing prochiral
carbonyl groups such as forming (S)-1-phenyl-1,2-ethanediol from
the corresponding ketone substrate, 2-hydroxyacetophenone. The
enzymes are, however, distinct in their catalytic properties, includ-
ing their pH dependency and substrate specificity spectrum. The
genes encoding SCR2, SCR3, CPADH, and SCR1 are sequential
in the cluster (Fig. 1) with intervals of several hundred base pairs.
Further studies are necessary to understand the metabolic roles of
these homologues and potential synergies among them.
The discovery of novel stereospecific carbonyl reductases of
anti-Prelog selectivity further demonstrates the diversity of stere-
ospecific oxidoreductases in microorganisms. Such enzymes pro-
vide a basis for elucidating the molecular mechanism of enzyme-
mediated asymmetric reactions involving stereo-recognition be-
tween protein and chiral molecule, and the mechanism of electron
transfer between functional groups of chiral molecule and key
amino acid residues in the enzyme. Apart from their unique
value for study of the mechanism of stereospecific oxidoreduction
reaction, these novel carbonyl reductases with anti-Prelog stereo-
preference discovered by genomic mining have much potential to
generate chiral alcohols useful as intermediates in fine chemical
synthesis.
Asymmetric reduction and stereoselectivity assay
Asymmetric reduction of 2-hydroxyacetophenone by the purified
enzymes was carried out at 30 ◦C for 6 h with shaking in a reaction
mixture comprising 0.1 M potassium phosphate buffer (pH 6.5),
1 g L-1 2-hydroxyacetophenone, NADPH (7 mM) and 0.5 mg of the
purified enzyme in a total volume of 0.5 mL. The reaction products
were extracted with ethyl acetate and the organic layer was used
for analysis. The optical purity of the reaction products were
analyzed by HPLC using a Chiralcel OB–H column (4.6 ¥ 250 mm,
Daicel Chemical Ind., Ltd., Japan). Enantiomers were eluted with
hexane and 2-propanol (9 : 1) at a flow rate of 0.5 mL min-1. The
effluent was monitored at 215 nm, and the areas under each peak
were integrated.16 To further confirm the product of 1-phenyl-
1,2-ethanediol but not byproducts of the reduction, the reaction
product was analyzed by 1H-NMR (for assay details see the ESI†).
Asymmetric reductions of various carbonyl compounds, in-
cluding aryl ketones, aliphatic ketones, a- and b-ketoesters, by
the enzymes were carried out at 30 ◦C for 6 h with shaking
in a reaction mixture comprising 0.1 M potassium phosphate
buffer (pH 6.5), NADPH (7 mM) and 0.5 mg of the purified
enzyme in a total volume of 0.5 mL. The reaction products were
extracted with ethyl acetate or hexane and the organic layer was
used for analysis. The reaction products were analyzed by chiral
HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB–H
column (4.6 mm ¥ 250 mm; Daicel Chemical Ind. Ltd., Japan)
or chiral GC (7890A, Agilent, USA) equipped with FID detector
and Chrompack Chirasil-Dex CB chiral capillary column (25 m ¥
0.25 mm; Varian, USA) (for assay details see the ESI†).
Acknowledgements
We thank the Wellcome Trust Sanger Institute Pathogen
Genomics group for Candida parapsilosis sequencing database
(http://www.sanger.ac.uk/sequencing/Candida/parapsilosis/).
We thank Dr R. Zhang for useful discussions and comments on
the manuscript.
Homology modeling, alignment, and flexible docking
The crystal structure of CPADH (PDB code: 3ctm) was
used. The homology modeling of SCRs was carried out by
HOMA (Homology Modeling Automatically) (http://www-
nmr.cabm.rutgers.edu/HOMA/). The structure alignment of
CPADH and SCRs was performed with the Deep View Swiss-Pdb
Viewer 4.0 program. All docking calculations were accomplished
This work was supported by the National Institutes of General
Medical Science Protein Structure Initiative program, grant U54
GM074958 (to G.T.M), the National Natural Science Foundation
of China (NSFC) (No. 30800017), the National Key Basic
Research and Development Program of China (973 Program)
(No. 2009CB724706 and 2011CB710802), and the Program of
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