The catalytic promiscuity of a microbial 7α-hydroxysteroid dehydrogenase. Reduction of non-steroidal carbonyl compounds

Article abstract of DOI:10.1016/j.steroids.2011.05.001

A thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis ATCC 25285 was found to catalyze the reduction of various benzaldehyde analogues to their corresponding benzyl alcohols. The enzyme activity was dependent upon the substituent on the benzene ring of the substrates. Benzaldehydes with electron-withdrawing substituent usually showed higher activity than those with electron-donating groups. Furthermore, this enzyme was tolerant to some organic solvents. These results together with previous studies suggested that 7α-hydroxysteroid dehydrogenase from B. fragilis might play multiple functional roles in biosynthesis and metabolism of bile acids, and in the detoxification of xenobiotics containing carbonyl groups in the large intestine. In addition, its broad substrate spectrum offers great potential for finding applications not only in the synthesis of steroidal compounds of pharmaceutical importance, but also for the production of other high-value fine chemicals.

Full text of DOI:10.1016/j.steroids.2011.05.001

Steroids 76 (2011) 1136–1140
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Steroids
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The catalytic promiscuity of a microbial 7␣-hydroxysteroid dehydrogenase.
Reduction of non-steroidal carbonyl compounds
Yang Liua,b, Tong Lva,b, Jie Renb, Min Wanga, Qiaqing Wub, Dunming Zhub,∗
a Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
b National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A thermostable 7␣-hydroxysteroid dehydrogenase from Bacteroides fragilis ATCC 25285 was found to
catalyze the reduction of various benzaldehyde analogues to their corresponding benzyl alcohols. The
enzyme activity was dependent upon the substituent on the benzene ring of the substrates. Ben-
zaldehydes with electron-withdrawing substituent usually showed higher activity than those with
electron-donating groups. Furthermore, this enzyme was tolerant to some organic solvents. These results
together with previous studies suggested that 7␣-hydroxysteroid dehydrogenase from B. fragilis might
play multiple functional roles in biosynthesis and metabolism of bile acids, and in the detoxification
of xenobiotics containing carbonyl groups in the large intestine. In addition, its broad substrate spec-
trum offers great potential for finding applications not only in the synthesis of steroidal compounds of
pharmaceutical importance, but also for the production of other high-value fine chemicals.
© 2011 Elsevier Inc. All rights reserved.
Received 9 January 2011
Received in revised form 13 April 2011
Accepted 1 May 2011
Available online 10 May 2011
Keywords:
7␣-Hydroxysteroid dehydrogenase
Benzaldehydes
Bioreduction
Catalytic promiscuity
Multi-potency
1. Introduction
ketoprofen and the tobacco-specific nitrosamine NNK [6–11]. 17␤-
Hydroxysteroid dehydrogenase (17␤-HSDH) from the filamentous
Hydroxysteroid dehydrogenases, which catalyze the reversible
oxidoreduction at different positions of the natural substrates
steroid nucleus, are pyridine nucleotide-dependent oxidoreduc-
tases, belonging to aldo-keto reductases or the short-chain
dehydrogenases/reductases [1,2]. This group of enzymes play
critical roles in the biosynthesis and inactivation of all steroid
hormones. Besides being involved in steroidogenesis and steroid
metabolism, hydroxysteroid dehydrogenases have also exhib-
ited activity toward the reduction of some nonsteroidal carbonyl
compounds [3]. For example, 3␣-hydroxysteroid dehydrogenase
(3␣-HSDH) from Comamonas testosteroni mediates the oxidore-
fungus Cochliobolus lunatus reduces 3-keto groups of 4,5-dihydro
steroids, 20-keto groups, and most efficiently, 17-keto groups of
steroidal substrates. It is also active toward quinones, menadione,
p-nitrobenzaldehyde, etc. [12]. However, to the best of our knowl-
edge, there is up to now no report about the alternative funtion
of 7␣-hydroxysteroid dehydrogenase for the reduction of nons-
teroidal carbonyl compounds except our recent study [13].
The catalytic promiscuous property of hydroxysteroid dehydro-
genases [14], together with their primary function of catalyzing
the interconversion between the oxo group and hydroxyl coun-
terpart of sterically bulky steroids, suggests that they might be a
group of potentially useful enzymes for applications both in the
synthesis of steroidal compounds of pharmaceutical importance,
and for the production of other high-value fine chemicals. How-
ever, their use in organic synthesis as a biocatalyst has been largely
unexplored. They have mainly been employed for the selective
modification of bile acids and other steroid derivatives [15–19]. A
preparative-scale regio- and stereo-specific oxidation of hydroxy
groups and reduction of keto functions at C(3) of several bile
acids, catalyzed by 3␣-hydroxysteroid dehydrogenase (3␣-HSDH)
which was isolated from the cells of Pseudomonas pauctmobilis
as a crude enzyme, has been reported [20]. Recently, Monti and
co-workers reported the hydroxysteroid dehydrogenases(HSDHs)-
catalyzed one-pot enzymatic synthesis of 12-ketoursodeoxycholic
acid (3␣,7␤-dihydroxy-12-oxo-5␤-cholanoic acid), a key inter-
mediate for the synthesis of ursodeoxycholic acid, from cholic
duction at position
3 of a great variety of C19–27 steroids
[4,5]. It also accepts as substrates a wide spectrum of non-
steroidal carbonyl compounds, such as p-nitrobenzaldehyde,
p-nitroacetophenone, metyrapone and the metyrapone-based
insecticide NKI 42255 [5]. 11␤-Hydroxysteroid dehydrogenase
type 1 (11␤-HSDH1) physiologically catalyzes the interconver-
sion of receptor-active 11-hydroxy glucocorticoids (cortisol) to
their receptor-inactive 11-oxo metabolites (cortisone) [6,7]. Mean-
while, it is capable of playing the role of carbonyl reductase in the
detoxification of aldehydes, ketones and quinones, for example,
p-nitrobenzaldehyde, p-nitroacetophenone, metyrapone, oracin,
∗
Corresponding author. Tel.: +86 22 84861962; fax: +86 22 84861996.
E-mail address: zhu dm@tib.cas.cn (D. Zhu).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.05.001

Y. Liu et al. / Steroids 76 (2011) 1136–1140
1137
acid [21]. With this in mind, in our continuing project of
O
searching for synthetically useful enzymes for asymmetic ketone
reduction, a thermostable 7␣-hydroxysteroid dehydrogenase from
Bacteroides fragilis ATCC 25285 has drawn our attention [13].
This enzyme reduces sterically demanding native substrate 7-
keto-lithocholic acid to cheno-deoxycholic acid in gastrointestinal
tract and it is relatively thermostable, retaining 95% of initial
activity after 1 h at 65 ◦C [22]. Therefore, this 7␣-hydroxysteroid
dehydrogease was produced as recombinant protein by over-
expression in E. coli. Its substrate specificity and stereoselectivity
toward reduction of various non-steroidal carbonyl compounds
have been examined. In our previous paper we reported that
7␣-hydroxysteroid dehydrogenase from B. fragilis catalyzed the
reduction of aromatic and sterically demanding aliphatic ␣-
ketoesters to the corresponding ␣-hydroxyesters in essentially
optically pure form, and also demonstrated its synthetic application
in the preparation of ethyl (R)-2-hydroxy-3-methylbutyrate, ethyl
(R)-2-hydroxy-3,3-dimethylbutyrate and ethyl (R)-2-hydroxy-2-
(3,5-diflurophenyl)acetate [13]. Herein, we would like to present
that the microbial 7␣-hydroxysteroid dehydrogenase can also
reduce some aldehydes to their corresponding alcohols, further
revealing its multi-functional properties, i.e. catalytic promiscuity
[14].
Η
OH
7α−HSDH
X
X
NAD+
NADH
D-Gluconic Acid
D-Glucose
GDH
Scheme 1. Reduction of benzaldehyde analogues catalyzed by 7␣-hydroxysteroid
dehydrogenase with a cofactor recyle system of GDH and d-glucose.
7.0). The mixture was shaken for 12 h at 30 ◦C, and was then
extracted with methyl tert-butyl ether (800 ␮L). The organic
extract was dried over anhydrous sodium sulfate and sub-
jected to HPLC analysis to measure the conversion. The products
were identified by comparison with authentic samples in HPLC
analysis.
2.4. Preparative scale reduction of benzaldehyde derivatives
catalyzed by 7˛-hydroxysteroid dehydrogenase
The preparative scale reactions were performed for 4-
2. Materials and methods
(trifluoromethyl)benzaldehyde,
pentafluorobenzaldehyde,
4-acetoxybenzaldehyde and 4-cyanobenzaldehyde. The pro-
cedure was as follows (using 4-(trifluoromethyl)benzaldehyde
as an example): 4-(trifluoromethyl)benzaldehyde (0.6 mmol in
300 ␮L DMSO), glucose (540 mg), glucose dehydrogenase (11 U),
NAD (60 mg) and 7␣-hydroxysteroid dehydrogenase (31 U) were
mixed in 30 mL of potassium phosphate buffer (100 mM, pH 7.0).
The reaction mixture was stirred at 30 ◦C. TLC was used to monitor
the reaction. When the reduction was completed, the mixture was
extracted with methyl tert-butyl ether (30 mL). The organic extract
was dried over anhydrous sodium sulfate and the solvent was
removed to give the product 4-(trifluoromethyl)benzyl alcohol
(94 mg, yield 90%). The product was identified by comparing its
1H NMR data with those reported in the literature [23]. Pentaflu-
orobenzaldehyde (84 mg, yield 72%) [24]. 4-Acetoxybenzaldehyde
(50 mg, yield 51%) [25]. 4-Cyanobenzaldehyde (66 mg, yield 85%)
[26].
2.1. Materials
The HPLC analysis was performed on an Agilent 1200
high-performance liquid chromatography system with a Eclipse
XDB-C18 column (4.6 × 150 mm). A mixture of water and methanol
(v/v, 40/60) was used as eluent. All the benzaldehydes were
obtained from commercial sources. The benzyl alcohols were either
purchased from commercial sources or prepared by the reduction
of the corresponding benzaldehydes using sodium borohydride in
methanol. 7␣-Hydroxysteroid dehydrogenase and d-glucose dehy-
drogenase were prepared as previously reported. A unit of activity
(U) was defined as the enzyme that converts 1 micromolar of
NADH in 1 min. The specific activity for methyl benzoylformate was
0.32 U/mg [13].
2.2. Reduction of various benzaldehydes catalyzed by
7˛-hydroxysteroid dehydrogenase
3. Results and discussion
The reaction procedure was as follows: glucose (18 mg), glucose
dehydrogenase (0.35 U), NAD (2 mg), 7␣-hydroxysteroid dehydro-
genase (1.1 U) and benzaldehyde (0.02 mmol, dissolved in 10 ␮L
of DMSO) were mixed in 1 mL of potassium phosphate buffer
(100 mM, pH 7.0). The mixture was shaken for 12 h at 30 ◦C, and
was then extracted with methyl tert-butyl ether (800 ␮L). The
organic extract was dried over anhydrous sodium sulfate and sub-
jected to HPLC analysis to measure the conversion. The products
were identified by comparison with authentic samples in HPLC
analysis.
The 7␣-hydroxysteroid dehydrogenase from B. fragilis ATCC
25285 was prepared as recombinant enzyme according to the
method reported in the literature [13,22]. The obtained 7␣-
hydroxysteroid dehydrogenase was assayed for activity toward
various benzaldehyde analogues. The reaction was carried at 1 mL
scale and the co-factor NADH was regenerated with d-glucose
dehydrogenase and d-glucose, as shown in Scheme 1. The conver-
sion was measured by HPLC analysis, and the product was identified
by comparison with authentic sample in HPLC analysis. The results
are presented in Table 1.
2.3. 7˛-Hydroxysteroid dehydrogenase-catalyzed reduction of
4-cyanobenzaldehyde in reaction media with different co-solvents
From the results, it can be seen that 7␣-hydroxysteroid
dehydrogenase catalyzed the reduction of various benzaldehyde
derivatives, affording the corresponding alcohols. For benzalde-
hyde and its derivatives with alkyl, methoxy and dimethylamino
substituents, the coversions were low to medium, while the ben-
zaldehyde analogues with electron-withdrawing substituents such
as chloro, bromo, trifluoromethyl and cyano groups, were reduced
to the corresponding benzyl alcohols in high conversions. This
showed that the enzyme had higher activity toward benzaldehydes
with electron-withdrawing group than those with electron-
donating substituent. The stonger electron-donating group was,
4-Cyanobenzaldehyde was chosen as substrate for the reduction
in reaction media with co-solvent such as dimethyl sulfox-
ide, methanol, isopropyl alcohol, methyl tert-butyl ether, ethyl
acetate, butyl acetate and toluene. The reaction procedure was
as follows: glucose (18 mg), glucose dehydrogenase (0.35 U), NAD
(2 mg), 7␣-hydroxysteroid dehydrogenase (1.1 U) and benzalde-
hyde (0.02 mmol, dissolved in 0.1 mL of organic solvent) were
mixed in 0.9 mL of potassium phosphate buffer (100 mM, pH

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Y. Liu et al. / Steroids 76 (2011) 1136–1140
Table 1
Table 2
Reduction of various benzaldehydes catalyzed by 7␣-hydroxysteroid
7␣-HSDH-catalyzed reduction of 4-cyanobenzaldehyde in reaction media with dif-
dehydrogenase.
ferent co-solvents.
Entry Benzaldehyde
Product
Conversion (%)a
57
Solvent in buffera
Conversion (%)b
No solvent
DMSO
MeOH
i-PrOH
MTBE
Ethyl acetate
Butyl acetate
Toluene
93
90
94
92
94
79
63
85
OH
O
1
2
OH
O
49
52
OH
O
O
a
Potassium phosphate buffer (100 mM, pH 7.0) containing 10% (v/v) of organic
solvent except where indicated otherwise.
3
b
Determined by HPLC analysis.
OH
4
5
6
46
38
1
mined the activity of this enzyme. The electronic factor might be
due to the increased electron deficiency of the recipient carbonyl
group undergoing reduction, which facilitates the H− transfer from
NADH to the carbonyl group.
OH
OH
O
O
O
N
O
N
Introduction of organic solvents, which improve the solubility
of hydrophobic substrates, is increasingly used as a strategy to
overcome the difficulty in the biocatalytic reaction of the poorly
water-soluble substrates in aqueous media. Enzymes with ability
to tolerate organic solvents is thus of great interest from both scien-
tific and practical points of view. Thermostable enzymes usually are
tolerant of organic solvent to some extent. The 7␣-hydroxysteroid
dehydrogenase from B. fragilis ATCC 25285 is a relatively highly
thermostable enzyme. It would be interesting to test its tolerance of
organic solvents. As such, 4-cyanobenzaldehyde was chosen as sub-
strate to examine the activity of 7␣-HSDH in potassium phosphate
buffer with various organic solvents. The results are summarized
in Table 2. From Table 2, it can be seen that toluene, ethyl acetate,
especially, butyl acetate in reaction medium decreased the activity
of 7␣-HSDH, while dimethylsulfoxide, methanol, isopropanol and
methyl t-butyl ether had little effect on the activity of the enzyme.
The results showed that 7␣-hydroxysteroid dehydrogenase from B.
fragilis ATCC 25285 is highly tolerant of organic solvents.
OH
O
7
8
9
55
79
85
F
F
OH
O
Cl
Cl
Br
OH
O
Br
F
OH
O
F
F
F
F
10
94
87
F
The biocatalytic reduction of a few benzaldehyde derivatives
was performed at preparative scale and the product benzyl alco-
hols were isolated and characterized by comparing their 1H NMR
data with those in the literature (Table 3). 4-Acetoxybenzylalcohol
was obtained in 51% yield, while other benzyl alcohols, 4-
trifloromethylbenzyl alcohol, 2,3,4,5,6-pentafluorobenzyl alcohol
and 4-cyanobenzyl alcohol were prepared in high yields.
F
F
F
F
F
F
F
OH
O
11
12
F
F
F
O
O
OH
O
Therefore, it has been demonstrated that 7␣-hydroxysteroid
dehydrogenase from B. fragilis ATCC 25285 not only catalyze the
interconversion of hydroxyl/oxo groups of bile acids, but also cat-
alyze the reduction of nonsteroidal carbonyl compounds such as
ketones, benzaldehydes and keto-esters [13]. Some hydroxysteroid
dehydrogenases, e.g. 3␣-hydroxysteroid dehydrogenase, 11␤-
hydroxysteroid dehydrogenase type 1 and 17␤-hydroxysteroid
dehydrogenase, have showed activity toward the reduction of some
nonsteroidal carbonyl compounds. Thus it is commonly accepted
that these enzymes play important roles in the detoxification of
various xenobiotics that contain reactive carbonyl groups, besides
their primary functions in steroidogenesis and steroid metabolism
[6]. 7␣-Hydroxysteroid dehydrogenase is a common class of bile
acid hydroxysteroid dehydrogenases found in nature, having been
detected in numerous genera of bacteria [27–35] and in mam-
malian liver [36]. The Bacteroides are the predominant genera in the
large intestine [37]. Therefore, it is reasonable to believe that 7␣-
hydroxysteroid dehydrogenase also play multiple functional roles
in biosynthesis and metabolism of bile acids, and in the detoxifica-
tion of xenobiotics containing carbonyl groups.
99
O
O
OH
O
13
100
N
N
a
Determined by HPLC analysis.
the lower the conversion of the substrate was. With respect to
benzaldehydes containing fluoro, chloro and bromo group, the con-
version of 4-bromobenzaldehyde was the highest despite fluorine
is the strongest electron-withdrawing group. This might be due
to the lager size of bromine. Other benzaldehydes with relatively
large electron-withdrawing substituents were also reduced with
higher conversion. These results indicated that 7␣-hydroxysteroid
dehydrogenase showed higher activity toward benzaldehydes with
electron-withdrawing group and when the substituent’s abilities
of withdrawing electron were about the same, steric factor deter-

Y. Liu et al. / Steroids 76 (2011) 1136–1140
1139
Table 3
Preparative scale reduction of some benzaldehydes catalyzed by 7␣-hydroxysteroid dehydrogenase.
Benzaldehyde
Product
Reaction time (h)
42
Isolated yield (%)
90
OH
O
F
F
F
F
F
F
F
F
F
F
F
OH
O
42
72
F
F
F
F
F
O
O
OH
O
21
25
51
85
O
O
OH
O
N
N
4. Conclusions
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The present study has for the first time demonstrated that
7␣-hydroxysteroid dehydrogenase from B. fragilis catalyzes the
reduction of various benzaldehyde analogues to their correspond-
ing benzyl alcohols. The enzyme activity is dependent upon the
substituent on the benzene ring of the substrates. Usually, electron-
withdrawing substituent increases the enzyme activity while the
enzyme is less active toward substrates with electron-donating
groups. Moreover, this enzyme shows good tolerance of organic
solvents. These results together with previous studies [13,21,22]
have demonstrated that 7␣-hydroxysteroid dehydrogenase from B.
fragilis not only reduces the native substrate 7-keto-lithocholic acid
to cheno-deoxycholic acid, but also catalyze the reduction of nons-
teroidal carbonyl compounds, suggesting the alternative functions
of this enzyme in the detoxification of xenobiotics containing car-
bonyl groups in the large intestine. In addition, the broad substrate
spectrum of 7␣-hydroxysteroid dehydrogenase provides opportu-
nity for finding applications in the construction of chiral alcohol
building blocks for the production of high-value fine chemicals, as
well as the synthesis of steroidal compounds of pharmaceutical
importance.
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We thank the Chinese Academy of Sciences for support
from the Knowledge Innovation Program (KSCX2-YW-G-031 and
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Commission (09ZCKFSH01000), Ministry of Science and Technol-
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(2008BAI63B07) and National Key Basic Research and Development
Program (2011CB710801).
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