J. Biol. Macromol., 4(1) 33-38 (2004)
ArticleMolecular and enzymatic properties of 7α-hydroxysteroid dehydrogenase from Pseudomonas sp.
B-0831

Shigeru Ueda1, Masayuki Oda2, 3, Shigeyuki Imamura1, and Masatake Ohnishi2
1

Dept. Diagnostics Research & Development, Div. Fine Chemical & Diagnostics, Asahi Kasei Pharma
Corporation, 632-1, Mifuku, Ohito-cho, Shizuoka 410-2321, Japan,
2
Dept. Cellular Macromolecule Chemistry, Graduate School of Agriculture, The Prefecture University of
Kyoto, 1-5, Shimogamo Nakaragi-cho, Sakyo-ku, Kyoto 606-8522, Japan.
Received January 12, 2004; Accepted January 28, 2004
A novel 7α-hydroxysteroid dehydrogenase (7α-HSD) was purified from Pseudomonas sp. B-0831.
The molecular weight of the purified enzyme was 25 k on SDS-PAGE and 108 k on gel filtration
analysis, suggesting that the enzyme exists as a tetramer of an identical subunit, similar to those of
7α-HSD from E. coli HB101 and Eubacterium sp. VPI 12708. 7α-HSD from P. sp. B-0831 showed
high NAD+-dependence and was able to catalyze the oxido-reduction of 7α-hydroxy bile acids
including glycine and taurine conjugates. The Km value for cholic acid was determined to be 0.25 mM,
which is similar to that for chenodeoxycholic acid and is about four times smaller than those for the
conjugates of cholic acid. The kcat values for the conjugates were determined to be about 60-70% ofthat for free cholic acid. In addition to NAD+, 7α-HSD from P. sp. B-0831 can utilize thio-NAD+ to thesame extent.
Keywords: 7α-hydroxysteroid dehydrogenase, nucleotide cofactor, Pseudomonas sp. B-0831, purification,
steady-state kinetics, substrate specificity
Introduction
Many members of the human intestinal
microflora can modify the glycine and taurine
conjugates of the primary bile acids, such as cholicacid and chenodeoxycholic acid. Microbial biotransformations of bile acids include hydrolysis of
the conjugate amide bond, 7-dehydroxylation, andthe dehydrogenation of the hydroxyl groups at
positions 3, 7, and 12 of the steroid skeleton. The
major reaction of physiological significance is the
7α-dehydroxylation of cholic acid and chenodeoxycholic acid, producing deoxycholic acid andlithocholic acid, respectively [1]. 7α-Hydroxysteroid dehydrogenase (7α-HSD) (EC 1.1.1.159),
catalyzing the dehydrogenation of a hydroxyl
group at position 7 of the steroid skeleton of bile
acids, was found in numerous bacteria and mammalian liver [2-10]. The enzymes elicited by thesebacteria have alkaline pH optima for bile acid oxidation. The two 7α-HSDs, the NAD+-dependent
3

To whom all correspondence should be addressed.
Tel & Fax: +81-75-703-5673,
E-mail: oda@kpu.ac.jp

enzyme from E. coli HB101 and the NADP+dependent enzyme from Eubacterium sp. VPI
12708 were purified to homogeneity andextensively characterized [10-13].
We previously reported that a bacterium,
Pseudomonas sp. B-0831, grown on cholic acidproduces dual nucleotide cofactor-specific 3αhydroxysteroid dehydrogenase (3α-HSD) [14].
The substrate specificity and the nucleotide
cofactor dependency of the 3α-HSD have been
analyzed [15, 16]. Besides, the bacterium wasfound to produce another enzyme with an amountsmaller than the 3α-HSD. In the present study, we
purified this enzyme to electrophoretic homogeneity and characterized its molecular and enzymatic
properties. This enzyme was revealed to be 7αHSD and has unique properties in comparison withother 7α-HSDs.
The abbreviations used are: 7α-HSD, 7α-hydroxysteroid dehydrogenase; 3α-HSD, 3α-hydroxysteroid dehydrogenase; APAD+, 3-acetylpyridine
adenine dinucleotide; PCMB, p-chloromercuribenzoate; SDR, short-chain dehydrogenase/reductase.

Molecular and enzymatic properties of 7α-HSD

Microorganism and cultivation
Pseudomonas sp. B-0831, isolated from soil in
Nerima Ward of Tokyo and classified in the genus
Pseudomonas, was grown in a 20-liter medium of
1% peptone, 1% yeast extract, 1% meat extract,
0.1% K2HPO4, and 0.05% cholic acid adjusted to
pH 7.5 at 37°C for 20 h under aerobic conditions
using a 30-liter jar fermentor.
Enzyme assay
The assays were carried out as described
previously [15]. The substrate concentrations forthe determination of the relative catalytic activity
and the steady-state kinetic constants were 1.0 mM
and 2.5 mM, respectively. One unit of the enzyme
activity was defined as the amount catalyzing theconsumption of 1 µmol of cholic acid per min.
Purification
The cells were harvested by centrifugation at
5,000 × g for 30 min and washed with 20 mM
Tris-HCl buffer, pH 8.0. The washed cells weresuspended in 2 liter of the same buffer containing
0.025% (w/v) lysozyme and 0.1 mM EDTA, andthe suspension was incubated for 30 min at 37°C.
After the mixture was centrifuged to remove celldebris and to obtain the cell-free extract, theresultant supernatant was fractionated withammonium sulfate. The precipitation occurring at
45% saturation was collected by centrifugation at
8,000 × g for 30 min and dialyzed against 10 mM
Tris-HCl buffer, pH 8.0. The dialyzed enzyme
solution was loaded onto a DEAE-Sepharose(Amersham Biosciences) column (2.5 x 10 cm)
and eluted with a 300 ml linear KCl gradient from0 to 0.5 M at a flow rate of 1 ml/min. After the
enzyme fractions were dialyzed against 10 mM
KH2PO4-NaOH buffer, pH 7.5, the enzyme
solution was loaded onto a Red-Sepharose(Amersham Biosciences) column (1.2 x 10 cm)

and eluted with a 100 ml linear KCl gradient from0 to 0.5 M at a flow rate of 0.5 ml/min. The eluted
7α-HSD fractions were concentrated with amembrane filter apparatus equipped with an
XM-50 (Amicon) and dialyzed against 10 mM
KH2PO4-NaOH buffer, pH 7.5.
Analytical methods
SDS-PAGE was performed by PhastSystem(Amersham Biosciences) using PhastGel Gradient8-25. Proteins in gels were stained by Coomassie
brilliant blue R-250. The isoelectric point (pI) of
the enzyme was estimated by the isoelectric
focusing method with a pH 3-10 carrier ampholyte[17]. The protein concentration was determined by
the method of Lowry with bovine serum albuminas the standard [18]. Gel filtration was performed
on a Sephadex G-150 column (1.5 x 85 cm)
equilibrated with a buffer containing 50 mM
Tris-HCl, pH 8.0, and 0.2 M NaCl. The following
proteins were used as standards to calibrate thecolumn; cytochrome C (13 k), chymotrypsin (25 k),
ovalbumin (45 k), bovine serum albumin (68 k),
and catalase (240 k).
Results
Purification
7α-HSD was produced in the culture mediumof P. sp. B-0831 together with 3α-HSD. Partial
separation of 7α-HSD from 3α-HSD was achievedby ammonium sulfate fractionation, and subsequent DEAE-Sepharose column chromatography
enabled each enzyme to be separated completely,
250

0.6

200

0.5
0.4

150

0.3
100

0.2

50

0.1

0

0
20

40

60

80

100

[KCl] (M)Materials and Methods
Materials
All steroids were obtained from Sigma. NAD+,
NADP+, and thio-NAD+ were purchased from
Oriental Yeast Co., Ltd., in Japan, and 3-acetylpyridine adenine dinucleotide (APAD+) was
purchased from Sigma. All other chemicals wereof the highest commercial quality available.

Activity (units/ml)

34

120

Fraction number

Figure 1. Elution profile on DEAE-Sepharosecolumn chromatography. Each 2 ml fraction wascollected. The enzymatic activities to catalyzecholic acid of respective fractions are indicated byopen circles, and the KCl concentration to elute thesample is indicated by linear line.

Ueda et al.

k

M

1

35

5.5

2 (A)

(B)

94

Log of MW

67
43
30
20.1
14.4

5.0

4.5

4.0
1

2

3

Ve/Vo
Figure 2. Molecular weight determination of the purified 7α-HSD. (A) Coomassie-stained polyacrylamide gel after SDS-PAGE under reducing conditions. Lane M, markers; Lane 1, 7α-HSD from P. sp.
B-0831 (0.5 µg); Lane 2, recombinant 3α-HSD from P. sp. B-0831 (1.8 µg). (B) A standard curve obtainedby plotting the log of molecular weights vs. relative elution volume (Ve/Vo) of standard proteins (○) on gel
filtration analysis. The Ve/Vo of 7α-HSD (●) was plotted on the standard curve to determine the native
molecular weight.

Table 1. Purification of 7α-HSD from P. sp. B-0831
Step
Cell-free extract
Ammonium sulfate
DEAE-Sepharose
Red-Sepharose

Protein
(mg)
28600
296
60
0.52

Activity(units)
4000
3200
2090
493

with the minor and major peaks containing
3α-HSD and 7α-HSD, respectively (Fig. 1). The
procedure of affinity chromatography using a
Red-Sepharose column gave a 27-fold purification
factor, resulting in nearly a single band at about 25
k on Coomassie stained gel after SDS-PAGE (Fig.
2A). The purification procedures of 7α-HSD aresummarized in Table 1. The pI of the enzyme wasdetermined to be 4.6. The gel filtration analysis of
the purified 7α-HSD gave a calculated molecularweight of about 108 k (Fig. 2B). The results of
SDS-PAGE and gel filtration indicated that the
enzyme exists in a tetrameric form.
Nucleotide cofactor dependency
To analyze the effects of nucleotide cofactors
on enzyme activity, the kcat values for oxidation of
1.0 mM cholic acid were determined in the presence of various cofactors. The relative activities,
in which the kcat values were normalized by setting
the value with NAD+ to 100%, are summarized inTable 2. The results clearly show that 7α-HSD
from P. sp. B-0831 is NAD+-dependent, which isin contrast to the dual nucleotide cofactor-specific

Specific activity(units/mg)
0.14
10.8
35
941

Purification(fold)
1
77
250
5881

Yield
(%)
100
80
52.3
12.3

3α-HSD produced by the same bacterium. In
addition, 7α-HSD can utilize an NAD+ analogue,thio-NAD+, with the same extent of activity.
Substrate specificity
The enzyme was active toward the substrates,
each of which has a hydroxy group at position 7 ofthe steroid skeleton in the α orientation, andinactive toward the 7α-hydroxy-lacking substrates,
deoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, and androsterone. The steady-state kinetic rate parameters of 7αTable 2. Effect of cofactors on the 7α-HSDcatalyzed reaction for cholic acid
Cofactor
Relative activity (%)
+
NAD
100
+
thio-NAD
93
APAD+
4.9
+
NADP
0.7
Assays were carried out at 37°C in 40 mM TrisHCl, pH 9.5, 1.0 mM cholic acid, 0.05 units/ml
3α-HSD, and 1.0 mM cofactor.

Molecular and enzymatic properties of 7α-HSD

36The effect of various compounds on 7α-HSD
was evaluated (Table 4). The enzyme activity wasstrongly inhibited by CuCl2 and FeCl3 but was
minimally affected by a sulfhydryl-blocking
reagent, p-chloromercuribenzoate (PCMB).

hydroxysteroid dehydrogenation, Km and kcat, were
determined (Table 3). The Km value for cholic acidwas similar to that for chenodeoxycholic acid andabout four times smaller than those for the conjugates of cholic acid. The kcat values for the conjugates were about 60-70% of that for cholic acid.

Table 4. Effect of chemicals on the 7α-HSDcatalyzed reaction for cholic acid

Table 3. Steady-state kinetic parameters for 7αsteroid substrates
Substrate

Km (mmol/l)

Addition
Relative activity (%)
None (control)
100
2 mM MgCl2111
2 mM CaCl2108
2 mM CuCl222 mM ZnCl251
2 mM FeCl382 mM NaF
98
1 mM EDTA
98
0.02 mM PCMB78
The respective compounds were added into the
enzyme assay solution, described in Table 2.

kcat (1/s)

Cholic acid
0.25
423
Chenodeoxycholic acid
0.27
487
Glycocholic acid
1.20
254
Taurocholic acid
1.13
288
Assays were carried out at 37°C in 40 mM
Tris-HCl, pH 8.0, 0.025% nitro tetrazolium blue,
0.4% Triton X-100, 2.5 units/ml diaphorase, 1.0mM NAD+, and 2.5 mM substrate.
Experimental error was within 10%.

Discussion
7α-HSD-producing microorganisms often
have produced other steroid catabolic enzymes [3,4, 7, 19, 20]. So far, 7α-HSD from E. coli HB101
and Eubacterium sp. VPI 12708 have beensequenced and classified as members of the shortchain dehydrogenase/reductase (SDR) superfamily[11, 12]. The molecular weight analysis of 7αHSD from P. sp. B-0831 shows that the subunit
weight (25 k) is slightly smaller than those from E.
coli (28 k) and Eubacterium sp. (32 k), while the

120

(A)

100
80
60
40
20
0
2

4

6

8

pH

10

12

Relative activity (%)

Relative activity (%)

Effects of pH, temperature, and chemicals on
enzyme activity
The effect of pH on the oxidation of cholicacid was determined over a pH range of 5-11 asshown in Fig. 3A. The optimum pH range wasfound to be between 8 and 10.
As shown in Fig. 3B, the enzyme was stablebelow 50°C. After incubation at 55°C and 60°C for
10 min, the remaining activities were 55% and10%, respectively. The incubation at 37°C for 60
min resulted in no loss of activity in the pH rangeof 7 to 10.

120

(B)

100
80
60
40
20
0
0

20

40

60

80

Temperature (°C)

Figure 3. Effects of pH and temperature on the 7α-HSD-catalyzed reaction for cholic acid. (A) Optimization of pH. The kcat values were measured over a pH range of 5-11. The following buffer was used at therespective pH: acetate-sodium acetate (○), KH2PO4-NaOH (●), Tris-HCl (▲), dimethylglutaric acid-NaOH(∆), glycine-NaOH (□), and Na2HPO4-NaOH (◊). The relative activities were normalized by setting the kcat
value at pH 7.5 to 100%. (B) Thermal stability. The enzyme was incubated at various temperatures for 10
min in 10 mM Tris-HCl buffer, pH 8.0, followed by the measurement of kcat. The relative activities werenormalized by setting the kcat value at 30°C to 100%.

Ueda et al.
homo tetramer form of the mature enzyme is thesame as these two enzymes [10, 11]. 7α-HSD fromP. sp. B-0831 is NAD+-dependent and insensitive
to PCMB (Tables 2 and 4). These characteristics
are similar to those of 7α-HSD from E. coli
HB101 and in contrast to those of 7α-HSD from
Eubacterium sp. VPI 12708, which is NADP+dependent and PCMB-sensitive. The activity of
7α-HSD from P. sp. B-0831 was inhibited by someheavy-metal ions as well as 7α-HSDs from E. coli
HB101 and Eubacterium sp. VPI 12708. The
steady-state kinetic properties of 7α-HSD from P.
sp. B-0831 were found to be unique (Table 3) incomparison with the other two 7α-HSDs [10, 11].
The Km value of 7α-HSD from P. sp. B-0831 forcholic acid (0.25 mM) is larger than that of the
Eubacterium enzyme (11 µM) and smaller thanthat of the E. coli enzyme (1.2 mM). The kcat valueof the E. coli enzyme for cholic acid is nearly eighttimes larger than that for chenodeoxycholic acid,
while both of the corresponding values of 7α-HSD
from P. sp. B-0831 are similar. The kcat values of
the E. coli enzyme for the conjugates of cholic acidare less than one-twentieth that of cholic acid itself,while the corresponding values of 7α-HSD from P.
sp. B-0831 are about 60-70%. This comparatively
equal kcat values for free cholic acid and the
conjugates of cholic acid suggest that 7α-HSD
from P. sp. B-0831 is appropriate for thequantitative analysis of 7α-hydroxy bile acids.
In the metabolism of the primary bile acids,
cholic acid and chenodeoxycholic acid are dehydroxylated by intestinal microflora to deoxycholicacid and lithocholic acid, respectively. In addition,
another pathway for the 7-dehydroxylation system
has been proposed in Eubacterium sp. VPI 12708,
an intestinal anaerobic bacterium, in which 3αHSD is related to the initial step of this pathway[21]. 3α-HSD from Comamonas testosteroni is
known to participate in not only bile acidmetabolism but also steroid decomposition, which
is important for the defense strategy [22, 23]. We
recently showed the close similarity in thesubstrate specificity between 3α-HSDs from P. sp.
B-0831 and C. testosteroni [15, 16]. The
characterization of different HSDs from the same
bacterium can help us to understand the more
precise biological roles of the respective enzymes.
In comparison with 7α-HSD and 3α- HSD from P.

37

sp. B-0831, the subunit molecular weights are
similar, while the native oligomeric forms aredifferent. The nucleotide cofactor specificities aredifferent from each other: NAD+-dependent 7αHSD and dual nucleotide cofactor-specific 3αHSD. The Km values of 7α-HSD for cholic acidand its conjugates are larger than those of 3α-HSD.
The slightly decreased kcat values for the cholicacid conjugates relative to that for cholic acid aresimilar in both of the HSDs.
The present study shows that 7α-HSD from P.
sp. B-0831 can utilize an NAD+ analogue, thioNAD+ (Table 2). The activity difference of 7αHSD with the two cofactors, NAD+ and thioNAD+, is much smaller than that of 3α-HSD.
Using the advantage of the higher molar extinction
coefficient of thio-NAD+, 3α-HSD with thioNAD+ has been used in the assay system for themeasurement of total bile acids in serum ascommercially available reagents [24]. From apractical standpoint, 7α-HSD with thio-NAD+ is
considered to be useful for future clinical diagnostics in the measurement of the primary bile acids.
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Communicated by Hiroshi Ueno