J. Biol. Macromol., 4(1) 23-28 (2004)

ArticleSteady-state kinetic properties of 3α-hydroxysteroid dehydrogenase from Pseudomonas sp.
B-0831: Steroid substrate specificity and nucleotide cofactor dependency

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 December 16, 2003; Accepted January 7, 2004
We investigated the steroid substrate specificity of 3α-hydroxysteroid dehydrogenase (3α-HSD)
from Pseudomonas sp. B-0831. Similar to 3α-hydroxysteroid dehydrogenase/carbonyl reducetase(3α-HSD/CR) from Comamonas testosteroni, the recombinant 3α-HSD from P. sp. B-0831 catalyzed
the oxidation of the antibiotic fusidic acid with the lowest Km value among 3α-hydroxysteroid
substrates examined in this study, while the kcat value was 17% of that for one of the other substrates,
androsterone. The enzyme showed the utility of cofactor analogues, such as thio-NAD+,3-acetylpyridine adenine dinucleotide (APAD+), and nicotinamide hypoxanthine dinucleotide(deamino-NAD+). Comparing the effects of cofactors, NAD+ and thio-NAD+, on the kcat values, NAD+
was preferred for cholic acid, while thio-NAD+ was preferred for fusidic acid. These results suggestthat the enzyme is adaptable to various substrates and cofactors, thus generating the broad substratespecificity with each different reaction mode.
Keywords: 3α-hydroxysteroid dehydrogenase, nucleotide cofactor, Pseudomonas sp. B-0831,
steady-state kinetics, substrate specificity
Introduction
3α-Hydroxysteroid dehydrogenase (3α-HSD)
catalyzes the reversible inter-conversion ofhydroxy and oxo groups at position 3 of the steroid
nucleus. Prokaryotic 3α-HSDs have been
described in several microorganisms and belong to
the short-chain dehydrogenase/reductase (SDR)
superfamily (EC 1.1.1.50) [1-5]. Prokaryotic 3αHSDs are supposed to participate in steroid
degradation by their steroid utilization as a solecarbon source [6]. 3α-HSD from Comamonas(Pseudomonas) testosteroni, which has beenstudied extensively among bacterial 3α-HSDs, isan NAD+ dependent enzyme and shows broad
substrate specificity to various steroids, C19 to C27[6, 7]. In addition, since it can catalyze the carbonyl reduction of a variety of non-steroidal aldehydes
3

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

and ketones, it was named as 3α-hydroxysteroid
dehydrogenase/carbonyl reductase (3α-HSD/CR)[8]. C. testosteroni was able to grow on steroids asa sole carbon source and the enzyme, 3α-HSD/CR,
was induced by these steroids, such as testosterone
and progesterone [6, 9].
We previously cloned 3α-HSD from Pseudomonas sp. strain B-0831 and expressed it in E.
coli [10]. The amino acid sequence shares about
50% identity with 3α-HSD/CR from C. testosteroni ATCC11996 [7]. Unlike NAD+-dependent 3αHSD/CR from C. testosteroni, 3α-HSD from B0831 has the ability to use not only NAD+ but also
The abbreviations used are: 3α-HSD, 3α-hydroxysteroid dehydrogenase; SDR, short-chain dehydrogenase/reductase; 3α-HSD/CR, 3α-hydroxysteroid
dehydrogenase/carbonyl reductase; TBA, total bile
acids; APAD+, 3-acetylpyridine adenine dinucleotide; NTB, nitro tetrazolium blue; NAAD+, nicotinicacid adenine dinucleotide; deamino-NAD+, nicotinamide hypoxanthine dinucleotide.

24

Steroid substrate specificity of 3α-HSD

NADP+. In clinical diagnostics, the enzyme has
been used for the measurement of total bile acids(TBA) in serum [11]. Furthermore, a highly
sensitive and unique enzyme cycling method for
TBA assay has been developed using theadvantage of the higher molar extinction
coefficient of an NAD+ analogue, thio-NAD+, andthe assay system has been used for commerciallyavailable reagents [12]. Since other NAD+
analogues also have unique properties, their use in
the application of the assay system is considered
practical. For instance, 3-acetylpyridine adenine
dinucleotide (APAD+) can alter the equilibrium of
NAD+ related enzyme reactions in favor of
substrate oxidation [13].
In the present study, we analyzed thedehydrogenase activity of 3α-HSD from P. sp.
B-0831 toward various steroid substrates (Fig. 1)
and compared the results with those of 3α-HSD/
CR from C. testosteroni. Using the results of thisstudy together with the previous reports, we wereable to elucidate the biological roles of steroids
and steroid decomposition in prokaryotes. In
addition, we evaluated the effects of the cofactors,
NAD+ and its analogues, on the catalytic activity.
Materials and Methods
Materials
NAD+, NADP+, and nicotinic acid adenineOdinucleotide (NAAD+) were purchased from
Oriental Yeast Co., Ltd., in Japan. Nicotinamide
hypoxanthine dinucleotide (deamino-NAD+),
APAD+, thio-NAD+, and all steroids used in thisstudy (Fig. 1) were purchased from Sigma. Allother chemicals were of the highest commercial
quality available. Recombinant 3α-HSD from
Pseudomonas sp. strain B-0831 was expressed in E.
coli and purified as described previously [10]. The
protein purity was determined to be over 95% by
SDS-PAGE analysis, and the protein concentration
was determined by the method of Lowry [14].
Steady-state kinetic study
The oxidataion reactions were carried out at
37°C in 1 ml solution containing 40 mM Tris-HCl,
pH 8.5, 0.025% nitro tetrazolium blue (NTB),
0.4% Triton X-100, 2.5 units/ml diaphorase, and
1.0 mM cofactor, NAD+ or thio-NAD+, in thepresence of either 1.0 mM substrate for thedetermination of kcat or various concentrations of
substrate for the determination of Km [10]. The
lower solubility steroids, such as androsterone,
lithocholic acid, and testosterone, were dissolved
in methanol and were added to the assay mixture inwhich the final solvent concentration did notexceed 2% (v/v). Other steroids were dissolved in
distilled water and adjusted to neutral pH. The
reaction was started by adding the enzyme and

OH

COOH

COR

HO

HO

H

Androsterone

H

HO

OH

Cholic acid (R: OH)
Glycocholic acid (R: NHCH2COOH)
Taurocholic acid (R: NH(CH2)2SO3H)

H

OH

Chenodeoxycholic acid

HOOC

CH3
CH3

HO
CH3

COOH

COOH

CH3
HO
HO

H

OH

Ursodeoxycholic acidHO

H

Lithocholic acid

Figure 1. Structures of steroid substrates used in this study.

H
CH3

Fusidic acid

O
O
H3C

Ueda et al.

stopped according to necessity by adding 2 ml of
0.5% SDS after incubation for 5 min. The
formation of formazan dye at 550 nm wasmeasured using a Shimadzu UV-2200 spectrophotometer and calculated using a molar extinction
coefficient of 16,700 mol-1 l cm-1. The Km and Ki
values were calculated by the nonlinear leastsquares method using Taylor expansion [15].
Quantitative analysis of cholic acid
Assays were carried out at 37°C in 1 ml
solution of 3α-HSD containing 40 mM Tris-HCl,
pH 8.0, 0.025% NTB, and 0.4% Triton X-100 witheither 1.0 mM NAD+, 2.5 units/ml diaphorase (EC1.6.99.2) (Asahi Kasei Pharma), or 1.0 mM
NADP+, 2.5 units/ml NADPH diaphorase (EC1.6.99.1) (Asahi Kasei Pharma). The reaction wasstarted by adding 0.05 ml of various concentrationsof cholic acid in distilled water and stopped byadding 2 ml of 0.5% SDS after incubation for 15
min. The amount of cholic acid was determined bymeasuring the formation of formazan dye at 550
nm.
Relative catalytic activity for various cofactors
Assays were carried out at 37°C in 40 mM
Tris-HCl, pH 9.5, 1.0 mM cholic acid, 0.05
units/ml 3α-HSD, and 1.0 mM cofactor. The kcat
values for the oxidation reaction were determinedby measuring the reduction of a cofactor at 340 nm
for NAD+, deamino-NAD+, and NAAD+; at 400

25

nm for thio-NAD+; and at 365 nm for APAD+.
Results
Steroid substrate specificity
The recombinant 3α-HSD from Pseudomonas
sp. B-0831 was shown to be active among avariety of steroid substrates, such as androsterone(C19 steroid), cholic acid (C24 steroid), and fusidic
acid (C27 steroid) (Table 1), while the 3-oxosteroid
reduction of testosterone was not observed. This
result was similar to the substrate specificity of
3α-HSD/CR from Comamonas testosteroni. The
steady-state kinetic constants for the 3α-hydroxysteroid oxidation of various steroid substrates
using NAD+ or thio-NAD+ as a cofactor were
determined and summarized in Table 1. The kcat
values for bile acids including their conjugates,
such as glycocholic acid and taurocholic acid werequite similar. Comparing the activity toward thesteroid substrates used in this study, the enzyme
oxidized steroid antibiotic fusidic acid more
effectively than androsterone and bile acids.
Quantitative analysis of cholic acid by using NAD+or NADP+ as a cofactor
3α-HSD from P. sp. B-0831 was found to be adual nucleotide cofactor-specific enzyme (Ueda etal., manuscript in preparation). In order to analyze
the different effects of these dual cofactors, NAD+and NADP+, on 3α-hydroxysteroid dehydrogenation, the quantitative analysis of cholic acid wasTable 1. Steady-state kinetic parameters for 3α-steroid substrates
Substrate

CofactorKm(µmol/l)kcat(1/sec)kcat/Km(l/µmol·sec)

Androsterone

NAD+

210

134

0.64

Cholic acid
Cholic acid
Chenodeoxycholic acid
Ursodeoxycholic acid
Lithocholic acid
Glycocholic acid
Taurocholic acidNAD+thio-NAD+
NAD+
NAD+
NAD+
NAD+
NAD+

31
84
63
69
68
38
43

75
35
76
82
72
77
74

2.41
0.41
1.21
1.19
1.06
2.02
1.72

6.2
37

23
25

3.75
0.67

Fusidic acid
NAD+
Fusidic acidthio-NAD+
Experimental error was within 10%.

Steroid substrate specificity of 3α-HSD

26carried out in the presence of either NAD+ or
NADP+. As shown in Fig. 2A, 0.1 units/ml enzyme
was enough for NAD+ to catalyze 2.0 mM cholicacid under the present reaction conditions, while
2.5 units/ml enzyme was needed for NADP+.
When either 0.5 units/ml enzyme with NAD+ or
2.5 units/ml enzyme with NADP+ was used, thesame standard curves, where the absorbancechange at 550 nm increased in proportion to theamount of cholic acid, were obtained (Fig. 2B).

0.5

A550

0.4
0.3
0.2
0.1

(A)

0
0

0.5

1

1.5

2

2.5

3α-HSD (units/ml)

Table 2. Effects of cofactors on the 3α-HSDcatalyzed reaction for cholic acid

0.5

Cofactor
Relative activity (%) aNAD+ b100+bthio-NAD
47
APAD+
42
+
deamino-NAD
36
NAAD+
0
a
The kcat values were normalized by setting thevalue with NAD+ to 100%.
b
The kcat values were taken from Table 1.

A550

0.4
0.3
0.2
0.1

(B)
0
0

0.5

1

1.5

sensitivity, as described above. It should be notedthat the effects of the cofactors, NAD+ andthio-NAD+, on the kcat values for cholic acid andfusidic acid were different from each other. Whenthio-NAD+ was used, the kcat value for cholic acidwas decreased to 47%, while that for fusidic acidwas similar (Table 1), indicating that the catalytic
function for each substrate can be modulated by
the cofactor in a different manner. From a practicalstandpoint, since the lower catalytic activity with
thio-NAD+ for cholic acid can be compensated by
the amount of enzyme used, the present results
supported the notion that the enzymatic analysis
using thio-NAD+ is applicable and attractive.
We also analyzed the effects of other oxidized
cofactor analogues on the kcat values and compared
the relative catalytic activity (Table 2). It was
found that 3α-HSD from P. sp. B-0831 can utilize
not only thio-NAD+ but also APAD+ and deaminoNAD+, while it can not utilize NAAD+, a precursorof NAD+, as a cofactor.

2

2.5

Cholic acid (mM)+

Figure 2. Effects of nucleotide cofactors, NAD(circle) and NADP+ (triangle), on the 3α-hydroxysteroid dehydrogenation of cholic acid by 3α-HSD
from P. sp. B-0831. (A) Correlation between the
enzyme concentration and the catalytic efficiency
toward 2.0 mM cholic acid. (B) Standard curve todetermine the amount of cholic acid by 0.5
units/ml enzyme with NAD+ and 2.5 units/ml
enzyme with NADP+.
Effects of NAD+ anologues on catalytic activity
An NAD+ analogue, thio-NAD+, has been used
for the TBA assay system, mainly due to its higher

Discussion
Although 3α-HSD is known to play a central
role in steroid metabolism, the information
regarding its molecular function is limited. The
steady-state kinetics of substrate specificity inprokaryotic 3α-HSDs has been reported only for
3α-HSD/CR from C. testosteroni [6, 7]. This
enzyme was shown to catalyze antibiotic fusidic
acid with the lowest Km value (6.1 µM), comparedwith those of general steroids, such as androsterone (18.4 µM) and cholic acid (31.1 µM) [7].
As well, the Km value of 3α-HSD from Pseudomonas sp. strain B-0831 for fusidic acid was 6.2

Ueda et al.

µM, lower than those for androsterone and cholicacid (Table 1). The steady-state kinetic constantsfor each bile acid, including its conjugates, such as
glycocholic acid and taurocholic acid were similar,
indicating the eligibility of the enzyme for themeasurement of TBA in serum.
3α-HSD from B-0831 has the ability to use
not only NAD+ but also NADP+, which is different
from NAD+-dependent 3α-HSD/CR from C.
testosteroni. The capability of NADP+ utilization
was confirmed by the quantitative analysis ofcholic acid as shown in Fig. 2B. In clinicaldiagnostics, 3α-HSD has been used for themeasurement of TBA in serum [11]. In this regard,
the dual cofactor specificity of the enzyme could
be advantageous. However, the present study
revealed that, when NADP+ is used as a cofactor, alarger amount of the enzyme and a longer reaction
time are needed (Fig. 2A). Therefore, it could be
difficult to use NADP+ for practical application.
The catalytic activity of 3α-HSD from B-0831
was largely changed by the types of cofactors(Tables 1 and 2). The subtle structural differences
between NAD+ and its analogues can drastically
influence the activity, in which the activity with
NAAD+ was completely lost. These results
indicate that the type of cofactor can modulate thefine catalytic function of the enzyme. At the pointof application, the activity difference using the
NAD+ analogues, such as thio-NAD+ and APAD+,
was found to be small. Therefore, the advantagesof these NAD+ analogues, resulting in higher
sensitivity and a larger substrate concentration
range, are useful and attractive, especially forclinical diagnostics.
Prokaryotic 3α-HSDs were shown to be
induced by steroids such as testosterone andprogesterone [6, 9, 16]. These steroids have been
considered to be potent inducers and not substratesof 3α-HSD [7]. Recently, two regulator genes for
enzyme expression, repA and repB, which act as
repressors by binding to the operator of the
enzyme gene and mRNA, respectively, wereidentified in C. testosteroni [17, 18]. The presenceof steroids resulted in the liberation of thesebindings such that translation could proceed. The
testosterone induction was reported to lead to an
approximately 6-fold elevation of resistance of C.

27

testosteroni ATCC11996 against fusidic acid [19].
These results are consistent with the results
showing that 3α-HSD/CR from C. testosteroni cancatalyze fusidic acid but cannot catalyze testosterone [7], similar to 3α-HSD from P. sp. B-0831
observed in this study. 3α-HSD-producing prokaryotes, isolated even from human sources, canutilize steroids as a sole carbon source [9]. They
often have produced other steroid catabolic
enzymes, such as 3β,17β-HSD from C. testosteroni [16] and 7α-HSD from P. sp. B-0831 [20].
Thus, the catabolic pathway could be related to thedecomposition of the steroids, which should also
be important for the defense strategy.
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Communicated by Keiko Kitagishi