J. Biol. Macromol., 4(1) 29-32 (2004)

ArticleCarbonyl reductase activity of a pluripotent enzyme, 3α-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 December 20, 2003; Accepted January 14, 2004
We found that 3α-hydroxysteroid dehydrogenase (3α-HSD) from Pseudomonas sp. B-0831 cancatalyze the carbonyl reduction of non-steroid substrates, such as metyrapone and p-nitrobenzaldehyde, in addition to the activity of 3α-hydroxysteroid dehydrogenase. The results of an
inhibition study in metyrapone reduction by androsterone and cholic acid indicated that metyrapone
bound to the same catalytic site as the steroids. The Km values for the carbonyl reduction wererelatively higher than those for the oxidoreduction at position 3 of the steroid nucleus. It should be
noted that the kcat value of metyrapone using NADPH as a cofactor was less than 1% of that with
NADH, indicating that the cofactor binding modulates the catalytic activity. The present resultclearly showed that 3α-HSD from P. sp. B-0831 has pluripotent substrate specificity and can be
named 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR).
Keywords: 3α-hydroxysteroid dehydrogenase/carbonyl reductase, nucleotide cofactor,
pluripotent substrate specificity, Pseudomonas sp. B-0831, steady-state kinetics
Introduction
3α-Hydroxysteroid dehydrogenase (3α-HSD)
is an NAD(P)+-dependent enzyme which catalyzes
the reversible inter-conversion of hydroxy and oxogroups at position 3 of the steroid nucleus and has
been found in many mammalian cells andmicroorganisms [1-3]. Besides oxidation of 3αhydroxysteroids and the reduction of corresponding 3-oxostertoids, 3α-HSD from Comamonas(Pseudomonas) testosteroni, which has beenstudied extensively among bacterial 3α-HSDs, iscapable of catalyzing the carbonyl reduction of
non-steroidal xenobiotic ketones and aldehydes,
such as the potent cytochrome P-450 inhibitor,
metyrapone, and p-nitrobenzaldehyde (Fig. 1). Dueto this pluripotent substrate specificity, the enzyme
was named 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) [4].
3

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

The carbonyl reductase activity has beenfound in several HSDs such as 11β-HSD type I
from mouse liver [5-7]. Carbonyl reduction issignificant in the detoxification process ofpotentially toxic ketone or aldehyde compounds inhumans and animals [8]. In contrast, the physiological role of prokaryotic carbonyl reductase
remains obscure. The structural similarity of C.
testosteroni 3α-HSD/CR to the mammalian
enzymes shown in an antibody cross-reactivity
study and a crystal structural analysis [6, 9] evokes
the idea that both prokaryotic and mammalian
3α-HSD/CRs have evolved from the sameancestral protein for the catabolism of a variety ofcarbonyl compounds [10].
In the present study, we found the carbonyl reThe abbreviations used are: 3α-HSD, 3α-hydroxysteroid dehydrogenase; 3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase; NTB,
nitro tetrazolium blue; Ki, inhibitor constant; AKR,
aldo-keto reductase; SDR, short-chain dehydrogenase/reductase.

Carbonyl reduction by 3α-HSD

30ductase activity of 3α-HSD from Pseudomonas sp.
strain B-0831 to reduce non-steroid compounds
such as metyrapone and p-nitrobenzaldehyde, andthe substrate binding site to be similar to that for
3α-hydroxysteroid dehydrogenation. We analyzed
the steady-state kinetics of this activity, together
with the effects of the cofactor type on the activity.
N

O CH3

N

CH3

Metyrapone

H
O

HO C
H2

NO2

p-Nitrobenzaldehyde

NO2p-Nitrobenzyl alcohol

Figure 1. Structures of non-steroid substrates usedin this study.
Materials and Methods
Materials
The substrates, metyrapone, p-nitrobenzaldehyde, and p-nitrobenzyl alcohol (Fig. 1), wereobtained from Sigma. The nucleotides, NAD+,
NADH, and NADPH, were purchased from
Oriental Yeast Co., Ltd., in Japan. All otherchemicals were of the highest commercial quality
available. Recombinant 3α-HSD from Pseudomonas sp. strain B-0831 was prepared as described
previously [3, 11].

Steady-state kinetic study
Assays were carried out at 37°C in 40 mM
Tris-HCl, pH 8.5, in the presence of 0.3 mM
NAD(P)H for reduction reaction, and in thepresence of 1 mM NAD+, 0.025% nitro tetrazolium
blue (NTB), 0.4% Triton X-100, and 2.5 units/ml
diaphorase (Asahi Kasei) for oxidation reaction.
The reaction pH was chosen due to the stabilitiesof the enzyme and the chemicals, NTB and NAD+.
The carbonyl reductase activity was determined bymeasuring the decrease of NAD(P)H at 340 nm
using a molar extinction coefficient of 6,220 mol-1
l cm-1. The substrates were dissolved in methanol
and added in the assay mixture within 5% (v/v). In
the assay for substrate specificity, 5.0 mM
substrate was used for the determination of kcat,
and various substrate concentrations were used forthe determination of Km. In the inhibition study,
0.4 mM to 4.0 mM substrate, metyrapone, wasused in the absence or presence of 1.0 mM
inhibitor, cholic acid and androsterone. The Km andKi values were calculated by the nonlinear leastsquares method using Taylor expansion [12].
Results and Discussion
The steady-state kinetic analysis revealed that
3α-HSD from Pseudomonas sp. strain B-0831 isable to catalyze the carbonyl reduction of nonsteroid substrates, metyrapone and p-nitrobenzaldehyde (Table 1). No reverse oxidation of pnitrobenzyl alcohol, the hydroxy product of pnitrobenzaldehyde, could be observed, nor could
3α-HSD/CR from C. testosteroni. The Km values
for the carbonyl reduction are relatively higher

Table 1. Steady-state kinetic parameters for 3α-HSD from Pseudomonas sp. B-0831 with non-steroid andsteroid substrates
Substrate

Cofactor

Metyrapone
NADH
Metyrapone
NADPH
p-Nitrobenzaldehyde
NADH
p-Nitrobenzyl alcohol
NAD+
Androsterone b
NAD+b
Cholic acid
NAD+
Fusidic acid b
NAD+a
Not determined due to the small kcat value.
b
Data were taken from Ueda et al. [3].

Km(µmol/l)
3370
n.d.a
4910
210
31
6.2

kcat
(1/sec)kcat/Km(l/µmol·sec)

13.3
0.058
1.12
No activity detectable
134
75
23

3.94 x 10-3–
2.28 x 10-4
0.64
2.41
3.75

Ueda et al.

1/v (l/µmol•min)than those for the oxidoreduction at position 3 ofthe steroid nucleus and are on the same order of
those of 3α-HSD/CR from C. testosteroni [13]. It
should be noted that the kcat value for metyrapone
using NADPH as a cofactor was less than 1% ofthat with NADH. Considering that the binding
affinity of 3α-HSD to NADH is higher than that of
NADPH (Ueda et al., manuscript in preparation),the cofactor binding can modulate the different
catalytic activity. A similar correlation between thecofactor preference and the catalytic activity was
also observed in the NADPH preference enzyme,
mouse liver 11β-HSD, and the NADH-dependent
enzyme, C. testosteroni 3α-HSD/CR [7, 13].
In order to determine the substrate binding site
for the carbonyl reduction, in comparison with thatfor the oxidoreduction of the steroid nucleus, we
conducted the inhibition study by using androsterone and cholic acid as the steroid inhibitor. As
shown in Fig. 2, the carbonyl reduction was
dramatically inhibited by these substrates following the competitive nature, suggesting that bothsteroids and metyrapone bind to the catalytically
active site on the 3α-HSD. The inhibitor constants(Ki) of metyrapone reduction by androsterone andcholic acid were determined to be 0.174 mM and
0.118 mM, respectively. These values are similar
to the Ki value of metyrapone reduction bycorticosterone for mouse liver 11β- HSD, which is

3

2

1

0
-1

0

1

2

3

1/[S] (l/mmol)

Figure 2. Lineweaver-Burk plot for the competitive inhibition of metyrapone reduction by androsterone and cholic acid. The carbonyl reductase
activity of 3α-HSD was assayed for the metyrapone concentration range from 0.4 mM to 4.0 mM
in the absence (circle) or presence of 0.1 mM
inhibitor, androsterone (square) and cholic acid
(triangle).

31

approximately 0.1 mM [7].
Carbonyl-reducing enzymes have been foundin several HSDs and classified into two protein
superfamilies: the aldo-keto reductase (AKR)
superfamily and the short-chain dehydrogenase/reductase (SDR) superfamily. In SDRs, much of
the carbonyl reductase activity has been found in
mammalian enzymes, such as 11β-HSD, 3α,20βHSD, and 17β-HSD [10]. Particularly in 11β-HSD,
carbonyl reductase activity has been estimated to
be involved with detoxification of biologically
active carbonyl compounds [8]. On the other hand,the physiological function of the prokaryotic
enzymes, such as 3α-HSD/CR from C. testosteroni
and 3α,20β-HSD/CR from Streptomyces hydrogenans, which also have the function of carbonyl
reduction, is still obscure. Recently, putative
3α-HSD/CR genes were found in plant pathogen,such as Xantomonas axonopodis, Xantomonas
campestris, and Ralstonia solanacearum [14, 15].
We found that 3α-HSD/CR from P. sp. B-0831 canalso catalyze antibiotic fusidic acid with lower Km
value (Table 1), similar to 3α-HSD/CR from C.
testosteroni [13]. These findings support the notionthat the steroid detoxification function is one of theresources for those prokaryotes to establish theinfectious or symbiotic relationship with theircorresponding hosts.
In conclusion, this study has shown that
3α-HSD from P. sp. B-0831 can catalyze thereduction of xenobiotic carbonyl compounds withhigher Km values than those for the steroids. It has
pluripotent substrate specificity, similar to 3αHSD/CR from C. testosteroni, and can be named
3α-HSD/CR.
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Pseudomonas sp. B-0831: Steroid substrate32

Carbonyl reduction by 3α-HSD

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Communicated by Kikuko Watanabe