Hydroxylation of oleanolic acid to queretaroic acid by cytochrome P450 from Nonomuraea recticatena

Article abstract of DOI:10.1271/bbb.60126

A gene for cytochrome P450 (moxA) from Nonomuraea recticatena, coexpressed with camAB for pseudomonad redox partners in Escherichia coli, hydroxylated oleanolic acid to produce queretaroic acid. When we used the P450-induced whole-cell as a catalyst, only a small amount of queretaroic acid was produced, probably due to poor permeability of oleanolic acid into the E. coli cell. In an alternative approach with the cell-free reaction system, the conversion ratio increased up to 17%.

Full text of DOI:10.1271/bbb.60126

Biosci. Biotechnol. Biochem., 70 (9), 2299–2302, 2006
Note
Hydroxylation of Oleanolic Acid to Queretaroic Acid
by Cytochrome P450 from Nonomuraea recticatena
y
Yoshikazu FUJII, Shinji HIROSUE, Tadashi FUJII, Naoki MATSUMOTO,
Hitosi AGEMATU, and Akira ARISAWA
Bioresource Laboratories, Mercian Corp., Nakaizumi 1808, Iwata-shi, Shizuoka 438-0078, Japan
Received March 9, 2006; Accepted May 22, 2006; Online Publication, September 7, 2006
[doi:10.1271/bbb.60126]
A gene for cytochrome P450 (moxA) from Nonomur-
29
30
aea recticatena, coexpressed with camAB for pseudomo-
nad redox partners in Escherichia coli, hydroxylated
oleanolic acid to produce queretaroic acid. When we
used the P450-induced whole-cell as a catalyst, only a
small amount of queretaroic acid was produced, prob-
ably due to poor permeability of oleanolic acid into the
E. coli cell. In an alternative approach with the cell-free
reaction system, the conversion ratio increased up to
H C
CH R
3
2
19
18
20
21
22
1
2
1
3
11
25
CH3
2
6
17
CH3
COOH
28
1
4
1
6
1
4
9
6
2
10
8
7
15
1
7%.
CH3
27
3
5
Key words: cytochrome P450; oleanolic acid; queretaro-
ic acid; hydroxylation
HO
H C
CH3
24
3
23
Cytochromes P450 (P450s) are a superfamily of
oxidative hemoproteins that catalyze an enormous
variety of oxidative conversions of organic compounds,
including hydroxylation, epoxidation, phenol coupling,
heteroatom oxidation, dehalogenation, and C–C bond
cleavage via successive hydroxylations. The ability of
P450s to catalyze difficult oxidation with high regio- and
stereoselectivity makes them attractive for numerous
biotechnological applications.
Fig. 1. Structure of Oleanolic Acid (R=H) and Queretaroic Acid
(R=OH).
oleanolic acid at its C-30 of the methyl group to
produce queretaroic acid as the main product (Fig. 1).
Queretaroic acid is an ingredient in herb drugs and has
been reported as to its physiological activity. Here we
report the novel hydroxylation of oleanolic acid to
queretaroic acid by a bacterial P450, P450moxA, with a
cell-free reaction system.
The gene coding P450moxA (moxA) and its down-
stream ferredoxin gene (moxB) were cloned together as
genes responsible for compactin hydroxylation.
P450moxA is putatively classified into the CYP105
family from its amino acid sequence identity (49%) to
4
)
We developed a useful bacterial P450-expression
system with Escherichia coli that harbors a plasmid
carrying genes for a P450 and Pseudomonas redox
1
,2)
partners (camAB).
So far we have collected 213
bacterial P450 genes and constructed a P450 library to
explore their ability to catalyze useful oxidative con-
versions.3
1
5,16)
)
Oleanolic acid is a triterpene isolated from some
plants that has various physiological activities such as
1
7)
P450sca-2 (CYP105A3).
4
)
5)
6)
7,8)
anti-tumor, anti-fungal, insecticidal, anti-HIV,
10,11)
E. coli BL21 (DE3) was transformed with expression
vector pET11a (Novagen, San Diego, CA) carrying
moxAB and camAB, named pMoxAB-CamAB. P450-
induced E. coli cells were prepared as described
previously, except for ethanol-glycerol treatment. It
has been reported that ethanol and glycerol enhanced
proper folding of the P450 protein.
tigation of concentrations of ethanol and glycerol to
optimize expression of P450moxA, we added 750 ml of
9)
diuretic, blood sugar level depression,
liver pro-
tection,1 and anti-inflammatory activities.13,14) Hence
oleanolic acid can be a suitable model compound worth
converting to its hydroxyl derivatives with superior
physiological properties. We screened P450s of the P450
library for oleanolic acid hydroxylation activity and
found that P450moxA, a P450 from rare actinomycete
Nonomuraea recticatena IFO 14525, hydroxylated
2)
3
)
1
8,19)
After inves-
y
To whom correspondence should be addressed. Tel: +81-538-21-1134; Fax: +81-538-21-1135; E-mail: fujii-ysk@mercian.co.jp

2300
Y. FUJII et al.
In the hydroxylation of oleanolic acid by P450moxA,
only 3.3% of oleanolic acid was converted to queretaroic
acid (Fig. 3A). In an alternative approach to improve the
conversion reaction, we constructed a cell-free reaction
system as follows: E. coli cells expressing P450moxA
prepared from 1.5 ml of culture as described above were
suspended in 300 ml of 50 mM Tris–HCl buffer (pH 7.4)
containing 20% glycerol, and lysed using Bug-Buster
reagent (Novagen, Darmstadt, Germany) with Benzo-
nase nuclease (Novagen) and lysozyme (1.2 mg). To the
cell lyzate, we added 300 ml of CamA-CamB solution
prepared in advance from E. coli expressing camAB in
the same manner as with the P450moxA solution,
2.6 mM NADH, 2.6 mM NADPH, 38 mM D-glucose,
6.25 U/ml glucose dehydrogenase, 0.1 mg/ml catalase,
0.5% randomly substituted methyl-ꢀ-cyclodextrin
(Wacker Chemie, Munich, Germany) and 200 mg/ml
oleanolic acid in a final volume of 800 ml. The reaction
mixture was incubated at 30 C for 20 h with shaking.
Fig. 2. Reduced CO Difference Spectrum of P450moxA Expressed in
E. coli BL21 (DE3).
ꢀ
With this cell-free reaction system, P450moxA success-
fully hydroxylated oleanolic acid to queretaroic acid
with a conversion ratio of 17% (Fig. 3C). This increased
conversion activity achieved by the cell-free reaction
system perhaps indicates that oleanolic acid, due to its
low penetration in the cell, cannot reach P450moxA
before cell disruption. Another possibility is an im-
proved electron transfer system supported by the
NAD(P)H regeneration system. This possibility, how-
ever, can be excluded considering our preliminary result
that conversion activity was decreased by the cell-free
reaction system when we used diclofenac as a substrate
that can penetrate into the cell for P450boxA-catalyzed
Cell extract of E. coli BL21 (DE3) expressing P450moxA was
prepared. A typical reduced CO difference spectrum of P450moxA
showed a Soret band at about 450 nm.
ethanol and 2.5 ml of 50% glycerol to 25 ml of the
ꢀ
culture, which was further incubated for 20 min at 22 C
prior to the addition of isopropyl-thio-ꢀ-D-galactopyr-
anoside (IPTG).
The expression of P450moxA in E. coli was verified
2
0)
by reduced CO difference spectral assay. The CO-
binding form of P450moxA in the reduced state with
sodium dithionite showed the distinctive CO complex
spectrum maximum at 450 nm that is characteristic of
P450s (Fig. 2). The expression level of P450moxA was
estimated at 2.5 mM in the culture of E. coli harboring
pMoxAB-CamAB.
0
*
4 -C hydroxylation (data not shown). No queretaroic
acid production was detected in control cell lysate
prepared from E. coli cells without moxA (Fig. 3B). In
this bioconversion, we also observed three other
products as minor components (Fig. 3C). The chemical
structures and pharmacological significance of these
products were not determined.
The P450moxA-induced E. coli cells were collected
from 1.5 ml of culture and suspended in 1.5 ml of CV
buffer (50 mM potassium phosphate buffer, pH 7.4,
containing 10% glycerol, 2 mM dithiothreitol, and
To obtain a larger amount of the product, large-scale
conversion was carried out in the same manner. The
product was extracted with 100 ml of ethyl acetate from
the reaction mixture (330 ml) and purified by preparative
HPLC on an Inertsil PREP-ODS column (250 ꢁ 20 mm
1
mM EDTA). Oleanolic acid dissolved in 30 ml of
acetone was added to a final concentration of 200 mg/ml
to the cell suspension. After incubation in a 24-well
ꢀ
microplate at 28 C on a shaker for 24 h, the product
ꢀ
was extracted with 1.5 ml of ethyl acetate. The organic
extract was evaporated under reduced pressure to
dryness. The residue was reconstituted with 200 ml of
methanol for HPLC analysis.
The HPLC analysis was performed with a Shimadzu
LC-2010C HPLC system (Shimadzu, Tokyo) with a
diode array ultraviolet detector set at 210 nm, fitted with
a Develosil ODS-HG-5 column (150 ꢁ 4:6 mm i.d.,
Nomura Chemical, Aichi, Japan). The sample (10 ml)
was chromatographed with a linear gradient of acetoni-
trile concentration in 5 mM phosphate buffer (pH 5.0)
from 50% (v/v) to 85% (v/v) for 10 min, 85% (v/v)
i.d.; GL Sciences, Tokyo) at 220 nm at 40 C with
MeOH/water (70:30 [v/v]) as the mobile phase at a flow
rate of 7.0 ml/min. A fraction containing queretaroic
acid was evaporated under reduced pressure to give
3.0 mg of queretaroic acid as a white powder.
The identification of the product was based on
analysis of NMR spectroscopy and electrospray ioniza-
tion mass spectrometry (ESI-MS). The mass spectrum of
the product showed 16 mass units more than that of
oleanolic acid, indicating that the product was a
*
P450boxA is a cytochrome P450 encoded by boxA (GenBank
Accession no. AB180845) cloned from compactin hydroxylating
Streptomyces sp. TM-7.
until 15 min, and 50% (v/v) until 20 min at a flow rate of
ꢀ
1
.0 ml/min at 35 C.

Hydroxylation of Oleanolic Acid by Cytochrome P450
2301
2
1
00
00
A
OA
QA
0
2
1
00
B
OA
00
0
2
1
00
C
00
0
OA
QA
0
5
10
15
20
Retention time (min)
Fig. 3. Bioconversion of Oleanolic Acid (OA) to Queretaroic Acid (QA) by Whole Cells (A), a Cell-Free Reaction System without P450moxA (B),
and a Cell-Free Reaction System with P450moxA (C).
Products detected by P450moxA reaction in the cell-free reaction system of oleanolic acid are indicated by arrow. Column, a Develosil ODS-
ꢀ
HG-5 (150 ꢁ 4:6 mm i.d., Nomura Chemical, Aichi, Japan); flow rate, 1.0 ml/min; column temperature, 35 C; detection, 210 nm; elution, an
acetonitrile concentration linear-gradient in 5 mM phosphate buffer (pH 5.0) from 50% (v/v) to 85% (v/v) for 10 min, 85% (v/v) until 15 min,
and 50% (v/v) until 20 min.
1
the literature values.22)
monohydroxylated derivative. The H NMR spectrum
(
in methanol-d6) of the product showed downfield
The whole-cell reaction using P450-expressed E. coli
strains is useful in a search for their hydroxylation
methylene protons at ꢁ 3.49, which corresponded to a
carbon (ꢁ 66.4) in the HMQC experiment. In the HMBC
spectrum, this methylene carbon showed a correlation to
methyl group protons (ꢁH 0.88, ꢁC 27.9). Two methyl
groups at the 23-position (ꢁH 0.97, ꢁC 28.2) and the 24-
position (ꢁH 0.77, ꢁC 16.4) were identified on the basis of
HMBC correlations to C-3 (ꢁH 3.14, ꢁC 79.8). These
results indicate that either the C-29 or the C-30 methyl
group was hydroxylated. C-29-hydroxylation gives
3
)
ability, but it is a precondition that the compound to
be hydroxylated permeates the E. coli cells. In this
study, we found that even a compound with a poor
permeability, like oleanolic acid, can be hydroxylated at
higher efficiency by the cell-free reaction system. The
cell-free reaction system requires some laborious han-
dling to perform (e.g., cell disruption and the addition
of reagents to reconstitute the electron transport and
NAD(P)H regeneration) as compared with the whole-
cell reaction system. Still, it should be useful tool to
detect products that cannot be detected by the whole cell
reaction due to poor substrate permeability.
2
1)
mesembryanthemoidigenic acid, while C-30-hydrox-
2
2)
ylation gives queretaroic acid. Comparing the oxy-
genated carbon signal (ꢁC 66.4 in methanol-d ) of the
6
product with C-29 (ꢁC 73.9 in pyridine-d5) of mesem-
bryanthemoidigenic acid or C-30 (ꢁC 65.5 in pyridine-
d5) of queretaroic acid, whose data are reported in the
literature, the product appeared to be queretaroic acid.
Finally the product was identified as queretaroic acid
Queretaroic acid is likely to be produced from
oleanolic acid by P450 hydroxylation in several plants,
including Clerodendron serratum Moon. It is interesting
that an actinomycete P450, P450moxA, catalyzes the
same hydroxylation as botanical P450 does. This finding
might indicate functional sharing of botanical P450s, at
(3ꢀ, 30-dihydroxy-12-oleanen-28-oic acid) by compar-
1
ison of the C NMR spectral data in pyridine-d with
3
5

2302
Y. FUJII et al.
least partly, with bacterial P450s. To the best of our
knowledge, this is the first report that shows that an
actinomycete P450 catalyzes the same hydroxylation as
a botanical P450.
11) Yoshikawa, M., and Matsuda, H., Antidiabetogenic
activity of oleanolic acid glycosides from medicinal
foodstuffs. Biofactors, 13, 231–237 (2000).
1
2) Yim, T. K., Wu, W. K., Pak, W. F., and Ko, K. M.,
Hepatoprotective action of an oleanolic acid-enriched
extract of Ligustrum lucidum fruits is mediated through
an enhancement on hepatic glutathione regeneration
capacity in mice. Phytother. Res., 15, 589–592 (2001).
References
1
)
)
Arisawa, A., and Kumeda, A., Patent WO2003/087381
Oct. 23, 2003).
13) Ismaili, H., Tortora, S., Sosa, S., Fkih-Tetouani, S.,
Ilidrissi, A., Della, L. R., Tubaro, A., and Aquino, R.,
Topical anti-inflammatory activity of Thymus willdeno-
wii. J. Pharm. Pharmacol., 53, 1645–1652 (2001).
14) Giner-Larza, E. M., Manez, S., Recio, M. C., Giner, R.
M., Prieto, J. M., Cerda-Nicolas, M., and Rios, J. L.,
Oleanolic acid, a 3-oxotriterpene from Pistacia, inhibits
leukotriene synthesis and has anti-inflammatory activity.
Eur. J. Pharmacol., 428, 137–143 (2001).
(
2
Peterson, J. A., Lorence, M. C., and Amarneh, B.,
Putidaredoxin reductase and putidaredoxin: cloning,
sequence determination, and heterologous expression
of the proteins. J. Biol. Chem., 265, 6066–6073 (1990).
Agematu, H., Matsumoto, N., Fujii, Y., Kabumoto, H.,
Doi, S., Machida, K., Ishikawa, J., and Arisawa, A.,
Hydroxylation of testosterone by bacterial cytochromes
P450 using the Escherichia coli expression system.
Biosci. Biotechnol. Biochem., 70, 307–311 (2006).
Kinoshita, K., Yang, Y., Koyama, K., Takahashi, K., and
Nishino, H., Inhibitory effect of some triterpenes from
cacti on 32Pi-incorporation into phospholipids of HeLa
cells promoted by 12-O-tetradecanoylphorbol-13-ace-
tate. Phytomedicine, 6, 73–77 (1999).
3
)
)
15) Fujii, T., Hirosue, S., Narita, T., Kobayashi, Y., Jodo, O.,
and Ishiki, K., Japan Kokai Tokkyo Koho, P2002-
247984A (Feb. 23, 2001).
4
16) GenBank accession no. AB180844.
17) Watanabe, I., Nara, F., and Serizawa, N., Cloning,
characterization and expression of the gene encoding
cytochrome P-450sca-2 from Streptomyces carbophilus
involved in production of pravastatin, a specific HMG-
CoA reductase inhibitor. Gene, 163, 81–85 (1995).
18) Kusano, K., Waterman, M. R., Sakaguchi, M., Omura,
T., and Kagawa, N., Protein synthesis inhibitors and
ethanol selectively enhance heterologous expression of
P450s and related proteins in Escherichia coli. Arch.
Biochem. Biophys., 367, 129–136 (1999).
19) Kagawa, N., and Cao, Q., Osmotic stress induced by
carbohydrates enhances expression of foreign proteins in
Escherichia coli. Arch. Biochem. Biophys., 393, 290–296
(2001).
20) Omura, T., and Sato, R., The carbon monoxide-binding
pigment of liver microsomes. I. Evidence for its
hemoprotein nature. J. Biol. Chem., 239, 2370–2378
(1964).
5
)
)
Tang, H. Q., Hu, J., Yang, L., and Tan, R. X., Terpenoids
and flavonoids from Artemisia species. Planta Med., 66,
391–393 (2000).
6
Marquina, S., Maldonado, N., Garduno-Ramirez, M. L.,
Aranda, E., Villarreal, M. L., Navarro, V., Bye, R.,
Delgado, G., and Alvarez, L., Bioactive oleanolic acid
saponins and other constituents from the roots of
Viguiera decurrens. Phytochemistry, 56, 93–97 (2001).
Kashiwada, Y., Nagao, T., Hashimoto, A., Ikeshiro, Y.,
Okabe, H., Cosentino, L. M., and Lee, K. H., Anti-AIDS
agents 38: anti-HIV activity of 3-O-acyl ursolic acid
derivatives. J. Nat. Prod., 63, 1619–1622 (2000).
Ma, C., Nakamura, N., Hattori, M., Kakuda, H., Qiao, J.,
and Yu, H., Inhibitory effects on HIV-1 protease of
constituents from the wood of Xanthoceras sorifolia.
J. Nat. Prod., 63, 238–242 (2000).
7
8
9
)
)
)
21) Shao, C., Kasai, R., Xu, J., and Tanaka, O., Saponins
from leaves of Acanthopanax senticosus Harms., Ciwu-
jia. II. Structures of Ciwujianosides A1, A2, A3, A4, and
D3. Chem. Pharm. Bull., 37, 42–45 (1989).
Alvarez, M. E., Maria, A. O., and Saad, J. R., Diuretic
activity of Fabiana patagonica in rats. Phytother. Res.,
16, 71–73 (2002).
1
0) Assefa, H., Nimrod, A., Walker, L., and Sindelar, R.,
Enantioselective synthesis and complement inhibitory
assay of A/B-ring partial analogues of oleanolic acid.
Bioorg. Med. Chem. Lett., 11, 1619–1623 (2001).
22) Yang, Y., Kinoshita, K., Koyama, K., Takahashi, K.,
Kondo, N., and Yuasa, H., New triterpenes from
Machaerocereus eruca. J. Nat. Prod., 61, 456–460
(1998).