Biocatalytic synthesis of dihydroxynaphthoic acids by cytochrome P450 CYP199A2

Article abstract of DOI:10.1271/bbb.90624

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was found to exhibit oxidation activity towards three hydroxynaphthoic acids. Whole cells of the recombinant Escherichia coli strain expressing CYP199A2 efficiently catalyzed the re

Full text of DOI:10.1271/bbb.90624

Biosci. Biotechnol. Biochem., 73 (12), 2796–2799, 2009
Note
Biocatalytic Synthesis of Dihydroxynaphthoic Acids
by Cytochrome P450 CYP199A2
y
Toshiki FURUYA and Kuniki KINO
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University,
3
-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan
Received August 25, 2009; Accepted September 16, 2009; Online Publication, December 7, 2009
[doi:10.1271/bbb.90624]
CYP199A2, a bacterial P450 monooxygenase from
P450 from Rhodopseudomonas palustris, as an oxida-
tion biocatalyst. We have found that CYP199A2
oxidized 2-naphthoic acid to 7- and 8-hydroxy-2-
Rhodopseudomonas palustris, was found to exhibit
oxidation activity towards three hydroxynaphthoic
acids. Whole cells of the recombinant Escherichia coli
strain expressing CYP199A2 efficiently catalyzed the
regioselective oxidation of 1-, 3-, and 6-hydroxy-2-
naphthoic acids to produce 1,7-, 3,7-, and 6,7-dihydroxy-
naphthoic acid respectively. These results suggest that
CYP199A2 might be a useful oxidation biocatalyst for
the synthesis of dihydroxynaphthoic acids.
1
4)
naphthoic acids (Fig. 1A).
CYP199A2 was also
reported to exhibit oxidation activity towards a variety
of aromatic carboxylic acids, including para-substituted
benzoic acids, indolecarboxylic acids, and a quinoline-
1
5–17)
carboxylic acid.
Here we examined the oxidation
activity of CYP199A2 towards hydroxynaphthoic acids
and investigated the application of the enzyme in the
synthesis of dihydroxynaphthoic acids.
Key words: CYP199A2; cytochrome P450; hydroxyla-
tion; naphthoic acid; oxidation
The CYP199A2 gene was coexpressed with the
palustrisredoxin gene from R. palustris and the putidar-
edoxin reductase gene from Pseudomonas putida to
provide the redox partners of CYP199A2 in Escherichia
Dihydroxynaphthoic acids are important chemicals
found in organisms and they exhibit biological activities.
For example, 1,4-dihydroxy-2-naphthoic acid is an
intermediate in the menaquinone biosynthetic pathway
1
4,17)
coli.
were prepared as described previously.
Whole cells of the recombinant E. coli strain
1
4,17)
Whole-cell
assays were performed as substrates with 1-, 3-, and 6-
hydroxy-2-naphthoic acids, which are commercially
available (Wako Pure Chemicals, Osaka, Japan), and
1
)
in various microorganisms. Also, 1,4-dihydroxy-2-
naphthoic acid has been reported to stimulate the growth
of bifidobacteria and thus to improve conditions in the
with 7- and 8-hydroxy-2-naphthoic acids, which were
14)
2)
human intestine. Recently, an alternative menaquinone
biosynthetic pathway was found in some microorgan-
isms, including Helicobacter pylori, Campylobacter
biocatalytically synthesized using CYP199A2.
The
other structural isomers, 4- and 5-hydroxy-2-naphthoic
acids, are commercially unavailable. The reaction
mixture (250 ml) contained whole cells of the recombi-
nant E. coli strain (50 g of wet cells per liter), a substrate
(1 mM), dimethyl sufoxide (1% v/v), and potassium
phosphate buffer (50 mM, pH 7.5) containing glycerol
(10% v/v). The reactions were performed in 1.5-ml
3
)
jejuni, and Streptomyces coelicolor. In this alternative
pathway, 5,8-dihydroxy-2-naphthoic acid (1,4-dihy-
droxy-6-naphthoic acid) was identified as an intermedi-
4
)
ate, instead of 1,4-dihydroxy-2-naphthoic acid. On the
other hand, 6,7-dihydroxy-2-naphthoic acid was report-
ed to exhibit inhibitory potency towards HIV-1 inte-
grase.5 Derivatives of 3,5-, 3,6-, and 3,7-dihydroxy-
naphthoic acids have been found to exhibit inhibitory
potency towards pp60c-src tyrosine kinase. Further-
more, dihydroxynaphthoic acids and their derivatives
should find use in numerous industrial applications,
ꢀ
microtubes at 30 C with vigorous shaking. High-
)
performance liquid chromatography (HPLC) analysis
was performed using a HPLC system (1100 series,
Agilent, Palo Alto, CA) with an XTerra MS C18 IS
column (4:6 mm ꢁ 20 mm; particle size, 3.5 mm; Waters,
6)
1
4)
Milford, MA), as described previously. In brief, the
reaction mixture was acidified by the addition of HCl
(pH 1.5–2), and was extracted with ethyl acetate (1 ml).
The extract (10 ml) was injected into the HPLC system.
Mobile phases A and B were composed of an acetoni-
trile/methanol/potassium phosphate buffer (10 mM,
pH 2.7) mixture at a ratio of 2.5:2.5:95 and of
acetonitrile, respectively. The samples were eluted with
7
,8)
including dyes and optical materials.
Cytochrome P450 monooxygenases (P450s) regio-
and stereoselectively introduce one oxygen atom derived
from molecular oxygen into organic compounds under
mild reaction conditions. P450s catalyze a variety of
reactions, including hydroxylation of aliphatic and
aromatic carbons, oxidation of organic nitrogen and
9
,10)
sulfur, epoxidation, and Baeyer-Villiger oxidation.
0% B for 3 min, followed by a linear gradient of 0% to
ꢂ1
Hence they are of considerable interest as biocatalysts
for the synthesis of industrial chemicals and pharma-
ceuticals.1
70% B for 9 min at a flow rate of 1 ml min
.
Compounds were detected spectrophotometrically at
220 and at 250 nm. The reaction products were isolated
by the HPLC system with a fraction collector (1200
series, Agilent) and an XTerra MS C18 column
1–13)
In this paper, we report a synthetic approach to
dihydroxynaphthoic acids using CYP199A2, a bacterial
y
To whom correspondence should be addressed. Fax: +81-3-3232-3889; E-mail: kkino@waseda.jp

Biocatalytic Synthesis of Dihydroxynaphthoic Acids
OH
2797
A
B
COOH
HO
COOH
COOH
+
2
-Naphthoic acid
7-Hydroxy-
8-Hydroxy-
2
-naphthoic acid
2-naphthoic acid
OH
OH
COOH
HO
COOH
1
-Hydroxy-
1,7-Dihydroxy-
2
-naphthoic acid
COOH
OH
2-naphthoic acid
HO
COOH
C
D
OH
3,7-Dihydroxy-
2-naphthoic acid
3
-Hydroxy-
2
2
-naphthoic acid
COOH
HO
HO
COOH
HO
6
-Hydroxy-
6,7-Dihydroxy-
2-naphthoic acid
-naphthoic acid
Fig. 1. Biocatalytic Oxidation of 2-Naphthoic Acid (A), 1-Hydroxy-2-naphthoic Acid (B), 3-Hydroxy-2-naphthoic Acid (C), and 6-Hydroxy-2-
naphthoic Acid (D) by CYP199A2.
(
4:6 mm ꢁ 250 mm; particle size, 3.5 mm; Waters), as
H-6 was observed, suggesting that the substrate was
oxidized at the C-7 position. In the HMBC spectrum,
H- C long-range couplings from H-5 to C-4, C-8a, and
C-7 were observed. Based on these observations, this
product was identified as 3,7-dihydroxy-2-naphthoic acid
(Fig. 1C). Furthermore, in the COSY spectrum of the
monooxygenated 6-hydroxy-2-naphthoic acid product
corresponding to a peak at 6.1 min (Fig. 2C), only one
1
4)
described previously. Mass analysis was performed
using a Thermo Finnigan LTQ FT (Waltham, MA) with
an electrospray ionization source. NMR analysis was
performed using a Bruker ADVANCE600 (Billerica,
MA).1 The protein concentration was measured using a
Coomassie protein assay kit (Pierce, Rockford, IL) with
1
13
1
4)
4)
1
8)
a bovine serum albumin standard. The P450 concen-
tration was measured based on CO-reduced difference
1
1
H- H coupling between H-3 and H-4 was observed,
suggesting that the substrate was oxidized at the C-7 or
ꢂ1
ꢂ1
spectra using an extinction coefficient of 91 mM cm
9)
at 450 nm.1
the C-8 position. In the HMBC spectrum, H- C long-
range couplings from H-5 to C-4, C-8a, and C-7 and from
H-8 to C-1, C-4a, and C-6 were observed. Based on these
observations, this product was identified as 6,7-dihy-
droxy-2-naphthoic acid (Fig. 1D).
1
13
We examined the oxidation activity of CYP199A2
towards 1-, 3-, and 6-hydroxy-2-naphthoic acids using
the whole cells. As shown in Fig. 2, HPLC analysis of
the reactions of CYP199A2 with 1-, 3-, and 6-hydroxy-
2
-naphthoic acids showed one major peak besides the
We also examined the oxidation activity of
CYP199A2 towards 7- and 8-hydroxy-2-naphthoic
acids, which are products of 2-naphthoic acid oxidation
by CYP199A2. CYP199A2 did not exhibit any activity
towards 7-hydroxy-2-naphthoic acid. In contrast, the
recombinant E. coli strain expressing CYP199A2 con-
sumed 60% of 8-hydroxy-2-naphthoic acid in 180 min,
and HPLC analysis of the reaction showed several small
peaks besides the substrate peak (data not shown). We
also confirmed that the recombinant E. coli cells carry-
ing the void vector did not consume 8-hydroxy-2-
naphthoic acid. We have reported that whole cells
expressing CYP199A2 produced 0.29 mM 7-hydroxy-2-
naphthoic acid and 0.44 mM 8-hydroxy-2-naphthoic acid
from 1 mM 2-naphthoic acid. The apparently insuffi-
cient conversion yield might be attributed partly to
further transformation of 8-hydroxy-2-naphthoic acid by
CYP199A2.
We examined the time courses of the synthesis of
1,7-, 3,7-, and 6,7-dihydroxynaphthoic acids by the
CYP199A2 whole-cell catalyst. As shown in Fig. 3A,
the CYP199A2 whole-cell catalyst (50 g of wet cells per
liter) converted 1 mM 1-hydroxy-2-naphthoic acid to
substrate peak. These new peaks were not detected
following reactions with recombinant E. coli cells
carrying the void vector without the CYP199A2 gene.
The compounds corresponding to the new peaks were
confirmed to be monooxygenation products of the
hydroxynaphthoic acids based on precise determination
of their mass values. Further, the products were analyzed
by H NMR, C NMR, and two-dimensional NMR
spectroscopy. In the correlation spectroscopy (COSY)
spectrum of the monooxygenated 1-hydroxy-2-naphthoic
acid product corresponding to a peak at 7.1 min in
Fig. 2A, H- H couplings between H-3 and H-4 and
between H-5 and H-6 were observed, suggesting that the
substrate was oxidized at the C-7 position. In the
heteronuclear multiple bond correlation (HMBC) spec-
trum, H- C long-range couplings from H-5 to C-4,
C-8a, and C-7 were observed. Based on these observa-
tions, this product was identified as 1,7-dihydroxy-2-
naphthoic acid (Fig. 1B). Similarly, in the COSY
spectrum of the monooxygenated 3-hydroxy-2-naphthoic
acid product corresponding to a peak at 6.3 min
1
13
1
1
1
7)
1
13
1
1
(
Fig. 2B), only one H- H coupling between H-5 and

2798
T. FURUYA and K. KINO
5
4
3
2
1
00
00
00
00
00
0
A
B
C
7
.1 min
8
.5 min
(Substrate)
5
10
10
10
15
15
15
min
4
00
00
6
.3 min
3
2
00
7
.7 min
(Substrate)
1
00
0
5
.1 min
min
3
00
6
2
00
00
0
6
.7 min
(
Substrate)
1
5
min
Fig. 2. HPLC Chromatograms of the Reactions of CYP199A2 with 1-Hydroxy-2-naphthoic Acid (A), 3-Hydroxy-2-naphthoic Acid (B), and
6-Hydroxy-2-naphthoic Acid (C).
Compounds were detected at 220 nm (A and B) and at 250 nm (C). Peaks at 7.1 min in A, at 6.3 min in B, and at 6.1 min in C were found to
correspond to 1,7-, 3,7-, and 6,7-dihydroxy-2-naphthoic acid respectively.
1
,7-dihydroxy-2-naphthoic acid in 60 min. The initial
rate of hydroxylation was estimated to be 26 mol (mol
studied bacterial P450 enzyme, P450cam, and the Y96F
mutant have shown hydroxylation rates of 0.7 and
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
ꢂ1
P450) min
Similarly, the CYP199A2 biocatalyst converted 1 mM
-hydroxy-2-naphthoic acid to 3,7-dihydroxy-2-naph-
(560 nmol (g of wet cells) min ).
99.7 mol (mol P450) min
respectively towards
2
0)
naphthalene in vitro, although there has been no report
concerning bacterial P450s that exhibit activities towards
naphthoic acids and hydroxynaphthoic acids, except for
3
thoic acid in 90 min (Fig. 3B). The initial rate of
hydroxylation was estimated to be 13 mol (mol
1
4)
CYP199A2 (and CYP199A1 ).
ꢂ1
ꢂ1
ꢂ1
ꢂ1
P450) min
(290 nmol (g of wet cells) min ).
Furthermore, the CYP199A2 biocatalyst produced
In conclusion, we found that CYP199A2 exhibited
oxidation activity towards three hydroxynaphthoic
acids. Whole cells expressing CYP199A2 efficiently
catalyzed the oxidation of 1-, 3-, and 6-hydroxy-2-
naphthoic acids to produce 1,7-, 3,7-, and 6,7-dihy-
droxynaphthoic acid respectively. It is interesting to
note that CYP199A2 regioselectively oxidized these
hydroxynaphthoic acids at the C-7 position, although
this enzyme generated two products, 7- and 8-hydroxy-
2-naphthoic acids, from 2-naphthoic acid. CYP199A2
provides a new, easy and environmentally friendly
synthetic approach to the dihydroxynaphthoic acids
from commercially available inexpensive hydroxynaph-
thoic acids. This efficient synthetic method might pave
the way to new industrial and pharmaceutical applica-
tions of these chemicals.
0
.70 mM 6,7-dihydroxy-2-naphthoic acid from 1 mM
-hydroxy-2-naphthoic acid in 180 min (Fig. 3C). The
6
initial rate of hydroxylation was estimated to be 4.9 mol
ꢂ1 ꢂ1 ꢂ1 ꢂ1
(
mol P450) min (110 nmol (g of wet cells) min ).
The conversion yield of 6,7-dihydroxy-2-naphthoic acid
was relatively low (70%). There is a possibility that an
additional reaction(s) might have occurred, since we
observed that the reaction generated a brown com-
pound(s) that was not extracted with ethylacetate.
The CYP199A2 whole-cell catalyst oxidized 1-, 3-,
and 6-hydroxy-2-naphthoic acids at rates of 26, 13, and
ꢂ1
ꢂ1
4
.9 mol (mol P450) min
respectively. Using the
same whole cells, the oxidation rate of 2-naphthoic acid
was previously estimated to be 13 mol (mol
P450) min
group adjacent to the carboxyl group on the hydroxy-2-
naphthoic acid molecules does not have unfavorable
effect on oxidation activity. For comparison, the well-
ꢂ1
ꢂ1 17)
. These results suggest that the hydroxyl
The data concerning mass and NMR analyses are
shown below.
1
1,7-Dihydroxy-2-naphthoic acid: H NMR (600 MHz,
[D6]DMSO): ꢀ ¼ 7:14 (dd, J ¼ 8:7, 2.0 Hz, 1H; H-6),

Biocatalytic Synthesis of Dihydroxynaphthoic Acids
2799
1
3
,7-Dihydroxy-2-naphthoic acid: H NMR (600 MHz,
D6]DMSO): ꢀ ¼ 7:17 (dd, J ¼ 8:8, 2.4 Hz, 1H; H-6),
7.19 (d, J ¼ 2:4 Hz, 1H; H-8), 7.24 (s, 1H; H-4), 7.65 (d,
1
.8
.6
.4
.2
0
A
B
C
[
0
0
0
0
1
3
J ¼ 8:8 Hz, 1H; H-5), 8.33 (s, 1H; H-1); C NMR
(
600 MHz, [D ]DMSO): ꢀ ¼ 110:3 (C-8), 111.8 (C-4),
6
1
1
1
½
16.0 (C-2), 123.2 (C-6), 128.3 (C-5), 128.8 (C-7),
31.1 (C-1), 132.8 (C-4a), 154.4 (C-8a), 154.7 (C-3),
0
72.6 (C-2 ); MS (ESI) (m=z): Calcd for C11H7O4
ꢂ
M ꢂ Hꢃ : 203.0344, found: 203.0346.
1
6
,7-Dihydroxy-2-naphthoic acid: H NMR (600 MHz,
0
0
0
30
60
90
120
150
300
Time (min)
[D6]DMSO): ꢀ ¼ 7:19 (s, 1H; H-5), 7.29 (s, 1H; H-8),
7
.65 (d, J ¼ 8:5 Hz, 1H; H-4), 7.69 (d, J ¼ 8:5 Hz, 1H;
1
.8
.6
.4
.2
0
13
H-3), 8.28 (s, 1H; H-1);
C NMR (600 MHz,
0
0
0
0
[
(
(
D6]DMSO): ꢀ ¼ 110:2 (C-5), 111.7 (C-8), 123.3
C-3), 125.8 (C-2), 126.5 (C-4), 128.6 (C-8a), 129.3
C-1), 132.1 (C-4a), 148.4 (C-7), 150.0 (C-6), 168.8 (C-
0
ꢂ
2 ); MS (ESI) (m=z): Calcd for C H O ½M ꢂ Hꢃ :
1
1
7
4
2
03.0344, found: 203.0345.
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Fig. 3. Synthesis of 1,7-Dihydroxy-2-naphthoic Acid (A), 3,7-Dihy-
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1
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1
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7
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1
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1
3
1
H; H-5); C NMR (600 MHz, [D6]DMSO): ꢀ ¼ 113:1
1
(
(
(
C-8), 116.6 (C-2), 126.0 (C-4), 128.6 (C-6), 129.5
C-3), 134.8 (C-8a), 137.3 (C-5), 139.0 (C-4a), 163.4
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ꢂ
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2
0
for C H O ½M ꢂ Hꢃ : 203.0344, found: 203.0346.
1
1
7
4