Hydroxylation of various molecules including heterocyclic aromatics using recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes

Article abstract of DOI:10.1016/S0040-4020(02)01253-X

Various molecules, in which heterocyclic aromatics are linked with phenyl or benzyl groups, were converted to their corresponding cis-dihydrodiols by recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes. Heterocyclic aromatic

Full text of DOI:10.1016/S0040-4020(02)01253-X

Tetrahedron 58 (2002) 9605–9612
Hydroxylation of various molecules including heterocyclic
aromatics using recombinant Escherichia coli cells expressing
modified biphenyl dioxygenase genes
c
d
e
d
Norihiko Misawa,a,b Kazutoshi Shindo, Haruko Takahashi, Hikaru Suenaga, Kazuo Iguchi,
,
*
Hiroshi Okazaki,a Shigeaki Harayamab and Kensuke Furukawae
aCentral Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
bMarine Biotechnology Institute, 3-75-1, Heita, Kamaishi-shi 026-0001, Japan
cDepartment of Food and Nutrition, Japan Women’s University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
dTokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
eGraduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan
Received 14 August 2002; accepted 27 September 2002
Abstract—Various molecules, in which heterocyclic aromatics are linked with phenyl or benzyl groups, were converted to their
corresponding cis-dihydrodiols by recombinant Escherichia coli cells expressing modified biphenyl dioxygenase genes. Heterocyclic
aromatic compounds with substituted phenyl or aliphatic moieties were also biotransformed to the hydroxylated products by the cells. Many
of the converted products were novel compounds. These compounds are potentially useful as versatile starting materials for the chemical
synthesis of pharmaceuticals and biologically active organic molecules. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
dioxygenase).13 These genes have frequently been
expressed in Escherichia coli, and used to oxidize a variety
of arenes to their corresponding cis-diols in significant
yields.5,6,10 The substrates for the toluene-dioxygenase-
mediated cis-dihydroxylation include a great number of
monocyclic aromatics with benzene skeleton, whose
hydrogen(s) is (are) substituted preferentially with smaller
group(s) such as methyl, ethyl, vinyl, carboxyl, and
halogenated groups.2,3 Polycyclic-fused-aromatic hydro-
carbons including naphthalene and phenanthrene have also
been transformed to their corresponding cis-diols by
naphthalene and phenanthrene dioxygenases.2,3,8,14 Linked
aromatics such as biphenyl and its chlorinated substituents
have been converted to the cis-diols by biphenyl dioxygen-
ase.3,9,13,15 cis-Diol formation from dicyclic (bicyclic)- or
tricyclic-fused heterocyclic aromatics (heteroaromatics)
such as quinoline, dibenzofuran and phenanthridine by
arene dioxygenase has also been reported.2,3,16 These
bioconversion experiments have been performed by world-
wide research groups for over three decades. However, no
reports exist that describe cis-diol formation from a
heterocyclic aromatic molecule, in which a heteroaromatic
ring is linked with a bulky group such as phenyl group.
Since the first report by Gibson’s group in 1968 that
cyclohexadiene-cis-diols were generated through microbial
oxidation of benzene and toluene,1 numerous cis-dihydro-
diols (cis-diols) have been produced. To date over two
hundred structurally diverse arene-cis-diols have been
generated by bacterial dioxygenation enzymes.2,3 Since
the late 1980’s these cis-diols have been shown to be used as
chiral synthons for the enantiospecific synthesis of versatile
natural products and therapeutic agents.2–7
The enzymes vital to dihydrodiol formation consist of
ferredoxin, ferredoxin reductase, and an iron–sulfur protein
composed of large (a) and small (b) subunits. Introduction
of both molecular oxygen atoms into an aromatic nucleus
occurs in an NAD(P)H-dependent manner.2,8 The genes
coding for the four components of the enzyme were isolated
first from Pseudomonas pseudoalcaligenes KF707 (bi-
phenyl dioxygenase),9 and then from many other bacterial
species including P. putida F1 (toluene dioxygenase),10
Pseudomonas sp. strain 9816-4 (naphthalene dioxygen-
ase),11 Nocardioides sp. strain KP7 (phenanthrene
dioxygenase),12 and Burkholderia cepacia LB400 (biphenyl
In this article we describe the bioconversion of a variety of
such heterocyclic aromatic molecules using the cells of an
E. coli transformant that carries biphenyl dioxygenase genes
modified with DNA shuffling. The resulting products from
the cis-dihydroxylation described should be important as
Keywords: heterocyclic aromatics; cis-dihydrodiol; biphenyl dioxygenase;
Escherichia coli.
*
Corresponding author. Tel.: þ81-193-26-6537; fax: þ81-193-26-6584;
e-mail: norihiko.misawa@mbio.jp
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01253-X

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N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
Table 1. Bioconversion of various aromatic compounds by several recombinant E. coli cells
Substrates
Plasmids
pKF6622 (BDO)
pHA171 (PDO)
pJHF3051 (TDO)
pKF2072 (modified BDO)
n-Butylbenzene
t-Butylbenzene
Biphenyl
4-Methoxybiphenyl
Diphenylmethane
1-Methoxynaphthalene
Anthracene
Phenanthrene
Fluorene
Dibenzofuran
Dibenzothiophene
Carbazole
Xanthene
–
–
–
–
–
77
65
100
58
16
9
42
–
–
48
89
63
–
35
–
–
–
–
–
72
14
75
64
72
–
13
–
–
–
–
–
–
49
87
100
68
94
62
13
–
–
13
8
–
11
–
–
–
–
–
Phenothiazine
Acridine
–
–
6
–
–
Phenanthridine
1-Phenylpyrrole
1-Phenylpyrazole
3-Methyl-1-phenylpyrazole
2-Phenylpyridine
4-Phenylpyrimidine
2-Phenylindole
2-Phenylbenzoxazole
2-Phenylbenzothiazole
3-Phenyl-1-indanone
2-Phenylquinoline
90
–
–
–
–
–
–
–
–
–
75
–
–
–
23
–
–
–
–
–
100
–
63
–
–
23
45
81
93
53
–
100
47
100
14
100
71
100
36
97
89
–
–
–
PDO, phenanthrene dioxygenase; TDO, toluene dioxygenase; BDO, biphenyl dioxygenase; the number denotes the conversion ratio (%).
chiral intermediates for the chemical synthesis of various
therapeutic agents and agrochemicals, since many of them
include heterocycles in their molecular structure.
amino acids compared with those of BphA1 (KF707) and
BphA1 (LB400), respectively. In this BphA1 (2072),
His255, Val258, Gly268, and Phe277 of BphA1 (KF707)
were changed to Gln255, Ile258, Ala268, and Tyr277 of
BphA1 (LB400), respectively.21
2. Results and discussion
2.2. Biotransformation of various aromatic compounds
with recombinant E. coli cells
2.1. Construction of evolved biphenyl dioxygenase genes
Three distinctive genes encoding arene dioxygenases exist.
These are the phdABCD genes encoding phenanthrene
dioxygenase (plasmid pHA171) derived from the marine
bacterium Nocardioides sp. strain KP7,12 the todC1C2BA
genes coding for toluene dioxygenase (plasmid pJHF3051)
derived from P. putida F1,22,23 and the bphA1A2A3A4 genes
encoding biphenyl dioxygenase (plasmid pKF6622) from P.
pseudoalcaligenes KF707.9,16 Bioconversion (biotrans-
formation) experiments were performed with various
aromatic compounds as substrates, using the E. coli cells
expressing the three arene dioxygenase-encoding genes as
well as the E. coli cells expressing the shuffled biphenyl
dioxygenase genes (plasmid pKF2072). The substrates and
cells were co-cultured for 2–3 days, after which the ratio of
the products converted from the substrates was measured by
high performance liquid chromatography (HPLC). Results
are shown in Table 1. E. coli (pHA171) preferentially
converted tricyclic-fused aromatics such as anthracene,
phenanthrene, and phenanthridine. The structures of the
converted products have been described previously.16 Both
strains of E. coli (pJHF3051) and E. coli (pKF6622) were
incapable of transforming tricyclic-fused aromatics except
for anthracene in the latter strain, but capable of converting
monocyclic aromatic hydrocarbons with one large sub-
stituent such as n-butyl benzene, t-butyl benzene, biphenyl,
The bphA1 gene encodes the large subunit (ion–sulfur
protein) of biphenyl dioxygenase from P. pseudoalcaligenes
KF707 and B. cepacia LB400. Shuffled bphA1 gene variants
were constructed by DNA shuffling of both the correspond-
ing genes, bphA1 (KF707) and bphA1 (LB400).17,18
Shuffled variants were digested with SacI and BglII, and
inserted into plasmid pJHF18DMulI just upstream of the
bphA2A3A4BC genes of P. pseudoalcaligenes KF707,17,19
and then introduced into E. coli. The resultant transformants
were assayed for the production of ring meta-cleavage
products (yellow) from polychlorinated biphenyls (PCBs).17
One was found that has the double-intensified meta-
cleavage activity compared with the E. coli strains carrying
bphA1 (KF707) and bphA1 (LB400) solely. This evolved
bphA1-carrying E. coli strain was also found to convert
benzene, toluene, and ethylbenzene, compared to the E. coli
strains expressing the bphA1 parent genes, which showed
negligible conversion.20 Plasmid DNA was prepared from
the cells including the evolved bphA1 gene, and then the
1.43 kb PpuMI fragment was eliminated to disrupt the bphB
and bphC genes. The resultant plasmid carrying the shuffled
(evolved) bphA1<bphA2A3A4 genes, which code for
holoenzyme of biphenyl dioxygenase, was designated
pKF2072. The amino acid sequences of the modified
BphA1 [BphA1 (2072)] was different by four and fifteen

N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
9607
Figure 1. Bioconversion of various heteroaromatic compounds, in which heterocyclic molecules are linked with phenyl group, using the cells of E. coli
carrying the modified biphenyl dioxygenase genes. The number below arrows shows the conversion ratio. All the converted products were novel compounds
except for 2.
and diphenylmethane. E. coli (pHA171) did not perform
these conversions. It is notable that a difference existed
between E. coli (pJHF3051) and E. coli (pKF6622) in their
ability to convert heterocyclic forms, in which hetero-
aromatic molecules are linked with phenyl group (below
1-phenylpyrrole in Table 1), the latter being able to convert
many of these compounds. E. coli (pKF2072) was found to
have an extended substrate specificity, in comparison with
the parent strain E. coli (pKF6622). Specifically, this E. coli
strain harboring pKF2072 transformed all of the hetero-
cyclic compounds (below 1-phenylpyrrole in Table 1)
significantly, including 1-phenylpyrazole, 2-phenyl-
pyridine, and 4-phenylpyrimidine that E. coli (pKF6622)
did not convert. These cells of E. coli(pKF2072) were used
for further experiments.
2.3. Biotransformation of heteroaromatic compounds
including phenyl or benzyl moieties
Biphenyl is a linked form of two phenyl groups. Although
not reported to date it could be considered that one phenyl
group linked with a heterocyclic-aromatic ring could also
act as a substrate for biphenyl dioxygenase. 1-Phenyl-
pyrrole, 1-phenylpyrazole, 3-methyl-1-phenylpyrazole,
2-phenylpyridine, 4-phenylpyrimidine, 2-phenylindole,
2-phenylbenzoxazole, 2-phenylbenzothiazole, 3-phenyl-1-
indanone, and 2-phenylquinoline, the structures of which
are shown in Fig. 1, were used as examples of such
substrates for bioconversion experiments. The structures of
products converted through the cells of E. coli (pKF2072)
were determined through HRMS (EI) and 1H and 13C NMR
Figure 2. Application of modified Mosher’s method in determination of the absolute configuration of (1S,2R)-3-(2-quinolyl)-3,5-cyclohexadiene-1,2-diol (10)
and (4R,5S)-2-(2-hydroxyphenyl)-4,5-dihydro-1,3-benzoxazole-4,5-diol (15). The assignments of H-5–H-8 in 10 were obscured by the overlappings of
naphthalene signals of 2NMA.

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N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
with previously reported NMR data.24 Only this compound,
among many of the examined heterocyclic compounds with
phenol or benzyl moiety, was converted not to cis-diol but to
a compound with hydroxyl group in a heterocycle ring by
the E. coli transformant. We consider that the pyrazole ring
may have stronger affinity for the active site facilitating an
oxygenation reaction in this enzyme compared with a
phenyl ring. It is likely that substitution of one methyl group
in pyrazole ring reduces its affinity as shown in an example
of 3.
2.4. Biotransformations of other heteroaromatic
compounds
We further examined the ability of the modified biphenyl
dioxygenase enzyme via bioconversion experiments.
Heteroaromatic compounds with phenyl moieties, in
which methyl or hydroxyl groups are substituted, and
heteroaromatic compounds with one aliphatic chain instead
of phenyl or benzyl moieties were used as substrates. These
substrates were converted to the hydroxylated forms, as
shown in Fig. 4.
Figure 3. Bioconversion of several heteroaromatic compounds, in which
heterocyclic molecules are linked with benzyl group, using the cells of E.
coli carrying the modified biphenyl dioxygenase genes. The number below
arrows shows the conversion ratio. These converted products were novel
compounds.
2.4.1. 2-p-Tolylpyridine. The molecular formula of the
product (14) was determined to be C12H11NO through its
analysis including 2D spectral analysis. All the products
except for the product derived from 1-phenylpyrazole were
the corresponding cis-1,2-dihydrodiol forms (Fig. 1).
Taking into account the characteristics of the dioxygenase
enzymes,3 the absolute diastereomeric forms of the products
are also illustrated in Fig. 1. The absolute stereochemistry of
3-(2-quinolyl)-3,5-cyclohexadiene-1,2-diol (10) (a conver-
sion product of 2-phenylquinoline) was determined as a
representative by 1H NMR analysis of diastereomeric esters
formed with (R) and (S)-methoxy-(2-naphthyl)acetic acid
(2NMA). D(dR2dS) Values are summarized in Fig. 2. The
sign of Dd are systematically arranged right and left sides to
the 2NMA planes. From these results, the absolute
configurations of C-10 and C-20 in 10 were demonstrated
to be S and R, respectively, as expected. It was also
surprising that heteroaromatic compounds, in which phenyl
group are linked with more bulky dicyclic-fused hetero-
aromatics, were efficiently converted (6–10) (Fig. 1).
Several heteroaromatic compounds including benzyl
moieties (1-benzylimidazole, 4-benzylisothiazole, and
2-benzylpyridine), instead of phenyl moieties, were also
examined in the same manner. The converted products were
identified as the corresponding cis-1,2-dihydrodiol forms as
shown in Fig. 3. These results show that the recombinant E.
coli (pKF2072) possessing the modified biphenyl dioxy-
genase enzyme is able to convert a broad range of
heteroaromatic compounds with phenol and benzyl moieties
into cis-diols.
1
HRMS (EI) and H and 13C NMR spectral data. In DQF
COSY spectrum of 14, signals due to p-tolyl moiety and
vicinal spin network H-4 (d 7.29)–H-5 (d 7.15)–H-6 (d
8.11) in the pyridine ring were observed. From these
The molecular formula of product (2) converted from
1-phenylpyrazole was determined to be C9H8N2O by
1
HRMS (EI) as well as H and 13C NMR spectral data. In
1
the H NMR spectrum, signals derived from the phenyl
moiety of 1-phenylpyrazole were completely preserved,
while only 2H signals of pyrazole ring was observed.
Consistent with its molecular formula, the replacement of a
phenolic OH function in pyrazole ring was proposed. The
position of the phenolic OH was determined to be C-4 by the
comparison of 13C NMR data of 2 with that of pyrazole
(Fig. 1). The structure of 2 was confirmed by the comparison
Figure 4. Bioconversion of several heteroaromatic compounds, in which
heterocyclic molecules are linked with substituted phenyl or aliphatic
groups, using the cells of E. coli carrying the modified biphenyl
dioxygenase genes. The number below arrows shows the conversion
ratio. 15, 17, and 18 were novel compounds.

N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
9609
findings, 14 was identified as 2-(4-methylphenyl)-3-pyridiol
(Fig. 4).
the biphenyl dioxygenase-mediated transformation of
aromatics to cis-diols has mainly been studied for
bioremediation of environmental pollutants, although
numerous conversion experiments have been performed
using toluene dioxygenases with an industrial purpose as
described in Section 1. This paper also describes in vitro
evolution of biphenyl dioxygenases through DNA shuffling,
which has extended their substrate specificity to hetero-
aromatic compounds. Similar approaches have been utilized
in the environmental field with PCB bioconversions.17,25–27
2.4.2. 2-(2-Hydroxyphenyl)benzoxazole. The molecular
formula of the product (15) was determined to be
C13H11NO4 through HRMS (EI) and 1H and 13C NMR
1
spectral data. Analysis by H–13C COSY and DQF COSY
spectra showed that 15 was dihydrodiol derivative of
benzoxazole ring. The 4,5-diol regiochemical assignment
1
was confirmed by the long range H–13C connectivities
observed between H-6 (d 5.95) and C-7a (d 145.5) and H-7
(d 6.57) and C-3a (d 135.8) in HMBC spectrum. Thus,
compound 15 was identified as 2-(2-hydroxyphenyl)-4,5-
dihydro-1,3-benzoxazole-cis-4,5-diol as shown in Fig. 4.
4. Experimental
4.1. General
1
The absolute stereochemistry of 15 was determined by H
NMR analysis of diastereomeric esters formed with (R) and
(S)-2NMA. D(dR2dS) values are summarized in Fig. 2. The
sign of Dd are systematically arranged right and left sides to
the 2NMA planes. From these results, the absolute
configurations of C-4 and C-5 in 15 were determined to
be R and S, respectively.
4.1.1. Plasmids, bacterial strains, and growth conditions.
Plasmids pHA171,12 pJHF3051,22,23 and pKF66229,16 were
described. E. coli BL21 (DE3)28 and E. coli JM10929 were
used as hosts for plasmid pHA171 and plasmids pJHF3051,
pKF6622, and pKF2072, respectively, and cultured in LB
medium29 or M9 medium29 at 308C or 378C. Ampicillin
(Ap) (50–150 mg/ml) was added when needed.
2.4.3. 3-n-Hexylthiophene. The molecular formula of the
product (16) was determined to be C10H18O2S through
HRMS (EI) and 1H and 13C NMR spectral data. Analysis by
1H, 13C and DQF COSY spectra determined that 16 was
4-hexyl-2,3-dihydro-2,3-thiophenediol as shown in Fig. 4.
4.1.2. General recombinant DNA techniques. Restriction
enzymes and T4 DNA ligase were purchased from Takara
Shuzo. DNA manipulation was done in E. coli as
described.29
2.4.4. 2-n-Butylbenzofuran. The molecular formulas of
both products (17, 18) were determined to be C12H14O2
4.1.3. DNA shuffling. The bphA1 genes were shuffled
between the corresponding genes of P. pseudoalcaligenes
KF707 and B. cepacia LB400 as described.17
1
through HRMS (EI) and H and 13C NMR spectral data.
Analyses by 1H, 13C and DQF COSY spectra revealed that a
phenolic OH function was replaced in benzofuran ring of
2-butylbenzofuran in 17 and 18. In the HMBC spectrum of
17, 1H–13C long range couplings from H-4 (d 7.24) to C-6
(d 152.4) and C-7a (d 155.4) were observed. Therefore, 17
was identified to be 2-butybenzo[b]furan-6-ol (Fig. 4). In
4.1.4. Chemicals and conversion experiments. E. coli
BL21 (DE3) harboring pHA171 or E. coli JM109 harboring
pJHF3051, pKF6622, or pKF2072 was grown in LB
medium containing 150 mg/ml of Ap at 308C with
reciprocal shaking (175 rpm) for 8 h. Five milliliters of
this culture was inoculated into 100 ml of M9 medium with
150 mg/ml of Ap, 10 mg/ml of thiamine, and 0.4% (w/v)
glucose in Erlenmeyer flask at 308C with reciprocal shaking
(175 rpm) for 16–17 h, of which the absorbance in OD
600 nm reaches approximately 1.1 mM (the final concen-
tration) of isopropyl b-D-thiogalactopyranoside (IPTG) was
added to the culture, and further cultivated for 4 h. The cells
were collected by centrifugation, washed once with M9
medium, and then resuspended in 100 ml of fresh M9
medium with 150 mg/ml of Ap, 10 mg/ml of thiamine, 0.4%
(w/v) glucose, and 1 mM (the final concentration) of IPTG,
along with 10 mg or 1 mM (the final concentration) of each
substrate, and cultivated in Erlenmeyer flask at 308C with
reciprocal shaking (175 rpm) for 2–3 days.
1
the HMBC spectrum of 18, H–13C long range couplings
from H-7 (d 7.21) to C-5 (d 152.1) and C-3a (d 129.7) were
observed. Therefore, 18 was determined to be 2-buty-
benzo[b]furan-5-ol (Fig. 4).
3. Conclusions
Enzyme-mediated formation of cyclohexadiene-cis-diols
from benzene rings in a wide range of heterocyclic-aromatic
compounds including phenyl and benzyl moieties has been
shown in this study for the first time. The enantiomerically
pure metabolites are easily prepared using the recombinant
bacterial cells that carry the modified biphenyl dioxygenase
genes, and purified by simple column chromatography, as
shown in Section 4. Such cis-diols including hetero-
aromatics seem to be very important as versatile starting
materials for the enantioselective chemical synthesis of
biologically active organic molecules, such as therapeutic
agents that include heterocycles in their molecular structure.
We have further shown the introduction of (a) hydroxyl
group(s) into heteroaromatic rings in several heteroaromatic
compounds including methyl- or hydroxyl-substituted
phenyl and aliphatic moieties. It has also been shown here
that the use of biphenyl dioxygenase is effective for the
synthesis of organic molecules of industrial profit. So far,
Substrates used in this study were purchased from Aldrich
Chemical Co., Wako Pure Chemical Co., or Kanto
Chemical Co. The respective substrates were dissolved in
small volume of ethanol and added to the culture.
4.1.5. Extractions and HPLC analysis of converted
products. To extract the converted products as well as the
substrates, an equal volume of methanol (MeOH) to
the cultured medium was added to the co-culture of the
transformed cells of E. coli, and mixed for 30 min. After

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N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
centrifugation to remove cells, the liquid phase was used for
high-pressure liquid chromatography (HPLC) analysis or
for further purification steps of the converted products. The
liquid phase (80 ml) was put through HPLC on a Puresil C18
column (4.6£250 mm, Waters) with a photodiode array
detector (model 996, Waters). It was developed at a flow
rate of 1 ml/min with solvent A (H2O–MeOH, 1:1) for
5 min, followed by a logarithm-shaped gradient (No. 3;
Waters) from solvent A to solvent B (MeOH–2-propanol,
6:4) for 15 min, and with solvent B for 13 min, and
monitored with max absorbance between 230–350 nm.
4.2.4. 3-(2-Pyridyl)-3,5-cyclohexadiene-cis-1,2-diol (4)
(product converted from 2-phenylpyridine). The crude
EtOAc extract (50 mg) was subjected to column chroma-
tography (CH2Cl2–MeOH¼40:1) to yield 10 mg of 4. MS
(EI) m/z 189 (Mþ). HRMS (EI) calcd for C11H11NO2 (Mþ),
1
189.0790; found 189.0793. H NMR (DMSO-d6) d: 4.34
(dd, 1H, J¼2.5, 5.5 Hz), 4.56 (d, 1H, J¼5.5 Hz), 5.89 (d,
1H, J¼10.2 Hz), 6.04 (ddd, 1H, J¼3.0, 5.5, 9.8 Hz), 6.92 (d,
1H, J¼5.5 Hz), 7.21 (dd, 1H, J¼4.9, 8.0 Hz), 7.63 (d, 1H,
J¼8.0 Hz), 7.75 (dd, 1H, J¼8.0, 8.0 Hz), 8.54 (d, 1H,
J¼4.9 Hz). 13C NMR (DMSO-d6) d: 65.2 (C-20), 71.0
(C-10), 119.7 (C-3), 122.1 (C-5), 123.4 (C-50), 124.0 (C-40),
135.5 (C-60), 137.7 (C-4), 138.1 (C-30), 149.3 (C-6), 156.7
(C-2).
4.2. Purification and identification of products
The liquid phase (1400 ml), which was obtained by the
procedure described above, was concentrated in vacuo, and
extracted with ethyl acetate (EtOAc) (500 ml£2). The
organic layer was concentrated in vacuo, and analyzed by
thin-layer chromatography (TLC) on silica gel (0.25 mm
E. Merck silica gel plates (60F-254)). The formed
products were purified through column chromatography
on silica gel (20£250 mm2, Silica Gel 60 (Merck)). Their
structures were analyzed by mass (MS) (MS (EI) and
HRMS (EI), JEOL DX303) and nuclear magnetic resonance
(NMR) (500 MHz, JEOL GX500) spectra. (R) and (S)-
2NMA esters were prepared in a manner reported by
Kusumi et al.30
4.2.5. 3-(4-Pyrimidinyl)-3,5-cyclohexadiene-cis-1,2-diol
(5) (product converted from 4-phenylpyrimidine). The
crude EtOAc extract (23.5 mg) was subjected to column
chromatography (CH2Cl2–MeOH¼30:1) to yield 6.6 mg of
5. MS (EI) m/z 190 (Mþ). HRMS (EI) calcd for C10H10N2O2
(Mþ), 190.0743; found 190.0751. 1H NMR (CDCl3) d: 4.54
(d, 1H, J¼6.0 Hz), 4.84 (d, 1H, J¼6.0 Hz), 6.16–6.24 (2H),
6.91 (d, 1H, J¼4.9 Hz), 7.52 (dd, 1H, J¼1.8, 5.5 Hz), 8.66
(d, 1H, J¼5.5 Hz), 9.11 (d, 1H, J¼1.8 Hz). 13C NMR
(CDCl3) d: 67.3 (C-20), 69.3 (C-10), 116.3 (C-5), 123.7
(C-50), 128.0 (C-40), 135.4 (C-30), 135.4 (C-60), 157.2 (C-4),
158.3 (C-2).
4.2.1. 3-(1H-1-Pyrrolyl)-3,5-cyclohexadiene-cis-1,2-diol
(1) (product converted from 1-phenylpyrrole). The
crude EtOAc extract (25 mg) was subjected to column
chromatography (hexane–EtOAc¼10:1) to yield 5.0 mg of
1. MS (EI) m/z 177 (Mþ). HRMS (EI) calcd for C10H11NO2
(Mþ), 177.0790; found 177.0791. 1H NMR (CDCl3) d: 4.44
(d, 1H, J¼6.1 Hz), 4.62 (ddd, 1H, J¼3.0, 3.0, 6.1 Hz), 5.71
(dd, 1H, J¼2.4, 9.8 Hz), 5.91 (d, 1H, J¼6.1 Hz), 5.97 (ddd,
1H, J¼2.4, 6.1, 9.8 Hz), 6.26 (dd, 2H, J¼2.4, 2.4 Hz), 6.99
(dd, 2H, J¼2.4, 2.4 Hz). 13C NMR (CDCl3) d: 68.3 (C-20),
70.70(C-10), 109.6 (C-40), 110.6 (C-3,4), 118.5 (C-2,5), 122.8
(C-5 ), 127.9 (C-60).
4.2.6. 3-(1H-2-Indolyl)-3,5-cyclohexadiene-cis-1,2-diol
(6) (product converted from 2-phenylindole). The crude
EtOAc extract (86 mg) was subjected to column chroma-
tography (hexane–EtOAc¼10:1) to yield 5 mg of 6. MS
(EI) m/z 227 (Mþ). HRMS (EI) calcd for C14H13NO2 (Mþ),
1
227.0947; found 227.0948. H NMR (DMSO-d6) d: 4.34
(2H), 4.70 (d, 1H, J¼5.5 Hz), 4.97 (d, 1H, J¼6.0 Hz), 5.78
(d, 1H, J¼9.2 Hz), 6.01 (ddd, 1H, J¼2.4, 5.5, 9.2 Hz), 6.50
(d, 1H, J¼5.5 Hz), 6.61 (s, 1H), 6.94 (dd, J¼7.9, 7.9 Hz),
7.060(dd, 1H, J¼7.9, 7.9 Hz). 13C NMR (DMSO-d6) d: 66.8
(C-2 ), 70.5 (C-10), 100.4 (C-3), 110.8 (C-7), 118.4 (C-40),
119.1 (C-5), 120.0 (C-4), 121.8 (C-6), 122.6 (C-50), 128.3
(C-3a), 131.0 (C-30), 133.0 (C-60), 137.3 (C-2), 137.7
(C-7a).
4.2.2. 1-Phenyl-4-hydroxy-pyrazole (2) (product con-
verted from 1-phenylpyrazole). The crude EtOAc extract
(55 mg) was subjected to column chromatography
(hexane–EtOAc¼2:1) to yield 7.0 mg of 2. MS (EI) m/z
160 (Mþ). HRMS (EI) calcd for C9H8N2O (Mþ), 160.0637;
4.2.7. 3-(1,3-Benzoxazol-2-yl)-3,5-cyclohexadiene-cis-
1,2-diol (7) (product converted from 2-phenylbenzoxa-
zole). The crude EtOAc extract (65.4 mg) was subjected to
column chromatography (CH2Cl2–EtOAc¼1:1) to yield
26.1 mg of 7. MS (EI) m/z 229 (Mþ). HRMS (EI) calcd for
C13H11NO3 (Mþ), 229.0739; found 229.0735. 1H NMR
(DMSO-d6) d:4.41 (m, 1H), 4.62 (dd, 1H, J¼5.5, 5.5 Hz),
4.97 (d, 1H, J¼5.5 Hz), 5.18 (d, 1H, J¼7.1 Hz), 6.08–6.15
(2H), 7.10 (d, 1H, J¼4.9 Hz), 7.33–7.40 (2H), 7.68 (dd, 1H,
J¼2.0, 6.7 Hz), 7.72 (dd, 1H, J¼2.0, 6.7 Hz). 13C NMR
(DMSO-d6) d: 64.2 (C-20), 70.4 (C-10), 110.5 (C-7), 119.6
(C-4), 121.9 (C-50), 125.4 (C-5), 126.5 (C-6), 126.5 (C-30),
129.1 (C-40), 139.4 (C-60), 141.6 (C-3a), 149.9 (C-7a), 162.7
(C-2).
1
found 160.0637. H NMR (CDCl3) d: 7.17 (m, 1H), 7.34–
7.38 (3H), 7.50–7.55 (3H). 13C NMR (CDCl3) d: 113.8
(C-5), 118.5 (C-20,60), 126.1 (C-40), 129.3 (C-30,50), 131.2
(C-3), 140.2 (C-10), 142.6 (C-4).
4.2.3. 3-(3-Methyl-1H-1-pyrazolyl)-3,5-cyclohexadiene-
cis-1,2-diol (3) (product converted from 3-methyl-1-
phenylpyrazole). The crude EtOAc extract (68 mg) was
subjected to column chromatography (CH2Cl2–
MeOH¼20:1) to yield 18 mg of 3. MS (EI) m/z 192 (Mþ).
HRMS (EI) calcd for C10H12N2O2 (Mþ), 192.0900; found
1
192.0898. H NMR (CDCl3) d: 2.28 (s, 3H), 4.54 (m, 1H),
4.81 (d, 1H, J¼6.0 Hz), 5.86 (dd, 1H, J¼3.0, 5.0 Hz), 6.00–
6.08 (2H), 6.15 (d, 1H, J¼2.5 Hz), 7.65 (d, 1H, J¼2.5 Hz).
13C NMR (CDCl3) d: 13.7 (5-CH3), 66.9 (C-10), 68.3 (C-20),
107.5 (C-4), 108.4 (C-40), 123.2 (C-50), 127.7 (C-60), 128.1
(C-3), 137.2 (C-30), 150.6 (C-5).
4.2.8. 3-(1,3-Benzothiazol-2-yl)-3,5-cyclohexadiene-cis-
1,2-diol (8) (product converted from 2-phenylbenzothia-
zole). The crude EtOAc extract (68 mg) was subjected to
column chromatography (CH2Cl2–EtOAc¼5:1) to yield
32.5 mg of 8. MS (EI) m/z 245 (Mþ). HRMS (EI) calcd for

N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
9611
1
C13H11NO2S (Mþ), 245.0511; found 245.0508. H NMR
(CDCl3) d: 4.51 (m, 1H), 5.00 (d, 1H, J¼6.1 Hz), 6.21 (dd,
1H, J¼4.9, 9.2 Hz), 6.26 (dd, 1H, J¼4.3, 9.2 Hz), 6.78 (d,
1H, J¼4.9 Hz), 7.34 (dd, 1H, J¼7.3, 7.3 Hz), 7.44 (dd, 1H,
J¼7.3, 7.3 Hz), 7.81 (d, 1H, J¼7.3 Hz), 0 7.94 (d, 1H,
J¼7.3 Hz). 13C NMR (CDCl3) d: 66.4 (C-1 ), 68.6 (C-20),
121.4 (C-7), 123.0 (C-4), 124.6 (C-60), 125.8 (C-6), 126.5
(C-5), 127.8 (C-40), 132.4 (C-30), 133.3 (C-50), 133.7 (C-7a),
153.0 (C-3a), 168.0 (C-2).
J¼5.5, 9.8 Hz), 8.42 (s, 1H), 8.70 0 (s, 1H). 13C NMR
(DMSO-d6) d: 30.8 (C-10), 67.8 (C-4 ), 68.5 (C-30), 120.0
(C-70), 123.0 (C-60), 129.5 (C-50), 137.3 (C-4), 140.7 (C-20),
145.6 (C-5), 158.8 (C-3).
4.2.13. 3-(2-Pyridylmethyl)-3,5-cyclohexadiene-cis-1,2-
diol (13) (product converted from 2-benzylpyridine).
The crude EtOAc extract (55 mg) was subjected to column
chromatography (CH2Cl2–MeOH¼50:1) to yield 5.6 mg of
13. MS (EI) m/z 203 (Mþ). HRMS (EI) calcd for
C12H13NO2 (Mþ), 203.0947; found 203.0947. 1H NMR
(DMSO-d6) d: 3.55–3.62 (2H), 3.82 (m, 1H), 4.03 (m, 1H),
5.57 (d, 1H, J¼4.9 Hz), 5.66 (dd, 1H, J¼3.0, 9.7 Hz), 5.80
(m, 1H), 7.21 (dd, 1H, J¼5.5, 6.0 Hz), 7.27 (d, 1H,
J¼7.6 Hz), 7.70 (ddd, 1H, J¼4.9, 6.0, 7.6), 8.46 0(d, 1H,
J¼5.5 Hz). 13C NMR (DMSO-d6) d: 42.0 (C-1 ), 68.3
(C-40), 68.7 (C-30), 120.06 (C-70), 121.4 (C-5), 123.2 (C-3),
123.2 (C-60), 129.6 (C-5 ), 136.6 (C-40), 140.4 (C-20), 148.9
(C-6), 159.9 (C-2).
4.2.9. 3-(cis-5,6-Dihydroxy-1,3-cyclohexadienyl)-1-inda-
none (9) (product converted from 3-phenyl-1-indanone).
The crude EtOAc extract (57 mg) was subjected to column
chromatography (CH2Cl2–EtOAc¼2:1) to yield 10 mg of
9. MS (EI) m/z 242 (Mþ). HRMS (EI) calcd for C14H14O3
(Mþ), 242.0943; found 242.0943. 1H NMR (CDCl3) d: 2.68
(dd, 1H, J¼3.1, 18.9 Hz), 3.01 (dd, 1H, J¼7.9, 18.9 Hz),
4.17–4.28 (3H), 5.70 (d, 1H, J¼4.9 Hz), 5.91 (m, 1H), 5.95
(m, 1H), 7.38 (dd, 1H, J¼7.3, 7.3 Hz), 7.45 (d, 1H,
J¼7.3 Hz), 7.58 (dd, 1H, J¼7.3, 7.3 Hz), 7.74 (d, 1H,
J¼7.3 Hz). 13C NMR (CDCl3) d:43.0 (C-3), 43.6 (C-2),
67.80 (C-10), 71.6 (C-20), 120.4 (C-40), 123.8 (C-4), 125.3
(C-5 ), 126.5 (C-7), 127.9 (C-5), 128.0 (C-60), 134.7 (C-6),
138.1 (C-3a), 142.7 (C-30), 156.1 (C-7a), 206.9 (C-1).
4.2.14. 2-(4-Methylphenyl)-3-pyridiol (14) (product con-
verted from 2-p-tolylpyridine). The crude EtOAc extract
(65 mg) was subjected to column chromatography (CH2Cl2)
to yield 9.0 mg of 14. MS (EI) m/z 185 (Mþ). HRMS (EI)
1
calcd for C12H11NO (Mþ), 185.0841; found 185.0838. H
4.2.10. 3-(2-Quinolyl)-3,5-cyclohexadiene-cis-1,2-diol
(10) (product converted from 2-phenylquinoline). The
crude EtOAc extract (55 mg) was subjected to column
chromatography (hexane–EtOAc¼10:1) to yield 12 mg of
10. MS (EI) m/z 239 (Mþ). HRMS (EI) calcd for
C15H13NO2 (Mþ), 239.0947; found 239.0947. 1H NMR
(CDCl3) d: 4.50 (dd, 1H, J¼3.0, 6.7 Hz), 5.08 (d, 1H,
J¼6.7 Hz), 6.23 (m, 1H), 6.78 (m 1H), 7.46 (dd, 1H, J¼6.7,
6.7 Hz), 7.49 (dd, 1H, 6.7, 6.7 Hz), 7.69 (d, 1H, J¼6.7 Hz),
7.73 (d, 1H, J¼6.7 Hz), 7.96 (d, 1H, J¼8.5 Hz), 8.07 (d, 1H,
J¼9.2 Hz). 13C NMR (CDCl3) d: 66.7 (C-10), 69.6 (C-20),
117.8 (C-3), 125.1 (C-40), 125.1 (C-50), 126.6 (C-7), 1260.8
(C-8a), 127.4 (C-8), 128.9 (C-5), 130.0 (C-6), 131.7 (C-6 ),
136.4 (C-30), 136.7 (C-4), 146.5 (C-4a), 157.1 (C-2).
NMR (DMSO-d6) d: 2.33 (s, 3H), 7.15 (dd, 1H, J¼4.3,
7.9 Hz), 7.21 (d, 2H, J¼7.9 Hz), 7.29 (d, 1H, J¼7.9 Hz),
7.91 (d, 2H, J¼7.9), 8.11 (d, 1H, J¼4.4 Hz), 10.06 (s, 1H).
13C NMR (DMSO-d6) d: 20.8 (10-CH3), 123.0 (C-5), 123.3
(C-4), 128.2 (C-20,60), 128.7 (C-30,40), 135.2 (C-40), 137.0
(C-10), 140.1 (C-6), 144.4 (C-2), 152.2 (C-3).
4.2.15. 2-(2-Hydroxyphenyl)-4,5-dihydro-1,3-benzoxa-
zole-cis-4,5-diol (15) (product converted from of 2-(2-
hydroxyphenyl)benzoxazole). The crude EtOAc extract
(40 mg) was subjected to column chromatography
(CH2Cl2–EtOAc¼10:1) to yield 7.8 mg of 15. MS (EI)
m/z 245 (Mþ). HRMS (EI) calcd for C13H11NO4 (Mþ),
1
245.0688; found 245.0691. H NMR (DMSO-d6) d: 4.50
(2H), 5.22 (d, 1H, J¼5.5 Hz), 5.33 (d, 1H, J¼6.7 Hz), 5.95
(d, 1H, J¼10.0 Hz), 6.57 (dd, 1H, J¼2.4, 10.0 Hz), 7.00 (dd,
1H, J¼7.3, 7.3 Hz), 7.04 (d, 1H, J¼8.6 Hz), 7.39 (dd, 1H,
J¼7.3, 8.6), 7.79 (d, 1H, J¼7.3 Hz), 10.92 (s, 1H). 130C
NMR (DMSO-d6) d: 64.4 (C-4), 71.3 (C-5), 111.0 (C-2 ),
112.5 (C-7), 116.9 (C-60), 120.0 (C-40), 126.0 (C-30), 132.3
(C-500), 135.8 (C-3a), 136.0 (C-6), 145.5 (C-7a), 156.2
(C-1 ), 158.8 (C-2).
4.2.11. 3-(1H-1-Imidazolylmethyl)-3,5-cyclohexadiene-
cis-1,2-diol (11) (product converted from 1-benzyl-
imidazole). The crude EtOAc extract (65 mg) was sub-
jected to column chromatography (CH2Cl2–MeOH¼7:1) to
yield 2.0 mg of 11. MS (EI) m/z 192 (Mþ). HRMS (EI)
calcd for C10H12N2O2 (Mþ), 192.0900; found 192.0889. 1H
NMR (DMSO-d6) d: 3.72 (m, 1H), 4.00 (m, 1H), 4.62 (d,
1H, J¼15.9 Hz), 4.77 (d, 1H, J¼15.9 Hz), 5.58 (d, 1H,
J¼5.5 Hz), 5.75 (dd, 1H, J¼3.0, 9.1 Hz), 5.82 (m, 1H), 6.89
(s, 1H), 7.010 (s, 1H), 7.061 (s, 1H). 13C NMR (DMSO-d6) d:
48.40 (C-1 ), 66.6 0(C-3 ), 68.2 (C-40), 120.5 (C-70), 122.3
(C-6 ), 131.4 (C-5 ), 137.8 (C-20).
4.2.16. 4-Hexyl-2,3-dihydro-2,3-thiophenediol (16) (pro-
duct converted from 3-n-hexylthiophene). The crude
EtOAc extract (37.5 mg) was subjected to column chroma-
tography (hexane–EtOAc¼5:1) to yield 10.0 mg of 16. MS
(EI) m/z 202 (Mþ). HRMS (EI) calcd for C10H18O2S (Mþ),
202.1028; found 202.1023. 1H NMR (CDCl3) d: 0.88 (t, 3H,
J¼7.3 Hz), 1.20–1.28 (6H), 1.45 (m, 2H), 2.16 (t, 2H,
J¼7.3 Hz), 4.53 (s, 1H), 5.54 (s, 10H), 5.82 (s, 1H). 13C NMR
(CDCl3) d: 13.8 (C-60), 22.6 (C-5 ), 27.8 (C-20), 28.8 (C-30),
29.2 (C-10), 31.7 (C-40), 79.5 (C-3), 81.7 (C-2), 117.8 (C-5),
137.3 (C-4).
4.2.12. 3-(4-Isothiazolylmethyl)-3,5-cyclohexadiene-cis-
1,2-diol (12) (product converted from 4-benzylisothia-
zole). The crude EtOAc extract (34 mg) was subjected to
column chromatography (CH2Cl2–MeOH¼40:1) to yield
8.0 mg of 12. MS (EI) m/z 209 (Mþ). HRMS (EI) calcd for
1
C10H11NO2S (Mþ), 209.0511; found 209.0505. H NMR
(DMSO-d6) d: 3.52 (d, 1H, J¼16.5 Hz), 3.62 (d, 1H,
J¼16.5 Hz), 3.78 (dd, 1H, J¼6.0, 6.0 Hz), 4.03 (m, 1H),
4.61 (d, 1H, J¼6.7 Hz), 4.66 (d, 1H, J¼6.0 Hz), 5.55 (d, 1H,
J¼5.5 Hz), 5.68 (dd, 1H, J¼3.0, 9.8 Hz), 5.80 (dd, 1H,
4.2.17. 2-Butylbenzo[b]furan-6-ol (17) and 2-butylbenzo-
[b]furan-5-ol (18) (products converted from 2-n-butyl-
benzofuran). The crude EtOAc extract (45 mg) was

9612
N. Misawa et al. / Tetrahedron 58 (2002) 9605–9612
Iglewski, B., Kaplan, S., Eds.; American Society for
Microbiology: Washington, DC, 1990; pp 121–132.
11. Gibson, D. T.; Resnick, S. M.; Lee, K.; Brand, J. M.; Torok,
D. S.; Wackett, L. P.; Schocken, M. J.; Haigler, B. E.
J. Bacteriol. 1995, 177, 2615–2621.
subjected to column chromatography (hexane–
EtOAc¼10:1) to yield 17 mg of 17 and 5 mg of 18.
17 MS (EI) m/z 190 (Mþ). HRMS (EI) calcd for C12H14O2
(Mþ), 190.0994; found 190.0991. 1H NMR (CDCl3) d: 0.91
(t, 3H, J¼7.3 Hz), 1.37 (m, 2H), 1.67 (m, 2H), 2.67 (m, 2H),
4.81 (s, 1H), 6.24 (s, 1H), 6.67 (dd, 1H, J¼2.4, 7.9 Hz), 6.88
(s, 1H), 7.24 (d, 10H, J¼7.9 Hz). 13C NMR (CDCl3) d: 13.8
(C-40), 22.1 (C-3 ), 28.1 (C-10), 29.8 (C-20), 98.1 (C-7),
101.5 (C-3), 111.4 (C-5), 120.2 (C-4), 122.6 (C-3a), 152.4
(C-6), 155.4 (C-7a), 158.9 (C-2).
12. Saito, A.; Iwabuchi, T.; Harayama, S. J. Bacteriol. 2000, 182,
2134–2141.
13. Erikson, B. D.; Mondello, F. J. J. Bacteriol. 1992, 174,
2903–2912.
14. Chun, H.-K.; Ohnishi, Y.; Misawa, N.; Shindo, K.; Hayashi,
M.; Harayama, S.; Horinouchi, S. Biosci. Biotechnol.
Biochem. 2001, 65, 1774–1781.
18 MS (EI) m/z 190 (Mþ). HRMS (EI) calcd for C12H14O2
15. Gibson, D. T.; Cruden, D. L.; Haddock, J. D.; Zylstra, G. J.;
Brand, J. M. J. Bacteriol. 1993, 175, 4561–4564.
16. Shindo, K.; Ohnishi, Y.; Chun, H. K.; Takahashi, H.; Hayashi,
M.; Saito, A.; Iguchi, K.; Furukawa, K.; Harayama, S.;
Horinouchi, S.; Misawa, N. Biosci. Biotechnol. Biochem.
2001, 65, 2472–2481.
1
(Mþ), 190.0994; found 190.0994. H NMR CDCl3) d:0.91
(t, 3H, J¼7.3 Hz), 1.37 (m, 2H), 1.67 (m, 2H), 2.67 (m, 2H),
4.58 (s, 1H), 6.23 (s, 1H), 6.76 (dd, 1H, J¼2.0, 8.0 Hz), 6.84
(d, 1H, J¼2.0 Hz), 7.21 (d, 1H, J¼8.0 Hz). 13C NMR
(CDCl3) d: 13.8 (C-40), 22.1 (C-30), 28.2 (C-10), 29.8 (C-20),
101.7 (C-3), 101.7 (C-4), 111.0 (C-7), 111.5 (C-6), 129.7
(C-3a), 149.8 (C-7a), 152.1 (C-5), 161.6 (C-2).
17. Kumamaru, T.; Suenaga, H.; Mitsuoka, M.; Watanabe, T.;
Furukawa, K. Nat. Biotechnol. 1998, 16, 663–666.
18. DNA sequence of bphA1 (KF707) and bphA1 (LB400) is
shown in DDBJ/Genbank accession number M83673 and
M86348, respectively.
Acknowledgements
19. DNA sequence of the bphA2A3A4BC genes from P.
pseudoalcaligenes KF707 is also available in DDBJ/Genbank
accession number M83673. bphA2, bphA3, and bphA4 encode
the iron–sulfur protein small subunit, ferredoxin, and
ferredoxin reductase of biphenyl dioxygenase, respectively.
20. Suenaga, H.; Mitsuoka, M.; Ura, Y.; Watanabe, T.; Furukawa,
K. J. Bacteriol. 2001, 183, 5441–5444.
We wish to thank Dr Paul D. Fraser (Royal Holloway
University of London) for careful reading of the manuscript.
We also wish to thank Hiroaki Ohno, Miki Hayashi, and
Yuko Miwa (Kirin Brewery Co., Ltd, Japan) for valuable
technical contributions. The toluene dioxygenase genes
included in plasmid pJHF3051 were kindly provided by Dr
D. T Gibson.
21. DNA sequence of the modified bphA1 gene (2072) has been
assigned DDBJ/Genbank accession number AB085748.
22. Zylstra, G. J.; Gibson, D. T. J. Biol. Chem. 1989, 264,
14940–14946.
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