FEMS MicrobiologyLetters 113 (1993) 297-302
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
297
FEMSLE 05672
Oxidation of carbazole to 3-hydroxycarbazole
by naphthalene 1,2-dioxygenase and biphenyl
2,3-dioxygenase
Sol M. R e s n i c k *, D a n i e l S. T o r o k 1 a n d D a v i d T. G i b s o n
The Department of Microbiology and Center for Biocatalysis, College of Medicine, The University of Iowa, Iowa City, 1.4 52242, USA
(Received 27 July 1993; revision received 18 August 1993; accepted 22 August 1993)
Abstract: Naphthalene 1,2-dioxygenasefrom Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenasefrom Beijerinckia sp. B8/36
oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole.Toluene dioxygenase from Pseudomonas putida F39/D did
not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis-dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenasefrom Pseudomonas sp.
NCIB 9816-4.3-Hydroxycarbazoleis presumed to result from the dehydration of an unstable cis-dihydrodiol.
Key words: Carbazole; Biotransformation; Naphthalene dioxygenase; Biphenyl dioxygenase; Pseudomonas
Introduction
Carbazole is the major tricyclic aromatic Nheterocyclic compound in coal tar creosote [1]
and has been detected as an environmental pollutant in both soil and groundwater [2,3]. Several
organisms have been reported to degrade carbazole [4-7] and the structures of some carbazole
degradation products have recently been reported [7].
Since bacterial dioxygenases often catalyze the
initial oxidation of aromatic hydrocarbons and
* Corresponding author. Tel: (319) 335-7980; Fax: (319) 3359999.
1 Present address: National Institutes of Health, Building 5,
Room B1 31, Bethesda, MD 20892, USA.
related heterocycles, we examined carbazole oxidation by strains containing toluene dioxygenase
( T D O ) from P s e u d o m o n a s p u t i d a
F39/D
( P p F 3 9 / D ) [8], naphthalene 1,2-dioxygenase
(NDO) from Pseudomonas sp. N C I B 9816-4 [9],
and biphenyl dioxygenase (BPO) from Beijerinckia sp. B 8 / 3 6 [10]. We report here the oxidation of carbazole to 3-hydroxycarbazole by strains
expressing N D O and BPO activity.
Material and Methods
Cultivation o f bacteria
Bacterial strains used in this study are listed in
Table 1. Strains 9816/11, P p F 3 9 / D and B 8 / 3 6
were grown in a mineral salts medium (MSB) [14]
298
(800 ml in 2.8-I Fernbach flasks) containing 0.2%
pyruvate at 30°C with shaking at 200 rpm. Their
respective dioxygenases, NDO, T D O and BPO,
were induced during the log phase of growth with
0.05% salicylate, toluene and m-xylene vapors.
Cultures were harvested in late log phase by
centrifugation, washed in MSB, and used for
transformation studies.
E. coli strains JM109(pDTG141) and JM
109(pKK223-3) were grown in a 5.0 1 Bioflo II
Fermentor (New Brunswick, Inc.) containing 4.0 1
MSB supplemented with 0.2% glucose, 100 mg
1-1 ampicillin, 1 mM thiamine, and 0.5 g 1 l
additional ammonium sulfate. Cultures were
grown with vigorous agitation and aeration (600
rpm, 2 1 a i r / m i n ) at 37°C. Additions of glucose,
to a concentration of 0.3%, were made at 2, 4, 5,
and 6 h and a pH of 7.2 was maintained by
addition of 10 N sodium hydroxide. When the
turbidity of the culture at 600 nm reached 8.010.0 (approximately 6 h growth), the temperature
was reduced to 30°C and isopropyl-/3-D-thiogalactoside (IPTG) and ferrous ammonium sulfate
were added to give concentrations of 50 mM and
0.03%, respectively. After 1.0 h IPTG-induction,
the cells were used in carbazole transformation
experiments described below.
Oxidation of carbazole
Induced ceils of strains 9816/11, B 8 / 3 6 and
P p F 3 9 / D were suspended in 800 ml of MSB to
give a turbidity of approximately 1.0 at 600 nm.
Pyruvate (0.1%) was provided as an energy source.
The cell suspension was transferred to a sterile
2.8-I Fernbach flask which contained 200 mg carbazole. Carbazole was delivered to the flask from
an acetone stock solution (10 g 1-~) and the
solvent was allowed to evaporate prior to the
addition of the cell suspension. The flask and its
contents were incubated with shaking (200 rpm)
at 30°C for 20 h at which time excess solid substrate was removed by filtration through glass
wool, and cells were removed by centrifugation
(9000 × g , 10 rain).
IPTG-induced cells of JM109(pDTG141) and
JM109(pKK223-3) (turbidity 10.0-13.0 at 600 nm)
were incubated with 0.025% carbazole for 20 h as
described above except that 0.2% glucose was
provided as an energy source.
Isolation and identification of carbazole oxidation
products
The clear supernatant solutions were extracted
four times with an equal volume of sodium hydroxide-washed ethyl acetate. The combined ethyl
Table 1
Bacterial strains used in this study
Strain
Relevant phenotype a
P. putida F39/D
Mutant which oxidizes toluene to (+)-cis-(1S,2R)dihydroxy-3-methylcyclohexa-3,5-diene(cis-toluene
dihydrodioD
Mutant which oxidizes biphenyl to ( + )-cis-(1S,2R)dihydroxy-3-phenylcyclohexa-3,5-diene(cis-biphenyl
dihydrodiol)
Mutant which oxidizes naphthalene to
( + )-cis-(1R,2S)-dihydroxy-l,2-dihydronaphthalene
(cis-naphthalene dihydrodiol)
JM109 containing structural genes for naphthalene
dioxygenase (nahAaAbAcAd) in pKK223-3; IPTGinducible; Amp r
JM109 containing the expression vector pKK223-3; tac
promoter; Amp r
Beijerinckia sp. B8/36
Pseudomonas sp. NCIB 9816/11
E. coli JM109(pDTG141)
E. coli JM109(pKK223-3)
Amp r, resistant to ampicillin.
Reference
8
10, 11
12
13
Pharmacia LKB Biotechnology
(Piscataway, N J)
299
were obtained with a Hewlett-Packard model 5970
mass selective detector. High resolution mass
spectra were recorded (by Dr. Lynn Teesch, HRMS facility, University of Iowa) on a VG ZAB-HF
mass spectrometer equipped with direct inlet
probe.
Proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker WM-360 spectrometer at 360.14 MHz
and 90.56 mHz, respectively, and are reported as
ppm with respect to TMS. Two-dimensional
NOESY NMR spectra were obtained on a Bruker
MSL-300 spectrometer at 300.17 MHz.
acetate solutions were dried over anhydrous
sodium sulfate and concentrated at 30°C under
reduced pressure.
Thin layer chromatography (TLC) of extracts
was performed on silica gel 60 F254 sheets (E.
Merck, no. 5735). The solvent was chloroformacetone (80:20). Compounds were visualized by
observing quenching of fluorescence under shortwave UV light (254 nm) and by exposure to
iodine vapor. Carbazole oxidation products were
isolated by preparative layer chromatography
(PLC) using multiple elution (2.0 mm silica, 5
times in chloroform-acetone [95:5]). Compounds
were extracted from the silica with chloroformmethanol (8:2).
The N-methyl derivative of carbazole and the
N-methyl, methyl-ether of its hydroxylated
metabolite were prepared by treatment with
sodium hydride in tetrahydrofuran followed by
addition of methyl iodide. After 4 h stirring, the
reactions were quenched by dropwise addition of
water, extracted with methylene chloride, dried
over sodium sulfate, and concentrated for analyses.
Gas chromatography-mass spectrometry (GCMS) was performed on a Hewlett-Packard (HP)
model 5890 gas chromatograph equipped with a
HP Ultra-1 capillary column (25 m × 0.2 mm with
0.33 /xm film thickness). The temperature program used was from 150-275°C at 10°C min -1.
Temperatures of the injection port and detector
were 220°C and 280°C, respectively, and helium
was the carrier gas (0.5 ml min-a). Mass spectra
S
Results and D i s c u s s i o n
Incubation of induced ceils of strains 9816/11
and B8/36 with carbazole led to the formation of
a neutral metabolite which gave an Rf value of
0.4 when analyzed by TLC. In contrast, tolueneinduced cells of strain P p F 3 9 / D did not oxidize
carbazole. The product formed by strains 9816/11
and B8/36 was purified by PLC to give a palebrown solid (6-10 mg 1-~ reaction) which was
chromatographically pure (TLC). GC-MS analysis
(Fig. 1) showed a single compound (R t 11.7 rain)
with a molecular ion (M +) at m / z 183 and
fragment ions at m / z 154 (M-29), 127 (M-56), 92
(M-91) and 77 (M-107) corresponding to possible
losses of COH or CHeN, C3H6N , CsHNO, and
C6HsNO , respectively. The accurate mass at m /
z 183 (calculated for Cl2H9ON was 183.0684,
4
183
lO0
o~
0f -
"o
t-
8O
8
60
N
9
1
.Q
<
.>_.
_~
M ÷ 1 83
4o
1154
I
20
II
o
I,
I
40
.11. . . .
=].
,
d
......
I,
.
I I
,,I
,
I
I
I
I
I
I
60
80
100
120
140
160
1/10
Mass/Charge
Fig. 1. Mass spectrum of hydroxycarbazole formed by naphthalene dioxygenase and biphenyl dioxygenase.
300
found 183.0695) identified the metabolite as a
hydroxycarbazole isomer. The ~H NMR spectra
of the hydroxycarbazole metabolites formed by
strains 9816/11 and B8/36 showed seven identical chemical shifts (6 ppm) and coupling constants (J in Hz) (assigned as numbered in Fig. 1)
in D6-acetone: 6.98 (dd, J = 8.6, 2.4 Hz, H-2),
7.10 (td, J = 7.5, 0.9 Hz, H-6), 7.30-7.40 (m, H-l,
H-7), 7.44 (d, J = 8.1 Hz, H-8), 7.53 (d, J = 2.4
Hz, H-4), 7.97 (d, J = 7 . 5 Hz, H-5). The H-9
proton signal was at 10.0 ppm (s) and the hydroxyl proton appeared as a broad singlet at 7.89
ppm and was dependent on sample concentration. The presence of a signal appearing as a
doublet (7.53 ppm) with a coupling constant of
2.4 Hz is indicative of an 'isolated proton' metacoupled with another aromatic proton. This suggested that the carbazole was substituted at either the 2- or 3-position with the signal of interest
arising from either the 1 or 4 proton, respectively.
The same hydroxycarbazole (HCZ) metabolite
was formed by the NDO expressed by
JM109(pDTG141). The involvement of NDO in
the reaction was confirmed in a separate experiment where JM109(pKK223-3), incubated under
identical conditions, showed no transformation
products from carbazole. Extraction of 4.0 1 of
the JM109(pDTG141) culture filtrate with ethyl
acetate followed by PLC resulted in the isolation
of a tan solid (60 rag) with identical properties to
the metabolite described above. In addition, the
~3C NMR for the compound in D6-acetone
showed signals at 6 105.6 (CH), 114.4 (CH), 111.9
(CH), 115.6 (CH), 118.7 (CH), 120.6 (CH), 123.6
(C), 124.5 (C), 125.9 (CH), 135.1 (C), 141.6 (C),
151.4 (C-O). The 13C NMR chemical shifts for
authentic 2-HCZ (D0-acetone) were 96.81 (CH),
108.7 (CH), 110.6 (CH), 116.6 (C), 118.9 (CH),
119.1 (CH), 120.9 (CH), 123.8 (C), 124.1 (CH),
140.2 (C), 141.9 (C), 156.8 (C-O). The difference
between carbon resonances, in conjunction with
the ]H NMR data suggested that the carbazole
metabolite was 3-HCZ.
To conclusively determine the position of the
hydroxyl group, the N-methyl-methoxy derivative
of the NDO-metabolite was synthesized and analyzed by NMR and GC-MS. The latter procedure
gave a M + at m / z 211 which corresponds to the
O-Me
5
6
4
N Nte
OCHa
2,s~
5
7
8
8
N
I
CH3
/
,
3.0
3.5
4.0
4.5
5.0
5.5
6.0
J
i :
i
pi
I
T
L
7,5
7.0
6.5
6.0
5.5
5,0
4.5
7.0
7.5
i~i
8.0
PPM
t IliJIJla*lllllllllJIJIJlJ,lJ*llll=JllIJIJllllllJJJlll
8.0
6.5
4.0
3.5
3.0
PPM
F i g . 2. I H N M R
spectrum
(top) and two-dimensional
NOESY
NMR (bottom) of the dimethylderivativeof 3-hydroxycarbazole formed by naphthalene dioxygenase.Correlations are
discussed in the text.
dimethyl derivative, N-methyl-methoxycarbazole.
The 1H NMR (C6D 6) showed signals (assigned as
numbered in Fig. 2) at ~ 3.02 (s, 3H, N-Me), 3.56
(s, 3H, O-Me), 6.93 (d, J = 8.8 Hz, H-l), 7.04 (d,
J = 8.2 Hz, H-8), 7.20-7.25 (m, H-2, H-6), 7.41 (t,
J = 8.2 Hz, H-7), 7.62 (d, J = 2.3 Hz, H-4), 8.03
(d, J = 7.8 Hz, H-5). The results of a 2-dimensional NOESY NMR experiment (for the detection of ~H-1H interactions 'through-space') are
shown in Fig. 2. The correlations between the
N-9-methyl signal (3.02 ppm) and the adjacent
H-1 and H-8 signals (6.93 and 7.04 ppm, respectively), and between the O-methyl signal (3.56
301
Grant T32 GM8365 awarded by the National
Institute of General Medical Sciences.
N
1%
References
OH OH
OH
N
"DJhydrodJol"
N
3-Hydroxycarbazole
Fig. 3. Proposed reaction for the formation of 3-hydroxycarbazole by naphthalene dioxygenase and biphenyl dioxygenase.
ppm) and the H-2 signal (7.2 ppm) and 'isolated'
proton signal (7.62 ppm) confirm the structure of
the metabolite as 3-HCZ. A dihydrodiol dehydrogenase mutant of the PCB-degrading Pseudomonas sp. LB400 also oxidized carbazole to
3-HCZ.
An explanation for the formation of 3-HCZ by
NDO and BPO, based on previous studies [1517], involves initial dioxygenation of carbazole to
form an unstable cis-carbazole-3,4-dihydrodiol
which undergoes specific loss of water to yield
3-HCZ (Fig. 3). However, since no carbazole dihydrodiol was detected, the possibility of direct
monooxygenation cannot be eliminated. Synthetic
3-HCZ has been prepared for determination of
potential anticancer activity [18]; hence, the biotransformation of carbazole to 3-HCZ is of interest since it provides a direct route to 3-HCZ.
Acknowledgements
This work was supported by U.S. Public Health
Service Grant GM29909 from the National Institute of General Medical Sciences. S.M.R. is the
recipient of a Predoctoral Fellowship in Biotechnology from U.S. Public Health Service Training
1 Mueller, J.G., Chapman, P.J. and Pritchard, P.H. (1989)
Creosote-contaminated sites: Their potential for bioremediation. Environ. Sci. Technol. 23, 1197-1201.
2 Goerlitz, D.F., Troutman, D.E., Godsy, E.M. and Franks,
B.J. (1985) Migration of wood-preserving chemicals in
contaminated groundwater in a sand aquifer at Pensacola,
Florida. Environ. Sci. Technol. 19, 955-961.
3 Mueller, J.G., Middaugh, D.P., Lantz, S.E. and Chapman,
P.J. (1991) Biodegradation of creosote and pentachlorophenol in contaminated groundwater: Chemical
and biological assessment. Appl. Environ. Microbiol. 57,
1277-1285.
4 Finnerty, W.R., Shockley, K. and Attaway, H. (1983) Microbial desulfurization and denitrogenation of hydrocarbons. In: Microbial enhanced oil recovery (Zajic, J.E. et
al., Eds.), pp. 83-91. PennWell Books, Tulsa, OK.
5 Grosser, R.J., Warshawsky, D. and Vestal, J.R. (1991)
Indigenous and enhanced mineralization of pyrene,
benzo[a]pyrene, and carbazole in soils. Appl. Environ.
Microbiol. 57, 3462-3469.
6 Gieg, L.M., Fedorak, P.M. and Barker, J.F. (1993)
Metabolic characteristics of a carbazole-degrading bacterium. Abstr. 93rd Gen. Meet. Amer. Soc. for Microbiol.,
Atlanta, GA Q-114, p. 367.
7 Ouchiyama, N., Zhang, Y., Omori, T. and Kodama, T.
(1993) Biodegradation of carbazole by Pseudomonas spp.
CA06 and CA10. Biosci. Biotech. Biochem. 57, 455-460.
8 Gibson, D.T., Hensley, M., Yoshioka, H. and Mabry, T.J.
(1970) Formation of (+)-cis-2,3-dihydroxy-l-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida.
Biochemistry. 9, 1626-1630.
9 Yen, K.-M. and Serdar, C.M. (1988) Genetics of naphthalene catabolism in Pseudomonads. CRC Crit. Rev. Microbiol. 15,247-267.
10 Gibson, D.T., Roberts, R.L., Wells, M.C. and Kobal, V.M.
(1973) Oxidation of biphenyl by a Beijerinckia species.
Biochem. Biophys. Res. Commun. 50, 211-219.
11 Ziffer, H., Kabuto, K., Gibson, D.T., Kobal, V.M. and
Jerina, D.M. (1977) The absolute stereochemistry of several cis-dihydrodiols microbially produced from substituted benzenes. Tetrahedron 33, 2491-2496.
12 Jerina, D.M., Daly, A.M., Jeffrey, A.M. and Gibson, D.T.
(1971) cis-l,2-Dihydroxy-l,2-dihydronaphthalene: A bacterial metabolite from naphthalene. Arch. Biochem. Biophys. 142, 394-396.
13 Suen, W.-C. (1991) Gene expression of naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4 in Escherichia coli. Ph.D. dissertation, The University of Iowa,
Iowa City, IA.
14 Stanier, R.Y., Palleroni, N.J. and Doudoroff, M. (1966)
302
The aerobic pseudomonads; a taxonomic study. J. Gen.
Microbiol. 43, 159-271.
15 Ensley, B.D., Ratzkin, B.J., Osslund, T.D., Simon, M.J.,
Wackett, L.P. and Gibson, D.T. (1983) Expression of
naphthalene oxidation genes in Escherichia coli results in
the biosynthesis of indigo. Science 222, 167-169.
16 Klecka, G.M. and Gibson, D.T. (1979) Metabolism of
dibenzo[1,4]dioxan by a Pseudomonas species. Biochem. J.
180, 639-645.
17 Laborde, A.L. and Gibson, D.T. (1977) Metabolism of
dibenzothiophene by a Beijerinckia species. Appl. Environ. Microbiol. 34, 783 790.
18 Langendoen, A., Koomen, G.-J. and Pandit, U.K. (1988) A
process for preparing hydroxyl derivatives of compounds
containing a carbazole, dibenzofurane or dibenzothiophene group. Eur. Pat. Appl. EP 257,701