Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR

Article abstract of DOI:10.1023/A:1008321610465

Spectrophotometric assays of Mycobacterium aurum MO1 cells extracts gave evidence of a soluble cytochrome P450, involved in the degradative pathway of morpholine, a waste product from the chemical industry. In order to get further information, the kinetics of the biodegradation of the sulfur analogue thiomorpholine was monitored by using in situ nuclear magnetic resonance (NMR). This technique allowed the identification of two intermediates: the sulfoxide of thiomorpholine resulting from S-oxidation and thiodiglycolic acid owing to ring cleavage. The S-oxidation (S ← SO) represents one of the well-known reactions catalyzed by cytochromes P450. The inhibitory effect of metyrapone, a cytochrome P450 inhibitor, on the thiomorpholine and morpholine degradative abilities of M. aurum MO1 confirmed the involvement of a cytochrome P450. These results and the decrease of the rate of formation of the first intermediate during the morpholine degradation, 2-(2-aminoethoxy) acetate, proved the key role of the cytochrome P450 in the early events of the biodegradation, i.e, in the C - N bond cleavage.

Full text of DOI:10.1023/A:1008321610465

Biodegradation 9: 433–442, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
433
Thiomorpholine and morpholine oxidation by a cytochrome P450
in Mycobacterium aurum MO1. Evidence of the intermediates by
in situ 1H NMR
Bruno Combourieu1, Pascal Poupin2, Pascale Besse1, Martine Sancelme1, Henri Veschambre1,
Nicole Truffaut2 & Anne-Marie Delort1,∗
1 Laboratoire de Synthe`se, Electrosynthe`se et Etude de Syste`mes a` Inte´rêt Biologique, UMR 6504 CNRS,
Universite´ Blaise Pascal, 63177 Aubie`re Cedex, France; 2Laboratoire de Ge´ne´tique Microbienne, Universite´
Technologique de Compie`gne, B.P. 649, 60206 Compie`gne, France
(∗author for correspondence, e-mail: amdelort@chimtp.univ-bpclermont.fr)
Key words: cytochrome P450, in situ 1H NMR, metyrapone, Mycobacterium, morpholine, thiomorpholine
Abstract
Spectrophotometric assays of Mycobacterium aurum MO1 cells extracts gave evidence of a soluble cytochrome
P450, involved in the degradative pathway of morpholine, a waste product from the chemical industry. In order to
get further information, the kinetics of the biodegradation of the sulfur analogue thiomorpholine was monitored by
using in situ nuclear magnetic resonance (NMR). This technique allowed the identification of two intermediates:
the sulfoxide of thiomorpholine resulting from S-oxidation and thiodiglycolic acid owing to ring cleavage. The
S-oxidation (S → SO) represents one of the well-known reactions catalyzed by cytochromes P450. The inhibitory
effect of metyrapone, a cytochrome P450 inhibitor, on the thiomorpholine and morpholine degradative abilities of
M. aurum MO1 confirmed the involvement of a cytochrome P450. These results and the decrease of the rate of
formation of the first intermediate during the morpholine degradation, 2-(2-aminoethoxy) acetate, proved the key
role of the cytochrome P450 in the early events of the biodegradation, i.e, in the C—N bond cleavage.
Introduction
Knapp & Brown 1988; Mazure & Truffaut 1994;
Poupin et al. 1998) or Arthrobacter (Dmitrenko et al.
1987), and more recently with Gram-negative bacte-
ria (Knapp et al. 1996). The biodegradative pathway
was not clearly established because no tool for direct
detection of the intermediates was available. How-
ever, in a recent work (Combourieu et al. 1998), two
metabolites of this pathway in Mycobacterium aurum
MO1 have been proven by using in situ 1H NMR
spectroscopy, which allows direct determination and
quantification of the intermediary compounds formed.
The identification of 2-(2-aminoethoxy) acetate and
glycolate as intermediates was in agreement with the
pathway suggested by Swain for M. chelonae (Swain
et al. 1991).
The heterocyclic, secondary amine morpholine,
mainly used in the manufacture of rubber additives
but with an ever-widening range of applications (cat-
alyst, solvent, antioxidant, ... ) (Mjos 1978; Anon
1989), is regularly released, via industrial effluents,
into the environment. Its presence in water, and even
in foods (Singer & Lijinski 1976), is of particu-
lar concern owing to its potential sensitivity towards
N-nitrosation producing the potent mutagen and car-
cinogen N-nitrosomorpholine (Anon 1989; Enzmann
et al. 1995).
In the literature, many research works have been
reported on the biodegradation of morpholine with
strains of the genera Mycobacterium (Cech et al. 1988;
Dmitrenko & Gvozdyak 1988, Knapp et al. 1982,
In order to understand the mechanism of this
biodegradative pathway in M. aurum MO1, we were

434
thus interested in giving evidences of the type of
enzymes involved.
dized. The 1H NMR technique, performed directly on
the incubation medium in H2O, allowed to prove the
formation of an oxidized product and to quantify it,
showing its possible involvement in the thiomorpho-
line degradation. The use of a cytochrome P450 in-
hibitor, metyrapone, added to the incubation medium
containing thiomorpholine or morpholine, showed an
inhibitory effect on the degradative properties of M.
aurum MO1, providing another proof of the key role
played by this enzyme in the biotransformations of the
two compounds.
Knapp (Knapp et al. 1982) and Mazure (Mazure
& Truffaut 1994) have shown that morpholine degra-
dation was associated with oxygen consumption, indi-
cating the possibility for involvement of a monooxy-
genase. Monooxygenases are widely distributed en-
zymes which catalyze dioxygen activation towards the
insertion of one oxygen atom from O2 into a sub-
strate, the other one being reduced into water. A great
number of these monooxygenases contain a heme pro-
tein called cytochromeP450. These cytochromes P450
are involved in various steps of the biosynthesis of
endogenous compounds (steroids, fatty acids...) (Wa-
terman et al. 1986; Jefcoate 1986) and in the oxidative
detoxification and elimination of many hydrophobic
xenobiotics (pollutants, drugs, pesticides ... ) (Wis-
locki et al. 1980, Guengerich 1990a). Because of
their wide distribution in living organisms and their
very important role in biochemistry, pharmacology
and toxicology, cytochromes P450 have been exten-
sively studied (Guengerich 1990b; Ortiz de Montel-
lano 1986; Ruckpaul & Rein 1984, 1990). Several bac-
terial cytochromes P450 (from Pseudomonas putida,
Streptomyces griseus, Bacillus megaterium) whose
cristalline structure was known, were used as models
for human cytochromes P450 (Fulco 1991, Asperger
& Kleber 1991). Several other bacteria genera also
contain cytochromes P450, for example Rhodococcus,
a genus very close to Mycobacterium (Asperger et al.
1990, Karlson et al. 1993, Nagy et al. 1995). Recently,
we demonstrated the involvement of a cytochrome
P450 in morpholine biodegradation by a strain of My-
cobacterium (Poupin and Truffaut 1996; Poupin et al.
1998). This prompted us to investigate the possible
role of cytochrome P450 in biodegradative abilities of
xenobiotics in Mycobacterium aurum MO1.
Materials and methods
Chemicals. Thiomorpholine, morpholine, metyrapone
and m-chloroperbenzoïc acid were purchased from
Aldrich Chemical (Sigma Aldrich Sarl, St. Quentin
Fallavier, France) and tetradeuterated sodium tri-
methylsilylpropionate (TSPd4) was purchased from
EurisoTop (St. Aubin, France).
Growth conditions. M. aurum MO1 cultures were
grown in 100 ml of Trypcase soy broth (bioMerieux,
Marcy l’Etoile, France) in 500-ml Erlenmeyer flasks
◦
incubated at 30 C with agitation at 200 rpm. They
were harvested after 48h of culture. For spectropho-
tometric assays, M. aurum MO1 was grown in min-
eral salts medium (Knapp buffer which contains per
liter of deionized water: KH2PO4 1 g, K2HPO4 1 g,
FeCl3.6H2O 4 mg and MgSO4.7H2O 40 mg, pH 6.6)
amended with 10 mM morpholine or 20 mM acetate.
When thiomorpholine was used for these experiments,
the cells were grown in rich medium, harvested by
centrifugation (8.000 × g, 10 min), and washed once
with mineral salts medium. They were then resus-
pended in this medium containing 5 mM thiomorpho-
line and incubated during 6 h in a gyratory shaker at
30 ◦C (200 rpm).
In this work, we bring evidence of the induction of
a soluble cytochrome P450 in M. aurum MO1 in the
presence of morpholine and thiomorpholine by clas-
sical spectrophotometric assays. In order to prove its
key role in the degradative pathways of xenobiotics
by this bacterium, we have studied first, by in situ 1H
NMR, the biodegradation of thiomorpholine, a sulfur
analogue of morpholine. M. aurum MO1 do not grow
on thiomorpholine (Mazure & Truffaut 1994) but is
able to degrade it. Thus, the accumulation of metabo-
lites was expected. Their identification should allow
us to have a better understanding of the biodegrada-
tive pathway of heterocyclic xenobiotics. Moreover,
the sulfur atom of thiomorpholine can be easily oxi-
Incubation with xenobiotics. Cells were har-
vested by centrifugation at 9,000 × g for 15 min
◦
at 5 C. The supernatant was eliminated, the pellet
was washed twice with Knapp buffer and finally re-
suspended in this buffer (5 g of wet cells in 50 ml
of buffer). The cells were incubated with 10 mM of
thiomorpholine or morpholine as the only source of
◦
energy in a 500 ml Erlenmeyer at 30 C with agita-
tion (200 rpm). Metyrapone (5 or 10 mM) was added
for the assays of inhibition. Incubation of cells under
the same conditions in the absence of substrate con-
stituted a negative control, as did incubation of the
substrate in the buffer without cells. Samples (1 ml)

435
were taken every hour for 12 h and from time to time
for 72 h. They were centrifuged at 12,000 × g for 5
min. The supernatants were isolated and immediately
frozen until NMR analysis.
Spectrophotometric analysis of a cytochrome
P450. Mycobacterial cells were harvested by centrifu-
gation (8,000 × g, 10 min) at 4 ◦C, washed and
resuspended in 50 mM phosphate buffer (pH 7.4) con-
taining 0.3 mM phenylmethylsulfonyl fluoride. Bac-
terial walls were broken by three passages through
a French pressure cells (SLM-Aminco) at 18,000 psi
and centrifuged at 30,000 × g at 4 ◦C for 30 min. The
obtained supernatant was reduced by sodium dithion-
ite, divided equally between two optically matched
cuvettes and the difference spectra was recorded in the
presence of CO (Peterson & Lu 1991). An extinction
coefficient of 91 mM−1.cm−1 was used to determine
the content of cytochrome P450 in crude extracts
(Omura & Sato, 1964). All photometric analyses were
determined using a Shimadzu UV 160A spectropho-
tometer. The protein concentration was determined by
the Bradford method (Bradford 1976).
Figure 1. Carbon monoxide difference spectra of cell extracts. Cul-
tures of M. aurum were grown with morpholine (spectrum A),
acetate (spectrum B), or induced by thiomorpholine (spectrum C);
the protein contents were 10, 10, and 11.5 mg/ml, respectively.
1H NMR spectroscopy.
Preparation of NMR samples: The supernatant (540
µl) was supplemented with 60 µl of a 8 mM solu-
tion of TSPd4 in D2O and adjusted to pH 10 with
4N NaOH. pH adjustment avoided changes in chem-
ical shifts. D2O was used for locking and shimming.
TSPd4 constituted a reference for chemical shift (0
ppm) and quantification.
Synthesis of the sulfoxide of thiomorpholine
(Madesclaire 1986). To a solution of thiomorpholine
(500 mg, 4.85 mmol) in dichloromethane (50 ml),
◦
stirred and cooled at −15 C, was added dropwise a
1H NMR spectra: 1H NMR spectra were performed
at 300.13 MHz with an Avance 300 DSX or at 400.◦13
MHz with a 400 AC Bruker spectrometers at 21 C
with 5 mm-diameter tubes containing 500 µl of sam-
ple; water was suppressed by saturation with a clas-
sical NOE Bruker program. 64 scans were collected.
No filter was applied before Fourier transformation
but a baseline correction was performed on spectra
before integration with Bruker software. Under these
conditions, the detection limit was in the range of 0.05
mM.
solution of m-chloroperbenzoïc acid (1.05 eq., 1 g)
in dichloromethane (25 ml). The solution was stirred
for another 3 h. The solvent was then removed and
the crude residue was purified by flash column chro-
matography (eluent: methanol/ethyl acetate 93/7). The
sulfoxide was obtained in 70% yield.
IR: Broad band 1070–1130 cm−1; MS (EI): 119
(M+.); 102; 71; 56.
NMR spectroscopy: the assignement of the different
signals was done using 1D and 2D (COSY 1H-1H and
1H-13C) experiments. In particular, an heteronuclear
1
correlation H-13C allowed us to assign the protons
Quantification of metabolites: The concentration
of metabolites was calculated as follows: [m] = (9 A0
× [TSPd4])/(b × Aref), where [m] is the concentra-
tion of metabolite m; Ao is the area of metabolite m
(HC-HD) and (HA-HB) attached respectively to C-2
and C-3 according to the 13C chemical shifts given
by Gallego et al. (1993). NMR 1H (400.13 MHz;
CD3OD) δ: 2.69 ppm (ddd, 2H, HB or HA); 2.87 ppm
(ddd, 2H, HC or HD ); 2.96 ppm (ddd, 2H, HA or HB);
3.39 ppm (ddd, 2H, HD or HC). Coupling constants:
1
resonance in the H NMR spectrum; [TSPd4] is the
concentration of the reference; Aref is the area of ref-
1
erence resonance in the H NMR spectrum; b is the
number of protons of metabolite m in the signal in-
tegrated; 9 is the number of protons resonating at 0
ppm.
JAC = 13.9 Hz, JAD = 2.4 Hz, JBC = 3.1 Hz; JBD
=
2.9 Hz. NMR 13C (100.61 MHz; CD3OD): 39.2 ppm
(C-2); 47.8 ppm (C-3).

436
1
Figure 2. Kinetics of thiomorpholine transformation by M. aurum MO1. The H NMR spectra were recorded at 400.13 MHz with a 400
◦
AC spectrometer at 21 C with 5-mm-diameter tubes; water was suppressed by saturation with a classical NOE Bruker programm. 64 scans
◦
were collected (90 pulse 11.6 µs, relaxation delay 4 s, acquisition time 1.786 s, 16 K data points). TM: thiomorpholine; S: sulfoxide; X:
thiodiglycolic acid.
Identification of metabolite X. After 24 h of
incubation with thiomorpholine, the supernantant con-
taining only the metabolite X was freeze-dried to be
analyzed by mass spectrometry (electronic impact).
MS (EI): 150 (M+.); 132, 104, 77, 60.
(Figure 1). When M. aurum MO1 cells were grown
on morpholine as sole source of carbon, nitrogen, and
energy a peak at 449 nm was present in the spec-
trum (Figure 1, spectrum A) indicating the presence
of a soluble cytochrome P450. Since this peak was
not observed in acetate-grown cells (Figure 1, spec-
trum B), the synthesis of this enzyme was induced by
morpholine. Using thiomorpholine as substrate (Fig-
ure 1, spectrum C), similar results were obtained. The
cytochrome P450 contents were about 70 pmol per
mg of protein in morpholine-grown cells extracts and
about 60 pmol per mg of protein in thiomorpholine-
induced cells. Other structurally related amines, such
as pyrrolidine and piperidine, also induced the synthe-
sis of a soluble cytochrome P450 (data not shown).
We can reasonably conclude that these cyclic amines
induced the synthesis of the same heme-containing
monooxygenase.
Results
Spectrophotometric evidence of the induction of a
cytochrome P450
In a previous work (Poupin & Truffaut 1996; Poupin
et al. 1998), we demonstrated the involvement of a
soluble cytochrome P450 in the degradation of mor-
pholine by Mycobacterium sp. strain RP1. To inves-
tigate the presence of such a monooxygenase in M.
aurum MO1, we measured the carbon monoxide dif-
ferential spectra of sodium dithionite-reduced soluble
fractions of morpholine-induced and uninduced cells

437
Degradation of thiomorpholine by M. aurum MO1
The supernatants of M. aurum MO1 resting cells (5 g
of wet cells in 50 ml of Knapp buffer) were analyzed
by 1H NMR spectroscopy as indicated in the Materials
and methods section. The spectra were compared with
those of the negative controls. Spectra collected at 0,
8, 12 and 22.5 h are presented in Figure 2.
In the spectrum obtained at time zero, three main
signals are visible: a singlet at 0 ppm that belongs to
the methyl groups of TSPd4, our reference, and two
pseudotriplets at 2.66 and 3.08 ppm that correspond
to CH2(a) and CH2(b) of thiomorpholine (Figure 8).
At 8 h, thiomorpholine was completely exhausted and
new signals were detected, in particular a group of four
signals, resonating at 2.86, 2.99, 3.10 and 3.39 ppm,
which seemed to belong to the same molecule (S)
because they first increased and then decreased simul-
taneously. At 22 h 30, the intermediate compound (S)
had disappeared. A new signal (X), a singlet resonat-
ing at 3.25 ppm, appeared at 8 h and its concentration
increased with time.
As the presence of a soluble cytochrome P450
had been previously confirmed by spectrophotomet-
ric experiments, a “classical” oxidation of the sulfur
atom of thiomorpholine was expected (Oldham 1989).
The pattern of the four signals (S) showed that they
were coupled. Moreover, their symmetry indicated
that the corresponding protons belonged to a ring.
So, the intermediate compound (S) could be a sulfox-
ide. In order to confirm this hypothesis, a chemical
reaction of oxidation was conducted with 1 eq. of
m-chloroperbenzoïc acid (m-CPBA) as the oxidant
◦
(Madesclaire 1986) at −15 C. After purification by
column chromatography, the structure of the obtained
product was confirmed by IR, mass spectrometry, 1H
and 13C NMR. After adjustment of the pH to 10 in
1
Figure 3. (A) H NMR spectrum of synthetic sulfoxide recorded at
1
300.13 MHz. (B) H NMR spectrum of thiomorpholine degradation
by M. aurum MO1 recorded at 400.13 MHz after 8 h of incubation.
(C) Chair conformation of sulfoxide.
1
the buffer, the H NMR spectrum of the synthesized
sulfoxide was recorded (Figure 3A). The resonances
corresponded perfectly to those of the biodegradative
product after 8 h of incubation (Figure 3B).
product was performed with the programm Batchmin
(Monte Carlo procedure). The conformation presented
on Figure 3C was found in 407 times out of 1000. In
this model, the sulfoxide preferentially takes a chair
conformation, where SO is axial. This preferential
SO position is in agreement with the conformational
analysis obtained by Gallego et al. on different sub-
stituted 1,4-thiazanes and their derivatives (Gallego et
al. 1993). The protons (A,B) and (C,D) were then non
equivalent; that is the reason why the 1H NMR spec-
trum obtained was complex. The coupling constants
The corresponding sulfone, which could be also
observed by the action of a cytochrome P450 (Ren-
wick 1989), was also synthesized from thiomorpho-
line (addition of 2.5 eq. of m-CPBA at room temper-
1
ature). None of the H NMR signals of the synthetic
sulfone corresponded to those observed in the spec-
tra obtained during the degradation of thiomorpholine
(data not shown).
The complexity of the 1H NMR signals of the sul-
foxide was quite surprising. A modelisation of the

438
determined by 1H NMR were the following ones: JAC
= 13.9 Hz, JAD = 2.4 Hz, JBC = 3.1 Hz, JBD = 2.9 Hz.
These values directly measured on the spectrum were
consistent with those obtained by calculation.
These results prove the presence in M. aurum MO1
of a cytochrome P450 which oxidizes thiomorpholine
into its corresponding sulfoxide.
pletely inhibited at a 10 mM concentration (Figure
4B). The same effect was observed for the thiodigly-
colic acid formation (Figure 4C). The direct effects of
metyrapone on the sulfoxide formation, first interme-
diate in the thiomorpholine biodegradation, confirmed
that a cytochrome P450 was involved in the first step
of this degradation.
The singlet resonating at δ: 3.25 ppm (metabolite
X) was assigned to thiodiglycolic acid as evidenced
by NMR and mass spectroscopy. The H NMR reso-
Inhibition of morpholine degradation by metyrapone
1
In a previous work (Combourieu et al. 1998), we have
shown that morpholine was degraded by M. aurum
MO1 in 10 h. Two intermediates of the metabolic path-
way have been identified: 2-(2-aminoethoxy) acetate
and glycolate (Figure 6).
nances of this commercial compound and of metabo-
lite X perfectly overlapped. Also the fragmentations
obtained from electronic impact spectrum were the
same for the two compounds.
The time courses of the concentrations of thiomor-
pholine, sulfoxide (S) and thiodiglycolic acid (X) are
presented in Figures 4A, B and C respectively. While
thiomorpholine was degraded, sulfoxide was pro-
duced, suggesting that sulfoxide is a primary metabo-
lite. After six hours of incubation the concentration
of sulfoxide was decreasing and that of thiodigly-
colic acid was increasing in parallel. It seemed that
sulfoxide was degraded into metabolite X. To check
this hypothesis, the cells were incubated with syn-
thetic sulfoxide; it was effectively metabolized into
thiodiglycolic acid (Figure 5).
In order to know how the cytochrome P450 was
involved in the first steps of the morpholine degra-
dation by M. aurum strain MO1, the same kind of
experiments as those made with thiomorpholine were
carried out, i.e., addition of different concentrations of
metyrapone (5 and 10 mM) in the incubation medium
containing 10 mM morpholine. The kinetics of the
morpholine degradation are reported on Figure 7.
As in the case of thiomorpholine, the addition of
metyrapone led to an inhibition of the degradative
reactions of morpholine which is concentration depen-
dent. With increasing concentrations of metyrapone,
the following effects were observed: (i) the rates of
morpholine degradation (Figure 7A) and of the in-
termediates formation (Figure 7B and C) decreased
and (ii) the appearance of the intermediates was de-
layed. The effects of metyrapone on the formation
of the intermediate compounds (especially on 2-(2-
aminoethoxy) acetate, which is the first metabolite)
indicated that the oxidative ring cleavage takes place
in the early events of morpholine degradation. These
observations confirm the results obtained with My-
cobacterium sp. strain RP1 (Combourieu et al. 1998,
Poupin et al. 1998).
Inhibition of thiomorpholine transformation by a
cytochrome P450 inhibitor: metyrapone
Another way to confirm the key role played by a
cytochrome P450 in the transformation of thiomor-
pholine was to add a selective inhibitor of this enzyme
in the incubation medium and to observe its effect on
the kinetics of thiomorpholine transformation.
Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propa-
none) was chosen as a specific cytochrome P450 in-
hibitor (Testa & Jenner 1981). After checking that
metyrapone did not affect the viability of M. aurum
MO1 (the addition of this chemical to the culture
medium did not prevent cell growth), experiments
were carried out with different concentrations of this
Discussion
1
compound and analyzed by H NMR. To the flasks
containing the cells (100 g/l), metyrapone (5 and 10
mM) and thiomorpholine (10 mM) were added. The
kinetics of thiomorpholine degradation are shown in
Figure 4.
The addition of metyrapone led to an inhibition
of the degradative reactions of thiomorpholine (Fig-
ure 4A). In the presence of metyrapone (5 mM), the
rate of sulfoxide formation decreased and was com-
In a previous work (Combourieu et al. 1998),
two intermediate compounds were identified, 2-(2-
aminoethoxy) acetate and glycolate, during morpho-
line degradation by M. aurum MO1, by using a new
approach: H NMR spectroscopy performed directly
on the incubation medium.
1
In this paper, we were interested in giving evi-
dences of the enzymes involved in this degradation.

439
Figure 4. Incubation of M. aurum MO1 cells with thiomorpholine (10 mM) in the presence of 5 mM (ꢀ) or 10 mM (1) metyrapone or in the
absence of metyrapone (×). Time courses for the concentrations of thiomorpholine (A), sulfoxide (B) and thiodiglycolic acid (C). Experimental
conditions are as in Figure 2.
M. aurum MO1 was not able to grow on this com-
pound but degraded it (Mazure & Truffaut 1994). At
the same time, the production of a cytochrome P450
was induced. The accumulation of metabolites was
then expected. The NMR spectra collected at differ-
ent incubation times showed that this strain is able
to completely transform thiomorpholine which disap-
1
pears in 8 h, at a rate of about 1.25 mM/h. The H
NMR technique also allowed us to identify sulfoxide
and thiodiglycolic acid as two intermediates of the
biodegradative pathway (Figure 8).
The transformation of thiomorpholine into its cor-
responding sulfoxide, as well as the oxygen consump-
Figure 5. Incubation of M. aurum MO1 cells with thiomorpho-
tion associated with morpholine and thiomorpholine
line-S-oxide (5 mM). Time courses for the concentrations of sul-
degradation (Mazure & Truffaut 1994), showed the
foxide (×) and thiodiglycolic acid (ꢀ).
presence of an activity due to a monooxygenase. How-
ever, if the cytochrome P450, put into evidence by
the spectrophotometric assays, can be responsible for
The presence of a soluble cytochrome P450 was
first shown by spectrophotometric assays: the CO-
difference spectrum of cell extracts of M. aurum MO1
grown on liquid mineral salts medium amended with
morpholine exhibited a typical peak at 449 nm. A sim-
ilar spectrum was observed when M. aurum MO1 cells
were induced by thiomorpholine but not when they
were grown on mineral salts medium amended with
acetate. Conclusively, M. aurum MO1 contains at least
one soluble cytochrome P450 inducible by morpholine
and thiomorpholine.
this oxidation reaction, many authors have shown in
the literature that it could also be catalyzed by flavin-
containing monooxygenases (Ziegler 1988). In order
to check what kind of monooxygenase was involved
in the degradation of thiomorpholine, the influence of
a selective cytochrome P450 inhibitor, metyrapone,
was studied. The inhibitory effects of metyrapone
on the thiomorpholine degradation and on the sul-
foxide formation proved the presence of an activity
due to a cytochrome P450 in the first step of the
biodegradative pathway (Figure 8). In the absence of
inhibitory effects, the sulfoxide is in its turn trans-
formed into thiodiglycolic acid which accumulates.
The synthesis of this metabolite implies three types of
In order to extend studies on the key role played
by this cytochrome P450 in the degradation of such
xenobiotics, the degradative pathway of thiomorpho-
line, a sulfur analogue of morpholine, was studied.

440
Figure 6. Biodegradative pathway of morpholine by M. aurum MO1.
Figure 7. Incubation of M. aurum MO1 cells with morpholine (10 mM) in the presence of 5 mM (ꢀ) or 10 mM (1) metyrapone or in the
absence of metyrapone (×). Time courses for the concentrations of morpholine (A), 2-(2-aminoethoxy)acetate (B) and glycolic acid (C ).
1
◦
Integrals were measured on H NMR spectra recorded at 300.13 MHz with a Avance 300 DSX spectrometer at 21 C with 5-mm-diameter
◦
tubes; water was suppressed by saturation with a classical NOE Bruker programm. 64 scans were collected (90 pulse 6.5µs, relaxation delay
5 s, acquisition time 4.561 s, 32 K data points).
reactions: opening of the ring, reduction of the cor-
responding sulfoxide and deamination. The first step
corresponds to the cleavage of the C-N bond leading
to 2-(2-aminoethylsulfinyl) acetate. The reduction of
this compound could be performed by a sulfoxide re-
ductase, this type of enzyme has been described in
procaryotes as well as in eukaryotes cells (Madesclaire
1988). It is not clear wether this reduction takes place
before or after deamination.
In order to confirm that the cytochrome P450 was
also involved in the oxidative catabolism of morpho-
line in M. aurum strain MO1, metyrapone was added
in the incubation medium containing morpholine. The
same inhibitory effects were observed on the morpho-
line biodegradation. The decrease of the intermediates
formation rates, in particular 2-(2-aminoethoxy) ac-
etate, evidenced that a cytochrome P450 was also
involved and that it took part in the early steps of
the degradation. Our results suggest the opening of
the ring in morpholine as well as in thiomorpholine.
The cytochrome P450 catalyzes an hydroxylation of
a C-atom adjacent to the amine group rather than
N-oxidation, because protons at the α position are
available (Guengerich 1990a). When the cells were
incubated with thiomorpholine thiodiglycolate was
detected, while glycolate accumulated in the pres-
ence of morpholine. However glycolate could result
from the cleavage of the ether bond of diglycolate
(White et al. 1996). In this case, glycolate would
result from deamination of 2-(2-aminoethoxy)acetate
and not from its cleavage in ethanolamine and glyco-
late. The biodegradative pathway of morpholine and
thiomorpholine would be similar to that of pyrrolidine
mentioned by Swain et al (1991) and would exclude
the ethanolamine branch previously proposed for mor-
pholine degradation (Swain et al. 1991). Note that for
thiomorpholine the sulfur atom is primary oxidized as
its higher reactivity is concerned.
In this work, we showed another example of the
detoxification role played by cytochrome P450. The
presence of such an enzyme in Mycobacterium au-
rum MO1 is not surprising as it was demonstrated in

441
Figure 8. First steps of thiomorpholine biodegradative pathway in M. aurum MO1.
very close bacterial genera and involved in cholesterol
References
metabolism of Streptomyces sp. (Horii et al. 1990),
the sulphonylureas-inducible cytochrome P-450 from
Streptomyces grisoleus (Omer et al. 1990) or the
ethyldipropyl carbamothioate – inducible cytochrome
P-450 from Rhodococcus sp. strain N186/21 (Nagy et
al. 1995).
In conclusion, this work demonstrated the pres-
ence of a soluble cytochrome P450 in M. aurum MO1
and its key role in the degradation of morpholine and
thiomorpholine, its sulfur analogue. The use of in
situ 1H NMR spectroscopy, performed directly on the
incubation medium in H2O, allowed us to identify un-
ambiguously some of the intermediates formed during
the degradation and to quantify them. This technique,
both qualitative and quantitative, is easy to apply and
could be used to investigate many other biodegradative
processes.
Anon (1989) Final report on the safety assessment of morpholine. J.
Amer. Coll. Toxicol. 8: 707–748
Asperger O, Wirkner K & Kleber HP (1990) Occurrence of cy-
tochrome P-450 in Rhodococci. Biocatalysis 4: 59–65
Asperger O & Kleber HP (1991) Distribution and diversity of bac-
terial cytochrome P450. In: Ruckpaul K & Rein H (Ed) Frontiers
in Biotransformation, Vol. 4 (pp 1–53)
Bradford MM (1976) A rapid and sensitive method for the quantifi-
cation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72: 248–254
Cech JS, Hartman P, Slosarek M & Chudoba J (1988) Isolation
and identification of a morpholine-degrading bacterium. Appl.
Environ. Microbiol. 54: 619–621
Combourieu B, Besse P, Sancelme M, Veschambre H, Delort AM,
Poupin P & Truffaut N (1998) Morpholine degradation pathway
of Mycobacterium aurum MO1: direct evidence of intermedi-
1
ates by in situ H nuclear magnetic resonance. Appl. Environ.
Microbiol. 64: 153–158
Dmitrenko GN, Gvozdyak PI & Udod VM (1987) Selection of de-
structor microorganisms for heterocyclic xenobiotics. Khimiya i
Teknologiya Vody 9: 442–445
Dmitrenko GN & Gvozdyak PI (1988) Destruction of morpholine
by mycobacteria. In: Proceedings of Conference on Microbi-
ological Methods for Protecting the Environment. Puschino,
USSR: Centre for Biological Research
Acknowledgements
Enzmann H, Zerban H, Kopp-Schnelder A, Loser E & Bannasch P
(1995) Effects of low doses of N-nitrosomorpholine on the devel-
opment of early stages of hepatocarcinogenesis. Carcinogenesis
16: 1513–1518
This work was supported by interdisciplinary pro-
grams of CNRS “PIRSEM” and “ECOTECH”.
We acknowledge Anne-Lise Etienne, coordinator
of the CNRS programs. We greatfully acknowledge
Jacques Guyot for the modelisation of the sulfoxide,
Michel Renard for mass spectroscopy experiments and
Tran Thi Minh Ngoc for her help. We thank J.S. Cech
for the gift of M. aurum MO1. This manuscript was
kindly corrected by Bernd Schöllhorn.
Fulco AJ (1991) P-450
tochromes: biochemistry and regulation. Annu. Rev. Pharmacol.
Toxicol. 31: 177–203
and other inducible bacterial P-450 cy-
BM−3
Gallego MT, Brunet E & Ruano JLG (1993) Conformational analy-
sis of methylthiazanes: the problem of the Me-C-Me gauche
interaction. J. Org. Chem. 58: 3905–3911
Guengerich FP (1990a) Enzymatic oxidation of xenobiotic chemi-
cals. Biochem. Mol. Biol. 25: 97–153
Guengerich FP (1990b) Chemical mechanisms of cytochromes P-
450 catalysis. Asia Pacific J. Pharmacol. 5: 253–268
Horii M, Ishizaki T, Paik SY, Manome T & Murooka Y (1990) An
operon containing the genes for cholesterol oxidase and a cy-

442
tochrome P450 like protein from Streptomyces sp. J. Bacteriol.
172: 3644–3653
Ortiz de Montellano PR (1986) Cytochromes P-450, Structure,
Mechanism and Biochemistry. Plenum Press, New York
Peterson JA & Lu JY (1991) Bacterial cytochromes P450: isolation
and identification. Methods Enzymol. 206: 612–620
Poupin P & Truffaut N (1996) Etude de la dégradation de la
morpholine par Mycobacterium sp. RP1. Colloque de la So-
ciété Française de Microbiologie “Microbiologie industrielle et
environnement” Avril 1996-Narbonne
Poupin P, Truffaut N, Combourieu B, Besse P, Sancelme M,
Veschambre H & Delort AM (1998) Degradation of morpho-
line by an environmental Mycobacterium strain involves a cy-
tochrome P450. Appl. Environ. Microbiol. 64: 159–165
Renwick AG (1989) Sulphoxides and sulphones. In: Danami LA
(Ed) Sulfur Containing Drugs and Related Organic Compounds.
Chemistry, Biochemistry and Toxicology, Vol. 1, Part B (pp 133–
153). Ellis Horwood Limited, Chichester
Jefcoate CR (1986) Cytochrome P-450 enzymes in sterol biosyn-
thesis and metabolism. In: Ortiz de Montellano PR (Ed) Cy-
tochromes P-450, Structure, Mechanism and Biochemistry.
Plenum Press, New York (pp 387–428)
Karlson U, Dwyer DF, Hooper SW, Moore ERB, Timmis KN
& Eltis LD (1993) Two independently regulated cytochromes
P-450 in a Rhodococcus rhodochrous strain that degrades 2-
ethoxyethanol and 4-methoxybenzoate. J. Bacteriol. 175: 1467–
1474
Knapp JS, Callely AG & Mainprize J (1982) The microbial degra-
dation of morpholine. J. Appl. Bacteriol. 52: 5–13
Knapp JS & Brown VR (1988) Morpholine biodegradation. Int.
Biode. 25: 299–306
Knapp JS, Emtiazi G, Yusoff S & Heron ST (1996) The utilization of
morpholine as a sole nitrogen source by Gram-negative bacteria.
Lett. Appl. Microbiol. 23: 334–338
Ruckpaul K & Rein H (1984) Cytochrome P-450, Akademie Verlag,
Berlin
Madesclaire M (1986) Synthesis of sulfoxides by oxidation of
thioethers. Tetrahedron 42: 5459–5495
Ruckpaul K & Rein H (1990) Frontiers in Biotransformation, Vol.
2. Akademie Verlag, Berlin
Madesclaire M (1988) Reduction of sulfoxides to thioethers. Tetra-
hedron 44: 6537–6580
Singer GM & Lijinsky W (1976) Naturally occuring nitrosable com-
pounds. I. Secondary amines in foodstuffs. J. Agric. Food Chem.
24: 550–553
Swain A, Waterhouse KV, Venables WA, Callely AG & Lowe SE
(1991) Biochemical studies of morpholine catabolism by an en-
vironmental Mycobacterium. Appl. Microbiol. Biotechnol. 35:
110–114
Mazure N & Truffaut N (1994) Degradation of morpholine by
Mycobacterium aurum MO1. Can. J. Microbiol. 40: 751–765
Mjos K (1978) Cyclic amines. In: Kirk-Othmer Encyclopedia of
Chemical Technology, Vol. 2, 3rd edn. (pp 298–308). Wiley
Interscience, New York
Nagy I, Schoofs G, Compernolle F, Proost P, Vanderleyden J & de
Mot R (1995) Degradation of the thiocarbamate herbicide EPTC
(S-ethyl dipropylcarbamothioate) and biosafening by Rhodococ-
cus sp. strain NI86/21 involve an inducible cytochrome P-450
system and aldehyde dehydrogenase. J. Bacteriol. 177: 676–687
Oldham HG (1989) Interactions of sulphur-containing xenobi-
otics with cytochrome(s) P-450 and glucuronyl transferases. In:
Danami LA (Ed) Sulfur Containing Drugs and Related Organic
Compounds. Chemistry, Biochemistry and Toxicology, Vol. 2,
Part B (pp 9–45). Ellis Horwood Limited, Chichester
Omer CA, Lenstra R, Little PJ, Dean C, Tepperman JM, Leto KJ,
Romesser JA & O’Keefe DP (1990) Genes for two herbicide-
inducible cytochromes P-450 from Streptomyces griseolus. J.
Bacteriol. 172: 3335–3345
Testa B & Jenner P (1981) Inhibitors of cytochrome P450s and their
mechanism of action. Drug Metab. Rev. 12: 1–117
Waterman MR, John ME & Simpson ER (1986) Regulation of
synthesis and activity of cytochrome P-450 enzymes in physio-
logical pathways. In: Ortiz de Montellano PR (Ed) Cytochromes
P-450, Structure, Mechanism and Biochemistry (pp 345–386).
Plenum Press, New York
White GF, Russell NJ & Tidswell E (1996) Bacterial scission of
ether bond. Microbiol. Rev. 60: 216–232
Wislocki PG, Miwa GT & Lu AYH (1980) Reactions catalyzed by
the cytochrome P-450 system. In: Jakoby WB (Ed) Enzymatic
Basis of Detoxification (pp 135–182)
Ziegler DM (1988) Flavin-containing monooxygenases: catalytic
mechanism and substrate specificities. Drug Metab. Rev. 19:
1–32
Omura T & Sato R (1964) The carbon monoxide-binding pigment
of liver microsomes. J. Biol. Chem. 239: 2379–2387