89
FEMS MicrobiologyLetters 3 (1978) 89-93
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
M E T A B O L I S M O F E T H Y L E N E BY M Y C O B A C T E R I U M
E 20
J.A.M. DE BONT * and W. HARDER
Department of Microbiology, University of Groningen, Kerklaan 30, Haren (Gr.), The Netherlands
Received 23 November 1977
1. Introduction
2.2. Inhibitor studies
Recently, Mycobacteria capable of utilizing
ethylene have been isolated [1,2]. Preliminary studies
with Mycobacterium E 20 indicated that this compound was metabolized via ethylene oxide, and that
the glyoxylate cycle was involved in the further
metabolism of the epoxide [3]. Ethylene oxide
therefore was probably metabolized via acetyl-CoA,
but the reactions leading to its formation from the
epoxide remained obscure.
This paper reports on investigations aimed at
elucidating this pathway.
The effect of fluoroacetate (20 mM final concentration) on the excretion of products by ethaneor ethylene-grown cells ofMycobacterium E 20 was
studied in 35 ml screw cap bottles containing 4 ml
of a washed suspension of organisms in 25 mM
Na-phosphate buffer, pH 7.2. The bottles were
incubated on a rotary shaker at 30°C; ethane or
ethylene was injected into the bottles and the change
in the concentration of ethane, ethylene and ethylene
oxide in the gas phase was measured by gas chromatography. Any accumulation of ethanol, acetaldehyde
or acetate was also measured by gas chromatography,
using samples of the supernatant fluid collected after
centrifugation (see 2.3).
2. Materials and Methods
2.1. Organism, media and culture conditions
2.3. Chemical estimations
Mycobacterium E 20 [ 1] was grown in a mineral
medium [4] in an ethylene-limited chemostat with
2.5 1 culture volume at a dilution rate of 0.015 h -l,
at pH 6.7 and 30°C. Ethylene was supplied to the
culture by passing 1.5 1/min air containing 1% (v/v)
ethylene over its surface. A fermenter vessel without
baffles was used [5] and the stirrer speed was 400
rpm. The organism was also grown on ethane or
ethanol in batch cultures in 2-1 erlenmeyer flasks
containing 500 ml mineral medium, as described
previously [3].
* Permanent address: Laboratory of Microbiology,
Agricultural University,Hesselinkvan Suchtelenweg4,
Wageningen, The Netherlands.
Ethylene oxide, acetaldehyde, ethanol, ethylene
and ethane were determined with a gas chromatograph, equipped with a heated duel-flame ionization
detector (Pye Unicam, Series 104) containing a glass
column (1.5 m X 0.4 cm) packed with Porapak Q.
Nitrogen (40 ml/min) was used as the carrier gas.
Column temperature was 80°C, except for ethanol
(120°C). Acetic acid was measured with a Packard
Becker model 417 gas chromatograph, equiped with
a flame ionization detector. A glass column (1 m X
0.4 cm) with 20% Tween 80 on Chromosorb W-AW
(80-100 mesh) was used at 170°C with nitrogen,
saturated with formic acid, as the carrier gas (60 ml/
min).
Protein was measured by the method of Lowry
et al. [6].
90
2.4. Enzyme assay
Cell-flee extracts were prepared by ultrasonic disintegration of washed cell suspensions in 50 mM Naphosphate buffer, pH 7.2, containing 2 mM cysteine
(6 X 30 sec), at 0°C. The extract was centrifuged at
15 000 g for 20 rain and the supernatant ( 6 - 1 2 mg
protein/ml) used for enzyme assays. Occasionally
extracts were dialysed overnight, at 4°C, against
50 mM Na-phosphate buffer pH 7.2, containing 2 mM
cysteine, before use. Ethylene oxide utilization by
extracts was followed in tubes with a Suba seal
containing a standard reaction mixture (1 ml) of the
following composition (mM): Tris-HC1 buffer pH
8.5, 50; NAD ÷, 0.5. CoA, 0.5; FAD, 0.1 ; concentrated dialysate, if required for optimal activity, 0.15
ml; ethylene oxide, 0.1 and cell-free extract. After
equilibration at 30°C, the reaction was started by
injecting cell-free extract into the tubes and the
decrease of the concentration of ethylene oxide
in the gas phase was measured. The ethylene oxide
concentration in the gas phase was kept in equilibrium
with that in solution by frequent shaking. Ethylene
oxide-dependent NADH formation was measured
spectrophotometrically at 340 nm and 30°C in
cuvettes (1 ml), sealed with Suba seals and gassed
with nitrogen gas, before the reaction was started by
adding 0.1 ml of an oxygen-free ethylene oxide
solution in water. The reaction mixture used in this
assay was the same as described above.
For the preparation of a dialysate, a cell-flee
extract (4 ml), prepared as described above, was
dialysed overnight at 4°C against water (31 ml). The
dialysate was concentrated to a final volume of 3 ml
in a rotary vacuum evaporator at 45°C and samples of
the resulting clear solution were used in the enzyme
assays.
2.5. Formation of[ 14C]acetyl-CoA from [ 14C]ethylene oxMe and its detection as [ 14C]eitrate
The formation of [14C]acetyl-CoAfrom [14C]ethylene oxide in cell-free extracts was measured by
converting the labelled acetyl-CoA into radioactive
citrate using oxaloacetate and citrate synthase (EC
4.1.3.7). The complete reaction mixture (1.5 ml)
placed in tubes with Suba seals consisted of (mM):
Tris-HC1 buffer pH 8.5, 50; NAD ÷, 0.5; CoA, 0.5;
FAD, 0.1 ; sufficient amount of dialysate (0.15 ml);
[14C]ethylene oxide (specific radioactivity, 5 ~tCi/
/~mole), 0.1 ; oxaloacetate, 2.0; fluorocitrate, 0.01;
citrate synthase, 10 units and cell-free extract, 0.15
ml. The reaction was started by injecting [14C]ethylene oxide and stopped, after incubation at 30°C
for 30 min, by injecting 0.15 ml of 1 N HzSO4 to
give a final pH of approximately 2. The mixture was
then extracted twice with 1 ml diethylether. The
ether layer was evaporated to dryness and the residue
was dissolved in 0.1 ml water. Radioactivity in this
solution was counted in a liquid scintillation counter
(Beckman LS 250) in 5 ml scintillation fluid (toluene
and 0.5% PPO), while radioactive citrate was detected
by applying aliquots of the solution on cellulose
plates over a 2.5/11 spot of 10 mM authentic citrate.
Chromatography was carried out with ether-formic
acid-water (7 : 2 : 1) as a solvent [7]. After developing the plates with bromocresol green, the citrate
spots were removed and radioactivity counted as
above.
2. 6. Chemicals
Ethylene (99%) and other gaseous substrates (commercial purity) were obtained from the Matheson
Co., East Rutherford, N.J., U.S.A. [14C]Ethylene
oxide (35 mCi/mmole) was obtained from the Radiochemical Centre, Amersham, U.K.
3. Results
3.1. Effect of fluoroacetate on the oxidation of
ethane and ethylene by resting cell suspensions
Resting cell suspensions ofMycobacterium E 20
grown on ethane or ethylene were allowed to oxidize
their respective growth substrate in the presence or
absence of 20 mM fluoroacetate and the accumulation of intermediary products was detected by gas
chromatography. In vivo, fluoroacetate is converted
to fluorocitrate, which is a potent inhibitor of
aconitate hydratase [8], and in this way the operation of the TCA cycle and the glyoxylate cycle in the
bacteria may be impeded. Ethane-grown cells
excreted acetate when oxidizing ethane in the
presence of the inhibitor (Fig. 1a), while the ethylene-
91
3.2. Conversion of ethylene oxide by cell-free extracts
100
Cell-free extracts of ethylene-grown cells were able
to catalyze the oxidation of ethylene oxide (Fig. 2).
The reaction was completely dependent upon the
presence of NAD ÷ and CoA, while the rate of conversion of the epoxide in the presence of NAD ÷ and
CoA was approximately doubled by including FAD in
the incubation mixture. The reaction was linear with
time for more than 30 min. In the absence of FAD,
the reaction could also be monitored by following the
ethylene oxide-dependent reduction of NAD ÷ under
anaerobic conditions. The rate of reduction of NAD ÷
was only linear with time for a few minutes and then
levelled off. However, the initial reduction rates were
such that the amount of ethylene oxide oxidized, and
that of NADH formed, were approximately
stoicheiometric. This ethylene oxide-dependent
reduction of NAD ÷ was strictly CoA-dependent and
e
o
E
:t
in
~o
75
o
no.
uJ
lm
n,
50
C.)
X
UJ
C3
Z
.<
(/I
~< 25
e¢
m
0.1
I
I
I
A
0
o
2
4
0
2
TIME (h)
Fig. 1. Effect of fluoroacetate on the excretion of intermediates by Mycobacterium E 20 grown on ethane (a) or
ethylene (b). Closed symbols refer to incubations without
inhibitor, open symbols are in the presence of inhibitor.
o, ethane; z~, acetate; D, ethylene; v, ethylene oxide. After
1.5 h (Fig. b) a fresh amount of ethylene was injected into
the bottle without the inhibitor.
m
e
O
E
z
w
o
0.05
m
z
grown cells excreted only ethylene oxide when
oxidizing ethylene (Fig. lb). Excretion of other
products such as ethanol or acetaldehyde, could not
be demonstrated. An attractive explanation for these
results is that ethylene oxide in ethylene catabolism
holds a position similar to that of acetate in ethane
oxidation; that is, both intermediates are end
products of a catabolic sequence that feeds into the
citric acid cycle via acetyl-CoA. Acetate is produced
from ethane through ethanol and acetaldehyde and is
then converted into acetyl-CoA by acetyl.CoA synthetase [3]. Likewise, ethylene oxide might thus be
converted into acetyl-CoA in one single enzyme-"
catalyzed reaction. Confirmation of this hypothesis
was sought by work with cell-free extracts.
m
._i
)2:
Iill
0
0
I
15
I
30
I
~5
TIME (mln)
Fig. 2. Effect of various cofactors on the conversion of
ethylene oxide by dialysed cell-free extracts of ethylenegrown cells of Mycobacterium E 20. The complete reaction
mixture (.) contained 0.15 ml concentrated dialysate to give
a saturating concentration of the unknown cofactor and 0.1
ml cell-free extract (0.9 tug.protein). Omissions from the com.
.
+
plete reaction mLxture: NAD (~); concentrated dialysate
(a); CoA (×) and FAD (o).
92
NADP ÷ did not replace NAD ÷. Cell-free extracts of
ethane-grown cells did not catalyse the oxidation of
ethylene oxide.
The following observations indicated that, apart
from NAD +, CoA and FAD, a fourth unknown dissociable cofactor was involved in the enzymic conversion of ethylene oxide: (i) The observed enzyme
activity in cell-free extracts (before dialysis) was not
proportional to the amount of extract added in that
addition of higher enzyme concentrations resulted in
disproportionally higher specific enzyme activities.
This behaviour indicates that a dissociable cofactor
may be present in the extract [9]. (ii) The specific
enzyme activity at relatively low protein concentrations in the reaction mixture (less than 1 mg protein/
ml) could be enhanced several-fold by adding boiled
crude extract of ethylene-grown cells to the reaction
mixture. Boiled extracts of ethanol-grown cells did
not have such an effect. (t/i) Enzyme activity in the
presence of NAD ÷, CoA and FAD was completely lost
upon dialysis of cell-free extracts. Activity could be
restored by adding boiled cell-free extract of ethylenegrown cells, but again boiled extract of ethanolgrown cells was ineffective. (iv) Enzyme activity of
dialysed extracts also could be restored by adding a
concentrated dialysate of ethylene-grown cells (see
Materials and Methods) to the system (Fig. 2).
Attempts to identify this heat stable, dialysable
cofactor remained unsuccessful.
3.3. Product o f the reaction
Since the oxidation of ethylene oxide was strictly
dependent upon CoA and NAD ÷, it seemed reasonable
to suppose that acetyl-CoA was the product of the
reaction under study. Experiments with [14C]ethylene
oxide were undertaken to support this assumption.
Cell-free extracts were incubated with the radioactive epoxide and the required cofactors. In addition
oxaloacetate and citrate synthase were included in
the reaction mixtures so that any acetyl-CoA formed
would be converted immediately into citrate. The
further metabolism of the citrate so formed was
prevented by including fluorocitrate in the reaction
mixtures. The results (Table 1) indicate that citrate
was indeed formed in the complete reaction mixtme
since a labelled product, which was extractable with '~
TABLE 1
Formation of labelled ether-extractable compounds from
[14C]ethylene oxide by cell-free extracts of ethylene-grown
Mycobacterium E 20 in the presence of oxaloacetate and
citrate synthase
Composition of
the reaction mixture
Complete
Extract boiled (5 min)
Minus citrate synthase
Minus oxaloacetate
Minus citrate synthase and oxaloacetate
Radioactivity in
ether-extracts
(dpm)
t=0
t=30
(min)
8400
34300
7500
8800
9800
9600
ether, was not formed in the absence of oxaloacetate
and/or citrate synthase. Furthermore, thin-layer
chromatography of the ether-extractable radioactive
compound showed that most radioactivity cochromatographed with citrate if reaction mixtures
were complete, while in all other instances almost no
radioactivity was contained in the citrate spot.
4. Discussion
Inhibition experiments with fluoracetate indicated
that, in Mycobacterium E 20, the metabolism of
ethylene is initiated by an epoxidation to ethylene
oxide. This is in accordance with previous observations
that ethylene oxide was a growth substrate for the
bacterium and that ethylene-dependent oxygen uptake by washed cell suspensions was only observed
with ethylene-grown cells [3]. The mechanism of this
epoxidation reaction is unknown, but may be
analogous to alkene epoxidation observed in Pseudomonas oleovorans, which is catalysed by a
hydroxylase [10,11 ].
Further metabolism of ethylene oxide may require
the presence of an epoxidase [ 12]. Enzymes of this
class catalyse the ring-opening of epoxides with
glutathion to give corresponding thioethers, or with
water to give a diol. Non-enzymic hydrolysis of
epoxides may also occur [13]. The possibility that
ethylene glycol was an intermediate in ethylene oxide
93
metabolism in Mycobacterium E 20 had become unlikely from previous experiments [3]. Since we were
also unable to observe thiolysis of ethylene oxide
with glutathion in cell-free extracts o f the organism
(unpublished results), it became evident that a
hitherto unknown type of enzymic conversion of the
epoxide might exist.
The results presented in this paper show that an
ethylene oxide-oxidizing enzyme system is present in
cell-free extracts of ethylene-grown cells of Mycobacterium E 20, which requires NAD ÷, CoA and an
unknown cofactor in the transformation of the
epoxide to acetyl-CoA (Fig. 2, Table 1). Operation of
the glyoxylate cycle as the main carbon assimilation
pathway during growth on ethylene [3] is fully compatible with the finding that acetyl-CoA is an intermediate in ethylene metabolism. At present it is not
clear whether one single enzyme or an enzyme complex is responsible for the oxidation of ethylene
oxide. Participation of such a complex is feasible
since an enzyme is known that catalyses an analogous
reaction, i.e. pyruvate dehydrogenase complex [ 14].
Although a decarboxylation reaction is not associated
with the epoxidase, the product of both enzymes is
acetyl-CoA, while the same cofactors namely FAD,
CoA and NAD ÷ are involved. Whether the epoxidase
is indeed similar to pyruvate dehydrogenase complex
remains to be elucidated.
The specific enzyme activity of the ethylene
oxide-oxidizing enzyme system in cell-free extracts
never exceeded 4 nmoles of substrate oxidized/mg
protein/minute. This specific activity is very low, but
since no activity was found in ethane- or ethanolgrown cells, it is concluded that the enzyme system
under study is indeed involved in the oxidation of
ethylene oxide in vivo. Specific activities of other
enzymes in ethylene-grown cells were also very low
[3], which doubtless is a reflection of the extremely
low growth rate ofMycobacterium E 20 on ethylene.
Acknowledgements
We are grateful to Dr. A.C. van der Linden for
many helpful discussions and to DSM, Geleen, The
Netherlands for financial support.
References
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