FEMS MicrobiologyLetters 68 (1990) 189-194
Published by Elsevier
189
FEMSLE 03945
Purification of an enzyme responsible for acetate formation
from acetyl coenzyme A in Selenomonas ruminantium
T o m a s A. Michel n a n d J o a n M. M a c y
Department of Animal Science, University of California. Davis. Davis, CA. U.S.A.
Received28 November 1989
Accepted I December 1989
Key words: Selenomonas ruminantium: Acetate formation: Enzyme purification
1. S U M M A R Y
2. I N T R O D U C T I O N
An enzyme, capable of catalyzing the ADP-dependent conversion of acetyl coenzyme A, propionyl coenzyme A and succinyl cocnzyme A into
the corresponding free acids, has been purified
from the strict tureen anaerobe Selenomonas
ruminantium. All three activities cochromatographed and have been purified 60-fold. As determined by gel filtration, the molecular weight of
this enzyme was 230000. Gel electrophoresis in
native gels resolved the purified protein fraction
into a major and three minor protein bands. The
relative mobility of the major band coincided with
the relative mobility of single bands that were
obtained when gels were stained for activity with
each of the three substrates. The physiological role
of this new enzyme is discussed.
Preliminary evidence suggested that in the strict
rumen anaerobe Selenomonas ruminantium HD4 a
single enzyme, which we originally termed acetate
thiokinase, performs the functions of both phosphotransaeetylase and acetate kinase [1]. Neither
of these two activities were detected in cell extracts prepared from cells that were forming
acetate as a major fermentation product [1]. We
describe here the purification of such an enzyme
from S. ruminatium. However, since it has not yet
been possible to measure stoichiometric A T P formation by this enzyme, we refer to it now as aeyl
CoA esterase. The enzyme catalyzes the ADP-dependent conversion of the coenzyme A thioesters
of acetate, propionate and succinate to the corresponding free acid.
3. M A T E R I A L S A N D M E T H O D S
Correspondence to: J.M. Macy, Department of Animal Science,
University of California at Davis, Davis, CA 95616, U.S.A.
i Present address: Departamento de Microbiologia, Facuhad
de Biol6gicas, Universidadde Barcelona, Barcelona, Spain.
3.1. Organism and growth conditions
S. ruminantium subsp, lactilytica strain HD4
was grown in batch culture in 19 iitre carboys on
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
190
modified Medium 10 as described [2], with glucose
(15 raM) as substrate. After all the glucose had
been used, fumarate (9 mM) was added and growth
was allowed to proceed until the cell density had
doubled (8-9 h). In the presence of fumarate, the
lactate formed during glucose fermentation was
metabolized to propionate and acetate [2].
3.2. Preparation of ce.l extracts
Cells (38 liters) were harvested in a Sharpies
centrifuge. The following steps were carried out at
4°C. The cell paste was washed twice with 50 mM
potassium phosphate buffer (pH 7.2), suspended
(1 g wet weight cells/3 mi buffer) in potassium
phosphate buffer (pH 7.2) containing 5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonylfluoride (PMSF) and 0.1 ~M
pepstatin A and passed through an Aminco French
press at 22000 psi. Ten mg each of deoxyribonuclease 1 and ribonuclease A were added
to the cell suspension just prior to breakage. After
low speed centrifugation the lysate was recentrifuged at 30000 x g for 60 min (high speed centrifugation). The supernatant of this step (crude
extract) was used as the starting material for enzyme purification.
3.3. Acyl CoA esterase assay
Acyl CoA esterase activity was assayed by following the formation of reduced acetyl pyridine
adenine dinucleotide (APADH + H +) in a coupled reaction with 2-oxoglutarate dehydrogenase.
The assay was carried out in 1 ml or 0.5 ml
cuvettes of 1 cm light path containing 50 mM
potassium phosphate buffer (pH 7.2), 1 mM
2-oxoglutarate, 2.5 mM MgCi 2, 1.25 mM EDTA,
0.4 mM APAD, 50 ~M acyl CoA and 50 mU of
2-oxoglutarate dehydrogenase (pig heart). Formation of ATP was assayed using the same reaction
mixture in a time course assay, after heating the
samples at 90°C for 5 min, either directly with
firefly luciferase [3] or by following increased production of APADH + H + after addition of
hexokinase and glucose-6-phosphate dehydrogenase.
The reverse reaction (i.e. ATP-dependent formation of acetyl CoA from acetate) was assayed
in tris[hydroxymethyl]aminoethane(TRIS), potassium phosphate, triethanolamine or 3-[N-morpho-
lino]propanesuifonicacid (MOPS) buffers (50 mM,
pH 6.6-7.8) containing 2.5 mM MgC! 2, 1.25 mM
EDTA, 2-10 mM sodium acetate, 2-10 mM ATP
and 2-5 mM CoA. Assays were done in a number
of ways: (a) by following the increase in A232 due
to formation of the thioester bond in acetyl CoA
[4], (b) by measuring the disappearance of acetate
using either enzymatic [5] or gas chromatographic
methods [6], (c) by following ADP formation in a
pyruvate kinase- anu lactate dehydrogenase-linked
enzymatic assay and (d) by following the formation of acetyl phosphate in a phosphotransacetylase-linked enzymatic assay [7].
3.4. Enzyme purification (steps described below)
All chromatographic steps were carried out
aerobically. Column effluents were monitored for
protein by measuring the A2s0, using a Gilson
Model 111 UV detector equiped with a flowthrough cell. Chromatographic supports were
swollen, packed and equilibrated following the
manufacturers recommendations. Protein was assayed by a dye binding assay with Rose Bengal
[8l.
Step 1: Ion exchange chromatography. The high
speed centrifugation supernatant (see above) was
passed over a column (3 x 5 cm) of DEAE-cellulose (Whatman) which had been equilibrated
with 10 volumes of distilled water. All protein that
passed through the column without interacting
with the anion exchanger was collected.
Step II: Ammonium sulfate fractionation. To the
DEAE flow-through fraction solid ammonium
sulfate was added to 50% saturation (314 g/I).
The solution was stirred for 20 min, and left
standing without stirring for another 20 min. The
precipitate was collected by centrifugation at
20000 x g for 20 rain and discarded. Saturated
ammonium sulfate solution (84.9 ml, pH 7.5) was
added to the supernatant (298 mi) until 65%
saturation was reached. The precipitate was collected by centrifugation (see above) and was suspended in 25 ml of 20 mM potassium phosphate
buffer (pH 7.2) containing 30% glycerol (v/v).
This preparation was desahed on a column of
Sephadex G-25, using the suspension buffer.
St~,,s I H and IV: Dye Iigand chromatography.
The protein from the previous step was loaded
191
onto a column (2.5 × 41.5 cm) of Cibacron Blue
F3GA agarose (Affi-gel Blue, Biorad) that had
been equilibrated with 20 mM potassium phosphate buffer (pH 7.2) containing 30% glycerol
(v/v). Acyl CoA esterase activity was eluted with
1600 ml of a linear potassium chloride gradient (0
to 0.8 M) in equilibration buffer, at a flow rate of
11.25 cm h-I. Fractions containing between 0.25
and 0.45 M potassium chloride were pooled and
concentrated on an Amicon XM-100A ultrafiltration membrane (100000 molecular weight cutoff).
After desalting on a column of Sephadex G-25,
the protein was loaded onto a second column
(1.5 x 9.0 cm) of Affi-gel Blue and chromatographed as described above using a scaled-down
elution schedule. Fractions containing between
0.22 and 0.41 M potassium chloride were pooled
and concentrated by uhrafiltration.
Step V: Gel fihration. The concentrated protein
from the previous step was loaded onto a column
(2.6 × 97 cm) of Sephacryl S-300 HR (Pharmacia)
equilibrated with 20 mM potassium phosphate
buffer (pH 7.2), containing 30cg glycerol (v/v) and
0.2 M potassium chloride. The column was developed at a flow rate of 4.2 cm h- n with the buffer
used to equilibrate the column. The acyl CoA
esterase activity eluted from this column as a
single symmetrical peak with minor tailing peaks
of higher elution volume. Only those fractions
corresponding to the major peak were pooled and
concentrated by uhrafihration to a final protein
concentration of 0.8 mg/ml.
3.5. Electrophoresis
The native purified enzyme was subjected to
discontinuous electrophoresis in 6, 7, 8 and 9~
polyacrylamide gels (10 x 7 cm, C = 5~) under
nondenaturing conditions (pH 8.8), according to
Andrews [9]. After stacking (50 V, constant voltage) gels were run at a constant voltage of 150 V
until the bromophenol blue tracking dye had reached about 1 cm from thq gel bottom. Gels were
stained using Coomassie Brilliant Blue R 250 [9]
or were silver stained (silver staining kit, Biorad).
3.6. Affinity staining
Portions of the gels were also specifically stained
for enzymatic activity. Gel lanes containing the
purified enzyme were excised longitudinally and
stained by using either 0.8% agarose overlays or
by direct incubation, in a reaction mixture similar
to that used to assay the enzyme. The reaction
mixtures for activity staining contained all components present in the assay mixtures: additionally,
0.4 mg/ml phenazine methosulfate (PMS) and 0.7
mg/ml 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetrazolium bromide (MT]') were present, and acyl
CoA concentrations were increased 8-fold. Gel
strips were incubated at room temperature in the
dark for up to 120 min.
3. 7. Molecular weight determination
Molecular weight was estimated using gel filtration of Sephacryl S-200, as described above for
chromatography on Sephacryl S-300 HR. The following proteins were used as molecular weight
markers: fumarase (204000), glucose oxidase
(154 000), hexokinase (102 000), hemoglobin
(68 000) and malate dehydrogenase (40 000).
Chemical reagents were from Sigma (Sigma
Chemical Co., St. Louis, MI) or were of comparable analytical quality. Electrophoretic reagents and
supplies were from Biorad (Biorad, Richmond,
CA).
4. RESULTS AND DISCUSSION
4.1. Purification of the enzyme
The acyl CoA esterase activity bound to
Cibacron Blue F3GA agarose from which it could
be eluted at relatively low ionic strength. Binding
to affinity matrices such as desulfo CoA-linked
[10] or AMP-linked agaroses [11] was not observed. The enzymes was also not stable at the pH
values necessary for binding to DEAE-cellulose
(pH 9.5) and CM-cellulose (pH 5.0).
The results from the enzymatic purification
steps are summarized in Table 1. The enzyme has
been purified approximately 60 fold. Propionyl
CoA and succinyl CoA esterase activities cochromatographed with the acetyl CoA esterase activity and have been purified to the same degree
(footnote. Table 1). Final yields of all three purified activities were only 2.6%. Major losses of
enzymatic activity occurred primarily during dye
192
Table 1
Purification of S. ruminantium acetyl CoA esteras¢ a
Purification
step
Total
(rag)
protein
Activity
pmols/min
Sp. activity
IU's
Recovery
7o
Purification
fold
Crude extract
DEAE
50-65~ (NH 4) 2SO4
Ist Affi-Bluecolumn
2nd Affi Bluecolumn
Sephacryl S-300 HR
7057.41
5242.02
878.90
44.28
4.97
3.02
4495.6
4010.1
3807.8
770.1
178.6
117.9
0.64
0,77
4.33
17.39
35.93
39.04
100
89.2
84.7
17.1
3.9
2.6
1.2
6.8
27.3
56.4
61.3
a propionyl CoA esterase activity and succinyl Coa esterase activities have been purified 58.2- and 634,4-fold. respectively.The
specific aclivity of the purified enzymewith these two substrates was 43.73 and 86.31 pmoi APAD + per mg protein OU" s), In the
order slated.
ligand chromatography, but were also unusually
high during the final gel filtration step. Only
negligible loss of acyl CoA esterase activity could
be detected after prolonged storage (several
months) at room temperature. The enzyme appeared very sensitive, however, to even moderate
mechanical stress. Inclusion of 30~ ( v / v ) glycerol
in the separation buffers improved the mechanical
stability of the enzyme significantly.
The first chromatographic step resulted in very
little purification and was included to remove
nucleic acids which interfered with the dye ligand
chromatography. Narrowing of the 55-607o ammonium sulfate cut had little effect on the purification factor obtained in subsequent chromatographic steps, while recovery was decreased significantly.
The acyl CoA esterase activity was eluted from
the second Affi-gel blue and Sephacryl S-300 H R
columns as a well resolved symmetrical peak. The
electrophoretic analysis on 7¢$ polyacrylamide gels,
of step I! of the enzyme purification and of the
purified thiokinase enzyme in shown in Fig. 1.
Electrophoresis of the iso!atcd protein resulted in
the formation of a major and three minor bands at
all fixed gel concentration used. The relative staining intensity of the major band increased if gels
contained 30~ ( v / v ) glycerol. Rechromatography
of the purified protein on Sephacryl $-300 HR,
resulted in tailing peaks of higher elution volume.
It i~ possible that all, or a fraction, of the additional bands observed after electrophoresis were
due to partial degradation of the enzyme or to the
disaggregation of subunits from the enzyme. It has
not yet been possible to elute the major band from
preparative gels and obtain enzymatically active
protein. Furthermore. extrusion of the major band
from polyacrylamide gels and reelectrophoresis resuited in a diffuse smear of protein; even when
gels were silver stained, the major band could no
longer be identified.
A single band was revealed when gel lanes
(T = 7~, 30f[ glycerol) containing the purified
protein fraction were stained for activity using
either acetyl CoA, propionyl C o A or succinyl CoA
as substrates. Furthermore. the single bands obtained from each of these activity stains always
had the same relative mobility as the major band
detected when these same or parallel control lanes
were stained with Coomassie Brilliant Blue. This
result, and the nearly identical purification factors
obtained for the acetyl CoA, propionyl C o A and
succinyl CoA esterase activities indicated that the
ADPodependent reactions which result in the format8ion of acetate, propionate and succinate from
their corresponding acyl CoA esters are all catalyzed by the same single enzyme.
4.2. Acyl CoA esterase, properties.
The molecular weight acetyl C o A esterase from
S. ruminantium has been determined to be 230000
by gel filtration. The physiological direction of the
acetyl CoA esterase reaction in S. ruminantium
appears to be only that of acetate formation from
acetyl CoA. it has not b~.en possible to measure
enzymatic activity in the opposite direction of
acetyl CoA formation from acetate. Enzymatic
activity was optimal at a pH of 7.2, and was
193
A
Omsn~
8
-,qsmmt3
could be detected when this nocleotide was replaced by G D P or by A M P and pyrophosphate.
In crude extracts, Km values determined for acetyl
CoA, propionyl CoA and A D P were 5.9 ttM, 28.5
ttM and 27.5/tM in the order stated [1]. Detailed
kinetic analyses have not yet been performed wi~h
the purified enzyme.
The reaction catalyzed by the enzyme purified
frum S. ruminantium is similar to that carried out
by acetate out by acetate thiokinases and by the
succinate thiokinase (succinyl CoA synthetase)
found in most aerobic bacteria and higher
organisms [12]. We have however, not yet been
able to demonstrate that ATP is stoichiometrically
formed from A D P by this enzyme. ATP formation
from A D P could not be experimentally verified,
since the enzyme appeared to have an endogenous
ATPase activity, possibly similar to that described
for purified succinate thiokinase [13|. A T P was
also quite rapidly hydrolized by the enzyme in the
absence of acetate and only traces of ATP could
be detected in the reverse direction in a time-course
assay. Thus the inability to detect stoichiometry
ATP formation by the enzyme could be mostly
due to methodological problems. On the other
hand, we have presented kinetic evidence, which
rules out the role of A D P as an activator of the
enzyme and which identifies this nucleotide as a
substrate in the reaction [1].
4.3. Acetyl CoA esterase, physiological function
Fig. 1. Discontinuous elcctrophoresis of the acyl CoA esterase
enzyme on a 7% polyacrylamide gel ( l O x 7 cm, C = 5~) under
nondenaluring conditions (pH 8.8) at two different stages of its
purification. Lane A, contains a sample (20 p g protein) of ~he
5 0 - 6 5 ~ ammonium sulfate cut of the crude exlract, and lane
B, contains the nature purified enzyme (8 lag protein). The gel
was stained using Coomassi¢ Brilliant Blue R 250. The arrows
(a and b, respectively) indicate the relative position of the
bands, which were identified as corresponding to the acetyl
CoA esterase enzyme after affinity staining. The apparent
slightly higher relative mobility of the acetate thiokinase enzyme in lane A is due to the fact thai the sample loaded on this
lane was not completelydesalted.
strongly inhibited by frcc CoA. It was not possible
to measure enzymatic activity in assays that did
not involve rapid removal of this reaction product.
The enzyme was specific for ADP: no activity
The role of the acetyl CoA esterase is to catalize a reaction that in most other acetate-forming
anaerobes is catalyzed by the two enzymes phosphotransacetylase and acetate kinase [14]; neither
of these two enzymes are present in S.
ruminantium. The specific activity of acetyl CoA
esterase, measured in chemostat cultures at slow
(0.11 h - I ) and fast (0.51 h -1) dilution rates, was
found to be 75.5 and 8L2 nmol A P A D ÷ reduced
r a i n - ' m g - ' , respectively. These levels were entirely sufficient to account for all of the acetate
formed at these dilution rates. Thus the acetyl
CoA esterase provides an alternate route for
acetate f u , nation from acetyl CoA in this
organism, and is most likely responsible for all of
the acetate formed by S. ruminantium. Based on
growth yield studies and fermentation product
194
a n a l y s i s it h a s to b e inferred that S. ruminantium
c o n s e r v e s e n e r g y b y s u b t r a t e level p h o s p h o r y l a tion in this reaction w h i c h u s e s A D P as a s u b strate [1]; however, final p r o o f for this w o u l d
require t h e d e m o n s t r a t i o n o f t h e f o r m a t i o n o f
stoichiometric amounts of ATP.
Physiological a n d kinetic evidence s u g g e s t t h a t
t h e e n z y m e purified f r o m S. ruminantium c o u l d
p l a u s i b l y be a n a c e t a t e thiokinase. If this is t h e
case, t h e e n z y m e o p e r a t e s in a physiological direction distinct f r o m t h a t o f a c e t a t e t h i o k i n a s e s (E.C.
6.2.1) f o u n d in o t h e r b a c t e r i a e x a m i n e d . T h e s e
o p e r a t e physiologically m o s t l y in t h e d i r e c t i o n o f
acetyi C o A s y n t h e s i s f r o m a c e t a t e a n d are A M P d e p e n d e n t e n z y m e s [15-19]. O n l y in s o m e p r o t o zoa, devoid o f m i t o c h o n d r i a [20,21], h a s a c e t a t e
t h i o k i n a s e b e e n s h o w n to f u n c t i o n physiologically
in t h e direction o f a c e t a t e f o r m a t i o n ; t h e n u c l e o tide specificity is also t h e s a m e as that o f t h e S.
rurninantium acyl C o A esterase e n z y m e .
REFERENCES
[I] Melville, S.B., Michel, T.A. and Macy, J.M. (1988) J.
Bacteriol. 170, 5298-5304.
12] Melville, S.B., Michel, T.A. and Macy, J.M (1987) FEMS
Microbiol. Len. 40, 289-293.
13] Wulff, K. and DiSppen W. (1985) in Methods of Enzymatic Analysis 3red Ed. (Bergmeyer, H,U., ed.), Vol.
VII, 357-360.
[4] Klotzsch, H.R. (1969) Methods Enzymol. 13, 381-386.
[5] Boehringer-Mannheim, Methods of Enzymatic Food
Analysis.
[6] Macy, J.M., Fan'and, J.R. and Montgomery, L. (1982)
Appl. Envimnm. Microbiol. 44, 1428-1434.
[7] Tubbs, P.K. and Garland. P.B. (1969) Meth. Enzymol.
Vol. 18, 535-551.
|81 Elliot. J.I. and Brewer. J.M. (1978) Arch. Biochem. Biophys. 190. 351-357.
[9] Andrews, A.T. (1986) Electrophoresis. Theory. Techniques
and Biochemical and Clinical Applications. 2nd ed.
Clarendon Press. Oxford.
I10] Smith. L.T. and Kaplan, N.O. (1979) Anal. Biochem. 95.
2-7.
tll] Scopes, R.K. (19821 Protein Purification. Principles and
Practice. Springer Verlag. New York.
[12] Bridget, W.A. (1974) in The Enzymes (Boyer, P.D. ed.),
Academic Press, new York.
[13] Grinnell, F.L. and Nishimura J,S. (1968) Biochemistry 8,
562-568.
[14] Gottschalk, G. (1986) Bacterial Metabolism. Springer
Verlag, New York.
[15] Klein, H.P. and Jahnk¢, L. (1979) J. Bacteriol. 137,
179-184.
[16] Kohler, H.P. and Zehnder, A.J.B. (1984) FEMS Microbiol. Lett. 21,287-292.
[17] Oberlies, G., Fuchs, G. and Thauer. R.K. (1980) Arch.
Microbiol. 128, 248-252.
[18] Sch~ifer, K., Bartowski, C. and Fuchs, O. (1986) Arch.
Microbiol. 146, 310-308.
[19] Ihlenfeldt, .~,4.J.A.and Gibson, J. (~ ~77) Arch. Microbiol.
133, 231-241.
[20] Milller, M. (1988) Ann. Rev. Microbiol. 42, 465-488.
[21] Reeves, R.E., Warren, LG., Susskind, B. and Lo, H.-S.
(1977) J. Biol. Chem. 252, 726-731.