Download Malonate decarboxylase of Pseudomonas putida is composed of

FEMS Microbiology Letters 169 (1998) 37^43
Malonate decarboxylase of Pseudomonas putida is composed of
¢ve subunits
Shigeru Chohnan, Tooru Fujio, Toshikazu Takaki, Masami Yonekura,
Hirofumi Nishihara, Yoshichika Takamura *
Department of Bioresource Sciences, School of Agriculture, Ibaraki University, 3-21-1 Chu-ou, Ami-machi, Ibaraki 300-0393, Japan
Received 23 June 1998; received in revised form 30 September 1998; accepted 1 October 1998
Abstract
Two different forms of malonate decarboxylase were purified from Pseudomonas putida. The active form was composed of
the five different subunits K (60 kDa), L (33 kDa), Q (28 kDa), N (13 kDa), and O (30 kDa) and the inactive form was composed
of the four subunits lacking the O subunit. The former catalyzed the decarboxylation of malonate to acetate, but the latter could
not, although it retained both activities of acetyl-CoA:malonate CoA transferase and malonyl-CoA decarboxylase. The N
subunit of the active form was acylated by the incubation with [2-14 C]malonyl-CoA, but the N subunit of the inactive form was
not labeled. From the above results and the N-terminal amino acid sequence analysis, it was concluded that the O subunit was
an essential subunit to function as malonyl-CoA:ACP transacylase, which was an indispensable component of the enzyme for
the cyclic decarboxylation of malonate. z 1998 Federation of European Microbiological Societies. Published by Elsevier
Science B.V. All rights reserved.
Keywords : Malonate decarboxylase ; Pseudomonas putida; Acyl carrier protein; Malonyl-CoA ; Acetyl-CoA
1. Introduction
Malonate decarboxylase, which catalyzes the decarboxylation of malonate to acetate and CO2 , has
been studied in various bacteria. Initial investigation
related to malonate metabolism was conducted by
using the partially puri¢ed preparation of Pseudomonas £uorescens [1]. Takamura and Kitayama [2] reported that the Pseudomonas putida malonate decarboxylase was an oligomeric enzyme with a molecular
* Corresponding author. Tel.: +81 (298) 888672;
Fax: +81 (298) 888672;
E-mail: [email protected]
mass of 170 kDa composed of three subunits K, L,
and Q, and they proposed the cyclic reaction mechanism that malonate was activated by acetyl-CoA:
malonate CoA transferase to malonyl-CoA that subsequently underwent decarboxylation by malonylCoA decarboxylase thereby regenerating the acetylCoA. Recently, malonate decarboxylases were discovered from Malonomonas rubra [3^5], Klebsiella
pneumoniae [3,6], Acinetobacter calcoaceticus, P. £uorescens, and P. putida [7,8]. The enzymes from Klebsiella, Acinetobacter, and Pseudomonas are composed
of four subunits K, L, Q, and N. The N subunit is the
acyl-carrier protein (ACP) being responsible for the
reaction sequence on cyclic decarboxylation of mal-
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 6 1 - 3
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S. Chohnan et al. / FEMS Microbiology Letters 169 (1998) 37^43
onate. These results were clearly di¡erent from our
early report with P. putida malonate decarboxylase
in its subunit composition and the cyclic mechanism
of the decarboxylation of malonate.
In this paper, we show that the P. putida enzyme
consists of ¢ve subunits K, L, Q, N, and O. The ¢fth
polypeptide, the O subunit, was newly discovered to
be an essential component of the malonate decarboxylase to catalyze the transfer of the acyl residue of
short chain acyl-CoAs to the N subunit, that was
most likely related to the ACP possessing 2P-(5Qphosphoribosyl)-3P-dephospho-CoA as its prosthetic
group [3,6].
2. Materials and methods
2.1. Growth of bacteria
P. putida IAM 1177 was cultivated aerobically at
30³C for 24 h on a medium containing 10 g neutralized malonate, 10 g NH4 Cl, 0.5 g KH2 PO4 , 1.5 g
K2 HPO4 , 0.2 g MgSO4 , 1 g yeast extract, and 5 g
peptone in 1 l of distilled water (pH 7.0). Sixteen
liters of bacterial culture was centrifuged at
12 000Ug for 20 min and the yield was about 77 g
cells (wet mass).
2.2. Puri¢cation of malonate decarboxylase
The crude extract of the enzyme was prepared by
sonic disruption of cells (77 g) suspended in 300 ml
of bu¡er A, 50 mM Tris-HCl (pH 7.2) containing
0.1 mM 2-mercaptoethanol and 10 mM MgSO4 , followed by centrifugation at 17 000Ug for 20 min. The
supernatant £uid was brought to 30% saturation
with ammonium sulfate and after stirring at 4³C
overnight, the suspension was centrifuged at
27 000Ug for 30 min. The supernatant £uid was
treated with additional ammonium sulfate until
45% saturation was achieved. The precipitated proteins were recovered by centrifugation and were dissolved in 64 ml of bu¡er A. The protein solution was
subjected to gel ¢ltration on a Sepharose 4B column
(3.4U127 cm) with 10-ml fractions at a £ow rate of
30 ml h31 . Fractions with malonate decarboxylase
activity were combined. The combined solution
(175 ml) was put on a column (2.5U16 cm) of
DEAE-Sepharose CL-6B previously equilibrated
with the bu¡er A. A linear gradient elution was conducted with 0^0.3 M NaCl in bu¡er A, the total
volume of the gradient being 500 ml. The 2-ml fractions were taken at a £ow rate of 30 ml h31 . To the
combined active fractions (40 ml), ammonium sulfate
was added to achieve 50% saturation. The resulting
precipitate was dissolved in the bu¡er A (6 ml) and
put on a column (2.5U120 cm) of Sephadex G-200
at a £ow rate of 12 ml h31 with 3-ml fractions collected. The enzyme fractions were combined and adjusted to 0.75 M ammonium sulfate, then centrifuged
at 27 000Ug for 20 min. The supernatant £uid (50
ml) was loaded onto a Butyl-Toyopearl 650S column
(1.5U30 cm) equilibrated with the bu¡er B (bu¡er A
containing 0.75 M ammonium sulfate). A linear gradient elution was done with 100% bu¡er B to 100%
bu¡er A, the total volume of the gradient being 400
ml. Fractions (2-ml) were taken at a £ow rate of
12 ml h31 . Proteins were measured by the Bradford
method [9], with bovine serum albumin as standard.
In column chromatography, protein elution patterns
were usually measured by 280 nm absorption. All
operations of the puri¢cation procedure were done
at 4³C.
2.3. Malonate decarboxylase assay (cyclic reaction)
(Fig. 1a)
The mixture was composed of 50 mM neutralized
malonate, 10 mM ATP, 2 WM acetyl-CoA, 50 mM
Tris-HCl (pH 7.2), 0.1 mM 2-mercaptoethanol,
10 mM MgSO4 , 2.5 U of acetate kinase (EC 2.7.2.1),
and the enzyme solution, in a total volume of 920 Wl.
After 20 min of incubation at 30³C, 480 Wl of 2.5 M
neutralized hydroxylamine was added, and the incubation was continued for an additional 20 min at
30³C. The reaction was terminated by adding 1.4 ml
of 10 mM ferric chloride dissolved in 25 mM trichloroacetic acid-1 M HCl. The OD540nm of acetohydroxamate formed was measured. The activity was
expressed as Wmol of the acetohydroxamate formed
per min under these conditions.
2.4. Acetyl-CoA:malonate CoA transferase and
malonyl-CoA decarboxylase assays
The mixture for the acetyl-CoA:malonate CoA
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39
transferase assay (Fig. 1b) was composed of 50 mM
malonate, 2 mM acetyl-CoA, 20 mM phthalate (pH
5.0), 1 mM MgSO4 , 1 mM monoiodo acetic acid to
inhibit the malonyl-CoA decarboxylase activity, and
the enzyme solution, in a total volume of 0.1 ml. The
mixture for the decarboxylase assay (Fig. 1c) was
composed of 2 mM malonyl-CoA, 20 mM potassium
phosphate (pH 6.0), 1 mM MgSO4 , and the enzyme
solution in a total volume of 0.1 ml. Both reactions
were conducted with the same procedure as described below. After 10 min of incubation at 40³C,
0.9 ml of bu¡er A was added to each tube and then
the reaction was terminated by removing the enzyme
protein through an ultra¢ltermembrane (Millipore
Mol-Cut II). The ¢ltrate was treated with citrate
synthase (EC 4.1.3.7) to remove the acetyl-CoA remaining in the solution [10^12]. The malonyl-CoA in
the mixture was measured by the acyl-CoA cycling
method [10^12]. The transferase and decarboxylase
activities were expressed as Wmol of the malonylCoA formed and decreased per min under these conditions, respectively.
2.5. Measurement of molecular mass
The molecular mass of the enzyme was measured
by gel ¢ltration with a column of Sephadex G-200
(2.5U120 cm) [13]. The molecular masses of
subunits of the enzyme were measured by SDSPAGE [14].
2.6. Labeling of the d subunit with
[2-14C]malonyl-CoA
The enzyme was incubated with 42 WM [2C]malonyl-CoA (49.5 mCi mmol31 ) in 50 mM
Tris-HCl (pH 7.2) containing 10 mM MgSO4 for
20 min at 30³C. The reaction mixture was treated
at 95³C for 5 min with SDS gel-loading bu¡er without 2-mercaptoethanol. The 14 C-labeled enzyme
specimens were analyzed by an imager (AMBIS
4000; AMBIS) following SDS^15% PAGE.
14
2.7. Chemicals and enzyme
Acetyl-CoA and malonyl-CoA were obtained from
Sigma; [2-14 C]malonyl-CoA (speci¢c activity, 49.5
mCi mmol31 ) from NEN; acetate kinase and citrate
Fig. 1. Postulated reaction mechanism of malonate decarboxylase
from P. putida. (a) Cyclic reaction of malonate decarboxylase. I,
malonyl-CoA:ACP transacylase; II, acetyl-S-ACP :malonate ACP
transferase; III, malonyl-S-ACP decarboxylase. (b) Acetyl-CoA :
malonate CoA transferase. (c) Malonyl-CoA decarboxylase. The
enzymes II and III catalyze the reactions (b) and (c), respectively.
synthase from Boehringer Mannheim. All other materials were reagent grade or better.
3. Results
3.1. The subunit composition of the active and inactive
forms of the P. putida malonate decarboxylase
The results of the overall puri¢cation of the P.
putida malonate decarboxylase are summarized in
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Table 1
Puri¢cation of malonate decarboxylase from Pseudomonas putida
Puri¢cation step
Protein (mg)
Total activity (units)
Speci¢c activity (units mg31 )
Puri¢cation (fold)
Recovery (%)
Crude extract
Ammonium sulfate
Sepharose 4B
DEAE-Sepharose
Sephadex G-200
Butyl-Toyopearl
8710
1920
927
304
175
19
12665
8819
5232
2940
2349
393
1.4
4.6
5.6
9.7
13.4
20.7
1.0
3.3
4.0
6.9
9.6
14.8
100.0
69.6
41.3
23.2
18.5
3.1
Table 1. Speci¢c activity of the puri¢ed enzyme obtained in the ¢nal step was 20.7 U mg protein31 with
14.8-fold increase and over all recovery was 3.1% in
the enzyme activity. In the ¢nal puri¢cation step,
using Butyl-Toyopearl 650S chromatography, the
elution pro¢le of the protein showed two peaks, i.e.
one of them was eluted with 0.46^0.50 M of ammonium sulfate (Fraction A) and the other with 0.41^
0.43 M of that (Fraction B) (Fig. 2a). The malonate
decarboxylase activity was detected in the minor
peak, Fraction B (19 mg as protein), whereas no
malonate decarboxylase activity was detected in the
major peak, Fraction A (102 mg as protein). Puri¢ed
active and inactive protein both showed a single protein band on native PAGE (Fig. 2b); however, they
were distinctly di¡erent in the subunit composition
as well as in enzymatic activity. The SDS-PAGE of
the active enzyme showed ¢ve protein bands with
molecular masses of 60 (K), 33 (L), 30 (O), 28 (Q),
and 13 kDa (N) (Fig. 2c). Composition of the ¢ve
di¡erent subunits of the active form was assigned
to 1:1:1:1:1 by a densitometric analysis, although
the N subunit was stained only faintly with Coomassie brilliant blue. On the other hand, the inactive
enzyme was composed of the four subunits lacking
the O subunit. Each active and the inactive enzyme
was eluted in a single peak of protein after the Sephadex G-200 column chromatography, corresponding to molecular masses of 165 and 130 kDa, respectively. The above results indicated that the native
malonate decarboxylase from P. putida was composed of the ¢ve di¡erent subunits involving the O
subunit newly discovered in this study. The O subunit
was susceptible to dissociate from the complex of
other four subunits to yield the inactive form of
which subunit composition was identical with those
of enzymes consisting of four di¡erent subunits reported by Schmid et al. [6] and Byun and Kim [8].
Fig. 2. Puri¢cation of malonate decarboxylase. (a) Elution pro¢le of the enzyme on Butyl-Toyopearl 650S (see Section 2). (b) Native
PAGE (7.5%) of 10 Wg Fraction A (inactive form) (lane 2) and 10 Wg Fraction B (active form) (lane 1) on Butyl-Toyopearl 650S. (c)
SDS-PAGE (12.5%) of 10 Wg Fraction A (inactive form) (lane 2) and 10 Wg Fraction B (active form) (lane 1). Numbers on the left show
the molecular masses of size standards.
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41
3.2. Function of the e subunit
As shown in Table 2, the inactive form of malonate decarboxylase lacking the O subunit was not
able to catalyze the decarboxylation of malonate in
a cyclic manner. However, it still retained activities
of acetyl-CoA:malonate CoA transferase (Fig. 1b)
and malonyl-CoA decarboxylase (Fig. 1c). Since
the two speci¢c activities of the inactive form were
almost the same as those of the active form, the O
subunit therefore apparently must catalyze another
reaction required for the cyclic decarboxylation of
malonate.
The N-terminal amino acid sequence of the O subunit, 1-SSLFAFPGQGAQQVGMLQRLPEGCGQLLEE-30 was similar to those of malonyl-CoA:ACP
transacylases (EC 2.3.1.39), i.e. MdcH in gene cluster
encoding malonate decarboxylase from K. pneumoniae [15] and FabDs from Bacillus subtilis [16], Escherichia coli [17,18], and Haemophilus in£uenzae
[19]. Therefore, it was predicted that the O subunit
would have a function like the malonyl-CoA:ACP
transacylase in the fatty acid synthase system.
The labeling of N subunit (HS-ACP) with [214
C]malonyl-CoA was conducted, using both forms
of the enzyme. After incubation of the active form
with [2-14 C]malonyl-CoA, the N subunit became labeled by 14 C radioactivity (Fig. 3, lane 2). Furthermore, the labeled [2-14 C]malonyl-residue of N subunit
was completely released by incubating with an excess
amount of cold malonate (Fig. 3, lane 3). On the
other hand, the N subunit of catalytically inactive
enzyme did not label at all (Fig. 3, lane 4). These
results indicate that the O subunit was responsible for
the acylation of the N subunit (HS-ACP). The N subunit of the enzyme obtained in this study seems to be
deacylated during the puri¢cation, since the active
form did not represent the catalytic activity without
such short chain acyl-CoA as malonyl-CoA, acetyl-
Fig. 3. Labeling of the N subunit with [2-14 C]malonyl-CoA. Eight
Wg of active form enzyme (lane 2) or inactive form enzyme (lane
4) was incubated with 42 WM [2-14 C]malonyl-CoA. Lane 1 is the
sample incubated without the enzyme. Sample of lane 3 was incubated with 8 Wg of active form enzyme, 42 WM [2-14 C]malonylCoA, and 50 mM cold malonate. Numbers on the left show the
molecular masses of size standards.
CoA, propionyl-CoA, acetoacetyl-CoA, butylylCoA, or methylmalonyl-CoA as acyl donors.
3.3. Activation of the inactive form enzyme by the
active form enzyme and malonyl-CoA
The inactive form was e¡ectively activated by the
incubation with the active form and malonyl-CoA to
show the cyclic decarboxylation of malonate (Table
3). After the incubation of 45 pmol of the inactive
form with 5 pmol of active form and malonyl-CoA,
it was activated to show 95% of the enzyme activity
of 50 pmol of the active form. Likewise, 2.5 and
1 pmol of the active form activated the inactive enzyme to show 68 and 41% maximal activity, respectively. The above results indicate that a catalytic
amount of the active form is able to activate the
Table 2
Comparison of transferase and decarboxylase activities between active and inactive malonate decarboxylases
Form
Cyclic reaction (units mg31 )
Transferasea (units mg31 )
Decarboxylaseb (units mg31 )
Subunits
Active form
Inactive form
20.7
6 0.1
2.4
1.9
0.8
1.5
K, L, Q, N, and O
K, L, Q, and N
a
b
Acetyl-CoA:malonate CoA transferase.
Malonyl-CoA decarboxylase.
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S. Chohnan et al. / FEMS Microbiology Letters 169 (1998) 37^43
Table 3
Activation of the inactive form enzyme by the active form enzyme and malonyl-CoAa
Added enzyme (pmol)
Cyclic reaction
Active form
(units)
(%)
0.22
0.22
0.22
0.22
0.21
0.21
0.17
0.09
0.06
6 0.001
100
100
100
100
95
95
68
41
27
6 0.5
b
50
35
25
15
10
5
2.5
1
0.5
0
Inactive form
c
0
15
25
35
40
45
47.5
49
49.5
50
a
After the incubation of the mixture containing the enzymes of the
amount listed in table and 2 WM malonyl-CoA at 30³C for 10 min,
the cyclic reaction was initiated by adding 50 mM malonate.
b
50 pmol of the active form enzyme corresponds to 8.1 Wg.
c
50 pmol of the inactive form enzyme corresponds to 6.6 Wg.
inactive enzyme with the consequence that the O subunit, which is loosely associated with the complex of
the other four subunits, is able to catalyze the transacylation of malonyl-residue to the N subunit of inactive form. Not only malonyl-CoA, but also acetylCoA, propionyl-CoA, acetoacetyl-CoA, butylylCoA, or methylmalonyl-CoA was an active donor
of acyl residue to the N subunit (data not shown).
The most prominent activation was triggered by the
addition of either one of malonyl-CoA or acetylCoA. The results established that the O subunit is
an indispensable protein with the function of malonyl-CoA:ACP transacylase to form the acyl-S-ACP
that is an essential intermediate for cyclic decarboxylation of malonate.
4. Discussion
In this study, we demonstrated that malonate decarboxylase from Pseudomonas putida IAM 1177 is
an oligomeric enzyme consisting of ¢ve di¡erent subunits K (60 kDa), L (33 kDa), Q (28 kDa), N (13 kDa),
and O (30 kDa) and that the O subunit newly discovered was responsible for the acylation of the N subunit (HS-ACP). Hoenke et al. [15] suggested the existence of such a catalytic protein (MdcH) required
for the acylation of the N subunit, based on the de-
duced amino acid sequence of the gene encoding
malonate decarboxylase from K. pneumoniae. The
Klebsiella enzyme, however, was composed of the
four subunits K (MdcA), L (MdcD), Q (MdcE), and
N (MdcC) lacking MdcH [6]. There are several discrepancies between Pseudomonas enzyme and Klebsiella enzyme in point of the subunit composition
and the necessity of the O subunit. The O subunit
was susceptible to dissociation from the intact malonate decarboxylase consisting of the ¢ve subunits,
resulting in the inactive enzyme consisting of four
subunits. Even by the careful procedure of puri¢cation, the dissociation of O subunit occurred and signi¢cant loss of the enzyme activity was observed.
The native enzyme preparation seemed to become a
mixture of the two forms with four and ¢ve subunits
during the puri¢cation. Butyl-Toyopearl chromatography was an e¡ective tool to separate the native
enzyme protein composed of the ¢ve subunits from
the inactive form composed of the four subunits.
These phenomena account for 83% loss of the cyclic
decarboxylation activity at the step of the ButylToyopearl 650S column chromatography.
In the earlier report from this laboratory [2], the
subunit composition of this enzyme was mistakenly
estimated to be three, K, L, and Q. This reason might
be due to the experimental facts that the N subunit
was stained faintly with Coomassie brilliant blue and
that a presence of small amount of the O subunit
brought a considerable malonate decarboxylase activity (Table 3).
Although all malonate decarboxylases composed
of the four subunits from K. pneumoniae [6], A. calcoaceticus, P. £uorescens, and P. putida [8] were reported to be activated by short chain acyl-CoAs, the
inactive form of Pseudomonas enzyme was not activated at all in the presence of malonyl-CoA. For the
activation of the inactive enzyme, the catalytic
amount of the O subunit involved in the active
form was required.
Previously, we reported [2] that malonate was decarboxylated in a cyclic manner by two steps involving acetyl-CoA and malonyl-CoA as metabolic intermediates and that short chain acyl-CoAs as
propionyl-CoA, acetoacetyl-CoA, butylyl-CoA, and
methylmalonyl-CoA were also recognized as a substrate [10]. However, this study shows the reaction
mechanism proposed by us is in need of review, be-
FEMSLE 8462 17-11-98
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cause the subunits O and N were identi¢ed as malonyl-CoA:ACP transacylase and ACP in P. putida malonate decarboxylase, respectively. The cyclic mechanism of P. putida malonate decarboxylase seems to
closely resemble with the proposed catalytic mechanism of K. pneumoniae enzyme [6] except for the
indispensable role of the O subunit. In P. putida,
malonate decarboxylase, free malonate is decarboxylated to acetate in cyclic manner as follows (Fig. 1a).
First the N subunit (HS-ACP) is acylated to acyl-SACP by the O subunit in the presence of acyl-CoAs
as active acyl donors. Second, the acyl residue on the
N subunit is replaced to malonate. Then the malonyl
residue on the N subunit subsequently undergoes the
decarboxylation, thereby generating acetyl-S-ACP
by the subunit(s) among subunits K, L, and/or Q.
Thus, the O subunit is an integral part of the puri¢ed
malonate decarboxylase to trigger decarboxylation
of malonate in cyclic fashion. The cloning and sequencing of the gene cluster encoding malonate decarboxylase from P. putida will be published in another report.
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