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Plant Physiol. (1990) 92, 73-
Received for publication (^) April 24, 1989
and in revised form August 29, 1989
Purification and Characterization (^) of Acetyl-CoA
Carboxylase from the Diatom (^) Cyclotella cryptica
Paul G. (^) Roessler Biotechnology Research (^) Branch, Solar Energy Research Institute, Golden, Colorado 80401
ABSTRACT Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl-CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a mo- lecular weight of approximately 740 kilodaltons and appears to be composed of four identical (^) biotin-containing subunits. The enzyme has maximal activity at pH (^) 8.2, but (^) enzyme stability is greater at pH 6.5. Km values for MgATP, (^) acetyl-CoA, and (^) HC were determined to be 65, 233, and (^750) micromolar, respectively. The purified enzyme is strongly inhibited by (^) palmitoyl-CoA, and is inhibited to a lesser extent by (^) malonyl-CoA, ADP, and (^) phos- phate. Pyruvate stimulates enzymatic activity to a (^) slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxyphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar cleth- odim and 100 micromolar haloxyfop, respectively.
taining subunits are commonly observed, leading several in-
vestigators to suggest that plant acetyl-CoA carboxylases are
multifunctional proteins able to catalyze both steps of the
reaction (biotin carboxylation and carboxyl transfer to acetyl-
CoA) (8).
Little is known about the allosteric regulation of acetyl-
CoA carboxylase activity in higher plants. Adenylate nucleo-
tides (AMP, ADP, and free ATP) have been shown to inhibit
the enzyme from several higher plants (2, 3, 15, 20). Acetyl-
CoA carboxylase from maize is also inhibited by malonyl-
CoA and palmitoyl-CoA (15). Free coenzyme A has been
shown to inhibit the enzyme from maize (15) but to stimulate
the activity of the enzyme from spinach (1 1). Citrate, free
Mg2+, K+, and glycine have also been reported to stimulate
acetyl-CoA carboxylase from various plant sources (6, 12-
The research described in this (^) report was carried out in
order to further our understanding ofacetyl-CoA carboxylases
from eukaryotic microalgae and to compare the properties of
acetyl-CoA carboxylase from the diatom Cyclotella cryptica
with those of higher plant acetyl-CoA carboxylases.
Acetyl-CoA carboxylase is a biotin-containing enzyme that
catalyzes the formation of malonyl-CoA, which is one of the
initial steps of fatty acid biosynthesis. Previous research has
indicated that changes in the activity ofthis enzyme may play
a role in the (^) accumulation of lipids when the diatom Cyclo- tella cryptica is grown under (^) silicon-limiting conditions (19).
It was therefore of interest to investigate the properties of
acetyl-CoA carboxylase from this alga. Although acetyl-CoA
carboxylase has been purified from several higher plants, the
enzyme has not previously been purified from an algal source.
Acetyl-CoA carboxylases from higher plants have several
characteristics in common, including an alkaline pH opti-
mum and an absolute requirement for ATP and divalent
metal cations. Early studies indicated that plant acetyl-CoA
carboxylases had complex subunit structures (three to six
peptides having different mol wt), but more recent studies
suggest that these earlier attempts to characterize the enzyme
were subject to error due to the effects of endogenous prote-
olytic activity. Recent investigations have suggested simpler
subunit structures. High mol wt (200-240 kD), biotin-con-
' (^) This research was supported by the Biofuels and (^) Municipal Waste Division of the U.S. Department of Energy under FWP BF8 1. (^2) Present address: Department of Botany and Plant Pathology,
Michigan State University, East (^) Lansing, MI 48824.
MATERIALS AND METHODS
Organism and Growth Conditions
Cyclotella cryptica Reimann, Lewin, and Guillard strain
T13L was obtained from the Culture Collection of Marine
Phytoplankton at the Bigelow Laboratory for Ocean Sciences
(W. Boothbay Harbor, ME). Cells were cultured in 2 L
polycarbonate bottles as described previously (18). Cultures
were bubbled with 0.5% CO2 in air (500 mL/min) and main-
tained at 25°C under constant illumination from fluorescent
lamps (photon flux density at the vessel surface averaged over
3600 =^85 ,umol quanta- m-2.s ').
Materials
Analytical grade haloxyfop and clethodim were kindly pro-
vided by J. Secor (Dow Chemical Co., Walnut Creek, CA)
and A. Rendina (Chevron Chemical Co., Richmond, CA),
respectively.
Analytical Methods
Protein was quantified by the Coomassie blue dye-binding
method (Bio-Rad) using bovine y-globulin as a standard.
Radioactivity was determined by liquid scintillation counting
in a Beckman model LS9000 scintillation counter, using the
73
Plant Physiol. Vol. 92, 1990
H# routine for quench correction. Biofluor (Dupont/New
England Nuclear) or Optifluor (Packard) was used as the
scintillation cocktail for all isotope studies.
Electrophoresis
SDS-PAGE was performed as described by Laemmli (10)
with a 7.5% separating slab gel. Proteins were transferred to
nitrocellulose membranes with a semidry electroblotting ap-
paratus (American Bionetics) using the buffer system of
Shafer-Nielsen et al. (23). Proteins were detected by the Bio-
Rad Biotin-Blot procedure, using the manufacturer's proto-
col. Biotin-containing proteins were detected by the same
procedure, except that the protein biotinylation step was
omitted.
Nondenaturing discontinuous PAGE was performed on 5
mm diameter tube gels (5%tota1/5%bis separating gel) with a
Hoefer model DE 102 unit, using the manufacturer's protocol.
Assay for Acetyl-CoA Carboxylase Activity
Acetyl-CoA carboxylase activity was measured by the in-
corporation of ['4C]bicarbonate into acid- and heat-stable
material (malonyl-CoA) using a modification ofthe procedure
of Sauer and Heise (20). The reaction mixture (final volume
= 0.3 mL) contained 100 mm Tricine buffer (pH 8.2), 0.5 mM
acetyl-CoA, 1 mm ATP, 2 mM MgCl2, 10 mm KCI, and 10
mM (^) ['4C]NaHCO3 (specific activity = (^1) 1.1 MBq/mmol). The
reaction was initiated by the addition of enzyme and termi-
nated after 10 min at 3O°C by the addition of 0.3 mL of 2 N
HCI. A^ portion (0.5 mL) of the acidified solution was trans-
ferred to a scintillation vial and heated at 70°C until dry (
h). The residue was dissolved in 0.3 mL of 0.2 N HCI prior to
the addition of scintillation cocktail. Control assays were
carried out in the absence of acetyl-CoA in order to correct
for nonspecific radioactivity, which was typically less than 5%
of the acetyl-CoA-dependent "1C incorporation. One unit of
activity is defined as the amount of enzyme required to
catalyze the formation of 1 ,umol of malonyl-CoA per minute
under standard assay conditions.
The reaction product was analyzed by the procedure de-
scribed by Thomson and Zalik (24), which includes an alka-
line hydrolysis step to cleave the thioester linkage to coenzyme
A. The reaction product comigrated with authentic ["`C]
malonate (Sigma Chemical Co.) on silica gel TLC plates (J.
T. Baker Co., Si250) developed in water-saturated diethyl
ether:formic acid (7:1, v:v) (24) and on Whatman No. 1 paper
developed in^ isobutyric acid:NH40H:H20 (66:1:33, v:v:v)
Preparation of Cell-Free Extracts
Cells were harvested (^) by centrifugation at (^) 5,000g for 5 min and washed with MCD buffer (100 mm Mes containing 10
mM K-citrate and 2 mm DTT, pH 6.5). The cells were then
suspended in^10 to^25 mL^ of^ MCD^ buffer^ and^ passed through a French pressure cell at 15,000 (^) psi. The (^) pressate was centri- fuged at 37,000g for 20 min. The supernatant was diluted
with MCD buffer so that the total protein concentration was
below (^5) mg/mL; this is referred to as the crude extract. Cell
disruption and all subsequent purification steps were carried
out at 4C.
Purification Procedure
(NH4)2SO4 Fractionation
A saturated solution of ice-cold (NH4)2SO4 was added to
the crude extract with stirring to yield a 30% saturated solu-
tion, which^ was^ then^ centrifuged at 8000g for 5 min. The
precipitate was^ discarded^ and^ the supernatant solution was
subjected to^ further^ fractionation by the addition of
(NH4)2SO4 to 43, 50, and 60% saturation. All precipitates
were discarded except for the one obtained at 60% saturation,
which was dissolved in 5 to 10 mL of MCD buffer and used
for the gel filtration chromatography step.
Gel Filtration Chromatography
The solution obtained after (NH4)2SO4 fractionation was
loaded onto a 90 x 2.2 cm column of Biogel A- 1.5 m (Bio-
Rad) and eluted with MCD buffer at a flow rate of 20 mL/h.
The fractions containing the highest acetyl-CoA carboxylase
activity (numbers 41-57, 4 mL each) were combined and
used in the affinity chromatography step.
Affinity Chromatography
The naturally occurring biotin molecules found in acetyl-
CoA carboxylase allow affinity chromatography through col-
umns containing covalently bound avidin. Monomeric avi-
din-agarose was prepared by incubation of 10 mg tetrameric
chicken egg avidin (Calbiochem) in 4 mL 50 mm Hepes (pH
8.0) with 2 mL ofAffi-gel 10 (Bio-Rad) for 4 h at 4°C, followed
by treatment of the gel with 6 M guanidine-HCl (pH 2.15) for
16 h at 20°C. Noncovalently attached avidin subunits were
removed from the column by washing with several column
volumes of 6 M guanidine-HCl. The column was prepared for
use by passing four column volumes of MKD buffer (100 mm
Mes buffer with 100 mM KCI and 2 mM DTT) containing 0.
mg biotin/mL through the column to saturate the biotin-
binding sites followed by 10 column volumes of 0.1 M glycine
(pH 2) to remove exchangeable biotin (9). Portions of the gel
filtration-purified solution were passed through a 2 mL col-
umn of monomeric avidin-agarose, followed by washing
with 15 mL of MKD buffer. Acetyl-CoA carboxylase was
eluted from the column with MKD buffer containing 0.5 mg
biotin/mL.
RESULTS
Acetyl-CoA carboxylase was purified from Cyclotella cryp-
tica by a simple procedure utilizing (NH4)2S04 precipitation,
gel filtration^ chromatography, and^ affinity chromatography
with monomeric avidin-agarose. This procedure resulted in a
nearly homogeneous preparation having a specific activity of up to 14.6 ,mol malonyl-CoA formed mg protein'. minI', which represented an increase in (^) specific activity of (^) approxi-
mately 600-fold. The results from a typical purification are
shown in Table I.
Gel filtration chromatography of C. cryptica acetyl-CoA
(^74) Roessler
Plant Physiol. Vol. 92, 1990
tion by the use of a desalting column, enzymatic activity was
completely lost and could not be restored by the readdition
of NaCl or KCI. Partially purified acetyl-CoA carboxylase
from C. cryptica could be stored frozen at -20°C for at least 3
weeks with little loss in activity. However, affinity-purified
acetyl-CoA carboxylase was less stable, losing all activity upon
freezing and a substantial portion (30%) ofthe inititial activity
after 24 h at 4°C.
The activity of the enzyme was dependent upon the pres-
ence of divalent metal cations, with Mg2' being the most
effective cation tested. When ATP was included at a concen-
tration of 1 mm, maximal acetyl-CoA carboxylase activity
was observed at a Mg2' concentration of 2 mm (Fig. 3). Above
this concentration, enzyme activity declined^ to a^ slight^ extent.
Mn2+ was able partially to replace the Mg2' requirement, but
2 mm MnCl2 yielded only 20% of the activity observed in the
presence of 2 mm^ MgCl2.^ The enzyme was inactive when
CO2+ (2 mM) was the only divalent metal cation present.
Purified acetyl-CoA carboxylase from^ C.^ cryptica^ exhibited
Michaelis-Menten kinetics with^ respect to MgATP,^ acetyl-
CoA, and NaHCO3. The apparent Km values^ for^ these sub-
strates were determined to be 65, 233, and 750 (^) gM, respec-
tively. Citrate (1 mM) did^ not^ affect^ the Km^ of^ the^ enzyme^ for
acetyl-CoA or the Vma of the reaction using different^ concen-
trations of acetyl-CoA as substrate (data not^ shown).^ Pro-
pionyl-CoA was a poor substrate for^ C.^ cryptica^ acetyl-CoA
carboxylase; 0.5 mM propionyl-CoA was carboxylated at only
one-tenth the rate of 0.5 mM acetyl-CoA.
The effects of various cellular metabolites on the activity of
purified acetyl-CoA carboxylase are shown in Table II. Ma-
lonyl-CoA, ADP, and orthophosphate, which are all products
ofthe reaction, substantially inhibited acetyl-CoA carboxylase
activity. The inclusion of 1 mM ADP and 1 mM^ NaH2PO4 at
me same timc^ LA *w +^ sS1sCA07^ LA_0_I,.:_^ -^ 4-,1^ --A
carboxylase ac
reaction by 84
creasing enzyn
._^ c 0
co
L
I-
0
0 0
5
4
3
2
OE- 0
Figure 3. Effect CoA carboxylas were added per
Table II. Effects of Various Compounds on the Activity of Acetyl- CoA Carboxylase Purified from Cyclotella cryptica The values shown are the mean values obtained from two to four separate experiments. Additions Relative Activity ± SD None looa 1 mM malonyl-CoA 16.0 ±^ 1. (^100) AM malonyl-CoA 59.7 ± 6. 1 mM NaH2PO4 53.0 ± 4. 1 mM ADPb 51.3 ± 2. 1 mM NaH2PO4 + 1 mM ADPb 36.2 ± 0. 1 mM AMP 94.8 ± 1. (^100) yM palmitoyl-CoA 21.8 ± 4. 10 Mm palmitoyl-CoA 64.7 ± 8. 100AMCoA 98.1±3. 1 mM 3-phosphoglycerate 94.4 ± 0. 1 mM phosphoenolpyruvate 84.1 ± 8. 1 mM pyruvate 148.8 ± 10. 1 mm glucose-1-phosphate 103.1 ± 9. 1 mM glucose-6-phosphate 100.1 ± 8. 1 mM fructose-6-phosphate 94.6 ± 7. 1 mm (^) fructose-1,6-bisphosphate 99.7 ± 8. 1 mM citrateb 107.0 ± 8. 1 mm acetate 94.1 ± 6. 1 mM NADH 98.3 ± 5. 1 mM NADPH 87.3 ± 7. 1 mM NAD+ 100.0 ± 0. 1 mM NADP+ 94.5 ± 5.
a 100% relative activity ranged from 6.7 x^1 0-4 to^ 1.2^ x^ 10-3 units/
mL reaction mixture for the different experiments. 2.0 x^ 10-4 to 3.
X 1 0-4 units of affinity-purified enzyme were added per reaction,
depending on the experiment. b^ Additional MgCI2 (1 mM) was also
included in^ these assays to overcome^ the^ effects^ of^ Mg2+^ chelation.
e resuitec in^ a o4,o recuction in^ acetyi-LoA centration of (^100) Mm. Free palmitate was also quite (^) inhibitory; 'tivity, while 1 mM^ malonyl-CoA inhibited^ the^100 AM palmitate (which is approximately three times^ higher
M%. Palmitoyl-CoA was a^ strong inhibitor, de-^ than the solubility limit of palmitate in water)^ inhibited
natic activity by 78% when^ included^ at a^ con-^ malonyl-CoA formation by 44%. The^ photosynthetic/glyco-
lytic intermediates^ 3-phosphoglycerate, phosphoenolpyru-
vate, fructose- 1 ,6-bisphosphate, glucose- 1-phosphate, glucose-
6-phosphate, and^ fructose-6-phosphate had^ little effect^ on
acetyl-CoA carboxylase activity, but 1 mM pyruvate was
shown to consistently stimulate enzymatic activity by approx-
imately 50%. CoA, which stimulates the activity of certain
higher plant acetyl-CoA carboxylases (11) while inhibiting
others (15), had no effect on the enzyme from C. cryptica.
Citrate, acetate, AMP, NADH, NADPH, NAD+, and^ NADP+
(1 mM) likewise did^ not^ alter^ acetyl-CoA carboxylase activity
substantially.
Since acetyl-CoA carboxylase has recently been shown to
be the site of action of monocot-specific aryloxyphenoxypro-
pionic acid and cyclohexanedione herbicides^ (1, 16, 21), it
was of interest to determine the^ effects^ of^ these^ herbicides^ on
the activity of^ purified C.^ cryptica^ acetyl-CoA carboxylase.
2 4 6 8 10 12 The cyclohexanedione herbicide clethodim inhibited C. cryp-
tica acetyl-CoA carboxylase activity in a dose-dependent man-
[MgCI2 ]^ (mM) (^) ner, with 50% inhibition occurring at a concentration of 23
t of MgC12 on the activity of purified C. cryptica acetyl- AM (Fig. 4). Conversely, the aryloxyphenoxypropionic acid
e. A total of 6.1 x^1 0 4units^ of^ affinity-purified enzyme herbicide^ haloxyfop did not affect^ enzymatic^ activity^ at^ con-
reaction. centrations^ up^ to^100 MM.
76 Roessler
ACETYL-CoA CARBOXYLASE FROM CYCLOTELLA CRYPTICA
100
80
0
z 0
60
40
20
0 I 10 100
[CLETHODIM] (MM)
Figure 4. Inhibition of purified C. cryptica acetyl-CoA carboxylase by clethodim. A freshly prepared 20 mm clethodim stock solution (in ethanol) was diluted into 50 mm Tricine (pH 8.2) immediately prior to the assay. The mean values (±SD) from two separate experiments are shown. The^ mean^ value^ for^ 100% relative^ activity^ for^ the^ two experiments was^ 6.5^ units/mg protein.^ An^ average^ of 6.3^ x^1 04 units of (^) affinity-purified enzyme were added per reaction.
DISCUSSION
Many properties of acetyl-CoA carboxylase from the^ dia-
tom Cyclotella cryptica are similar to those of acetyl-CoA
carboxylases isolated from various higher plants. Like the enzyme from^ higher plants, C.^ cryptica acetyl-CoA carboxyl- ase is a large (about 740 kD) protein composed of^ several subunits. The molecular mass ofacetyl-CoA carboxylase from wheat germ (4), avocado (12), castor seed (6), maize (15), and cultured parsley cells^ (5) was^ reported to be^ 700, 650,^ 528, 500, and 420 kD, respectively. C.^ cryptica acetyl-CoA carbox- ylase appears to be a tetrameric protein composed of four identical biotin-containing subunits, each having a molecular mass of about 185 kD. This suggests that the subunits are multifunctional (^) peptides containing domains responsible for both biotin carboxylation and^ subsequent carboxyl transfer to acetyl-CoA. Acetyl-CoA carboxylase from maize^ is also composed of multiple identical subunits (15), but in this case the subunits are (^) only 60 to 61 kD. Acetyl-CoA carboxylase from cultured parsley cells is^ composed oftwo^ equal subunits, each having a molecular mass of 220 kD (5). Although the mol wt of native acetyl-CoA carboxylase from soybean and oil seed rape are not known, these^ enzymes have^ been^ shown to be composed of identical subunits having molecular mass
of 240 and 220 kD, respectively (2, 8). Another variety of
soybean ("Wayne")^ exhibited^ a more^ complex^ subunit^ struc- ture, however (2). Acetyl-CoA carboxylase from C. cryptica and the (^) majority of acetyl-CoA carboxylases from higher plants therefore appear to be similar in that they are composed of multiple, but identical, subunits. The number of subunits in the various holoenzymes can vary substantially, however. It is rather surprising that so much diversity in the structure of this^ ubiquitous enzyme exists^ among different^ plants. It is interesting to^ note that^ the^ subunit^ structure^ of C.^ cryptica acetyl-CoA carboxylase is^ more^ similar^ to^ acetyl-CoA carbox-
ylase from brewer's yeast^ (which^ is^ composed^ of four^ identical 150 kD subunits [22]) than to^ the acetyl-CoA^ carboxylases described thus far^ from^ higher plants. The activity of all acetyl-CoA carboxylases studied to date has been shown to be dependent upon the presence ofdivalent metal cations. C. cryptica acetyl-CoA carboxylase also exhib- ited this characteristic (Fig. 3). It has been demonstrated by several researchers (6, 12, 13, 20) that MgATP is the actual substrate for acetyl-CoA carboxylase, and it is assumed that this is also the^ case^ for^ acetyl-CoA^ carboxylase^ from^ C. cryptica. In^ addition, free^ Mg2+ stimulates^ acetyl-CoA^ carbox- ylase activity^ in^ several higher plants (6, 12,^ 13,^ 20).^ Based^ on the increase in^ activity of C. cryptica acetyl-CoA carboxylase
due to concentrations of^ Mg2> that^ exceeded the^ ATP^ concen-
tration in^ the assay mixture^ (Fig. 3), it^ appears^ that free^ Mg2+ also stimulates the activity of the C.^ cryptica enzyme. The Km values obtained for C.^ cryptica acetyl-CoA carbox- ylase for acetyl-CoA, MgATP, and^ bicarbonate (233,^ 65,^ and 750 uM, respectively) are similar to the^ Km values^ reported for higher plant acetyl-CoA carboxylases. A^ survey of Km values for acetyl-CoA carboxylases from several higher plants, (in- cluding spinach [12, 20], avocado [12], maize [15], wheat [7], parsley [5], soybean [2], barley [16], and castor seed [6]) yielded ranges of 26 to (^320) tLM for acetyl-CoA, 21 to 460 uM for (^) MgATP, and 0.86 to 8 mm for bicarbonate. Unlike the case for acetyl-CoA carboxylase from^ avocado^ and^ spinach (12), citrate did not affect the Vma of the reaction when the enzyme was supplied with^ different concentrations^ of^ acetyl- CoA. The activity of C. cryptica acetyl-CoA carboxylase can^ be modulated by several metabolites. As is the case with^ acetyl- CoA carboxylase from several higher plants (2, 3, 15, 20), the diatom enzyme is^ inhibited^ by ADP. For the^ higher plants examined, this^ inhibition^ appears to^ be^ competitive^ with respect to ATP.^ C.^ cryptica acetyl-CoA carboxylase^ is^ also inhibited by orthophosphate and^ malonyl-CoA. It is^ not^ clear whether the inhibitory effects of ADP, phosphate, and^ ma- lonyl-CoA are due to true allosteric mechanisms or^ simply to shifts in the thermodynamic equilibrium of the reaction. Nonetheless, it is clear that acetyl-CoA carboxylase activity would be higher during periods of photosynthesis due to the combined effects of increased pH and Mg" levels and de- creased ADP and orthophosphate levels within the chloroplast (the presumed location of^ the^ enzyme). The strongest inhibitor of C. cryptica acetyl-CoA carbox- ylase activity tested^ was^ palmitoyl-CoA,^ which^ inhibited^ the reaction by 35 and 78% at concentrations of^ 10 and 100 ,uM, respectively. It appears that the acyl component is at^ least partially responsible for this inhibition since free palmitate also inhibited enzymatic activity quite strongly. Palmitoyl- CoA was also reported to inhibit maize leaf^ acetyl-CoA car- boxylase activity (15), with^ nearly complete inhibition^ occur- ring at a concentration of 37.5 (^) gM. The low concentration of acyl-CoA (or free fatty acids) required to inhibit acetyl-CoA carboxylase suggests that this inhibition may be physiologi- cally relevant under conditions when acyl chains are not incorporated into membrane lipids or exported from the chloroplast at rates comparable to their rates of synthesis. The only metabolite tested that stimulated the activity of
10 - 23.3 (^) pM x
77
