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This document reports the purification and characterization of acetyl-CoA carboxylase from the diatom Cyclotella cryptica. The study compares the properties of this enzyme with those of higher plant acetyl-CoA carboxylases and investigates the effects of various cellular metabolites and herbicides on its activity.
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Plant Physiol. (1990) 92, 73-
Received for publication (^) April 24, 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.
The research described in this (^) report was carried out in
catalyzes the formation of malonyl-CoA, which is one of the
a role in the (^) accumulation of lipids when the diatom Cyclo- tella cryptica is grown under (^) silicon-limiting conditions (19).
' (^) 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.
Organism and Growth Conditions
respectively.
Analytical Methods
73
Plant Physiol. Vol. 92, 1990
mM (^) ['4C]NaHCO3 (specific activity = (^1) 1.1 MBq/mmol). The
of the acetyl-CoA-dependent "1C incorporation. One unit of
A. The reaction product comigrated with authentic ["`C]
T. Baker Co., Si250) developed in water-saturated diethyl
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
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
below (^5) mg/mL; this is referred to as the crude extract. Cell
Purification Procedure
tion, which^ was^ then^ centrifuged at 8000g for 5 min. The
gel filtration^ chromatography, and^ affinity chromatography
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-
(^74) Roessler
Plant Physiol. Vol. 92, 1990
ence of divalent metal cations, with Mg2' being the most
was observed at a Mg2' concentration of 2 mm (Fig. 3). Above
strates were determined to be 65, 233, and 750 (^) gM, respec-
me same timc^ LA *w +^ sS1sCA07^ LA_0_I,.:_^ -^ 4-,1^ --A
._^ 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.
depending on the experiment. b^ Additional MgCI2 (1 mM) was also
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
vate, fructose- 1 ,6-bisphosphate, glucose- 1-phosphate, glucose-
shown to consistently stimulate enzymatic activity by approx-
(1 mM) likewise did^ not^ alter^ acetyl-CoA carboxylase activity
pionic acid and cyclohexanedione herbicides^ (1, 16, 21), it
the activity of^ purified C.^ cryptica^ acetyl-CoA carboxylase.
[MgCI2 ]^ (mM) (^) ner, with 50% inhibition occurring at a concentration of 23
76 Roessler
ACETYL-CoA CARBOXYLASE FROM CYCLOTELLA CRYPTICA
100
80
0
z 0
60
40
20
0 I 10 100
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.
Many properties of acetyl-CoA carboxylase from the^ dia-
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
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
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