Purification and Characterization of Acetone Carboxylase from Xanthobacter Strain Py2
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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 8456–8461, August 1997 Biochemistry Purification and characterization of acetone carboxylase from Xanthobacter strain Py2 MIRIAM K. SLUIS AND SCOTT A. ENSIGN* Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300 Communicated by R. H. Burris, University of Wisconsin, Madison, WI, June 9, 1997 (received for review March 24, 1997) ABSTRACT Acetone metabolism in the aerobic bacte- aerobic, Gram-negative bacterium (14). The metabolism of rium Xanthobacter strain Py2 proceeds by a carboxylation acetone by Xanthobacter Py2 was recently shown to proceed by reaction forming acetoacetate as the first detectable product. aCO2-dependent pathway analogous to that discussed above In this study, acetone carboxylase, the enzyme catalyzing this (3). The carboxylation of acetone to form acetoacetate was reaction, has been purified to homogeneity and characterized. reconstituted in cell extracts with the addition of ATP (3). This Acetone carboxylase was comprised of three polypeptides with study provided the first direct evidence for the involvement of molecular weights of 85,300, 78,300, and 19,600 arranged in an an ATP-dependent carboxylase in bacterial acetone metabo- a2b2g2 quaternary structure. The carboxylation of acetone lism. In this study, acetone carboxylase has been purified to was coupled to the hydrolysis of ATP and formation of 1 mol homogeneity. The molecular properties of acetone carboxylase AMP and 2 mol inorganic phosphate per mol acetoacetate are described, and evidence for a novel mechanism of acetone formed. ADP was also formed during the course of acetone carboxylation coupled to ATP hydrolysis and AMP and inor- consumption, but only accumulated at low, substoichiometric ganic phosphate formation is presented. levels ('10% yield) relative to acetoacetate. Inorganic pyro- phosphate could not be detected as an intermediate or product MATERIALS AND METHODS of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of Growth of Bacteria and Preparation of Cell Extracts. ATP to form both ADP and AMP, with ADP accumulating to Xanthobacter strain Py2 was grown with 32 mM isopropanol as higher levels than AMP during the course of the assays. the carbon source in a 15-liter capacity Microferm fermentor Acetone carboxylase did not have inorganic pyrophosphatase (New Brunswick Scientific) as described (15, 16). Cells were activity. Acetone carboxylase exhibited a Vmax for acetone harvested after reaching an OD600 (measured using a Shi- carboxylation of 0.225 mmol acetoacetate formed min21zmg21 madzu UV160U spectrophotometer) between 2.5 and 4.0 by at 30°C and pH 7.6 and apparent Km values of 7.80 mM tangential-flow filtration with a Pellicon system (Millipore) (acetone), 122 mM (ATP), and 4.17 mM (CO2 plus bicarbon- and stored at 280°C. Frozen cell paste (98 g for the protocol ate). These studies reveal molecular properties of the first described below) was resuspended in 2 vol of buffer A [25 mM bacterial acetone-metabolizing enzyme to be isolated and 4-morpholinepropanesulfonic acid (Mops), pH 7.6y1mM suggest a novel mechanism of acetone carboxylation coupled DTTy1 mM benzamidine] containing 0.1 mM EDTA, 0.1 mM to ATP hydrolysis and AMP and inorganic phosphate forma- EGTA, and 0.2 mgyml lysozyme and DNase I. The cell tion. suspension was passed three times through a French pressure cell at 110,000 kPa and 4°C and clarified by centrifugation Acetone is a toxic molecule that is produced biologically by the (105,000 3 g for 1 hr at 4°C). fermentative metabolism of certain anaerobic bacteria and Purification of Acetone Carboxylase. Purification proce- during mammalian starvation (1, 2). Acetone is known to dures were performed at 4°C. The supernatant of the cell undergo further metabolic transformations in microbes and extract was applied to a 5 3 25-cm column of DEAE- higher organisms, and a variety of diverse bacteria have been Sepharose equilibrated in buffer A containing 20% glycerol, found to grow using acetone as a source of carbon and energy 0.1 mM EDTA, and 0.1 mM EGTA at a linear flow rate of 28 (see refs. 3–5 and references cited therein). Studies of acetone- cmyhr. The column was washed with 1,250 ml of buffer A utilizing bacteria have provided evidence for the existence of containing 20% glycerol, followed by 1,250 ml of buffer A two distinct pathways of acetone metabolism. For some aer- containing 20% glycerol and 90 mM KCl. Bound protein was obic bacteria, acetone metabolism has been proposed to fractionated with a 3-liter linear gradient from 90 mM KCl to proceed by an O2-dependent, monooxygenase-catalyzed oxi- 270 mM KCl. Active fractions were pooled, diluted 4-fold with dation producing acetol (hydroxyacetone) as the initial prod- buffer A containing 20% glycerol and applied to a 2.6 3 10-cm uct (4, 6, 7). For other bacteria, including all anaerobes, column of Macroprep ceramic hydroxyapatite (Bio-Rad). The acetone metabolism has been proposed to proceed by a column was washed with 160 ml of buffer A containing 10% CO2-dependent carboxylation-producing acetoacetate or an glycerol at 45 cmyhr. A 380-ml linear gradient from 0 to 45 mM acetoacetyl derivative as the initial product (8–10). While in of potassium phosphate in buffer A containing 10% glycerol vivo and in vitro studies have provided some evidence sup- was applied to the column. Active fractions were pooled and porting these proposed bacterial pathways (6–8, 11–13), the concentrated by ultrafiltration using a YM100 membrane enzymes responsible for initiating acetone catabolism have not (Amicon). The sample was chromatographed in 250-mg por- been purified to date. tions on a 2.6 3 64-cm Sephacryl S-300 gel filtration column One bacterium capable of using acetone as a source of equilibrated with buffer A containing 10% glycerol and 0.2 M carbon and energy is Xanthobacter strain Py2, an obligately KCl at a linear flow rate of 8.5 cmyhr. Active fractions from the five S-300 chromatography procedures were pooled, di- The publication costs of this article were defrayed in part by page charge luted 4-fold with buffer A containing 20% glycerol, and payment. This article must therefore be hereby marked ‘‘advertisement’’ in applied to a 2.6 3 11-cm HiLoad Q-Sepharose column. The accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y948456-6$2.00y0 Abbreviation: Mops, 4-morpholinepropanesulfonic acid. PNAS is available online at http:yywww.pnas.org. *To whom reprint requests should be addressed. 8456 Downloaded by guest on September 29, 2021 Biochemistry: Sluis and Ensign Proc. Natl. Acad. Sci. USA 94 (1997) 8457 column was washed with 130 ml of buffer A containing 20% The absorbance at 340 nm was recorded, subtracted from the glycerol and 120 mM KCl at a flow rate of 45 cmyhr. Acetone initial absorbance value, and the difference used to calculate carboxylase was eluted with an 800-ml linear gradient from 120 the amount of ADP present in the sample. After recording the to 270 mM KCl. Appropriate fractions were pooled, concen- A340, adenylate kinase (10 units) was added to cuvettes to trated by ultrafiltration, and frozen in liquid nitrogen. convert AMP to ADP according to Eq. 3: Assay of Acetone Consumption and Acetoacetate Forma- tion. Acetone consumption assays were performed in serum AMP 1 ATP 3 2 ADP. [3] vials (9 ml) containing ATP (0–25 mM), MgCl2 (1 mM in excess of ATP concentration), potassium acetate (80 mM), The cuvettes were incubated an additional 15 sec to allow 1 Mops (100 mM), and a source of enzyme (cell extract, column complete reaction of AMP and production of NAD accord- fractions, or purified enzyme) in a total volume of 1 ml at pH ing to Eqs. 1–3. The A340 was then recorded, subtracted from the A recorded prior to addition of adenylate kinase, and 7.6. Potassium bicarbonate and CO2 gas were added to ap- 340 used to calculate AMP present in the samples. To verify the propriate sealed assay vials in a ratio (1 mol CO2 to 4 mol bicarbonate) that maintained the pH of the solutions at 7.6. accuracy of these determinations, AMP and ADP were also The concentrations of total carbonate species varied between quantified from standards and samples by HPLC analysis as 0 and 50 mM. For assays lacking CO2 and for Km determination described by Seefeldt and Mortenson (19). The two methods studies, residual CO2 was removed by sparging buffers and gave results for AMP and ADP determination that agreed flushing sealed assay vials with CO2-free nitrogen. For CO2- within 2%. free assays, a KOH-impregnated filter trap (16) was included Continuous, Coupled Spectrophotometric Assay for ADP in the vials. Assays were initiated by the addition of acetone. and AMP Formation. Assays were performed as described Vials were incubated throughout the course of the assay in a above, but in stoppered cuvettes containing the additional shaking water bath at 30°C and 250 cycles per minute. At components (coupling enzymes, phosphoenolpyruvate, desired time points, 100 ml samples of the gas phase (for NADH) allowing AMP andyor ADP formation to be coupled analysis of acetone) and 1 ml samples of the liquid phase (for to the oxidation of NADH (see Eqs. 1–3). Cuvettes were analysis of acetone plus acetoacetate) were removed and preincubated for 5 min at 30°C with all assay components analyzed by gas chromatography as described (15). The time except acetone. Assays were initiated by the addition of course of consumption of other potential substrates was fol- acetone.