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 acetone by Xanthobacter Py2 was recently shown to proceed by reaction forming acetoacetate as the first detectable . aCO2-dependent pathway analogous to that discussed above In this study, acetone carboxylase, the 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- ␣2␤2␥2 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 (Ϸ10% 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 ␮mol acetoacetate formed min؊1⅐mg؊1 madzu UV160U spectrophotometer) between 2.5 and 4.0 by at 30°C and pH 7.6 and apparent Km values of 7.80 ␮M tangential-flow filtration with a Pellicon system (Millipore) (acetone), 122 ␮M (ATP), and 4.17 mM (CO2 plus bicarbon- and stored at Ϫ80°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 7.6͞1mM suggest a novel mechanism of acetone carboxylation coupled DTT͞1 mM benzamidine] containing 0.1 mM EDTA, 0.1 mM to ATP hydrolysis and AMP and inorganic phosphate forma- EGTA, and 0.2 mg͞ml 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 ϫ 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 ϫ 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- cm͞hr. 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 ϫ 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 cm͞hr. 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 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 ϫ 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 cm͞hr. 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 ϫ 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 0027-8424͞97͞948456-6$2.00͞0 Abbreviation: Mops, 4-morpholinepropanesulfonic acid. PNAS is available online at http:͞͞www.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 cm͞hr. 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 ϩ 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 ϩ 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 ␮l samples of the gas phase (for NADH) allowing AMP and͞or ADP formation to be coupled analysis of acetone) and 1 ␮l 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. Assays were monitored by following the absorbance lowed by gas chromatography in the same manner. change at 340 nm in a Shimadzu model UV160U spectropho- Determination of Phosphate. Phosphate produced during tometer containing a thermostated cell holder maintained at the time course of assays was quantified by a modified mo- 30°C. lybdophosphoric acid method (17). At desired time points, 25 Protein Characterization. SDS͞PAGE (12% T, 2.7% C ␮l liquid samples were removed from assay vials and added to running gel) was performed following the Laemmli procedure 175 ␮l of 100 mM Mops (pH 7.6) containing 3.5 mM MgCl2. (20). Electrophoresed proteins were visualized by staining with The samples were brought to 1 ml total volume by the addition Coomassie blue. The apparent molecular masses of polypep- of H2O (600 ␮l), 2.5 M HClO4 (100 ␮l), and 0.41 M Na2MoO4 tides based on SDS͞PAGE migration were determined by (100 ␮l) followed by vortexing. After 5-min incubation at room comparison with Rf values of standard proteins. The standards temperature, the A380 of the samples was recorded. The used were myosin (200 kDa), ␤-galactosidase (116.2 kDa), phosphate content of samples was determined by comparison phosphorylase b (97.4 kDa), BSA (68 kDa), ovalbumin (45 with a standard curve prepared with potassium phosphate. kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor Determination of Pyrophosphate. Pyrophosphate was de- (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5 kDa). termined after conversion to inorganic phosphate using inor- Polypeptide molecular weights were also determined using ganic pyrophosphatase. Twenty-five microliter samples were matrix-assisted laser desorption ionization–time-of-flight mass removed from assay vials and assayed as described for the spectrometry performed in linear mode with an acceleration phosphate analysis, except that inorganic pyrophosphatase voltage of 16 kV. The matrix was ␣-phenyl-4-acetamido- (0.5 unit) was present in the 175 ␮l of Mops͞MgCl2. After cinnamic acid, and the internal standard was BSA. The relative 30-sec incubation, the additional reagents were added and the subunit proportions were determined by integrated scanning A380 was measured as described above. Standards of pyrophos- densitometry of SDS͞PAGE gels with an Isco model 1312 gel phate were treated identically and shown to undergo quanti- scanner. The native molecular weight was estimated by HPLC tative conversion to inorganic phosphate by this method. gel filtration using an Ultraspherogel TSK 3000SW column Pyrophosphate was calculated on the basis of the difference in (0.75 ϫ 30 cm) developed with 25 mM Mops (pH 7.6) phosphate content of the pyrophosphatase-treated and non- containing 0.2 M NaCl and 0.05% 3-[(3-cholamidopropyl)di- treated samples. methylammonio]-1-propanesulfonate. The column was cali- Determination of ADP and AMP in Fixed-Time Point brated with apoferritin (443 kDa), ␤-amylase (200 kDa), and Assays. ADP was determined spectrophotometrically by mea- alcohol dehydrogenase (150 kDa). Metal analysis was per- ϩ suring the ADP-dependent oxidation of NADH to NAD formed with an inductively coupled plasma atomic emission using a modification of a coupled enzyme assay (18). Samples spectrophotometer at the Utah State University Soil and Plant (25 ␮l) were removed from assay vials at desired time points Analysis Laboratory. Protein concentrations were determined and added to sealed cuvettes containing 0.975 ml of an assay by a modified biuret assay (21) using BSA as the standard. mixture consisting of 2 mM phosphoenolpyruvate, 2 mM Induction of Acetone Carboxylase, 35S-Labeling, and Auto- MgCl2, 1 mM ATP, 0.2 mM NADH, 21 units each pyruvate radiography. Batch cultures (25 ml) grown with glucose as the kinase and lactate dehydrogenase, and 100 mM Mops buffer carbon source (OD ϭ 1.5) were induced by the addition of at pH 7.6. After mixing, the cuvettes were incubated for 15 sec 600 propylene oxide, acetone, or isopropanol (13.5 ␮mol each), at 30°C, which was a sufficient time for quantitative phosphor- labeled with [35S]methionine and cysteine, lysed, and autora- ylation of ADP and concomitant oxidation of NADH accord- diographed as described (22). ing to Eqs. 1 and 2: Data Analysis. Kinetic constants (Km and Vmax) were cal- ADP ϩ phosphoenolpyruvate 3 ATP ϩ pyruvate, [1] culated by fitting initial rate data to the Michaelis–Menten equation as described by Cleland (23) and using the software pyruvate ϩ NADH 3 lactate ϩ NADϩ. [2] SIGMAPLOT. Downloaded by guest on September 29, 2021 8458 Biochemistry: Sluis and Ensign Proc. Natl. Acad. Sci. USA 94 (1997)

RESULTS Purification and Characterization of Acetone Carboxylase. The soluble fractions of cell extracts prepared from cultures of Xanthobacter strain Py2 grown with acetone or isopropanol consistently exhibited specific activities for ATP-dependent acetone carboxylation of 0.04 to 0.05 unit͞mg. These rates are directly comparable to the rates of acetone consumption measured in actively growing cultures or resting-state whole cell suspensions of acetone- or isopropanol-grown Xan- thobacter strain Py2 (3). These results demonstrate that ace- tone carboxylase can be reconstituted in vitro at physiologically relevant rates and suggest that the enzyme will be amenable to purification in an active state. Acetone carboxylase was purified 4.2-fold from the soluble FIG. 1. SDS͞PAGE analysis of acetone carboxylase. Lanes: 1, fraction of cell extracts with a recovery of 56% and specific molecular weight standards (2 ␮g each); 2, cell extract (35 ␮g); 3, activity for acetone carboxylation of 0.206 unit͞mg of protein DEAE-Sepharose fraction (17 ␮g); 4, hydroxyapatite fraction (15 ␮g); (Table 1). As shown in Fig. 1, the purification resulted in the 5, Sephacryl S-300 fraction (11 ␮g); 6 and 7, Q-Sepharose fraction (5.1 ␮ enrichment of three polypeptides that migrated on SDS͞ and 7.2 g, respectively). PAGE with apparent molecular masses of 78.8, 68.4, and 23.0 the three subunits with a molecular mass of 183 kDa. The kDa. These bands are readily visible in the soluble fraction native molecular weight of the acetone carboxylase complex used as the source of enzyme for the purification (Fig. 1, lane was determined to be 353 kDa. Therefore, the native enzyme 2), but are not visible in cell extracts prepared from cultures is likely to have an ␣ ␤ ␥ subunit configuration. grown under conditions where acetone carboxylase is not 2 2 2 Acetone carboxylase activity was dependent upon the ad- expressed, e.g., with glucose or propylene as carbon sources dition of ATP and an additional divalent metal ion (e.g., (22). To further confirm the central roles of these polypeptides Mg2ϩ). The addition of Kϩ stimulated acetone carboxylase in acetone metabolism, cultures of glucose-grown Xan- approximately 2-fold over assays performed in its absence. thobacter Py2 were induced for acetone carboxylase activity by the addition of acetone or isopropanol, followed by pulse- Other nucleoside triphosphates (GTP, CTP, UTP, TTP) did labeling with 35S amino acids. As shown in the autoradiogram not support acetone carboxylation. Acetone carboxylase ac- presented in Fig. 2, induction with acetone or isopropanol tivity was stable for several days at 4°C over the pH range 6.5 resulted in the new and high level synthesis of the three to 8.0. The pH optimum for activity was 7.6. The -binding polypeptides that purify in association with acetone carboxy- protein avidin was not an inhibitor of acetone carboxylation. lase activity. In contrast, no synthesis of these polypeptides was The UV͞visible absorption spectrum of acetone carboxylase detected in the noninduced, glucose-grown cells (compare exhibited an absorption maximum at 281 nm. No additional lane 1 with lanes 3 and 4). As a control, the gel banding absorbance was present in the wavelength range from 300 to patterns of proteins synthesized in cells exposed to acetone and 800 nm. Metal analysis of three separate acetone carboxylase isopropanol are compared in Fig. 2 with those synthesized in preparations purified from three different batches of cells cells exposed to propylene oxide, which induces to high levels revealed the presence of significant quantities of Fe, Mn, and a distinct set of enzymes involved in aliphatic alkene and Zn. The stoichiometries, averaged for the three preparations epoxide metabolism (22). It is apparent from these results that and reported as mols of metal per mol of ␣2␤2␥2 complex are: acetone carboxylase is highly inducible and represents a sizable 0.70 Ϯ 0.089 Fe, 1.31 Ϯ 0.061 Mn, and 1.02 Ϯ 0.055 Zn. Dialysis percentage of total soluble protein in cultures grown (or of acetone carboxylase vs. buffers containing 2 mM EDTA or induced) with acetone or isopropanol, an observation that 1mM␣-␣Ј-dipyridyl did not decrease the metal contents, explains the low-fold purification required to obtain a homog- indicating that the metals are tightly bound. Likewise, dialysis enous preparation of the enzyme (Table 1). The high level of of acetone carboxylase vs. buffer containing 5 mM MgCl2 did expression of acetone carboxylase is presumably related to the not decrease the metal contents, demonstrating that the metals relatively low specific activity of the enzyme, i.e., high levels of are not readily exchangeable. These treatments did not affect acetone carboxylase are required to support cell growth at the the specific activity of acetone carboxylase. The addition of observed rates (3). exogenous Fe2ϩ,Mn2ϩ,Zn2ϩ,Ca2ϩ,Co2ϩ,Cu2ϩ,orNi2ϩ to Mass spectrometry was used to provide a more accurate assays did not stimulate acetone carboxylase activity above the estimation of polypeptide molecular weights. This analysis maximal levels obtained in the presence of Mg2ϩ alone. revealed molecular weights of 85.3, 78.3, and 19.6 kDa for the Of a number of other ketones evaluated as possible sub- three subunits. The staining intensities of the three polypep- strates for acetone carboxylase (butanone, 2-pentanone, 3- tides on SDS͞PAGE gave relative molar ratios of 1.0 (85.3- pentanone, 2-hexanone, and chloroacetone), only butanone kDa band), 1.0 (78.3-kDa band), and 1.2 (19.6-kDa band), was a under the assay conditions used for acetone suggesting a minimal core complex consisting of one each of consumption. Butanone was consumed in a CO2- and ATP-

Table 1. Purification of acetone carboxylase Specific Total protein, Total activity, activity, Purification, Recovery, Step Volume, ml mg units* units͞mg x-fold % Cell extract 211 4,480 222 0.0496 1 100 DEAE-Sepharose 562 1,500 208 0.139 2.8 93 Hydroxyapatite 15 1,250 188 0.151 3.0 85 Sephacryl S-300 121 650 129 0.198 4.0 58 Q-Sepharose 223 609 125 0.206 4.2 56

*Activity assays were performed as described in Materials and Methods using 0.2–0.6 mg protein, 10 mM ATP, 50 mM CO2 plus KHCO3, and 2 ␮mol acetone. A unit of activity is defined as 1 ␮mol of acetone degraded per minute at 30°C. Downloaded by guest on September 29, 2021 Biochemistry: Sluis and Ensign Proc. Natl. Acad. Sci. USA 94 (1997) 8459

FIG. 2. Gel electrophoretic profiles of proteins synthesized after exposure of glucose-grown Xanthobacter strain Py2 to acetone and isopropanol. The autoradiogram of a 14% SDS gel is shown. Each gel lane contains cell extract equal to 20 ␮g protein. Lanes: 1, no inducer added; 2, propylene oxide added as inducer; 3, acetone added as inducer; 4, isopropanol added as inducer.

dependent fashion and with a specific activity of 0.094 unit͞mg FIG. 3. Time course of acetone carboxylase-catalyzed acetone of protein, which is 46% of the rate observed with acetone. degradation and product formation. Assays were performed as de- Pyruvate, phosphoenolpyruvate, acetaldehyde, propionalde- scribed in Materials and Methods using 0.29 mg of purified acetone hyde, and propylene oxide were not substrates for acetone carboxylase, 10 mM ATP, 50 mM CO2 plus KHCO3, and 2 ␮mol carboxylase. acetone. At the indicated time points, individual assays were termi- Acetone Carboxylase–Catalyzed Nucleotide Hydrolysis. nated by removing the assay vial from the water bath and analyzing The identity and stoichiometry of an ATP hydrolysis prod- samples of the liquid and gas phase for substrate and products. Each uct(s) formed in the course of acetone carboxylation was time point is an average of measurements on duplicate vials. (A) investigated. As shown in Fig. 3A, the carboxylation of acetone Assays performed in the presence of 50 mM CO2 plus KHCO3.(B) Assays performed in the absence of CO2 and KHCO3.(Ⅺ), acetone; to acetoacetate was coupled to the formation of AMP as a (Ⅵ), acetoacetate; (E), inorganic phosphate; (Ç), AMP; (É), ADP; (F), stoichiometric (1:1 mol ratio) product. ADP was also detected pyrophosphate. as a product, but accumulated only at low, substoichiometric levels. Inorganic pyrophosphate could not be detected as an kinase, and lactate dehydrogenase (see Eqs. 1–3). The rates of intermediate or product of nucleotide hydrolysis. Rather, NADH oxidation are numerically equivalent to rates of phos- inorganic phosphate was identified as the sole inorganic phodiester bond hydrolysis since phosphate is the only inor- product of nucleotide hydrolysis. The amount of phosphate ganic hydrolysis product observed. produced was equal to the sum of the ADP formed plus twice With acetone and CO2 present, the initial rates of phos- the sum of AMP formed. Upon complete consumption of phodiester bond hydrolysis measured using this assay were acetone, the rates of AMP, ADP, and phosphate formation 0.487 ␮mol minϪ1⅐mgϪ1 in the presence of adenylate kinase decreased to the background rates observed when acetone and 0.0207 ␮mol minϪ1⅐mgϪ1 in the absence of adenylate carboxylase was incubated with ATP, but in the absence of kinase (Fig. 4, Traces 1A and 2A). These results agree with acetone and CO2. These background rates were between 5 and those presented in Fig. 3 showing AMP rather than ADP to be 10% of the initial rates shown in Fig. 3A. the predominant nucleotide product formed during acetone In the absence of CO2 and KHCO3, acetone carboxylase did carboxylation. The specific activity of AMP formation was not catalyze any detectable consumption of acetone (Fig. 3B). determined to be 0.233 ␮mol AMP formed minϪ1⅐mgϪ1, which However, the enzyme did catalyze the hydrolysis of ATP to is directly comparable to the specific activity of 0.206 ␮mol form both ADP and AMP at rates significantly higher that acetone consumed minϪ1⅐mgϪ1 measured in fixed time point those observed in the absence of acetone. Particularly intrigu- assays. In the absence of acetone, phosphodiester bond hy- ing was the observation that ADP accumulated to higher levels drolysis occurred at significantly lower rates than those ob- than AMP under these conditions. As observed for assays with served in the presence of acetone (Traces 3A and 4A). CO2 present, the inorganic product of phosphodiester bond In the presence of acetone and absence of CO2, phosphodi- hydrolysis was phosphate. ester bond hydrolysis rates of 0.584 and 0.189 ␮mol The addition of pyrophosphate or inorganic pyrophos- minϪ1⅐mgϪ1 were calculated with and without adenylate kinase phatase to assays had no effect on the rate of acetone in the assays, respectively (Traces 1B and 2B). The corre- carboxylation. Acetone carboxylase did not exhibit any detect- sponding initial rates of nucleotide formation are 0.189 ␮mol able pyrophosphatase activity, in either the absence or pres- ADP formed minϪ1⅐mgϪ1 and 0.197 ␮mol AMP formed Ϫ1 Ϫ1 ence of acetone and CO2. min ⅐mg . These results agree with the results presented in Continuous Spectrophotometric Assay of Acetone Carbox- Fig. 3, which show both AMP and ADP to be significant ylase-Catalyzed ATP Hydrolysis. The assays used to quantify products of acetone-dependent, CO2-independent ATP hy- acetone consumption and product formation in Fig. 3 suffer drolysis. As observed for the assays performed in the presence from several limitations, the most significant of which is of CO2, the rates of ATP hydrolysis were significantly lower in reliance on fixed time point measurements. A continuous assays performed in the absence of acetone (Traces 3B and spectrophotometric assay would be superior for measuring 4B). initial rates and obtaining steady-state kinetic data. An ap- Kinetic Characterization of Acetone Carboxylase– propriate assay has been developed that relies on coupling Catalyzed Reactions. Fig. 5 shows a plot of the rate of acetone carboxylase-catalyzed AMP and ADP formation to phosphodiester bond hydrolysis vs. acetone concentration in NADH oxidation using the coupling enzymes adenylate kinase coupled enzyme assays where ATP was saturating, and CO2 (excluded for measurements of ADP formation only; included plus bicarbonate were either saturating or completely absent. for measurements of AMP plus ADP formation), pyruvate Fitting these data to the Michaelis–Menten equation provided Downloaded by guest on September 29, 2021 8460 Biochemistry: Sluis and Ensign Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 5. Effect of acetone concentration on the rate of acetone carboxylase-catalyzed phosphodiester bond hydrolysis. NADH-linked, coupled enzyme assays were performed in stoppered cuvettes as FIG. 4. Continuous, spectrophotometric assay of acetone carbox- described using 0.026 mg of purified acetone carboxylase. All assay ylase-catalyzed ATP hydrolysis. NADH-linked, coupled enzyme as- cuvettes contained pyruvate kinase, adenylate kinase, and lactate says were performed in stoppered cuvettes as described using 0.091 mg dehydrogenase. Assays were initiated by the addition of acetone. Rates of purified acetone carboxylase. All assay cuvettes contained pyruvate were derived from the linear portions of progress curves of A340 vs. kinase and lactate dehydrogenase. Acetone (2 ␮mol) and adenylate time (typically within the first 100 sec of reaction). The low rate of kinase (10 units) were included where noted below. (A) Assays nucleotide hydrolysis occurring in the absence of acetone was sub- tracted for each rate. (å), assays containing 50 mM CO2 plus KHCO3. containing 50 mM CO2 plus KHCO3.(B) Assays lacking CO2 and bicarbonate. Traces 1A and 1B, assays with acetone and adenylate (●), assays without CO2 and KHCO3. kinase; Traces 2A and 2B, assays with acetone and without adenylate kinase; Traces 3A and 3B, assays without acetone and with adenylate Py2. The physiological function of acetone carboxylase is to kinase; Traces 4A and 4B, assays without acetone and without convert acetone, a toxic and recalcitrant organic molecule, to adenylate kinase. acetoacetate, which is a central metabolite that can undergo further metabolic transformations by conventional and well- Vmax values of 0.485 Ϯ 0.015 ␮mol phosphodiester bonds characterized biochemical pathways. Ϫ1 Ϫ1 hydrolyzed min ⅐mg in the presence of CO2 and 0.616 Ϯ Acetone carboxylase exhibited an obligate requirement for 0.012 ␮mol phosphodiester bonds hydrolyzed min Ϫ1⅐mgϪ1 in ATP as a . Acetone carboxylation is a thermodynam- the absence of CO2. The corresponding apparent Km values for ically unfavorable process (⌬G°Ј for acetone carboxylation acetone were 7.80 Ϯ 0.79 ␮M in the presence of CO2 and with bicarbonate is ϩ17.1 kJ͞mol), and the hydrolysis of ATP 7.68 Ϯ 0.48 ␮M in the absence of CO2. It is interesting that the to ADP (⌬G°ЈϭϪ31 kJ͞mol) would theoretically provide rate of acetone-dependent phosphodiester bond hydrolysis is sufficient energy to drive the carboxylation reaction. Interest- slightly faster in the absence of CO 2, whereas the Km for ingly, during the course of acetone carboxylation, AMP and acetone is unchanged by the presence or absence of CO 2. inorganic phosphate form as the products of ATP hydrolysis A plot of rate of phosphodiester bond hydrolysis vs. ATP (Fig. 3A) according to Eq. 4: concentration in coupled enzyme assays where acetone and CO2 were saturating also followed Michaelis–Menten kinetics, acetone ϩ CO2 3 acetoacetate ϩ AMP ϩ 2Pi. [4] providing a Vmax of 0.463 Ϯ 0.018 ␮mol phosphodiester bonds Ϫ1 Ϫ1 hydrolyzed min ⅐mg and an apparent Km for ATP of The carboxylation of acetone thus requires the hydrolysis of 0.122 Ϯ 0.014 mM. This value of Vmax is statistically equivalent both the ␣–␤ and ␤–␥ phosphodiester bonds of a single ATP to the value of 0.485 ␮mol minϪ1⅐mgϪ1 reported above for molecule. assays in which acetone concentrations were varied. It is reasonable to speculate that ATP hydrolysis plays a role Since nucleotide hydrolysis occurs in the absence of CO 2,it in activating acetone for CO2 addition through one or more was necessary to employ fixed time point assays, where acetone group transfer reactions. Possibly, a phosphoryl or pyrophos- consumption and acetoacetate formation could be monitored, phoryl group is transferred directly to acetone, or via the to determine kinetic parameters for CO2. A plot of rate of mediation of a phosphoryl- or pyrophosphoryl-enzyme inter- acetoacetate formation vs. total carbonate species (CO2 plus mediate. Acetone carboxylation presumably involves nucleo- Ϫ HCO3 ) in assays where acetone and ATP were present at philic attack of the carbanion of acetone on CO2 (or bicar- saturating concentrations provided a Vmax of 0.225 Ϯ 0.011 bonate). The carbanion might be formed by general base Ϫ1 Ϫ1 ␮mol acetoacetate formed min ⅐mg and an apparent Km for abstraction of a proton, but would be hard to generate and Ϫ CO2 plus HCO3 of 4.17 Ϯ 0.69 mM. This value of Vmax is highly unstable due to the high pKa of the methyl group. The slightly less than one-half of the Vmax values obtained for carbanion could be stabilized by keto to enol tautomerization, phosphodiester bond hydrolysis using the coupled enzyme and the enol tautomer could be further stabilized by the assays. This is expected, since each acetoacetate formed transfer of a phosphoryl (or other) group from ATP to the requires the hydrolysis of two, rather than one, phosphodiester oxygen atom of the enolate. Nucleophilic attack of the enol on bonds (Fig. 2). CO2 (or bicarbonate) with concomitant hydrolysis of the oxygen–phosphate bond would result in the formation of DISCUSSION acetoacetate. This hypothetical reaction scheme bears similarities to well- In this study, a soluble, multimeric acetone-utilizing enzyme characterized reactions involving the glycolytic intermediates has been purified and characterized from Xanthobacter strain pyruvate and phosphoenolpyruvate (24–26). The conversion Downloaded by guest on September 29, 2021 Biochemistry: Sluis and Ensign Proc. Natl. Acad. Sci. USA 94 (1997) 8461

of pyruvate to phosphoenolpyruvate is catalyzed by phos- Recently, Birks and Kelly (13) demonstrated acetone car- phoenolpyruvate synthase, which phosphorylates the enol tau- boxylase activity in cell extracts prepared from acetone-grown tomer of pyruvate at the expense of two high-energy phos- cultures of Rhodobacter capsulatus. This activity required ATP phoanhydride bonds as shown in Eq. 5 (24, 26): and was stimulated by CoA or acetyl-CoA (these cofactors had no effect on acetone carboxylase activity in Xanthobacter Py2). pyruvate ϩ ATP 3 phosphoenolpyruvate ϩ AMP ϩ Pi. [5] The levels of activity observed for R. capsulatus extracts were The hydrolysis of two phosphoanhydride bonds is required to much lower (200- to 1,000-fold) than those observed in cell drive the phosphorylation of pyruvate, which is an uphill extracts of Xanthobacter Py2 and, as noted by the authors, too reaction relative to a single ATP hydrolysis (⌬G°Ј for phos- low to be of physiological significance (13). Possibly, an phoenolpyruvate hydrolysis is Ϫ61.9 kJ͞mol). Based on the additional component not required by the Xanthobacter system same thermodynamic considerations, the hydrolysis of two is limiting in the assay, and͞or the enzyme is much less stable phosphoanhydride bonds may be required for the generation in vitro. Similar scenarios may apply to strain BunN and other of phosphoenolacetone or another activated acetone interme- anaerobes for which acetone carboxylation cannot be recon- diate as well. stituted in vitro. If group transfer from ATP to acetone does occur as part of In summary, this paper provides the first reported purifica- the catalytic mechanism it would appear that the intermediate tion and characterization of a catabolic acetone-metabolizing is not stable, since no detectable depletion of acetone was enzyme. The identification and characterization of this enzyme observed in assays performed in the absence of CO2 (Fig. 3B). fills a long-standing gap in our understanding of the microbial In these assays, acetone stimulated the hydrolysis of ATP by acetone cycle. The properties of acetone carboxylase reported Ϸ20-fold over assays performed in the absence of acetone, in this initial study suggest a novel mechanism of ATP- suggesting that a reaction of ATP with acetone is taking place. dependent acetone carboxylation that promises to reveal new Possibly, the activated acetone intermediate simply breaks insights into biological strategies for adding CO2 to organic back down to acetone if CO2 is not available for further substrates. reaction to form acetoacetate. Alternatively, acetone may induce a conformational change that activates the ATPase We thank Jinhua Feng and Prof. Robert Brown for performing mass activity of the enzyme. spectrometry of acetone carboxylase. This work was supported by It is interesting that both ADP and AMP accumulated as National Science Foundation Grant MCB9630081. significant products of acetone-dependent ATP hydrolysis in the absence of CO2 (Fig. 3). ADP was also detected as a minor 1. Davies, R. & Stephenson, M. (1941) Biochem. J. 35, 1320–1331. 2. Argile´s, J. P. (1986) Trends Biochem. Sci. 11, 61–65. hydrolysis product in assays conducted in the presence of CO2 (Fig. 2). These results demonstrate that acetone carboxylase 3. Sluis, M. K., Small, F. J., Allen, J. R. & Ensign, S. A. (1996) J. can catalyze the hydrolysis of both the ␣–␤ and ␤–␥ phos- Bacteriol. 178, 4020–4026. phodiester bonds of ATP. Possibly, ATP hydrolysis occurs by 4. Taylor, D. G., Trudgill, P. W., Cripps, R. E. & Harris, P. R. (1980) J. Gen. Microbiol. 118, 159–170. the sequential cleavage of the ␤–␥ and ␣–␤ phosphodiester 5. Janssen, P. H. & Schink, B. (1995) Arch. Microbiol. 163, 188–194. bonds of ATP with the generation of an ADP intermediate 6. Lukins, H. H. & Foster, J. W. (1963) J. Bacteriol. 85, 1074–1087. rather than by an initial cleavage of the ␣–␤ bond, which would 7. Vestal, J. R. & Perry, J. J. (1969) J. Bacteriol. 99, 216–221. generate AMP and pyrophosphate (or a pyrophosphorylated 8. Bonnet-Smits, E. M., Robertson, L. A., Van Dijken, J. P., Senior, intermediate) as the first hydrolysis products. Acetone carbox- E. & Kuenen, J. G. (1988) J. Gen. Microbiol. 134, 2281–2289. ylase did not exhibit any pyrophosphatase activity, and we were 9. Platen, H. & Schink, B. (1990) Biodegradation 1, 243–251. unable to detect pyrophosphate as an intermediate in the 10. Platen, H., Temmes, A. & Schink, B. (1990) Arch. Microbiol. 154, steady-state carboxylation reactions (Figs. 2 and 3). These 355–361. results argue against pyrophosphate as an intermediate in the 11. Janssen, P. H. & Schink, B. (1995) J. Bacteriol. 177, 277–282. reaction. However, we cannot rule out the possibility of a 12. Janssen, P. H. & Schink, B. (1995) Eur. J. Biochem. 228, 677–682. tightly or covalently bound pyrophosphoryl group as an inter- 13. Birks, S. J. & Kelly, D. J. (1997) Microbiology 143, 755–766. mediate in the reaction sequence. It is apparent that the 14. van Ginkel, C. G. & de Bont, J. A. M. (1986) Arch. Microbiol. 145, elucidation of the mechanistic details of acetone carboxylation 403–407. 15. Allen, J. R. & Ensign, S. A. (1996) J. Bacteriol. 178, 1469–1472. and the roles ATP hydrolysis play in this reaction will require 16. Small, F. J., Tilley, J. K. & Ensign, S. A. (1995) Appl. Environ. the application of a variety of kinetic, mechanistic, and spec- Microbiol. 61, 1507–1513. troscopic tools. 17. Boltz, D. F., Lueck, C. H. & Jakubiec, R. J. (1978) in Colorimetric Schink and coworkers (5, 10, 11, 27, 28) have studied acetone Determination of Nonmetals, eds. Boltz, D. F. & Howell, J. A. metabolism in several anaerobic bacteria and obtained in vivo (Wiley, New York), Vol. 8, pp. 342–343. and in vitro data supporting the existence of CO2-dependent 18. Kreuzer, K. N. & Jongeneel, C. V. (1983) Methods Enzymol. 100, pathways in which acetone is carboxylated to acetoacetate or 144–160. an acetoacetyl derivative (e.g., acetoacetyl-CoA). The enzy- 19. Seefeldt, L. C. & Mortenson, L. E. (1993) Protein Sci. 2, 93–102. matic activity believed to be responsible for acetone carbox- 20. Laemmli, U. K. (1970) Nature (London) 227, 680–685. ylation in a denitrifier designated strain BunN was studied in 21. Chromy, V., Fischer, J. & Kulhanek, V. (1974) Clin. Chem. 20, cell extracts. The carboxylation of acetone could not be 1362–1363. reconstituted in cell extracts in either the absence or presence 22. Ensign, S. A. (1996) Appl. Environ. Microbiol. 62, 61–66. of ATP and other cofactors (9, 12). Cell extracts of strain BunN 23. Cleland, W. W. (1979) Methods Enzymol. 63, 103–138. did catalyze two reactions believed to be relevant to acetone 24. Cooper, R. A. & Kornberg, H. L. (1974) in The Enzymes, ed. Boyer, P. (Academic, New York), Vol. 10, pp. 631–649. carboxylation: the ADP-dependent decarboxylation of aceto- 14 25. Chollet, R., Vidal, J. & O’Leary, M. H. (1996) Annu. Rev. Plant acetate and ADP-dependent exchange of CO2 into the Physiol. Plant Mol. Biol. 47, 273–298. carboxylate carbon atom of acetoacetate (9, 12). Notably, these 26. Cooper, R. A. & Kornberg, H. L. (1969) Methods Enzymol. 13, enzymatic activities were lacking or greatly reduced in cell 309–315. extracts prepared from cells grown with other carbon sources, 27. Platen, H. & Schink, B. (1989) J. Gen. Microbiol. 135, 883–891. providing evidence that they are associated with the acetone- 28. Platen, H., Janssen, P. H. & Schink, B. (1994) FEMS Microbiol. carboxylating enzyme (9, 12). Lett. 122, 27–32. Downloaded by guest on September 29, 2021