Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO 2 to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus was purified to homogeneity and compared to that of Xanthobacter autotrophicus strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (␣ 2 ␤ 2 ␥ 2 multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH 3 COCH 3 ؉ CO 2 ؉ ATP3CH 3 COCH 2 COO ؊ ؉ H ؉ ؉ AMP ؉ 2P i ), and kinetic properties (K m for acetone, 8 M; k cat ‫؍‬ 45 min ؊1 ). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of X. autotrophicus and sequence analysis of the R. capsulatus genome and were found to be clustered in similar operons consisting of the genes acxA (␤ subunit), acxB (␣ subunit), and acxC (␥ subunit). Transposon mutagenesis of X. autotrophicus revealed a requirement of 54 and a 54 -dependent transcriptional activator (AcxR) for acetonedependent growth and acetone carboxylase gene expression. A potential 54 -dependent promoter 122 bp upstream of X. autotrophicus acxABC was identified. An AcxR gene homolog was identified 127 bp upstream of acxA in R. capsulatus, but this activator lacked key features of 54 -dependent activators, and the associated acxABC lacked an apparent 54 -dependent promoter, suggesting that 54 is not required for expression of acxABC in R. capsulatus. These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetoneutilizing bacteria.In addition to its importance as an industrial solvent, acetone is a major fermentation product of certain anaerobic bacteria (19,51), an intermediate in the microbial metabolism of propane and isopropanol (7,32,49), and one of the ketone bodies produced under ketogenic conditions (i.e., fasting or diabetes) in mammals. Acetone is known to undergo metabolic transformations in mammals, where the physiological importance is not fully understood (4, 25), and in diverse microbes which are capable of growth using acetone as the primary source of carbon and energy (20). The mammalian metabolism of acetone is believed to be mediated largely by cytochrome P450 isozyme 2E1, sequentially producing acetol and methylglyoxal as gluconeogenic intermediates (8,11,28). The carbon atoms originating from acetone are incorporated into glucose in starved mice, suggesting that acetone may be an intermediate in the only mammalian pathway allowing net synthesis of glucose from fatty acids (4,25,26,30).Two distinct transformation...

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an ␣ 2 ␤ 2 ␥ 2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (Ϸ10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO 2 , acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a V max for acetone carboxylation of 0.225 mol acetoacetate formed min ؊1 ⅐mg ؊1at 30°C and pH 7.6 and apparent K m values of 7.80 M (acetone), 122 M (ATP), and 4.17 mM (CO 2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.Acetone is a toxic molecule that is produced biologically by the fermentative metabolism of certain anaerobic bacteria and during mammalian starvation (1, 2). Acetone is known to undergo further metabolic transformations in microbes and higher organisms, and a variety of diverse bacteria have been found to grow using acetone as a source of carbon and energy (see refs. 3-5 and references cited therein). Studies of acetoneutilizing bacteria have provided evidence for the existence of two distinct pathways of acetone metabolism. For some aerobic bacteria, acetone metabolism has been proposed to proceed by an O 2 -dependent, monooxygenase-catalyzed oxidation producing acetol (hydroxyacetone) as the initial product (4, 6, 7). For other bacteria, including all anaerobes, acetone metabolism has been proposed to proceed by a CO 2 -dependent carboxylation-producing acetoacetate or an acetoacetyl derivative as the initial product (8-10). While in vivo and in vitro studies have provided some evidence supporting these proposed bacterial pathways (6-8, 11-13), the enzymes responsible for initiating acetone catabolism have not been purified to date. One bacterium capable of using acetone as a source of carbon and energy is Xanthobacter strain Py2, an obligately aerobic, Gram-negative bacterium (14). The metabolism of acetone by Xanthobacter Py2 was recently shown to proceed by a CO 2 -dependent pathway analogous to that discussed above (3). The carboxylation of acetone to...

' Pseudomonas butanovora ' is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an alternative substrate for sBMO) was reconstituted in the soluble portion of cell extracts and was NADH-dependent. Butane monooxygenase was separated into three components which were obligately required for substrate oxidation.

The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO 2 -dependent pathway of acetone metabolism for this bacterium. The depletion of CO 2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO 2 and was prevented by the depletion of CO 2 . The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14 CO 2 into acid-stable products, while the degradation of glucose or ␤-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH 13 CO 3 specifically incorporated 13 C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-␤-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO 2 -and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO 2 -dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.A variety of aerobic and anaerobic bacteria are capable of growth by using acetone as a source of carbon and energy. For some aerobic bacteria, the metabolism of acetone has been proposed to proceed via an O 2 -and reductant-dependent hydroxylation reaction producing acetol-(1-hydroxyacetone) as the initial product (4,12,21,23). For anaerobic bacteria, the metabolism of acetone has been proposed to proceed via a CO 2 -dependent carboxylation reaction producing acetoacetate as the initial product as shown in the following equation (2,10,11,13,14,(16)(17)(18): CH 3 COCH 3 ϩ CO 2 3CH 3 COCH 2 COO Ϫ . The carboxylation of acetone is the reverse of acetoacetate decarboxylation, a terminal reaction catalyzed by acetoacetate decarboxylases in certain fermentative bacteria of the genus Clostridium (6, 26).Acetoacetate decarboxylation represents the thermodynamically favorable direction for...

Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria. One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2. Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO2 in these processes. Both classes of compounds are metabolized by carboxylation reactions that produce beta-keto acids as products. The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases. Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD+ as cofactors. In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD+ undergoes reduction. Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation. Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions. ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha. These studies have identified new roles for CO2 as a cosubstrate in the metabolism of two classes of important xenobiotic compounds. In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO2 to organic substrates.