Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

Abstract

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...

Acetone and Butanone Metabolism of the Denitrifying Bacterium "Aromatoleum aromaticum" Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

“…The hydrolysis product under these conditions was also exclusively AMP. This indicates a decoupled ATPase activity of the enzyme, which is similar but less prominent than that observed for acetophenone carboxylase and acetone carboxylases of alphaproteobacteria (10,21,22). This decoupled ATPase activity of Acx was increased up to 5-fold by addition of 1 mM MnCl 2 , whereas the same MnCl 2 concentration inhibited net carboxylation activity by 25%.…”

“…This is supported by the chromatographic detection of AMP and P i as the only hydrolysis products of ATP, the dependence of the coupled ATPase assay on myokinase, the stoichiometries determined in the ATPase assay (assay II) with limiting amounts of acetone, and the relative specific activities measured with the three different enzyme assays but differs from the values reported for other acetone carboxylases (Table 4). For example, the enzyme from X. autotrophicus was also reported to produce AMP as a final hydrolysis product from ATP, but in a stoichiometry of 1 ATP per acetone with transient formation of ADP (3,21,22). In contrast, acetophenone carboxylase was shown to produce ADP as the only hydrolysis product in a stoichiometry of 2 ATP hydrolyzed per acetophenone (10).…”

“…Acetone carboxylation (Acx) was initially characterized in the aerobic acetone-degrading alphaproteobacterium Xanthobacter autotrophicus and in the anoxygenic phototrophic bacterium Rhodobacter capsulatus (1)(2)(3)(21)(22)(23). The enzymes catalyzing acetone carboxylation in these two species were purified and biochemically characterized.…”

Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria

“…Propionic acid is also metabolized via the methylcitrate cycle (Br€ amer and Steinbüchel, 2001). The acetone carboxylase genes for acetone utilization and detoxification are also present in the genomes of Z. resiniphila (Sluis and Ensign, 1997;Boyd and Ensign, 2005). This bacterium could also utilize acetoin for carbon in the presence of acetoin dehydrogenase.…”

Carboxylases in Natural and Synthetic Microbial Pathways

“…On the other hand, some of the assimilatory carboxylases share an evolutionary origin. This seems to be the case for acetophenone and acetone carboxylase, which show high sequence similarity and have similar mechanistic features (21,22,70,109). Recently, genes with homology to those of phenylphosphate carboxylase have been identified as important for the anaerobic metabolism of benzene in the iron-reducing culture BF (1) as well as for naphthalene in the sulfate-reducing enrichment culture N47 (20).…”