Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus

Abstract

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer Desulfococcus biacutus. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO 2 . The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ؎ 36 mU mg ؊1 protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.A cetone is produced by bacterial fermentations, for example, by several Clostridium species (1). It is also produced in chemistry as a solvent and as an intermediate in the synthetic chemical industry. Aerobic degradation of methyl ketones was first observed with hydrocarbon-utilizing bacteria (2). Acetone is degraded by some aerobic bacteria (3) and mammalian liver cells via oxygenase-dependent hydroxylation to acetol (4). Carboxylation of acetone to acetoacetate as a means of acetone activation was first proposed for a methanogenic enrichment culture (5). The requirement of CO 2 as a cosubstrate for acetone degradation was also observed with the nitrate reducer Thiosphaera pantotropha (6) and with Rhodobacter capsulatus and other phototrophs (7). The reaction was studied with the nitrate-reducing strain Bun N under anoxic conditions, and it was concluded that acetoacetate was formed by the ATP-dependent carboxylation of acetone (8, 9).Attempts to measure an in vitro carboxylation of acetone at that time were unsuccessful. However, exchange of radioactively labeled CO 2 with the carboxyl group of acetoacetate was catalyzed by cell extracts of strain Bun N (10). A similar CO 2 -and ATPdependent activation reaction was observed with the aerobic bacterium Xanthobacter autotrophicus strain Py2 (11). A comparison between the acetone carboxylase of strain Py2 and the carboxylase of the phototrophic bacterium Rhodobacter capsulatus showed that they are ide...

Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus

“…2, Debia-MDR might also play a role in the oxidation of the proposed 3-hydroxybutanal intermediate to acetoacetaldehyde, as suggested by its high activity towards this reaction (Table 1). Moreover, the extremely reactive acetoacetaldehyde was previously shown (as dinitrophenylhydrazone adduct) to appear at low concentration in cell-free extracts of D. biacutus in the presence of acetone and CO [8]. Notably, the specific activity of Debia-MDR dehydrogenase for 3-hydroxybutanal oxidation to acetoacetaldehyde determined in vitro (83 nmol min −1 mg −1 ) is sufficient to explain the specific substrate turnover rate of D. biacutus during growth with acetone and sulfate (19 nmol min −1 mg −1 ) [34], though upstream and downstream processes may influence the rate of this reaction step in vivo.

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“…Desulfococcus biacutus strain KMRActS (DSM5651) was grown in sulfide-reduced, CO 2 /bicarbonate-buffered (pH 7.2), freshwater mineral-salts medium as described previously [7, 8]. The medium was supplemented with 5 mM acetone as sole carbon and energy source, and with 10 mM sulfate as electron acceptor.…”

“…Cell debris and intact cells were removed by centrifugation (16,000 × g , 10min, 4 °C), and the soluble protein fraction was separated from the membrane protein fraction by ultracentrifugation (104,000 × g , 1h, 4 °C). Cell-free extract of D. biacutus was prepared as described before [8]. …”

“…Correspondingly, no acetone carboxylase activity was detected in cell-free extracts, and no acetone carboxylases were found in the genome and proteome of this bacterium [7–9]. Experiments with dense cell suspensions and cell-free extracts suggested that acetone may be activated through a carbonylation or a formylation reaction, and that acetoacetaldehyde rather than acetoacetate may be formed as an intermediate [3, 8]. In cell-free extracts of acetone-grown D. biacutus cells, acetoacetaldehyde was trapped as its dinitrophenylhydrazone derivative and was identified by mass spectrometry, after reactions with acetone, ATP and CO as cosubstrates [8].…”

“…Experiments with dense cell suspensions and cell-free extracts suggested that acetone may be activated through a carbonylation or a formylation reaction, and that acetoacetaldehyde rather than acetoacetate may be formed as an intermediate [3, 8]. In cell-free extracts of acetone-grown D. biacutus cells, acetoacetaldehyde was trapped as its dinitrophenylhydrazone derivative and was identified by mass spectrometry, after reactions with acetone, ATP and CO as cosubstrates [8]. This reaction was not observed in cell-free extract of butyrate-grown cells, hence, the proposed acetone-activating enzyme, and most likely the entire acetone utilization pathway, is inducibly expressed in D. biacutus.…”