Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus

Abstract

Acetone degradation by cell suspensions of Desulfococcus biacutus was CO 2 dependent, indicating initiation by a carboxylation reaction, while degradation of 3-hydroxybutyrate was not CO 2 dependent. Growth on 3-hydroxybutyrate resulted in acetate accumulation in the medium at a ratio of 1 mol of acetate per mol of substrate degraded. In acetone-grown cultures no coenzyme A (CoA) transferase or CoA ligase appeared to be involved in acetone metabolism, and no acetate accumulated in the medium, suggesting that the carboxylation of acetone and activation to acetoacetyl-CoA may occur without the formation of a free intermediate. Catabolism of 3-hydroxybutyrate occurred after activation by CoA transfer from acetyl-CoA, followed by oxidation to acetoacetyl-CoA. In both acetone-grown cells and 3-hydroxybutyrate-grown cells, acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA residues and further metabolized through the carbon monoxide dehydrogenase pathway. Comparison of the growth yields on acetone and 3-hydroxybutyrate suggested an additional energy requirement in the catabolism of acetone. This is postulated to be the carboxylation reaction (⌬G؇ for the carboxylation of acetone to acetoacetate, ؉17.1 kJ ⅐ mol ؊1). At the intracellular acyl-CoA concentrations measured, the net free energy change of acetone carboxylation and catabolism to two acetyl-CoA residues would be close to 0 kJ ⅐ mol of acetone ؊1, if one mol of ATP was invested. In the absence of an energy-utilizing step in this catabolic pathway, the predicted intracellular acetoacetyl-CoA concentration would be 10 13 times lower than that measured. Thus, acetone catabolism to two acetyl-CoA residues must be accompanied by the utilization of the energetic equivalent of (at least) one ATP molecule. Measurement of enzyme activities suggested that assimilation of acetyl-CoA occurred through a modified citric acid cycle in which isocitrate was cleaved to succinate and glyoxylate. Malate synthase, condensing glyoxylate and acetyl-CoA, acted as an anaplerotic enzyme. Carboxylation of pyruvate or phosphoenolpyruvate could not be detected.The anaerobic metabolism of acetone appears to involve an initial carboxylation of acetone to acetoacetate (or acetoacetyl coenzyme A [acetoacetyl-CoA]), followed by thiolytic cleavage to two acetyl-CoA residues (3, 32-35). The carboxylation reaction has not been measured in vitro, and evidence has come from 14 C labelling studies (32,33,35). Carboxylation reactions appear to play a role in a number of anaerobic transformations (16, 44), but these reactions have not been well studied. The carboxylation of acetone to acetoacetate, CH 3 COCH 3 ϩ HCO 3), is an endergonic reaction (42). Thus, the energetic equivalent of about 1/3 mol of ATP (⌬GЊЈ of ATP hydrolysis, Ϫ50 kJ ⅐ mol Ϫ1 [42]) is required, but the mechanism of coupling is not known. In addition, attempts to measure the carboxylase in sulfate-reducing bacteria have remained unsuccessful (21).Many genera of sulfate-reducing bacteria are able to oxidize completely (to CO ...

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

“…lienii is a moderately thermophilic bacterium with a fermentative type of metabolism, which can reduce elemental sulfur to sulfide (Dahle and Birkeland 2006). Sequences of sulfate reducers such as Desulfococcus biacutus and Desulfobacterium cetonicum which are able to oxidize acetone to CO 2 (Janssen and Schink 1995a, b;Platen et al 1990) or other microorganisms in those genera were not detected in the clone libraries, which is in agreement with the observed accumulation of acetone in the MR. Within the class Proteobacteria, clones were only found in the delta (d) sub-division.…”

Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from Xanthobacter autotrophicus Strain Py2 and Rhodobacter capsulatus Strain B10

“…However, the in vitro rates observed for this enzyme were only ϳ10% of the corresponding in vivo rates, and substantial enzyme activity was lost during purification, suggesting that this enzyme may have additional cofactor requirements beyond those identified in the initial studies (14). In vivo and in vitro studies of strict anaerobes that grow with acetone, e.g., sulfate reducers, denitrifiers, and fermenters, support a similar CO 2 -dependent pathway, but all attempts to reconstitute acetone carboxylase activity in vitro for these strains have been unsuccessful (22)(23)(24)(37)(38)(39). Thus, it remains to be determined whether the putative acetone carboxylases of these organisms are structurally and functionally similar to those of X. autotrophicus, R. capsulatus, and Rhodococcus rhodochrous.…”

Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2

“…The carboxylation of acetone to produce acetoacetate has been proposed as the initial step in the metabolism of acetone in a variety of anaerobic bacteria (2,10,11,13,14,(16)(17)(18). For some aerobic bacteria, the metabolism of acetone has been proposed to proceed by hydroxylation, producing acetol rather than acetoacetate as the initial product of acetone degradation (4,12,21,23).…”

“…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).…”