Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids

Abstract

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30°C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 ,uM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation

“…Some butyrate can be transformed into butyryl-CoA by reverse reaction of butyrate kinase and phosphotransbutyrylase [19]. The K m values of CoA transferase for acetate and butyrate were determined to be 1.2 M and 0.6 M, respectively [12]. These high concentrations probably reflect a regulatory system that allows the cell to prevent the toxic effects caused by increased amounts of acids.…”

“…Both proteins have been purified to homogeneity. CoA transferase was found to consist of two different subunits, originally determined to possess a molecular mass of 23 and 25 kDa [12]. Sequencing data later revealed that these sizes rather were 22.7 and 23.7 kDa for strain ATCC 824 and 23.6 kDa for both subunits in DSM 792 [13][14][15][16].…”

PTS regulation domain‐containing transcriptional activator CelR and sigma factor σ54 control cellobiose utilization in Clostridium acetobutylicum

“…Bacterial strains and growth conditions C. acetobutylicum ATCC 824 wild-type and mutant strains used in this study are listed in Supporting Information Table S3. C. acetobutylicum strains were precultured anaerobically on clostridial growth medium (CGM) (Wiesenborn et al, 1989) to exponential growth phase. Erythromycin (30 lg/ml) or chloramphenicol (17 lg/ml) was added when needed.…”

Host-plasmid interactions in recombinant strains of Clostridium acetobutylicum ATCC 824

“…Product concentrations were determined by gas chromatography [13]. Enzymatic assays for the phosphotransbutyrylase [14], butyrate kinase [15], CoA-transferase [16], acetaldehyde dehydrogenase [4], and butanol dehydrogenase [4] were performed as previously described.…”