Caffeate respiration in the acetogenic bacterium Acetobacterium woodii: a coenzyme A loop saves energy for caffeate activation

Abstract

The anaerobic acetogenic bacterium Acetobacterium woodii couples reduction of caffeate with electrons derived from molecular hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions. Caffeate is activated to caffeyl coenzyme A (caffeyl-CoA) prior to its reduction, and the caffeate reduction operon encodes an ATP-dependent caffeyl-CoA synthetase that is thought to catalyze the initial caffeate activation. The operon also encodes a potential CoA transferase, the product of carA, which was thought to be involved in subsequent ATP-independent caffeate activation. To prove the proposed function of carA, we overproduced its protein in Escherichia coli and then purified it. Purified CarA drives the formation of caffeyl-CoA from caffeate with hydrocaffeyl-CoA as the CoA donor. The dependence of the reaction on caffeate and hydrocaffeyl-CoA followed Michaelis-Menten kinetics, with apparent K(m) values of 75 ± 5 μM for caffeate and 8 ± 2 μM for hydrocaffeyl-CoA. The enzyme activity had broad ranges of pH and temperature optima. In addition to being able to use caffeate, CarA could use p-coumarate and ferulate but not cinnamate, sinapate, or p-hydroxybenzoate as a CoA acceptor. Neither acetyl-CoA nor butyryl-CoA served as the CoA donor for CarA. The enzyme uses a ping-pong mechanism for CoA transfer and is the first classified member of a new subclass of family I CoA transferases that has two catalytic domains on one polypeptide chain. Apparently, CarA catalyzes an energy-saving CoA loop for caffeate activation in the steady state of caffeate respiration.

Fig 1

Purification of C-terminally His₆-tagged hydrocaffeyl-CoA:caffeate CoA transferase CarA of A. woodii. E. coli pET21a-carA was cultured at 37°C in LB medium. CarA was purified by affinity chromatography on Ni²⁺-NTA. Samples were separated on SDS-PAGE; proteins were stained with Coomassie blue or the recombinant His₆ tag was detected via Western blotting.

Fig 2

Heterologously produced and purified CarA catalyzes CoA transfer from hydrocaffeyl-CoA to caffeate. The assay mixture contained 1 ml 100 mM KP_i buffer (pH 7.5) and 0.15 μg CarA. (A) 250 μM caffeate and 45 μM hydrocaffeyl-CoA were added as indicated. (B) No enzyme added. (C) No caffeate added. The formation of caffeyl-CoA was followed spectrophotometrically at 346 nm.

Fig 3

Caffeate dependence of hydrocaffeyl-CoA:caffeate CoA transferase activity of CarA. The formation of caffeyl-CoA by purified recombinant CarA (0.15 μg/ml) was monitored at 346 nm.

Fig 4

Hydrocaffeyl-CoA dependence of hydrocaffeyl-CoA:caffeate CoA transferase activity of CarA. The formation of caffeyl-CoA by purified recombinant CarA (0.15 μg/ml) was monitored at 346 nm.

Fig 5

Kinetics of caffeyl-CoA formation. The assay mixture contained 1 ml 100 mM KP_i buffer (pH 7.5), 0.15 μg CarA, caffeate as indicated, and hydrocaffeyl-CoA at 10 μM (■), 20 μM (▲), and 30 μM (▼).

Fig 6

Substrate specificity of the hydrocaffeyl-CoA:caffeate CoA transferase CarA. (A) Structures of phenyl acrylates and other substrates used. (B) The assay mixture contained 1 ml 100 mM KP_i buffer (pH 7.5), 0.15 μg CarA, and 45 μM hydrocaffeyl-CoA. The reaction was started with the addition of 250 μM substrate. Formation of the corresponding CoA thioester was followed spectrophotometrically; 100% corresponds to 151 U/mg.

Fig 7

The carA gene neighborhood in A. woodii and putative similar operons in the genomes of selected anaerobic bacteria. A similar gene arrangement is found in Pelosinus and Holophaga (Firmicutes). The latter organisms and Thauera carry out the anaerobic metabolism of aromatic compounds that is known to involve a CoA transferase (36). The locus tags are shown below the gene names or annotations. Orthologs are drawn with the same pattern.

Fig 8

Model of caffeate respiration in A. woodii. The electron flow from molecular hydrogen to caffeate is shown. Ferredoxin is reduced by a bifurcating hydrogenase and reoxidized by the Rnf complex, which generates a sodium ion gradient across the cytoplasmic membrane. NADH serves as an electron donor for the caffeyl-CoA reducing complex, potentially encoded by carCDE, which might reduce another ferredoxin via electron bifurcation, fueling the Rnf complex. Caffeate is initially activated by CarB (A), whereas CarA catalyzes an energy-saving CoA loop in the steady state of caffeate respiration (B).