A caffeyl-coenzyme A synthetase initiates caffeate activation prior to caffeate reduction in the acetogenic bacterium Acetobacterium woodii

Abstract

The anaerobic acetogenic bacterium Acetobacterium woodii couples the reduction of caffeate with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism using sodium ions as coupling ions, but the enzymes involved remain to be established. Previously, the electron transfer flavoproteins EtfA and EtfB were found to be involved in caffeate respiration. By inverse PCR, we identified three genes upstream of etfA and etfB: carA, carB, and carC. carA encodes a potential coenzyme A (CoA) transferase, carB an acyl-CoA synthetase, and carC an acyl-CoA dehydrogenase. carA, -B, and -C are located together with etfA/carE and etfB/carD on one polycistronic message, indicating that CarA, CarB, and CarC are also part of the caffeate respiration pathway. The genetic data suggest an initial ATP-dependent activation of caffeate by CarB. To prove the proposed function of CarB, the protein was overproduced in Escherichia coli, and the recombinant protein was purified. Purified CarB activates caffeate to caffeyl-CoA in an ATP- and CoA-dependent reaction. The enzyme has broad pH and temperature optima and requires K(+) for activity. In addition to caffeate, it can use ρ-coumarate, ferulate, and cinnamate as substrates, with 50, 15, and 9%, respectively, of the activity obtained with caffeate. Expression of the car operon is induced not only by caffeate, ρ-coumarate, ferulate, and cinnamate but also by sinapate. There is no induction by ρ-hydroxybenzoate or syringate.

FIG. 1.

Arrangement and expression analysis of the car operon in A. woodii. (A) The car operon comprises five genes. (B) For transcriptional analysis, cDNA was used as a template for PCR (lanes c). DNA (lanes a) was used as a positive control, and RNA (lanes b) was used as a negative control. The primers and the expected fragments are indicated. The predicted fragment sizes are 730 bp (carA and carB), 560 bp (carB and carC), 640 bp (carC and carD), and 926 bp (carD and carE).

FIG. 2.

Expression and purification of C-terminally His₆-tagged caffeyl-CoA synthetase CarB of A. woodii. E. coli/pET21a-carB was cultured at 16°C. Samples were taken before (lane 1) and 18 h after (lane 2) induction with IPTG, and CarB was purified with Ni²⁺-NTA (lane 3; elution fraction 6). Samples were separated by SDS-PAGE, and proteins were stained with Coomassie blue. The arrow denotes the recombinant caffeyl-CoA synthetase.

FIG. 3.

K⁺ dependence of caffeyl-CoA synthetase activity of CarB. Caffeyl-CoA formation catalyzed by purified recombinant CarB was followed spectrophotometrically and was dependent on the K⁺ concentration.

FIG. 4.

Caffeate dependence of caffeyl-CoA synthetase activity of CarB. The formation of caffeyl-CoA by purified recombinant CarB was monitored at 346 nm. The caffeate dependence of the enzymatic activity was determined.

FIG. 5.

Structures of phenylacrylates and other substrates used in this study.

FIG. 6.

Induction of CarDE by various electron acceptors. A. woodii was grown on fructose in the absence (control) or presence of caffeate, sinapate, cinnamate, ρ-hydroxybenzoate, or syringate, and the cellular levels of CarD and CarE in the cell extract were determined by Western blotting.

FIG. 7.

Model of caffeate respiration in A. woodii. The flow of electrons from hydrogen to the acceptor caffeate is shown. Rnf couples the flow of electrons from reduced ferredoxin to NAD⁺, thereby generating a sodium ion gradient across the cytoplasmic membrane. Initial activation of caffeate is by CarB, whereas CarA catalyzes an energy-saving CoA loop in the steady state of caffeate respiration. CarC is similar to acyl-CoA dehydrogenases. In view of its physiological function, we suggest the name caffeyl-CoA reductase instead of hydrocaffeyl-CoA dehydrogenase.

FIG. 8.

Comparison of the caffeyl-CoA synthetase from A. woodii with acyl-CoA synthetases from representative organisms. se, Salmonella enterica SC4154; hs, Halobacterium salinarum VNG0484G; sc, Saccharomyces cerevisiae YAL054C; Aw, Acetobacterium woodii. Sequence motifs conserved among members of the AMP-forming family of enzymes are boxed and named A1 to A10.