The involvement of coenzyme A esters in the dehydration of ( R )‐phenyllactate to ( E )‐cinnamate by Clostridium sporogenes

Phenyllactate dehydratase from Clostridium sporogenes grown anaerobically on l ‐phenylalanine catalyses the reversible syn ‐dehydration of ( R )‐phenyllactate to ( E )‐cinnamate. Purification yielded a heterotrimeric enzyme complex (130 ± 15 kDa) composed of FldA (46 kDa), FldB (43 kDa) and FldC (40 kDa). By re‐chromatography on Q‐Sepharose, the major part of FldA could be separated and identified as oxygen insensitive cinnamoyl‐CoA:phenyllactate CoA‐transferase, whereas the transferase depleted trimeric complex retained oxygen sensitive phenyllactate dehydratase activity and contained about one [4Fe‐4S] cluster. The dehydratase activity required 10 µ m FAD, 0.4 m m ATP, 2.5 m m MgCl 2 , 0.1 m m NADH, 5 µ m cinnamoyl‐CoA and small amounts of cell‐free extract (10 µg protein per mL) similar to that known for 2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans . The N‐terminus of the homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequence identity) involved in carnitine metabolism in Escherichia coli . Both enzymes are members of an emerging group of CoA‐transferases which exhibit high substrate specificity but apparently do not form enzyme CoA‐ester intermediates. It is concluded that dehydration of ( R )‐phenyllactate to ( E )‐cinnamate proceeds in two steps, a CoA‐transfer from cinnamoyl‐CoA to phenyllactate, catalysed by FldA, followed by the dehydration of phenyllactyl‐CoA, catalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl‐CoA is regenerated. This demonstrates the necessity of a 2‐hydroxyacyl‐CoA intermediate in the dehydration of 2‐hydroxyacids. The transient CoA‐ester formation during the dehydration of phenyllactate resembles that during citrate cleavage catalysed by bacterial citrate lyase, which contain a derivative of acetyl‐CoA covalently bound to an acyl‐carrier‐protein (ACP).