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 o n L-phenylalanine catalyses the reversible syn-dehydration of (R)-phenyllac tate 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 sep arated and identified as oxygen insensitive cinnamoyl-CoA:phenyllactate CoA -transferase, whereas the transferase depleted trimeric complex retained ox ygen sensitive phenyllactate dehydratase activity and contained about one [ 4Fe-4S] cluster. The dehydratase activity required 10 mu m FAD, 0.4 mM ATP, 2.5 mM MgCl2, 0.1 mM NADH, 5 mu M cinnamoyl-CoA and small amounts of cell- free extract (10 mu g protein per mL) similar to that known for 2-hydroxygl utaryl-CoA dehydratase from Acidaminococcus fermentans. The N-terminus of t he homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequ ence identity) involved in carnitine metabolism in Escherichia coli. Both e nzymes are members of an emerging group of CoA-transferases which exhibit h igh substrate specificity but apparently do not form enzyme CoA-ester inter mediates. It is concluded that dehydration of (R)-phenyllactate to (E)-cinn amate proceeds in two steps, a CoA-transfer from cinnamoyl-CoA to phenyllac tate, catalysed by FldA, followed by the dehydration of phenyllactyl-CoA, c atalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl-CoA is regenerated. This demonstrates the necessity of a 2-hydro xyacyl-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 contai n a derivative of acetyl-CoA covalently bound to an acyl-carrier-protein (A CP).