title: The involvement of coenzyme A esters in the dehydration of (R)-phenyllactate to (E)-cinnamate by Clostridium sporogenes.
abstract: 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 microM FAD, 0.4 mM ATP, 2.5 mM MgCl2, 0.1 mM NADH, 5 microM cinnamoyl-CoA and small amounts of cell-free extract (10 microg 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).
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Dickert
authors.forename: A J
authors.initials: AJ
authors.lastname: Pierik
authors.forename: D
authors.initials: D
authors.lastname: Linder
authors.forename: W
authors.initials: W
authors.lastname: Buckel
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D002474:Cell-Free System; D002934:Cinnamates / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism*; D004798:Enzymes / Q000302:isolation & purification; D004952:Esters / Q000378:metabolism; D005285:Fermentation; D006836:Hydro-Lyases / Q000302:isolation & purification / Q000378:metabolism; D007773:Lactates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009876:Operon; D010666:Phenylpropionates / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D002934:Cinnamates; D004798:Enzymes; D004952:Esters; D007773:Lactates; D010666:Phenylpropionates; D009243:NAD; C017648:3-phenyllactic acid; C035253:3-phenylpropionic acid; C022903:cinnamoyl-coenzyme A; D006836:Hydro-Lyases; C411816:phenyllactate dehydratase; D003065:Coenzyme A