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: S Dickert, A J Pierik, D Linder, W Buckel
• Journal: European journal of biochemistry (Eur J Biochem) Vol. 267 Issue 12 Pg. 3874-84 (Jun 2000) ISSN: 0014-2956 [Print] England
• PMID: 10849007 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Acyl Coenzyme A
• Cinnamates
• Enzymes
• Esters
• Lactates
• Phenylpropionates
• NAD
• 3-phenyllactic acid
• 3-phenylpropionic acid
• cinnamoyl-coenzyme A
• Hydro-Lyases
• phenyllactate dehydratase
• Coenzyme A

Topics

• Acyl Coenzyme A (metabolism)
• Amino Acid Sequence
• Cell-Free System
• Cinnamates (metabolism)
• Clostridium (genetics, metabolism)
• Coenzyme A (metabolism)
• Enzymes (isolation & purification)
• Esters (metabolism)
• Fermentation
• Hydro-Lyases (isolation & purification, metabolism)
• Lactates (metabolism)
• Molecular Sequence Data
• NAD (metabolism)
• Operon
• Phenylpropionates (metabolism)