Publications

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

  Dickert S., Pierik A.J., Linder D., Buckel W.

  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 kD ... >> More

  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). << Less

  Eur. J. Biochem. 267:3874-3884(2000) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

  Bertsova Y.V., Serebryakova M.V., Anashkin V.A., Baykov A.A., Bogachev A.V.

  Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the fac ... >> More

  Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning. << Less

  Biochemistry (Mosc.) 89:241-256(2024) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.