Molecular characterization of phenyllactate dehydratase and its initiator from Clostridium sporogenes

The heterotrimeric phenyllactate dehydratase from Clostridium sporogenes , FldABC, catalyses the reversible dehydration of (R )-phenyllactate to (E )-cinnamate in two steps: (i) CoA-transfer from the cofactor cinnamoyl-CoA to phenyllactate to yield phenyllactyl-CoA and the product cinnamate mediated by FldA, a (R )-phenyllactate CoA-transferase; followed by (ii) dehydration of phenyllactyl-CoA to cinnamoyl-CoA mediated by heterodimeric FldBC, a phenyllactyl-CoA dehydratase. Phenyllactate dehydratase requires initiation by ATP, MgCl2 and a reducing agent such as dithionite mediated by an extremely oxygen-sensitive initiator protein (FldI) present in the cell-free extract. All four genes coding for these proteins were cloned and shown to be clustered in the order fldAIBC , which shares over 95% sequence identity of nucleotide and protein levels with a gene cluster detected in the genome of the closely related Clostridium botulinum Hall strain A. FldA shows sequence similarities to a new family of CoA-transferases, which apparently do not form covalent enzyme CoA-ester intermediates. An N-terminal Strep II-Tag containing enzymatically active FldI was overproduced and purified from Escherichia coli . FldI was characterized as a homodimeric protein, which contains one [4Fe-4S](1+ /2+) cluster with an electron spin S = 3/2 in the reduced form. The amino acid sequence as well as the chemical and EPR-properties of the pure protein are very similar to those of component A of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans (HgdC), which was able to replace FldI in the activation of phenyllactate dehydratase. Only in the oxidized state, FldI and component A exhibit significant ATPase activity, which appears to be essential for unidirectional electron transfer. Both subunits of phenyllactyl-CoA dehydratase (FldBC) show significant sequence similarities to both subunits of 2-hydroxyglutaryl-CoA dehydratase (HgdAB). The fldAIBC gene cluster resembles the hadAIBC gene cluster in the genome of Clostridium difficile and the hadABC,I genes in C. botulinum . The four subunits of these deduced 2-hydroxyacid dehydratases (65-81% amino acid sequence identity between the had genes) probably code for a 2-hydroxyisocaproate dehydratase involved in leucine fermentation. This enzyme could be the target for metronidazole in the treatment of pseudomembranous enterocolitis caused by C. difficile.