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.