Premium
Molecular characterization of phenyllactate dehydratase and its initiator from Clostridium sporogenes
Author(s) -
Dickert Sandra,
Pierik Antonio J.,
Buckel Wolfgang
Publication year - 2002
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2002.02867.x
Subject(s) - dehydratase , biology , biochemistry , clostridium sporogenes , carboxypeptidase a , escherichia coli , acetyl coa , peptide sequence , enzyme , carboxypeptidase , clostridium , gene , bacteria , genetics
Summary 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, MgCl 2 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‐ h ydroxy a cid d ehydratases (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 .