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Cited by 61Enzyme Structure and FunctionBiochemical and biochemical processesMicrobial metabolism and enzyme function

Molecular characterization of phenyllactate dehydratase and its initiator from Clostridium sporogenes

Dickert, Sandra, Pierik, Antonio J., Buckel, Wolfgang·2002·Molecular Microbiology
DOI 10.1046/j.1365-2958.2002.02867.x
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Abstract

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.

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Crossref · 10.1046/j.1365-2958.2002.02867.x

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  • A 15kb gene cluster in C. sporogenes harbors phenyllactate dehydratase (fldABC) and adjacent genes including fldH that are critical to reductive metabolism of aromatic amino acids including Phe.

    “Dodd and colleagues also characterized a 15kb gene cluster in C. sporogenes that harbors a phenyllactate dehydratase (fldABC) (Dickert et al., 2002), along with adjacent genes (e.g. fldH), that were shown to be critical to catalytic reductive metabolism of aromatic amino acids including Phe (Dodd et al., 2017).”

    From A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors (2020) · cites this paper as Dickert et al., 2002

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Authors
Dickert, S., Pierik, A. J., & Buckel, W.
Affiliations
Philipps University of Marburg (DE)
Corresponding author
Buckel, Wolfgang
ORCID
Venue
Molecular Microbiology
Publisher
Wiley
Published
2002-04
Volume
44
Issue
1
Pages
49-60
ISSN
  • 0950-382X (ISSN-L)
  • 1365-2958
DOI
10.1046/j.1365-2958.2002.02867.x
OpenAlex
W1898346422
Cited by
61
References
46
Citations by year
2026: 3 · 2025: 6 · 2024: 3 · 2023: 3 · 2022: 3 · 2020: 5 · 2019: 5 · 2018: 4 · 2017: 2 · 2016: 1 · 2015: 3 · 2014: 2 · 2013: 1 · 2012: 1
FWCI
1.64
Type
journal article
Language
en
Open access
closed
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor, 19 d)
Locations
  • Molecular Microbiology (publishedVersion)
  • PubMed (publishedVersion)
Funders
Deutsche Forschungsgemeinschaft
Topics
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Biochemical and biochemical processes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
Keywords
Clostridium sporogenes, gene clusters, [4Fe-4S] cluster, ATPase activity, electron transfer, Clostridium botulinum, Clostridium difficile, Acidaminococcus fermentans, protein purification
MeSH terms
Adenosine Triphosphate, Amino Acid Sequence, Bacterial Proteins, Cloning, Molecular, Clostridium, Hydro-Lyases, Molecular Sequence Data, Operon, Phylogeny, Recombinant Proteins, Magnesium Chloride, Sequence Alignment, Sequence Homology, Amino Acid, Dimerization

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