title: An allylic ketyl radical intermediate in clostridial amino-acid fermentation.
abstract: The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO(2), 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed to mediate this reaction catalysed by an iron-sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron-sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases and the related benzoyl-CoA reductases-present in anaerobically living bacteria-employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, D35032 Marburg, Germany.
authors.forename: Jihoe
authors.initials: J
authors.lastname: Kim
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Darley
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
mesh_terms: D000475:Alkenes / Q000378:metabolism*; D000838:Anions / Q000378:metabolism*; D016360:Clostridioides difficile / Q000201:enzymology / Q000378:metabolism*; D015256:Coenzyme A-Transferases / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005285:Fermentation; D006836:Hydro-Lyases / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000378:metabolism*
chemical_list: D000475:Alkenes; D000838:Anions; D015256:Coenzyme A-Transferases; D006836:Hydro-Lyases; D007930:Leucine