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- Title
An allylic ketyl radical intermediate in clostridial amino-acid fermentation.
- Authors
Kim, Jihoe; Darley, Daniel J.; Buckel, Wolfgang; Pierik, Antonio J.
- Abstract
The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO2, 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.
- Subjects
AMINO acids; FERMENTATION; LEUCINE; AMMONIA; SULFUR; PROTEINS; ENZYMES; ADENOSYLMETHIONINE; ELECTRON paramagnetic resonance
- Publication
Nature, 2008, Vol 452, Issue 7184, p239
- ISSN
0028-0836
- Publication type
Article
- DOI
10.1038/nature06637