Abstract
The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO2, 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions1. 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 proposed2 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 dehydratases3 and the related benzoyl-CoA reductases4—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.
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Acknowledgements
We thank R. K. Thauer for the use of his EPR spectrometer, and V. Schünemann and M. Bennati for the use of their freeze-quench instruments.
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The file contains Supplementary Figures S1-S3 with Legends, Supplementary Methods describing Synthesis and characterization of (labeled) substrates, Supplementary Tables S1-S5, Supplementary Discussion, and additional references. (PDF 980 kb)
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Kim, J., Darley, D., Buckel, W. et al. An allylic ketyl radical intermediate in clostridial amino-acid fermentation. Nature 452, 239–242 (2008). https://doi.org/10.1038/nature06637
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DOI: https://doi.org/10.1038/nature06637
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