An allylic ketyl radical intermediate in clostridial amino-acid fermentation

The human pathogenic bacterium Clostridium difficile thrives by the fermentation of L- leucine toammonia, CO2, 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions(1). 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(2) 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(3) and the related benzoyl- CoA reductases(4) - present in anaerobically living bacteria - employ ketyl radical intermediates. The absence of radical generators such as coenzyme B-12, S- adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.