AmericanChemicalSociety.com

Over the past forty years, the investigation into the mechanism of activation for the glycyl radical enzymes has lead to the identification of a diverse superfamily of enzymes that utilize S-adenosylmethionine (AdoMet or SAM) to generate an extremely reactive radical species.  This superfamily, termed the “AdoMet radical” or “Radical SAM” superfamily, subsequently employs the radical species for a diverse range of chemical transformations and has been found throughout all kingdoms of life.  The foundational discovery of this novel AdoMet reaction was made in 1976 while investigating the activation mechanism of the glycyl radical enzyme, pyruvate formate lyase (PFL).  It has been well established that the pyruvate formate lyase activating enzyme (PFL-AE) performs this activation by catalyzing the reductive cleavage of AdoMet to form a transient 5’-deoxyadenosyl radical and methionine.  In the case of PFL-AE, this highly reactive radical species serves to generate an essential glycyl radical on glycine 734 of PFL by hydrogen atom abstraction.  Given the elegant and extensive investigations that have been performed on PFL-AE, the present dogma has maintained that all activating enzymes work by a similar mechanism.  In this work, we examined the generation of the glycyl radical on the B₁₂-independent glycerol dehydratase (GD) by the glycerol dehydratase activating enzyme (GD-AE) from C. butyricum.  We show that, the reaction requires a reduced iron-sulfur cluster as well as AdoMet and also results in a glycyl radical that is similar to what has been reported for PFL activation.  However, analysis of the activation mechanism by HPLC and mass spectrometry demonstrates that 5’-deoxy-5’-(methylthio)adenosine and not 5’-deoxyadenosine is formed during the activation of the GD by the GD-AE.  These findings provide a new paradigm for a field of enzymes that have been heralded as one of the most diverse superfamilies found in Nature.