Radical S-adenosylmethionine (SAM) enzymes represent a diverse superfamily ofenzymes that are found across all kingdoms of life and their functional diversity is only now coming to light. Radical SAM activases are a subgroup of this superfamily that serve to activate the Glycyl Radical Enzymes (GREs) whose metabolic functions are also quite diverse. Prior to completing the work described herein, the catalytic diversity of the radical SAM superfamily has been proposed to stem in large part from the formation of a common, highly reactive, radical intermediate. The reaction intermediate that has been proposed to be common to all radical SAM enzymes is a 5-deoxyadenosyl radical and the chemical diversity of the radical SAM enzymes are discussed in Chapter I.In this dissertation we have characterized the radical SAM activase (glycerol dehydratase activating enzyme or GD-AE) for the B12-independent glycerol dehydratase (GD) from Clostridium butyricum. In Chapter II we demonstrate that activation of the GD by the GD-AE results in a glycyl radical, however, contrary to the current paradigm we also demonstrate that activation also results in the formation of 5-metylthioadenosine and 2-aminobutyrate. During this investigation, precedence for an alternative reductive cleavage mechanism was published byZhang et al. Their work and our observations imply that the transient radical species, generated as a results of the reductive cleavage of SAM, is also different in the GD-AE and would be a 3- amino-3carboxypropyl (ACP) radical. This is unprecedented for any radical SAM enzyme and in Chapter III we have used a deuterium labeling strategy and mass spectroscopy to further investigate the nature of the radical intermediate. These studies provide more evidence for the alternative reductive cleavage mechanism and insight into the intermediate.With a growing interest in the catalytic diversity of the radical SAM superfamily the implications of this work may be broad for additional radical SAM enzymes.