Abstract
Members of the radical S-adenosyl-L-methionine (rSAM) enzyme superfamily cleave SAM to generate the highly reactive 5'-deoxyadenosyl radical (dAdo·), where dAdo· initiates the reaction by an H-atom transfer from the substrate to form 5'-deoxyadenosine (dAdo) in nearly every member of the superfamily. However, in all rSAM enzymes, SAM also undergoes reductive cleavage to form dAdo in a reaction uncoupled from the product's formation. Herein, we examine the dAdo that is formed under catalytic conditions with the rSAM enzyme 7-carboxy-7-deazaguanine synthase (QueE), which catalyzes the radical-mediated transformation of 6-carboxy-5,6,7,8-tetrahydropterin (CPH(4)) to 7-carboxy-7-deazaguanine (CDG). We propose that the dAdo that is observed under catalytic conditions can be traced to multiple shunt pathways, which are not all truly uncoupled from catalysis. Indeed, in one case, we demonstrate that the dAdo can form due to the reductive quenching of the initially generated substrate radical by the very same reducing system used to reductively cleave SAM to initiate catalysis. The insights from this work are generally applicable to all members of the rSAM family, as they influence the choice of reducing system to avoid the non-productive shunt pathways that interfere with catalysis.