Spectroscopic, steady-state kinetic, and mechanistic characterization of the radical SAM enzyme QueE, which catalyzes a complex cyclization reaction in the biosynthesis of 7-deazapurines

7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH₄) to CDG in the third step of the biosynthetic pathway to all 7-deazapurines. Here we present a detailed characterization of QueE from Bacillus subtilis to delineate the mechanism of conversion of CPH₄ to CDG. QueE is a member of the radical S-adenosyl-l-methionine (SAM) superfamily, all of which use a bound [4Fe-4S]⁺ cluster to catalyze the reductive cleavage of the SAM cofactor to generate methionine and a 5′- deoxyadenosyl radical (5′-dAdo^•), which initiates enzymatic transformations requiring hydrogen atom abstraction. The ultraviolet-visible, electron paramagnetic resonance, and Mössbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a K_m of 20 ± 7 μM for CPH₄ and a k_cat of 5.4 ± 1.2 min^-1 for the overall transformation. The kinetically determined K_app for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a K_app for the divalent metal ion of 0.21 ± 0.03 mM. The SAM cofactor supports multiple turnovers, indicating that it is regenerated at the end of each catalytic cycle. The mechanism of rearrangement of QueE was probed with CPH₄ isotopologs containing deuterium at C-6 or the two prochiral positions at C-7. These studies implicate 5′-dAdo^• as the initiator of the ring contraction reaction catalyzed by QueE by abstraction of the H atom from C-6 of CPH₄.