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

• Published in: Biochemistry (Easton) Vol. 52; no. 1; pp. 188 - 198
• Main Authors: McCarty, Reid M, Krebs, Carsten, Bandarian, Vahe
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.01.2013
• Subjects: Bacillus subtilis - chemistry; Bacillus subtilis - enzymology; Bacillus subtilis - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Catalysis; Cyclization; Electron Spin Resonance Spectroscopy; Guanine - analogs & derivatives; Guanine - chemistry; Guanine - metabolism; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - metabolism; Kinetics; Protein Structure, Quaternary; Pterins - chemistry; Pterins - metabolism; S-Adenosylmethionine - metabolism; Spectroscopy, Mossbauer
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi301156w

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.