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 <i>Bacillus subtilis</i> to delineate the mechanism of conversion of CPH₄ to CDG. QueE is a member of the radical <i>S</i>-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 <i>K</i>_m of 20 ± 7 μM for CPH₄ and a <i>k</i>_cat of 5.4 ± 1.2 min^–1 for the overall transformation. The kinetically determined <i>K</i>_app for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a <i>K</i>_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

Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical <i>S</i>‑Adenosyl‑l‑methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration

Many cobalamin (Cbl)-dependent radical <i>S</i>-adenosyl-l-methionine (SAM) methyltransferases have been identified through sequence alignment and/or genetic analysis; however, few have been studied <i>in vitro</i>. GenK is one such enzyme that catalyzes methylation of the 6′-carbon of gentamicin X₂ (GenX₂) to produce G418 during the biosynthesis of gentamicins. Reported herein, several alternative substrates and fluorinated substrate analogs were prepared to investigate the mechanism of methyl transfer from Cbl to the substrate as well as the substrate specificity of GenK. Experiments with deuterated substrates are also shown here to demonstrate that the 6′-<i>pro</i>-<i>R</i>-hydrogen atom of GenX₂ is stereoselectively abstracted by the 5′-dAdo· radical and that methylation occurs with retention of configuration at C6′. Based on these observations, a model of GenK catalysis is proposed wherein free rotation of the radical-bearing carbon is prevented and the radical SAM machinery sits adjacent rather than opposite to the Me-Cbl cofactor with respect to the substrate in the enzyme active site

The Alpha Subunit of Nitrile Hydratase Is Sufficient for Catalytic Activity and Post-Translational Modification

Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from <i>Streptomyces rimosus</i> is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV–visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts

7‑Carboxy-7-deazaguanine Synthase: A Radical <i>S</i>‑Adenosyl‑l‑methionine Enzyme with Polar Tendencies

Radical <i>S</i>-adenosyl-l-methionine (SAM) enzymes are widely distributed and catalyze diverse reactions. SAM binds to the unique iron atom of a site-differentiated [4Fe-4S] cluster and is reductively cleaved to generate a 5′-deoxyadenosyl radical, which initiates turnover. 7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes a key step in the biosynthesis of 7-deazapurine containing natural products. 6-Carboxypterin (6-CP), an oxidized analogue of the natural substrate 6-carboxy-5,6,7,8-tetrahydropterin (CPH₄), is shown to be an alternate substrate for CDG synthase. Under reducing conditions that would promote the reductive cleavage of SAM, 6-CP is turned over to 6-deoxyadenosylpterin (6-dAP), presumably by radical addition of the 5′-deoxyadenosine followed by oxidative decarboxylation to the product. By contrast, in the absence of the strong reductant, dithionite, the carboxylate of 6-CP is esterified to generate 6-carboxypterin-5′-deoxyadenosyl ester (6-CP-dAdo ester). Structural studies with 6-CP and SAM also reveal electron density consistent with the ester product being formed in crystallo. The differential reactivity of 6-CP under reducing and nonreducing conditions highlights the ability of radical SAM enzymes to carry out both polar and radical transformations in the same active site

GenK-Catalyzed C‑6′ Methylation in the Biosynthesis of Gentamicin: Isolation and Characterization of a Cobalamin-Dependent Radical SAM Enzyme

The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical <i>S</i>-adenosyl-l-methionine (SAM) superfamily was previously predicted on the basis of bioinformatic analysis. A number of these are Cbl-dependent methyltransferases, but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the <i>in vitro</i> activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated <i>sp</i>³ carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that 1 equiv each of 5′-deoxyadenosine and <i>S</i>-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the <i>S</i>-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. On the basis of these results, one mechanistic possibility for the GenK reaction can be ruled out, and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed