7-Carboxy-7-deazaguanine Synthase: A Radical

Radical S-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

Radical SAM enzyme QueE defines a new minimal core fold and metal-dependent mechanism

7-carboxy-7-deazaguanine synthase (QueE) catalyzes a key S-adenosyl-L-methionine (AdoMet)- and Mg[superscript 2+]-dependent radical-mediated ring contraction step, which is common to the biosynthetic pathways of all deazapurine-containing compounds. QueE is a member of the AdoMet radical superfamily, which employs the 5′-deoxyadenosyl radical from reductive cleavage of AdoMet to initiate chemistry. To provide a mechanistic rationale for this elaborate transformation, we present the crystal structure of a QueE along with structures of pre- and post-turnover states. We find that substrate binds perpendicular to the [4Fe-4S]-bound AdoMet, exposing its C6 hydrogen atom for abstraction and generating the binding site for Mg[superscript 2+], which coordinates directly to the substrate. The Burkholderia multivorans structure reported here varies from all other previously characterized members of the AdoMet radical superfamily in that it contains a hypermodified ([β [subscript 6] over α [subscript 3]]) protein core and an expanded cluster-binding motif, CX[subscript 14]CX[subscript 2]C.United States. Dept. of Energy. Office of Biological and Environmental ResearchUnited States. Dept. of Energy. Office of Basic Energy SciencesNational Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473)National Center for Research Resources (U.S.) (5P41RR015301-10)National Institute of General Medical Sciences (U.S.) (8 P41 GM 103403-10)United States. Dept. of Energy (Contract DE-AC02-06CH11357

A Radical Intermediate in Bacillus subtilis QueE during Turnover with the Substrate Analogue 6‑Carboxypterin

7-Carboxy-7-deazaguanine (CDG) synthase (QueE), a member of the radical S-deoxyadenosyl-l-methionine (SAM) superfamily of enzymes, catalyzes a radical-mediated ring rearrangement required to convert 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) into CDG, forming the 7-dezapurine precursor to all pyrrolopyrimidine metabolites. Members of the radical SAM superfamily bind SAM to a [4Fe-4S] cluster, leveraging the reductive cleavage of SAM by the cluster to produce a highly reactive 5'-deoxyadenosyl radical which initiates chemistry by H atom abstraction from the substrate. QueE has recently been shown to use 6-carboxypterin (6-CP) as an alternative substrate, forming 6-deoxyadenosylpterin as the product. This reaction has been proposed to occur by radical addition between 5'-dAdo· and 6-CP, which upon oxidative decarboxylation yields the modified pterin. Here, we present spectroscopic evidence for a 6-CP-dAdo radical. The structure of this intermediate is determined by characterizing its electronic structure by continuous wave and pulse electron paramagnetic resonance spectroscopy

Biochemical and Spectroscopic Characterization of a Radical S‑Adenosyl‑l‑methionine Enzyme Involved in the Formation of a Peptide Thioether Cross-Link

Peptide-derived natural products are a class of metabolites that afford the producing organism a selective advantage over other organisms in their biological niche. While the polypeptide antibiotics produced by the nonribosomal polypeptide synthetases (NRPS) are the most widely recognized, the ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging group of natural products with diverse structures and biological functions. Both the NRPS derived peptides and the RiPPs undergo extensive post-translational modifications to produce structural diversity. Here we report the first characterization of the six cysteines in forty-five (SCIFF) [Haft, D. H. and Basu M. K. (2011) J. Bacteriol. 193, 2745-2755] peptide maturase Tte1186, which is a member of the radical S-adenosyl-l-methionine (SAM) superfamily. Tte1186 catalyzes the formation of a thioether cross-link in the peptide Tte1186a encoded by an orf located upstream of the maturase, under reducing conditions in the presence of SAM. Tte1186 contains three [4Fe-4S] clusters that are indispensable for thioether cross-link formation; however, only one cluster catalyzes the reductive cleavage of SAM. Mechanistic imperatives for the reaction catalyzed by the thioether forming radical SAM maturases will be discussed

A Radical Clock Probe Uncouples H Atom Abstraction from Thioether Cross-Link Formation by the Radical <i>S</i>‑Adenosyl‑l‑methionine Enzyme SkfB

Sporulation killing factor (SKF) is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by <i>Bacillus</i>. SKF contains a thioether cross-link between the α-carbon at position 40 and the thiol of Cys32, introduced by a member of the radical <i>S</i>-adenosyl-l-methionine (SAM) superfamily, SkfB. Radical SAM enzymes employ a 4Fe–4S cluster to bind and reductively cleave SAM to generate a 5′-deoxyadenosyl radical. SkfB utilizes this radical intermediate to abstract the α-H atom at Met40 to initiate cross-linking. In addition to the cluster that binds SAM, SkfB also has an auxiliary cluster, the function of which is not known. We demonstrate that a substrate analogue with a cyclopropylglycine (CPG) moiety replacing the wild-type Met40 side chain forgoes thioether cross-linking for an alternative radical ring opening of the CPG side chain. The ring opening reaction also takes place with a catalytically inactive SkfB variant in which the auxiliary Fe–S cluster is absent. Therefore, the CPG-containing peptide uncouples H atom abstraction from thioether bond formation, limiting the role of the auxiliary cluster to promoting thioether cross-link formation. CPG proves to be a valuable tool for uncoupling H atom abstraction from peptide modification in RiPP maturases and demonstrates potential to leverage RS enzyme reactivity to create noncanonical amino acids

Chemical and Biological Reduction of the Radical SAM Enzyme CPH₄ Synthase

The radical <i>S</i>-adenosyl-l-methionine (SAM) superfamily is a large and growing group of enzymes that conduct complex radical-mediated transformations. A one-electron reduction of SAM via the +1 state of the cubane [4Fe-4S] cluster generates a 5′-deoxyadenosyl radical, which initiates turnover. The [4Fe-4S] cluster must be reduced from its resting +2 state to the catalytically active +1 oxidation state by an electron. In practice, dithionite or the <i>Escherichia coli</i> flavodoxin (<i>Ec</i>FldA)/ferredoxin (flavodoxin):NADP⁺ oxidoreductase (Fpr)/NADPH system is used. Herein, we present a systematic investigation of the reductive activation of the radical SAM enzyme CDG synthase (<i>Bs</i>QueE) from <i>Bacillus subtilis</i> comparing biological and chemical reductants. These data show that either of the flavodoxin homologues encoded by the <i>B. subtilis</i> genome, <i>Bs</i>YkuN or <i>Bs</i>YkuP, as well as a series of small molecule redox mediators, supports <i>Bs</i>QueE activity. With dithionite as a reductant, the activity of <i>Bs</i>QueE is ∼75-fold greater in the presence of <i>Bs</i>YkuN and <i>Bs</i>YkuP compared to that in the presence of dithionite alone. By contrast, <i>Ec</i>FldA supports turnover to ∼10-fold greater levels than dithionite alone under the same conditions. Comparing the ratio of the rate of turnover to the apparent binding constant for the flavodoxin homologues reveals 10- and 240-fold preferences for <i>Bs</i>YkuN over <i>Bs</i>YkuP and <i>Ec</i>FldA, respectively. The differential activation of the enzyme cannot be explained by the abortive cleavage of SAM. We conclude from these observations that the differential activation of <i>Bs</i>QueE by Fld homologues may reside in the details of the interaction between the flavodoxin and the radical SAM enzyme