Nathan A. Bruender

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 Mg2+-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 first 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 Mg2+, which directly coordinates 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 (β6/α3) protein core and an expanded cluster-binding motif CX14CX2C.

The Radical S-Adenosyl-l-methionine Enzyme MftC Catalyzes an Oxidative Decarboxylation of the C-Terminus of the MftA Peptide

Ribosomally synthesized post-translationally modified peptides (RiPPs) are encoded in the genomes of a wide variety of microorganisms, in the proximity of open reading frames that encode enzymes that conduct extensive modifications, many of which are novel. Recently, members of the radical S-adenosyl-l-methionine (SAM) superfamily have been identified in these biosynthetic clusters. Herein, we demonstrate the putative radical SAM enzyme, MftC, oxidatively decarboxylates the C-terminus of the MftA peptide in the presence of the accessory protein MftB. The reaction catalyzed by MftC expands the repertoire of peptide-based radical SAM chemistry beyond the intramolecular cross-links.

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.

Active Site Architecture of a Sugar N-Oxygenase

KijD3 is a flavin-dependent N-oxygenase implicated in the formation of the nitro-containing sugar d-kijanose, found attached to the antibiotic kijanimicin. For this investigation, the structure of KijD3 in complex with FMN and its dTDP-sugar substrate was solved to 2.1 Å resolution. In contrast to the apoenzyme structure, the C-terminus of the protein becomes ordered and projects into the active site cleft [Bruender, N. A., Thoden, J. B., and Holden, H. M. (2010) Biochemistry 49, 3517-3524]. The amino group of the dTDP-aminosugar that is oxidized is located 4.9 Å from C4a of the flavin ring. The model provides a molecular basis for understanding the manner in which KijD3 catalyzes its unusual chemical transformation.

7-Carboxy-7-deazaguanine Synthase: A Radical S-Adenosyl-l-methionine Enzyme with Polar Tendencies

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.

Probing the catalytic mechanism of a C-3'-methyltransferase involved in the biosynthesis of D-tetronitrose.

D‐Tetronitrose is a nitro‐containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S‐adenosyl‐L‐methionine (SAM) to the C‐3′ carbon of dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D‐glucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N‐terminal motif contains a binding site for a structural zinc ion. The middle‐ and C‐terminal domains serve to anchor the SAM and dTDP–sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site‐directed mutagenesis, kinetic analyses, and X‐ray structural studies. In addition, two ternary complexes of the enzyme with bound S‐adenosyl‐L‐homocysteine and either dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐D‐glucose or dTDP‐3‐amino‐2,3,6‐trideoxy‐D‐galactose were determined to 1.5 or 1.6 Å resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C‐3′ carbon of the sugar.

The Creatininase Homolog MftE from Mycobacterium smegmatis Catalyzes a Peptide Cleavage Reaction in the Biosynthesis of a Novel Ribosomally Synthesized Post-translationally Modified Peptide (RiPP).

Most ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are processed by tailoring enzymes to create complex natural products that are still recognizably peptide-based. However, some tailoring enzymes dismantle the peptide en route to synthesis of small molecules. A small molecule natural product of as yet unknown structure, mycofactocin, is thought to be synthesized in this way via the mft gene cluster found in many strains of mycobacteria. This cluster harbors at least six genes, which appear to be conserved across species. We have previously shown that one enzyme from this cluster, MftC, catalyzes the oxidative decarboxylation of the C-terminal Tyr of the substrate peptide MftA in a reaction that requires the MftB protein. Herein we show that mftE encodes a creatininase homolog that catalyzes cleavage of the oxidatively decarboxylated MftA peptide to liberate its final two residues, including the C-terminal decarboxylated Tyr (VY*). Unlike MftC, which requires MftB for function, MftE catalyzes the cleavage reaction in the absence of MftB. The identification of this novel metabolite, VY*, supports the notion that the mft cluster is involved in generating a small molecule from the MftA peptide. The ability to produce VY* from MftA by in vitro reconstitution of the activities of MftB, MftC, and MftE sets the stage for identification of the novel metabolite that results from the proteins encoded by the mft cluster.

Crystal structure of AdoMet radical enzyme 7-carboxy-7-deazaguanine synthase from Escherichia coli suggests how modifications near [4Fe-4S] cluster engender flavodoxin specificity: QueE structural comparisons

7‐Carboxy‐7‐deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6‐carboxy‐5,6,7,8‐tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7‐deazaguanine natural products. QueE is a member of the S‐adenosyl‐L‐methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX3CXϕC motif, to bind a [4Fe‐4S] cluster, and a partial (β/α)6 TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster‐binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re‐haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin.

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

Sporulation killing factor (SKF) is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus. 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 S-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.