N-nitroso-containing small molecules, such as the bacterial natural product streptozotocin, are prominent carcinogens 1,2 and important cancer chemotherapeutics 3,4 . Despite this functional group's significant impact on human health, dedicated enzymes involved in N-nitroso assembly have not been identified. Here, we describe a metalloenzyme from streptozotocin biosynthesis (SznF) that catalyzes an oxidative rearrangement of the guanidine group of N ω -methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF uncovered two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. The discovery of this biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-heme iron-dependent enzymes to include N-N bond formation. We find biosynthetic gene clusters encoding SznF homologs are widely distributed among bacteria, including environmental organisms, plant symbionts, and human pathogens, suggesting an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites. Streptozotocin (SZN) (Zanosar®) is an N-nitrosourea natural product and approved cancer chemotherapeutic (Fig. 1a) 3,5 . SZN is also used to induce type I diabetes in animal models due to its toxicity towards pancreatic beta cells (> 28,500 PubMed references) 6 . Like other N-nitrosoureas, SZN exerts its activity in vivo by generating electrophilic DNA alkylating Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.

Fe 4 S 4 clusters are very common versatile prosthetic groups in proteins. Their redox property of being sensitive to O 2 -induced oxidative damage is, for instance, used by the cell to sense oxygen levels and switch between aerobic and anaerobic metabolisms, as exemplified by the fumarate, nitrate reduction regulator (FNR). Using the hydrogenase maturase HydE from Thermotoga maritima as a template, we obtained several unusual forms of FeS clusters, some of which are associated with important structural changes. These structures represent intermediate states relevant to both FeS cluster assembly and degradation. We observe one Fe 2 S 2 cluster bound by two cysteine persulfide residues. This observation lends structural support to a very recent Raman study, which reported that Fe 4 S 4 -to-Fe 2 S 2 cluster conversion upon oxygen exposure in FNR resulted in concomitant production of cysteine persulfide as cluster ligands. Similar persulfide ligands have been observed in vitro for several other Fe 4 S 4 cluster-containing proteins. We have also monitored FeS cluster conversion directly in our protein crystals. Our structures indicate that the Fe 4 S 4 -to-Fe 2 S 2 change requires large structural modifications, which are most likely responsible for the dimer-monomer transition in FNR.FeFe-hydrogenase | X-ray crystallography

[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN– ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (−S–CH2–NH–CH2–S–) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN– to produce a unique l-cysteine-Fe­(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric “SFe­(CO)2CN” precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.

The [Fe2S2]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed (Out) and buried (In) states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis and crystallographic and computational chemical studies to show that (i) the Out and In states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the In state due to a change in its pKa caused by cluster reduction and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes. Our findings illustrate a novel function of protonation resulting from electron transfer.

Edited by George DeMartinoSpeckle-type POZ protein (SPOP) is a ubiquitin ligase adaptor that binds substrate proteins and facilitates their proteasomal degradation. Most SPOP substrates present multiple SPOP-binding (SB) motifs and undergo liquid-liquid phase separation with SPOP. Pancreatic and duodenal homeobox 1 (Pdx1), an insulin transcription factor, is downregulated by interaction with SPOP. Unlike other substrates, only one SB motif has previously been reported within the Pdx1 C-terminal intrinsically disordered region (Pdx1-C). Given this difference, we aimed to determine the specific mode of interaction of Pdx1 with SPOP and how it is similar or different to that of other SPOP substrates. Here, we identify a second SB motif in Pdx1-C, but still find that the resulting moderate valency is insufficient to support phase separation with SPOP in cells. Although Pdx1 does not phase separate with SPOP, Pdx1 and SPOP interaction prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. Accordingly, we find that SPOP-mediated ubiquitination activity of Pdx1 occurs in the nucleoplasm and that highly efficient Pdx1 turnover requires both SB motifs. Our results suggest that the subnuclear localization of SPOP-substrate interactions and substrate ubiquitination may be directed by the properties of the substrate itself.

Cyclic peptides containing redox-stable thioether bridges might provide a useful alternative to disulfide-bridged bioactive peptides. We report the effect of replacing the disulfide bridge with a lanthionine linkage in a 16-mer cyclic peptide that binds to death receptor 5 (DR5, TRAIL-R2). Upon covalent oligomerisation, the disulfide-bridged peptide has previously shown similar behaviour to that of TNF-related apoptosis inducing ligand (TRAIL), by selectively triggering the DR5 cell death pathway. The structural and biological properties of the DR5-binding peptide and its desulfurised analogue were compared. Surface plasmon resonance (SPR) data suggest that these peptides bind DR5 with comparable affinities. The same holds true for dimeric versions of these peptides: the thioether is able to induce DR5-mediated apoptosis of BJAB lymphoma and tumorigenic BJELR cells, albeit to a slightly lower extent compared to its disulfide homologue. NMR analysis revealed subtle variation in the conformations of the two peptides and suggests that the thioether peptide is slightly less folded than its disulfide homologue. These observations could account for the different capability of the two dimers to cluster DR5 receptors on the cell surface and to trigger apoptosis. Nevertheless, our results suggest that the thioether peptide is a potential candidate for evaluation in animal models.