Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic ß-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB.

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon-carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys-Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys-Trp radical (Lys-Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.

Radical SAM Enzyme QmpB Installs Two 9-Membered Ring Sactionine Macrocycles during Biogenesis of a Ribosomal Peptide Natural Product.

We report the reaction catalyzed by QmpB, a new radical S-adenosylmethionine enzyme encoded by a ribosomal peptide natural product gene cluster in Streptococcus suis. Using isotopic labeling, site-directed mutagenesis, high-resolution mass spectrometry, and multidimensional NMR spectroscopy, we show that QmpB installs two 9-membered ring sactionine bridges, connecting a Cys residue with an upstream Asn via an α-thioether bridge, with the two macrocycles separated by a single residue. QmpB is only the second type II sactionine synthase characterized to date.

Macrocyclization via an Arginine-Tyrosine Crosslink Broadens the Reaction Scope of Radical S-Adenosylmethionine Enzymes.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an ascendant class of natural products with diverse structures and functions. Recently, we identified a wide array of RiPP gene clusters that are regulated by quorum sensing and encode one or more radical S-adenosylmethionine (RaS) enzymes, a diverse protein superfamily capable of catalyzing chemically difficult transformations. In this work, we characterize a novel reaction catalyzed by one such subfamily of RaS enzymes during RiPP biosynthesis: installation of a macrocyclic carbon-carbon bond that links the unactivated δ-carbon of an arginine side chain to the ortho-position of a tyrosine-phenol. Moreover, we show that this transformation is, unusually for RiPP biogenesis, largely insensitive to perturbations of the leader portion of the precursor peptide. This reaction expands the already impressive scope of RaS enzymes and contributes a unique macrocyclization motif to the growing body of RiPP architectures.

Radical Approach to Enzymatic ß-Thioether Bond Formation.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products that harbor diverse chemical functionalities, usually introduced via the action of a small number of tailoring enzymes. We have been interested in RiPP biosynthetic gene clusters that encode unusual metalloenzymes, as these may install as yet unknown alterations. Using a new bioinformatic search strategy, we recently identified an array of unexplored RiPP gene clusters that are quorum sensing-regulated and contain one or more uncharacterized radical S-adenosylmethionine (RaS) metalloenzymes. Herein, we investigate the reaction of one of these RaS enzymes and find that it installs an intramolecular ß-thioether bond onto its substrate peptide by connecting a Cys-thiol group to the ß-carbon of an upstream Asn residue. The enzyme responsible, NxxcB, accepts several amino acids in place of Asn and introduces unnatural ß-thioether linkages at unactivated positions. This new transformation adds to the growing list of Nature's peptide macrocyclization strategies and expands the already impressive catalytic repertoire of the RaS enzyme superfamily.

Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme.

Glycyl radical enzymes (GREs) utilize a glycyl radical cofactor to carry out a diverse array of chemically challenging enzymatic reactions in anaerobic bacteria. Although the glycyl radical is a powerful catalyst, it is also oxygen sensitive such that oxygen exposure causes cleavage of the GRE at the site of the radical. This oxygen sensitivity presents a challenge to facultative anaerobes dwelling in areas prone to oxygen exposure. Once GREs are irreversibly oxygen damaged, cells either need to make new GREs or somehow repair the damaged one. One particular GRE, pyruvate formate lyase (PFL), can be repaired through the binding of a 14.3 kDa protein, termed YfiD, which is constitutively expressed in E. coli. Herein, we have solved a solution structure of this 'spare part' protein using nuclear magnetic resonance spectroscopy. These data, coupled with data from circular dichroism, indicate that YfiD has an inherently flexible N-terminal region (residues 1-60) that is followed by a C-terminal region (residues 72-127) that has high similarity to the glycyl radical domain of PFL. Reconstitution of PFL activity requires that YfiD binds within the core of the PFL barrel fold; however, modeling suggests that oxygen-damaged, i.e. cleaved, PFL cannot fully accommodate YfiD. We further report that a PFL variant that mimics the oxygen-damaged enzyme is highly susceptible to proteolysis, yielding additionally truncated forms of PFL. One such PFL variant of ~ 77 kDa makes an ideal scaffold for the accommodation of YfiD. A molecular model for the rescue of PFL activity by YfiD is presented.

Mechanistic Investigations of Lysine-Tryptophan Cross-Link Formation Catalyzed by Streptococcal Radical S-Adenosylmethionine Enzymes.

Streptide is a ribosomally synthesized and post-translationally modified peptide with a unique cyclization motif consisting of an intramolecular lysine-tryptophan cross-link. Three radical S-adenosylmethionine enzymes, StrB, AgaB, and SuiB from different species of Streptococcus, have been shown to install this modification onto their respective precursor peptides in a leader-dependent fashion. Herein, we conduct detailed investigations to differentiate among several plausible mechanistic proposals, specifically addressing radical versus electrophilic addition to the indole during cross-link formation, the role of substrate side chains in binding in the enzyme active site, and the identity of the catalytic base in the reaction cycle. Our results are consistent with a radical electrophilic aromatic substitution mechanism for the key carbon-carbon bond-forming step. They also elaborate on other mechanistic features that underpin this unique and synthetically challenging post-translational modification.

Regiospecifically Fluorinated Polycyclic Aromatic Hydrocarbons via Julia-Kocienski Olefination and Oxidative Photocyclization. Effect of Fluorine Atom Substitution on Molecular Shape.

A modular synthesis of regiospecifically fluorinated polycyclic aromatic hydrocarbons (PAHs) is described. 1,2-Diarylfluoroalkenes, synthesized via Julia-Kocienski olefination (70-99% yields), were converted to isomeric 5- and 6-fluorobenzo[c]phenanthrene, 5-and 6-fluorochrysene, and 9- and 10-benzo[g]chrysene (66-83% yields) by oxidative photocyclization. Photocyclization to 6-fluorochrysene proceeded more slowly than conversion of 1-styrylnaphthalene to chrysene. Higher fluoroalkene dilution led to a more rapid cyclization. Therefore, photocyclizations were performed at higher dilutions. To evaluate the effect of fluorine atom on molecular shapes, X-ray data for 5- and 6-fluorobenzo[c]phenanthrene, 6-fluorochrysene, 9- and 10-fluorobenzo[g]chrysene, and unfluorinated chrysene as well as benzo[g]chrysene were obtained and compared. The fluorine atom caused a small deviation from planarity in the chrysene series and decreased nonplanarity in the benzo[c]phenanthrene derivatives, but its influence was most pronounced in the benzo[g]chrysene series. A remarkable flattening of the molecule was observed in 9-fluorobenzo[g]chrysene, where the short 2.055 Å interatomic distance between bay-region F-9 and H-8, downfield shift of H-8, and a 26.1 Hz coupling between F-9 and C-8 indicate a possible F-9···H-8 hydrogen bond. In addition, in 9-fluorobenzo[g]chrysene, the stacking distance is short at 3.365 Å and there is an additional interaction between the C-11-H and C-10a of a nearby molecule that is almost perpendicular.

Protection by extra virgin olive oil against oxidative stress in vitro and in vivo. Chemical and biological studies on the health benefits due to a major component of the Mediterranean diet.

We report the results of in vivo studies in Caenorhabditis elegans nematodes in which addition of extra virgin olive oil (EVOO) to their diet significantly increased their life span with respect to the control group. Furthermore, when nematodes were exposed to the pesticide paraquat, they started to die after two days, but after the addition of EVOO to their diet, both survival percentage and lifespans of paraquat-exposed nematodes increased. Since paraquat is associated with superoxide radical production, a test for scavenging this radical was performed using cyclovoltammetry and the EVOO efficiently scavenged the superoxide. Thus, a linear correlation (y = -0.0838x +19.73, regression factor = 0.99348) was observed for superoxide presence (y) in the voltaic cell as a function of aliquot (x) additions of EVOO, 10 µL each. The originally generated supoeroxide was approximately halved after 10 aliquots (100 µL total). The superoxide scavenging ability was analyzed, theoretically, using Density Functional Theory for tyrosol and hydroxytyrosol, two components of EVOO and was also confirmed experimentally for the galvinoxyl radical, using Electron Paramagnetic Resonance (EPR) spectroscopy. The galvinoxyl signal disappeared after adding 1 µL of EVOO to the EPR cell in 10 minutes. In addition, EVOO significantly decreased the proliferation of human leukemic THP-1 cells, while it kept the proliferation at about normal levels in rat L6 myoblasts, a non-tumoral skeletal muscle cell line. The protection due to EVOO was also assessed in L6 cells and THP-1 exposed to the radical generator cumene hydroperoxide, in which cell viability was reduced. Also in this case the oxidative stress was ameliorated by EVOO, in line with results obtained with tetrazolium dye reduction assays, cell cycle analysis and reactive oxygen species measurements. We ascribe these beneficial effects to EVOO antioxidant properties and our results are in agreement with a clear health benefit of EVOO use in the Mediterranean diet.

Crystal structures of 6-chloro-indan-1-one and 6-bromo-indan-1-one exhibit different inter-molecular packing inter-actions.

The two title compounds are analogs of 1-indanone that are substituted at the 6-position with chlorine and bromine. Although very similar in mol-ecular structure, the crystal structures are not isomorphous and reveal that 6-chloro-indan-1-one, C9H7ClO (I), and 6-bromo-indan-1-one, C9H7BrO (II), exhibit unique inter-molecular packing motifs. The mol-ecules of the chloro analog (I) pack with a herringbone packing motif of C-H⋯O inter-actions, whereas the bromo derivative (II) packs with offset face-to-face π-stacking, C-H⋯O, C-H⋯Br and Br⋯O inter-actions. Compound (II) was refined as a two-component non-merohedral twin, BASF 0.0762 (5).

The in vitro antitumor activity of arene-ruthenium(II) curcuminoid complexes improves when decreasing curcumin polarity.

The antitumor activity of ruthenium(II) arene (p-cymene, benzene, hexamethylbenzene) derivatives containing modified curcumin ligands (HCurcI=(1E,4Z,6E)-5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)hepta-1,4,6-trien-3-one and HCurcII=(1E,4Z,6E)-5-hydroxy-1,7-bis(4-methoxyphenyl)hepta-1,4,6-trien-3-one) is described. These have been characterized by IR, ESI-MS and NMR spectroscopy. The X-ray crystal structure of HCurcI has been determined and compared with its related Ru complex. Four complexes have been evaluated against five tumor cell lines, whose best activities [IC50 (µM)] are: breast MCF7, 9.7; ovarian A2780, 9.4; glioblastoma U-87, 9.4; lung carcinoma A549, 13.7 and colon-rectal HCT116, 15.5; they are associated with apoptotic features. These activities are improved when compared to the already known corresponding curcumin complex, (p-cymene)Ru(curcuminato)Cl, about twice for the breast and ovarian cancer, 4.7 times stronger in the lung cancer and about 6.6 times stronger in the glioblastoma cell lines. In fact, the less active (p-cymene)Ru(curcuminato)Cl complex only shows similar activity to two novel complexes in the colon cancer cell line. Comparing antitumor activity between these novel complexes and their related curcuminoids, improvement of antiproliferative activity is seen for a complex containing CurcII in A2780, A549 and U87 cell lines, whose IC50 are halved. Therefore, after replacing OH curcumin groups with OCH3, the obtained species HCurcI and its Ru complexes have increased antitumor activity compared to curcumin and its related complex. In contrast, HCurcII is less cytotoxic than curcumin but its related complex [(p-cymene)Ru(CurcII)Cl] is twice as active as HCurcII in 3 cell lines. Results from these novel arene-Ru curcuminoid species suggest that their increased cytotoxicity on tumor cells correlate with increase of curcuminoid lipophilicity.