Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci

Macrocycles represent a common structural framework in many naturally occurring peptides. Several strategies exist for macrocyclization, and the enzymes that incorporate them are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens <i>Streptococcus agalactiae</i> and <i>Streptococcus suis</i>. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides

The Polyene Natural Product Thailandamide A Inhibits Fatty Acid Biosynthesis in Gram-Positive and Gram-Negative Bacteria

<i>Burkholderia thailandensis</i> produces an impressive array of secondary metabolites, most with yet unknown targets. One of these metabolites is thailandamide, a linear polyene natural product that is constitutively synthesized by the corresponding <i>tha</i> gene cluster. Using broad bioactivity screens, we observed strong yet selective antibacterial activity by thailandamide against Gram-positive and cell wall-weakened Gram-negative bacteria. Bacterial cytological profiling and comparison with 10 antibiotics with known modes of action revealed a unique profile for thailandamide, suggesting a distinct mechanism of inhibition. To address the target of the drug, we obtained resistant mutants of <i>Bacillus subtilis</i> and mapped the resistant phenotype to <i>accA</i>, the product of which catalyzes the first committed step in fatty acid biosynthesis. Interestingly, the <i>tha</i> gene cluster encodes an <i>accA</i> homologue with a similar amino acid substitution. Heterologous expression showed that it confers resistance to otherwise susceptible <i>Escherichia coli</i> cultures, indicating that it provides immunity to thailandamide-producing <i>B. thailandensis</i> cells. Aside from moiramide B and andrimid, thailandamide represents only the second class of natural products that inhibits bacterial growth by targeting AccA/AccD

Roseochelin B, an Algaecidal Natural Product Synthesized by the <i>Roseobacter Phaeobacter inhibens</i> in Response to Algal Sinapic Acid

The secondary metabolome of the representative <i>Roseobacter</i>, <i>Phaeobacter inhibens</i>, was examined in response to algal sinapic acid. In addition to roseobacticides, sinapic acid induced the production of two new natural products, roseochelin A and B, which were characterized by NMR and X-ray crystallography. Functional assays showed that roseochelin B binds iron and is algaecidal against the algal host <i>Emiliania huxleyi</i>. It appears to be produced by a rarely observed combination of nonenzymatic and enzymatic transformations

Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine–Tryptophan Crosslink

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products with diverse activities and structures. RiPP classes are defined by the tailoring enzyme, which can introduce a narrow range of modifications or a diverse set of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs, have emerged as especially divergent. A map of all RaS-RiPP gene clusters does not yet exist. Moreover, precursor peptides remain difficult to predict using computational methods. Herein, we have addressed these challenges and report a bioinformatic atlas of RaS-RiPP gene clusters in available microbial genome sequences. Using co-occurrence of RaS enzymes and transporters from varied families as a bioinformatic hook in conjunction with an in-house code to identify precursor peptides, we generated a map of ∼15,500 RaS-RiPP gene clusters, which reveal a remarkable diversity of syntenies pointing to a tremendous range of enzymatic and natural product chemistries that remain to be explored. To assess its utility, we examined one family of gene clusters encoding a YcaO enzyme and a RaS enzyme. We find the former is noncanonical, contains an iron–sulfur cluster, and installs a novel modification, a backbone amidine into the precursor peptide. The RaS enzyme was also found to install a new modification, a C–C crosslink between the unactivated terminal δ-methyl group of Ile and a Trp side chain. The co-occurrence search can be applied to other families of RiPPs, as we demonstrate with the emerging DUF692 di-iron enzyme superfamily

Roseochelin B, an Algaecidal Natural Product Synthesized by the <i>Roseobacter Phaeobacter inhibens</i> in Response to Algal Sinapic Acid

The secondary metabolome of the representative <i>Roseobacter</i>, <i>Phaeobacter inhibens</i>, was examined in response to algal sinapic acid. In addition to roseobacticides, sinapic acid induced the production of two new natural products, roseochelin A and B, which were characterized by NMR and X-ray crystallography. Functional assays showed that roseochelin B binds iron and is algaecidal against the algal host <i>Emiliania huxleyi</i>. It appears to be produced by a rarely observed combination of nonenzymatic and enzymatic transformations

Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine–Tryptophan Crosslink

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products with diverse activities and structures. RiPP classes are defined by the tailoring enzyme, which can introduce a narrow range of modifications or a diverse set of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs, have emerged as especially divergent. A map of all RaS-RiPP gene clusters does not yet exist. Moreover, precursor peptides remain difficult to predict using computational methods. Herein, we have addressed these challenges and report a bioinformatic atlas of RaS-RiPP gene clusters in available microbial genome sequences. Using co-occurrence of RaS enzymes and transporters from varied families as a bioinformatic hook in conjunction with an in-house code to identify precursor peptides, we generated a map of ∼15,500 RaS-RiPP gene clusters, which reveal a remarkable diversity of syntenies pointing to a tremendous range of enzymatic and natural product chemistries that remain to be explored. To assess its utility, we examined one family of gene clusters encoding a YcaO enzyme and a RaS enzyme. We find the former is noncanonical, contains an iron–sulfur cluster, and installs a novel modification, a backbone amidine into the precursor peptide. The RaS enzyme was also found to install a new modification, a C–C crosslink between the unactivated terminal δ-methyl group of Ile and a Trp side chain. The co-occurrence search can be applied to other families of RiPPs, as we demonstrate with the emerging DUF692 di-iron enzyme superfamily

Roseobacticides: Small Molecule Modulators of an Algal-Bacterial Symbiosis

Marine bacteria and microalgae engage in dynamic symbioses mediated by small molecules. A recent study of <i>Phaeobacter gallaeciensis</i>, a member of the large roseobacter clade of α-proteobacteria, and <i>Emiliania huxleyi</i>, a prominent member of the microphytoplankton found in large algal blooms, revealed that an algal senescence signal produced by <i>E. huxleyi</i> elicits the production of novel algaecides, the roseobacticides, from the bacterial symbiont. In this report, the generality of these findings are examined by expanding the number of potential elicitors. This expansion led to the identification of nine new members of the roseobacticide family, rare bacterial troponoids, which provide insights into both their biological roles and their biosynthesis. The qualitative and quantitative changes in the levels of roseobacticides induced by the additional elicitors and the elicitors’ varied efficiencies support the concept of host-targeted roseobacticide production. Structures of the new family members arise from variable substituents at the C3 and C7 positions of the roseobacticide core as the diversifying elements and suggest that the roseobacticides result from modifications and combinations of aromatic amino acids. Together these studies support a model in which algal senescence converts a mutualistic bacterial symbiont into an opportunistic parasite of its hosts

Targeted Discovery of Cryptic Enediyne Natural Products via FRET-Coupled High-Throughput Elicitor Screening

Enediyne antibiotics are a striking family of DNA-cleaving natural products with high degrees of cytotoxicity and structural complexity. Microbial genome sequences, which have recently accumulated, point to an untapped trove of “cryptic” enediynes. Most of the cognate biosynthetic gene clusters (BGCs) are sparingly expressed under standard growth conditions, making it difficult to characterize their products. Herein, we report a fluorescence-based DNA cleavage assay coupled with high-throughput elicitor screening for the rapid, targeted discovery of cryptic enediyne metabolites. We applied the approach to Streptomyces clavuligerus, which harbors two such BGCs with unknown products, identified steroids as effective elicitors, and characterized 10 cryptic enediyne-derived natural products, termed clavulynes A–J with unusual carbonate and terminal olefin functionalities, with one of these congeners matching the recently reported jejucarboside. Our results contribute to the growing repertoire of enediynes and provide a blueprint for identifying additional ones in the future

Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of <i>E. coli</i> Ribonucleotide Reductase by Multifrequency EPR Spectroscopy

<i>E. coli</i> ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y₁₂₂•) within the β2 subunit to a cysteine residue (C₄₃₉) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y₁₂₂ → W₄₈ → Y₃₅₆ in β2, across the subunit interface to Y₇₃₁ → Y₇₃₀ → C₄₃₉ in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y₁₂₂• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH₂Y•). In the current study [¹⁵N]- and [¹⁴N]-NH₂Y₇₃₀• have been generated in H₂O and D₂O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH₂Y₇₃₀•. The <i>g</i> tensor (<i>g</i>_<i>x</i> = 2.0052, <i>g</i>_<i>y</i> = 2.0042, <i>g</i>_<i>z</i> = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH₂ moiety are consistent with an intramolecular hydrogen bond within NH₂Y₇₃₀•. This analysis is an essential first step in using the detailed structure of NH₂Y₇₃₀• to formulate a model for a PCET mechanism within α2 and for use of NH₂Y in other systems where transient Y•s participate in catalysis

Sources of Diversity in Bactobolin Biosynthesis by <i>Burkholderia thailandensis</i> E264

A series of deletion mutants in the recently identified bactobolin biosynthetic pathway defined the roles of several key biosynthetic enzymes and showed how promiscuity in three enzyme systems allows this cluster to produce multiple products. Studies on the deletion mutants also led to four new bactobolin analogs that provide additional structure–activity relationships for this interesting antibiotic family