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Muraymycins are peptidyl nucleoside antibiotics that contain two Cβ-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-β-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cβ-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cβhydroxylation of the Leu residue to generate (2S,3S)-β-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cβ-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2′-oxime (MTMox) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTMoxanalogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2′-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTMox32E(a Phe–Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTMox32Edisplayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR–GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

An efficient divergent synthetic strategy that leverages the natural product spectinomycin to access uniquely functionalized monosaccharides is described. Stereoselective 2′- and 3′-reduction of key spectinomycin-derived intermediates enabled facile access to all eight possible 2,3-stereoisomers of 4,6-dideoxyhexoses as well as representative 3,4,6-trideoxysugars and 3,4,6-trideoxy-3-aminohexoses. In addition, the method was applied to the synthesis of two functionalized sugars commonly associated with macrolide antibiotics—the 3-O-alkyl-4,6-dideoxysugar d-chalcose and the 3-N-alkyl-3,4,6-trideoxysugar d-desosamine.

We report the isolation and characterization of three new nybomycins (nybomycins B–D, 1–3) and six known compounds (nybomycin, 4; deoxynyboquinone, 5; α-rubromycin, 6; β-rubromycin, 7; γ-rubromycin, 8; and [2α(1E,3E),4β]-2-(1,3-pentadienyl)-4-piperidinol, 9) from the Rock Creek (McCreary County, KY) underground coal mine acid reclamation site isolate Streptomycessp. AD-3-6. Nybomycin D (3) and deoxynyboquinone (5) displayed moderate (3) to potent (5) cancer cell line cytotoxicity and displayed weak to moderate anti-Gram-(+) bacterial activity, whereas rubromycins 6–8displayed little to no cancer cell line cytotoxicity but moderate to potent anti-Gram-(+) bacterial and antifungal activity. Assessment of the impact of 3or 5cancer cell line treatment on 4E-BP1 phosphorylation, a predictive marker of ROS-mediated control of cap-dependent translation, also revealed deoxynyboquinone (5)-mediated downstream inhibition of 4E-BP1p. Evaluation of 1–9in a recently established axolotl embryo tail regeneration assay also highlighted the prototypical telomerase inhibitor γ-rubromycin (8) as a new inhibitor of tail regeneration. Cumulatively, this work highlights an alternative nybomycin production strain, a small set of new nybomycin metabolites, and previously unknown functions of rubromycins (antifungal activity and inhibition of tail regeneration) and also provides a basis for revision of the previously proposed nybomycin biosynthetic pathway.

Pathogenic bacteria scavenge ferric iron from the host for survival and proliferation using small-molecular chelators, siderophores. Here, we introduce and assess the gallium(III) complex of ciprofloxacin-functionalized desferrichrome (D2) as a potential therapeutic for bacterial infection using an in vitro assay and radiochemical, tracer-based approach. Ga-D2exhibits a minimum inhibitory concentration of 0.23 μM in Escherichia coli, in line with the parent fluoroquinolone antibiotic. Competitive and mutant strain assays show that Ga-D2relies on FhuA-mediated transport for internalization. Ga-D2is potent against Pseudomonas aeruginosa(3.8 μM), Staphylococcus aureus(0.94 μM), and Klebsiella pneumoniae(12.5 μM), while Fe-D2is inactive in these strains. Radiochemical experiments with E. colireveal that 67Ga-D2is taken up more efficiently than 67Ga-citrate. In naive mice, 67Ga-D2clears renally and is excreted 13% intact in the urine. These pharmacokinetic and bacterial growth inhibitory properties qualify Ga-D2for future investigations as a diagnosis and treatment tool for infection.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

The structures and bioactivities of three unprecedented fused 5-hydroxyquinoxaline/alpha-keto acid amino acid metabolites (baraphenazines A–C, 1–3), two unique diastaphenazine-type metabolites (baraphenazines D and E, 4and 5) and two new phenazinolin-type (baraphenazines F and G, 6and 7) metabolites from the Himalayan isolate Streptomycessp. PU-10A are reported. This study highlights the first reported bacterial strain capable of producing diastaphenazine-type, phenazinolin-type, and izumiphenazine A-type metabolites and presents a unique opportunity for the future biosynthetic interrogation of late-stage phenazine-based metabolite maturation.

Muraymycins are nucleoside antibiotics isolated from Streptomycessp. NRRL 30471 and several mutant strains thereof that were generated by random, chemical mutagenesis. Reinvestigation of two mutant strains using new media conditions led to the isolation of three new muraymycin congeners, named B8, B9, and C6 (1–3), as well as a known muraymycin, C1. Structures of the compounds were elucidated by HRMS and 1D and 2D NMR spectroscopic analyses. Complete 2D NMR assignments for the known muraymycin C1 are also provided for the first time. Compounds 1and 2, which differ from other muraymycins by having an elongated, terminally branched fatty acid side chain, had picomolar IC50values against Staphylococcus aureusand Aquifex aeolicusMraY and showed good antibacterial activity against S. aureus(MIC = 2 and 6 μg/mL, respectively) and Escherichia coliΔtolC(MIC = 4 and 2 μg/mL, respectively). Compound 3, which is characterized by an N-acetyl modification of the primary amine of the dissacharide core that is shared among nearly all of the reported muraymycin congeners, greatly reduced its inhibitory and antibacterial activity compared to nonacylated muraymycin C1, which possibly indicates this modification is used for self-resistance.

The structures of 12 new “enantiomeric”-like abyssomicin metabolites (abyssomicins M–X) from Streptomycessp. LC-6-2 are reported. Of this set, the abyssomicin W (11) contains an unprecedented 8/6/6/6 tetracyclic core, while the bicyclic abyssomicin X (12) represents the first reported naturally occurring linear spirotetronate. Metabolite structures were determined based on spectroscopic data and X-ray crystallography, and Streptomycessp. LC-6-2 genome sequencing also revealed the corresponding putative biosynthetic gene cluster.

Four new Y-type actinomycin analogues named Y6–Y9(1–4) were isolated and characterized from the scale-up fermentation of the Streptomycessp. strain Gö-GS12, as well as actinomycin Zp (5), which was, for the first time, isolated as a natural product. Structures of the new compounds were elucidated by the cumulative analyses of NMR spectroscopy and HRMS. The 4-hydroxythreonine on the β-ring of 1uniquely undergoes both a rearrangement by a 2-fold acyl shift and an additional ring closure with the amino group of the phenoxazinone chromophore, and the α-rings of 4and 5contain a rare 5-methyl proline. Compounds 2–5showed potent antibacterial activities against Gram-positive bacteria that correlated with cytotoxicity against representative human cell lines. The combination of a β-ring rearrangement and additional ring closure in 1rendered this actinomycin significantly less potent relative to the nonrearranged comparator actinomycin Y5and other actinomycins.

S-adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. We report the design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). Corresponding biochemical and structural studies demonstrate the AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Given this conservation in function and molecular recognition, the isosteres presented are anticipated to serve as useful surrogates in other AdoMet-dependent processes and may also be resistant to, and/or potentially even inhibit, other therapeutically relevant AdoMet-dependent metabolic transformations (such as the validated drug target AdoMet decarboxylase). This work also highlights the ability of the prototypical class I model methyltransferase DnrK to accept non-native surrogate acceptors as an enabling feature of a new high-throughput methyltransferase assay.

Teamwork: Defucogilvocarcin M (1, see scheme) was synthesized in a one‐pot, enzymatic reaction from acetyl‐CoA and malonyl‐CoA by a combination of 15 enzymes obtained from E. colias well as the gilvocarcin, jadomycin, and ravidomycin biosynthetic pathways. The mixture of enzymes was systematically reduced and varied to further delineate the sequence of reactions in the complex, post‐polyketide steps of gilvocarcin biosynthesis.

Teamwork: Defucogilvocarcin M (1, siehe Schema) wurde in einer Eintopfreaktion unter Einwirkung von 15 Enzymen aus E. coliund den Gilvocarcin‐, Jadomycin‐ und Ravidomycin‐Biosynthesewegen aus Acetyl‐CoA und Malonyl‐CoA synthetisiert. Die Enzymmischung wurde systematisch vereinfacht und variiert, um die komplexen Schritte der späten Gilvocarcin‐Biosynthese zu beleuchten.

In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia colistrain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.

We report the identification of the tergene cluster responsible for the formation of the p-terphenyl derivatives terfestatins B and C and echoside B from the Appalachian Streptomycesstrain RM-5-8. We characterize the function of TerB/C, catalysts that work together as a dual enzyme system in the biosynthesis of natural terphenyls. TerB acts as a reductase and TerC as a dehydratase to enable the conversion of polyporic acid to a terphenyl triol intermediate. X-ray crystallography of the apo and substrate-bound forms for both enzymes provides additional mechanistic insights. Validation of the TerC structural model via mutagenesis highlights a critical role of arginine 143 and aspartate 173 in catalysis. Cumulatively, this work highlights a set of enzymes acting in harmony to control and direct reactive intermediates and advances fundamental understanding of the previously unresolved early steps in terphenyl biosynthesis.

ABSTRACTMuraymycins are antibacterial natural products from Streptomycesspp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5′-C-glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assigned in vitroas a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.

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