A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Metabolomics and genomics in natural products research: complementary tools for targeting new chemical entities.

Covering: 2010 to 2021Organisms in nature have evolved into proficient synthetic chemists, utilizing specialized enzymatic machinery to biosynthesize an inspiring diversity of secondary metabolites. Often serving to boost competitive advantage for their producers, these secondary metabolites have widespread human impacts as antibiotics, anti-inflammatories, and antifungal drugs. The natural products discovery field has begun a shift away from traditional activity-guided approaches and is beginning to take advantage of increasingly available metabolomics and genomics datasets to explore undiscovered chemical space. Major strides have been made and now enable -omics-informed prioritization of chemical structures for discovery, including the prospect of confidently linking metabolites to their biosynthetic pathways. Over the last decade, more integrated strategies now provide researchers with pipelines for simultaneous identification of expressed secondary metabolites and their biosynthetic machinery. However, continuous collaboration by the natural products community will be required to optimize strategies for effective evaluation of natural product biosynthetic gene clusters to accelerate discovery efforts. Here, we provide an evaluative guide to scientific literature as it relates to studying natural product biosynthesis using genomics, metabolomics, and their integrated datasets. Particular emphasis is placed on the unique insights that can be gained from large-scale integrated strategies, and we provide source organism-specific considerations to evaluate the gaps in our current knowledge.

Characterization of a Copper-Chelating Natural Product from the Methanotroph Methylosinus sp. LW3.

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.

Discovery of the Biosynthetic Machinery for Stravidins, Biotin Antimetabolites.

Stravidins are peptide antibiotics produced by Streptomyces spp. Their antibacterial activity derives from an unusual amiclenomycin monomer, the warhead that inhibits biotin biosynthesis. Despite being discovered over five decades ago, stravidin biosynthesis has remained a mystery. Using our "metabologenomics" platform, we discover new stravidin analogues and identify the novel biosynthetic machinery responsible for their production. Analysis of the newly identified biosynthetic gene cluster (BGC) indicates the unusual amiclenomycin warhead is derived from chorismic acid, with initial steps similar to those involved in p-amino phenylalanine biosynthesis. However, a distinctive decarboxylation retains the nonaromatic character of a key ring and precedes a one-carbon extension to afford the warhead in its bioactive, untriggered state. Strikingly, we also identified two streptavidin genes flanking the new stravidin BGC reported here. This aligns with the known synergistic activity between the biotin-binding activity of streptavidin and the stravidins to antagonize both biotin biogenesis and bacterial growth.

Modular strategies for structure and function employed by marine cyanobacteria: characterization and synthesis of pitinoic acids.

Novel bioactive lipids were identified from a Guamanian cyanobacterium, the Pseudomonas aeruginosa quorum sensing inhibitor pitinoic acid A (1) and the anti-inflammatory pitinoic acids B (2) and C. The structure of 2 was confirmed by synthesis, which also allowed for biological evaluation. Since 2 is an ester of pitinoic acids A and C, it represents a prodrug strategy to liberate dual biological activity for the management of P. aeruginosa infections and their associated inflammation.

Marine cyanobacterial fatty acid amides acting on cannabinoid receptors.

Striking cAMP: certain fatty acid amides from marine cyanobacteria can mimic the endocannabinoids. Serinolamide B, a new analogue identified from a Guamanian sample, and malyngamide B, a representative member of a large class of cyanobacterial metabolites, can decrease forskolin-induced cAMP accumulation through the cannabinoid receptors.

Marine natural products: a new wave of drugs?

The largely unexplored marine world that presumably harbors the most biodiversity may be the vastest resource to discover novel 'validated' structures with novel modes of action that cover biologically relevant chemical space. Several challenges, including the supply problem and target identification, need to be met for successful drug development of these often complex molecules; however, approaches are available to overcome the hurdles. Advances in technologies such as sampling strategies, nanoscale NMR for structure determination, total chemical synthesis, fermentation and biotechnology are all crucial to the success of marine natural products as drug leads. We illustrate the high degree of innovation in the field of marine natural products, which in our view will lead to a new wave of drugs that flow into the market and pharmacies in the future.

Pitipeptolides C-F, antimycobacterial cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula from Guam.

Pitipeptolides A (1) and B (2) are cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula from Piti Bomb Holes, Guam. Additional analogues have now been isolated by revisiting larger collections of the same cyanobacterium. The four identified analogues, pitipeptolides C-F (3-6), are the tetrahydro analogue (3), an analogue with a lower degree of methylation (4) as well as two homologues (5 and 6) of pitipeptolide A. Their structures were elucidated using 2D NMR experiments, chiral HPLC analysis and comparison with pitipeptolide A. The identified analogues showed weaker cytotoxic activities compared to the two major parent compounds, pitipeptolides A (1) and B (2), against HT-29 colon adenocarcinoma and MCF7 breast cancer cells. On the other hand, pitipeptolide F (6) was the most potent pitipeptolide in a disc diffusion assay against Mycobacterium tuberculosis. The latter finding suggests that the structure of pitipeptolides could be optimized for selective antibacterial activity.

Malyngamide 3 and cocosamides A and B from the marine cyanobacterium Lyngbya majuscula from Cocos Lagoon, Guam.

Malyngamide 3 (1) and cocosamides A (2) and B (3) were isolated from the lipophilic extract of a collection of Lyngbya majuscula from Cocos Lagoon, Guam. The planar structures of compounds 1-3 were determined by spectroscopic methods. The absolute configuration of 1 was determined by modified Mosher's method, NOESY data, and comparison with lyngbic acid (4). The absolute configurations of 2 and 3 were assigned by enantioselective HPLC analysis and comparison with the closely related compound pitipeptolide A (5). Compounds 1-3 showed weak cytotoxicity against MCF7 breast cancer and HT-29 colon cancer cells.

Pitiprolamide, a proline-rich dolastatin 16 analogue from the marine cyanobacterium Lyngbya majuscula from Guam.

An unusual cyclic depsipeptide, pitiprolamide (1), was isolated from the marine cyanobacterium Lyngbya majuscula collected at Piti Bomb Holes, Guam. The structure was deduced using NMR, MS, X-ray crystallography, and enantioselective HPLC-MS techniques. Remarkably, proline represents half of the residues forming pitiprolamide (1). Other distinctive features include a 4-phenylvaline (dolaphenvaline, Dpv) moiety initially found in dolastatin 16 and the rare 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha) unit condensed in a unique sequence in one single molecule. Pitiprolamide (1) showed weak cytotoxic activity against HCT116 colon and MCF7 breast cancer cell lines, as well as weak antibacterial activities against Mycobacterium tuberculosis and Bacillus cereus.