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l‐(+)‐Muscarine (1)‐producing mushrooms pose a severe threat to human health as ingestion can result in circulatory collapse or even death. However, their metabolic profile is surprisingly poorly understood, including knowledge of poison release and potentially toxic congeners. In the mycelium of the 1‐producing fool's funnel mushroom Clitocybe rivulosa, we identified 4'‐phosphomuscarine (2) as the major natural product. Its structure was elucidated by high‐resolution mass spectrometry, nuclear magnetic resonance spectroscopy and by comparison with a synthesized reference. We also detected this previously overlooked phosphorylated compound in the fiber cap mushrooms Pseudosperma spectrale and Inocybe nitidiuscula. Studies on the activation of the muscarinic acetylcholine receptor M3 indicate only weak affinity of 2 to this target. Furthermore, we present biological evidence that muscaridine (3), a quaternary amine congener related to and co‐occurring with 1, does not activate the muscarinic acetylcholine receptor M3 on human embryonic kidney cells. Our work provides important insight into the metabolic profile and the pharmacology of some of the most poisonous mushrooms. As the harmless 2 can liberate the potentially fatal 1 by unspecific enzymatic ester cleavage, these results are highly relevant for emergency medicine to estimate the true toxicity of these mushrooms. [ABSTRACT FROM AUTHOR]

Various nonribosomal peptide synthetases (NRPSs) create structural and functional diversity by incorporating α‐hydroxy acids into peptide backbones. Trigonic acid, an unusual cyclopropanol‐substituted hydroxy acid, is the source of the molecular warhead of malleicyprol, a critical virulence factor of human and animal pathogens of the Burkholderia pseudomallei (BP) group. The process of selecting and loading this building block remained enigmatic as the NRPS module designated for this task is incomplete. Using a combination of bioinformatics, mutational analyses, targeted metabolomics, and in vitro biochemical assays, we show that two trans‐acting enzymes are required to load this central building block onto the modular assembly line. An adenylation‐thiolation didomain enzyme (BurJ) activates trigonic acid, followed by the translocation of the enzyme‐bound α‐hydroxy acid thioester by an FkbH‐like protein with a mutated phosphatase domain (BurH). This specialized gateway is the first reported direct loading of an α‐hydroxy acid onto a bona fide NRPS module in bacteria and expands the synthetic biology toolbox for the site‐specific incorporation of non‐canonical building blocks. Moreover, insight into the biochemical basis of virulence factor biosynthesis can provide a foundation for developing enzyme inhibitors as anti‐virulence therapeutics against BP pathogen infections. [ABSTRACT FROM AUTHOR]

Enzymes are increasingly recognized as valuable (bio)catalysts that complement existing synthetic methods. However, the range of biotransformations used in the laboratory is limited. Here we give an overview on the biosynthesis‐inspired discovery of novel biocatalysts that address various synthetic challenges. Prominent examples from this dynamic field highlight remarkable enzymes for protecting‐group‐free amide formation and modification, control of pericyclic reactions, stereoselective hetero‐ and polycyclizations, atroposelective aryl couplings, site‐selective C−H activations, introduction of ring strain, and N−N bond formation. We also explore unusual functions of cytochrome P450 monooxygenases, radical SAM‐dependent enzymes, flavoproteins, and enzymes recruited from primary metabolism, which offer opportunities for synthetic biology, enzyme engineering, directed evolution, and catalyst design. [ABSTRACT FROM AUTHOR]

In microbial interactions bacteria employ diverse molecules with specific functions, such as sensing the environment, communication with other microbes or hosts, and conferring virulence. Insights into the molecular basis of bacterial communication are thus of high relevance for ecology and medicine. Targeted gene activation and in vitro studies revealed that the cell‐to‐cell signaling molecule and disease mediator IQS (aeruginaldehyde) of the human pathogen Pseudomonas aeruginosa and related bacteria derives from the siderophore pyochelin. Addition of IQS to bacterial cultures (Burkholderia thailandensis) showed that the signaling molecule is captured by a congener of another siderophore family, malleobactin, to form a nitrone conjugate (malleonitrone) that is active against the IQS‐producer. This study uncovers complex communication processes with derailed siderophore functions, a novel nitrone bioconjugation, and a new type of antibiotic against Gram‐negative bacteria. Repurposing iron transporters: The siderophore pyochelin was found to be the true biosynthetic origin of IQS, a signaling molecule and virulence mediator of pathogenic bacteria such as Pseudomonas aeruginosa. In microbial interaction IQS is captured by another siderophore congener, malleobactin from Burkholderia thailandensis, to form an unusual nitrone conjugate that is active against the IQS producer. [ABSTRACT FROM AUTHOR]