A gene-encoded aldehyde tag repurposed from RiPP cyclophane-forming pathway.

Gene-encoded aldehyde tag technology has been widely utilized in protein bioorthogonal chemistry and biotechnological application. Herein, we report utilization of the promiscuous rSAM cyclophane synthase SjiB involved in triceptide biosynthesis as a dedicated and highly efficient formylglycine synthase. The new aldehyde tag sequence in this system, YQSSI, is biosynthetically orthogonal to the known aldehyde tag (C/S)x(P/A)xR. The potential use of SjiB/YQSSI aldehyde tag system was further validated in fluorescent labelling of model proteins.

Nature-Inspired Photocatalytic Azo Bond Cleavage with Red Light.

The emerging field of photoredox catalysis in mammalian cells enables spatiotemporal regulation of a wealth of biological processes. However, the selective cleavage of stable covalent bonds driven by low-energy visible light remains a great challenge. Herein, we report that red light excitation of a commercially available dye, abbreviated NMB+, leads to catalytic cleavage of stable azo bonds in both aqueous solutions and hypoxic cells and hence a means to photodeliver drugs or functional molecules. Detailed mechanistic studies reveal that azo bond cleavage is triggered by a previously unknown consecutive two-photon process. The first photon generates a triplet excited state, 3NMB+*, that is reductively quenched by an electron donor to generate a protonated NMBH•+. The NMBH•+ undergoes a disproportionation reaction that yields the initial NMB+ and two-electron-reduced NMBH (i.e., leuco-NMB, abbreviated as LNMB). Interestingly, LNMB forms a charge transfer complex with all four azo substrates that possess an intense absorption band in the red region. A second red photon induces electron transfer from LNMB to the azo substrate, resulting in azo bond cleavage. The charge transfer complex mediated two-photon catalytic mechanism reported herein is reminiscent of the flavin-dependent natural photoenzyme that catalyzes bond cleavage reactions with high-energy photons. The red-light-driven photocatalytic strategy offers a new approach to bioorthogonal azo bond cleavage for photodelivery of drugs or functional molecules.

Substrate-Controlled Catalysis in the Ether Cross-Link-Forming Radical SAM Enzymes.

Darobactin is a heptapeptide antibiotic featuring an ether cross-link and a C-C cross-link, and both cross-links are installed by a radical S-adenosylmethionine (rSAM) enzyme DarE. How a single DarE enzyme affords the two chemically distinct cross-links remains largely obscure. Herein, by mapping the biosynthetic landscape for darobactin-like RiPP (daropeptide), we identified and characterized two novel daropeptides that lack the C-C cross-link present in darobactin and instead are solely composed of ether cross-links. Phylogenetic and mutagenesis analyses reveal that the daropeptide maturases possess intrinsic multifunctionality, catalyzing not only the formation of ether cross-link but also C-C cross-linking and Ser oxidation. Intriguingly, the different chemical outcomes are controlled by the exact substrate motifs. Our work not only provides a roadmap for the discovery of new daropeptide natural products but also offers insights into the regulatory mechanisms that govern these remarkably versatile ether cross-link-forming rSAM enzymes.

Hijacking a Linaridin Biosynthetic Intermediate for Lanthipeptide Production.

Linaridins and lanthipeptides are two classes of natural products belonging to the ribosomally synthesized and posttranslationally modified peptide (RiPP) superfamily. Although these two RiPP classes share similar structural motifs such as dehydroamino acids and thioether-based cross-links, the biosynthesis of linaridins and lanthipeptides involved distinct sets of enzymes. Here, we report the identification of a novel lanthipeptide cypepeptin from a recombinant strain of Streptomyces lividans, which harbors most of the cypemycin (a prototypic linaridin) biosynthetic gene cluster but lacks the decarboxylase gene cypD. In contrast to the generally believed structure of cypemycin, multiple d-amino acids and Z-dehydrobutyrines were observed in both cypepeptin and cypemycin, and the stereochemistry of each amino acid was established by the extensive structural analysis in combination with genetic knockout and mutagenesis studies. Comparative analysis of cypemycin and cypepeptin showed that the aminovinyl-cysteine (AviCys) moiety of cypemycin plays an essential role in disrupting the cell integrity of M. luteus, which cannot be functionally substituted by the structurally similar lanthionine moiety.

Radical SAM-dependent ether crosslink in daropeptide biosynthesis.

Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C-C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H218O, apparent 18O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process in darobactin biosynthesis, involving a highly unusual oxygen insertion step from a water molecule and subsequent O-H and C-H activations. Genome mining analysis demonstrates the diversity of darobactin-like biosynthetic gene clusters, a subclade of which likely encode monocyclic products with only an ether linkage. We propose the name daropeptide for this growing family of ether-containing RiPPs produced by DarE enzymes.

Biosynthesis of cyclopropane in natural products.

Covering: 2012 to 2021Cyclopropane attracts wide interests in the fields of synthetic and pharmaceutical chemistry, and chemical biology because of its unique structural and chemical properties. This structural motif is widespread in natural products, and is usually essential for biological activities. Nature has evolved diverse strategies to access this structural motif, and increasing knowledge of the enzymes forming cyclopropane (i.e., cyclopropanases) has been revealed over the last two decades. Here, the scientific literature from the last two decades relating to cyclopropane biosynthesis is summarized, and the enzymatic cyclopropanations, according to reaction mechanism, which can be grouped into two major pathways according to whether the reaction involves an exogenous C1 unit from S-adenosylmethionine (SAM) or not, is discussed. The reactions can further be classified based on the key intermediates required prior to cyclopropane formation, which can be carbocations, carbanions, or carbon radicals. Besides the general biosynthetic pathways of the cyclopropane-containing natural products, particular emphasis is placed on the mechanism and engineering of the enzymes required for forming this unique structure motif.

Post-Translational Formation of Aminomalonate by a Promiscuous Peptide-Modifying Radical SAM Enzyme.

Aminomalonate (Ama) is a widespread structural motif in Nature, whereas its biosynthetic route is only partially understood. In this study, we show that a radical S-adenosylmethionine (rSAM) enzyme involved in cyclophane biosynthesis exhibits remarkable catalytic promiscuity. This enzyme, named three-residue cyclophane forming enzyme (3-CyFE), mainly produces cyclophane in vivo, whereas it produces formylglycine (FGly) as a major product and barely produce cyclophane in vitro. Importantly, the enzyme can further oxidize FGly to produce Ama. Bioinformatic study revealed that 3-CyFEs have evolved from a common ancestor with anaerobic sulfatase maturases (anSMEs), and possess a similar set of catalytic residues with anSMEs. Remarkably, the enzyme does not need leader peptide for activity and is fully active on a truncated peptide containing only 5 amino acids of the core sequence. Our work discloses the first ribosomal path towards Ama formation, providing a possible hint for the rich occurrence of Ama in Nature.

Characterization and Mechanistic Study of the Radical SAM Enzyme ArsS Involved in Arsenosugar Biosynthesis.

Arsenosugars are a group of arsenic-containing ribosides that are found predominantly in marine algae but also in terrestrial organisms. It has been proposed that arsenosugar biosynthesis involves a key intermediate 5'-deoxy-5'-dimethylarsinoyl-adenosine (DDMAA), but how DDMAA is produced remains elusive. Now, we report characterization of ArsS as a DDMAA synthase, which catalyzes a radical S-adenosylmethionine (SAM)-mediated alkylation (adenosylation) of dimethylarsenite (DMAsIII ) to produce DDMAA. This radical-mediated reaction is redox neutral, and multiple turnover can be achieved without external reductant. Phylogenomic and biochemical analyses revealed that DDMAA synthases are widespread in distinct bacterial phyla with similar catalytic efficiencies; these enzymes likely originated from cyanobacteria. This study reveals a key step in arsenosugar biosynthesis and also a new paradigm in radical SAM chemistry, highlighting the catalytic diversity of this superfamily of enzymes.

Linaridin natural products.

Covering1993 up to May 2020 Linaridins, defined as linear, dehydrated (arid) peptides, are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. To date, only a few members of the linaridin family have been characterized; however, in silico analysis has shown that this family of RiPPs is widespread in nature with high structural diversity. Unlike the case of most of the dehydroamino acid-containing RiPPs, such as lanthipeptides and thiopeptides, in which dehydroamino acids are produced by lanthipeptide dehydratase-like enzymes, in linaridins, dehydroamino acids are produced by a distinct set of enzymes with still unknown biochemistry. In this Highlight we have discussed the structural features, classification, biosynthesis, engineering, and widespread occurrence of linaridins and highlighted several intriguing issues in the maturation of this RiPP family.

Convergent evolution of the Cys decarboxylases involved in aminovinyl-cysteine (AviCys) biosynthesis.

S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) is a unique motif found in several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). Biosynthesis of AviCys requires flavin-dependent Cys decarboxylases, which are highly divergent among different RiPP classes. In this study, we solved the crystal structure of the cypemycin decarboxylase CypD. We show that CypD is structurally highly similar to lanthipeptide decarboxylases despite the absence of sequence similarities between them. We further show that Cys decarboxylases from four RiPP classes have evolved independently and form two major clusters. These results reveal the convergent evolution of AviCys biosynthesis and suggest that all the flavin-dependent Cys decarboxylases likely have a similar Rossmann fold despite their sequence divergences.