Genome Mining and Heterologous Expression Reveal Two Distinct Families of Lasso Peptides Highly Conserved in Endofungal Bacteria.

Genome mining identified the fungal-bacterial endosymbiosis Rhizopus microsporus-Mycetohabitans (previously Burkholderia) rhizoxinica as a rich source of novel natural products. However, most of the predicted compounds have remained cryptic. In this study, we employed heterologous expression to isolate and characterize three ribosomally synthesized and post-translationally modified peptides with lariat topology (lasso peptides) from the endosymbiont M. rhizoxinica: burhizin-23, mycetohabin-16, and mycetohabin-15. Through coexpression experiments, it was shown that an orphan gene product results in mature mycetohabin-15, albeit encoded remotely from the core biosynthetic gene cluster. Comparative genomics revealed that mycetohabins are highly conserved among M. rhizoxinica and related endosymbiotic bacteria. Gene knockout and reinfection experiments indicated that the lasso peptides are not crucial for establishing symbiosis; instead, the peptides are exported into the environment during endosymbiosis. This is the first report on lasso peptides from endosymbiotic bacteria.

Signatures of Mechanically Interlocked Topology of Lasso Peptides by Ion Mobility-Mass Spectrometry: Lessons from a Collection of Representatives.

Lasso peptides are characterized by a mechanically interlocked structure, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Their compact and stable structures have a significant impact on their biological and physical properties and make them highly interesting for drug development. Ion mobility - mass spectrometry (IM-MS) has shown to be effective to discriminate the lasso topology from their corresponding branched-cyclic topoisomers in which the C-terminal tail is unthreaded. In fact, previous comparison of the IM-MS data of the two topologies has yielded three trends that allow differentiation of the lasso fold from the branched-cyclic structure: (1) the low abundance of highly charged ions, (2) the low change in collision cross sections (CCS) with increasing charge state and (3) a narrow ion mobility peak width. In this study, a three-dimensional plot was generated using three indicators based on these three trends: (1) mean charge divided by mass (ζ), (2) relative range of CCS covered by all protonated molecules (ΔΩ/Ω) and (3) mean ion mobility peak width (δΩ). The data were first collected on a set of twenty one lasso peptides and eight branched-cyclic peptides. The indicators were obtained also for eight variants of the well-known lasso peptide MccJ25 obtained by site-directed mutagenesis and further extended to five linear peptides, two macrocyclic peptides and one disulfide constrained peptide. In all cases, a clear clustering was observed between constrained and unconstrained structures, thus providing a new strategy to discriminate mechanically interlocked topologies. Graphical Abstract ᅟ.

The B1 Protein Guides the Biosynthesis of a Lasso Peptide.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold that endows them with extraordinary stability and biologically relevant activity. However, the biosynthetic mechanism of these fascinating molecules remains largely speculative. Generally, two enzymes (B for processing and C for cyclization) are required to assemble the unusual knot-like structure. Several subsets of lasso peptide gene clusters feature a "split" B protein on separate open reading frames (B1 and B2), suggesting distinct functions for the B protein in lasso peptide biosynthesis. Herein, we provide new insights into the role of the RiPP recognition element (RRE) PadeB1, characterizing its capacity to bind the paeninodin leader peptide and deliver its peptide substrate to PadeB2 for processing.

Dual substrate-controlled kinase activity leads to polyphosphorylated lasso peptides.

Lasso peptides are characterized by their peculiar lariat knot-like structure. Except for maturation of this fold, post-translational modifications of lasso peptides are rare. However, we recently delineated the biosynthetic pathway of a post-translationally phosphorylated lasso peptide, paeninodin. In this study, further investigation of two kinases revealed their ability to transfer multiple phosphate groups onto precursor peptide substrates, ultimately leading to polyphosphorylated lasso peptides. We found that this polyphosphorylating activity depended on the identity of the phosphate donor and the sequence of the precursor peptide. Our investigations provide new insight into the remarkable strategies for chemical diversification employed by the lasso peptide biosynthetic machinery.

Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis.

Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin.

Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo- and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products.

The ring residue proline 8 is crucial for the thermal stability of the lasso peptide caulosegnin II.

Lasso peptides are fascinating natural products with a unique structural fold that can exhibit tremendous thermal stability. Here, we investigate factors responsible for the thermal stability of caulosegnin II. By employing X-ray crystallography, mutational analysis and molecular dynamics simulations, the ring residue proline 8 was proven to be crucial for thermal stability.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

The PqqD homologous domain of the radical SAM enzyme ThnB is required for thioether bond formation during thurincin H maturation.

Thurincin H is a 31-residue, ribosomally synthesized bacteriocin originating from the thn operon of Bacillus thuringiensis SF361. It is the only known sactipeptide carrying four thioether bridges between four cysteines and the α-carbons of a serine, an asparagine and two threonine residues. By analysis of the thn operon and use of in vitro studies we now reveal that ThnB is a radical S-adenosylmethionine (SAM) enzyme containing two [4Fe-4S] clusters. Furthermore, we confirm the involvement of ThnB in the formation of the thioether bonds present within the structure of thurincin H. Finally, we show that the PqqD homologous N-terminal domain of ThnB is essential for maturation of the thurincin H precursor peptide, but not for the SAM cleavage activity of ThnB.

Lasso peptides: an intriguing class of bacterial natural products.

Natural products of peptidic origin often represent a rich source of medically relevant compounds. The synthesis of such polypeptides in nature is either initiated by deciphering the genetic code on the ribosome during the translation process or driven by ribosome-independent processes. In the latter case, highly modified bioactive peptides are assembled by multimodular enzymes designated as nonribosomal peptide synthetases (NRPS) that act as a protein-template to generate chemically diverse peptides. On the other hand, the ribosome-dependent strategy, although relying strictly on the 20-22 proteinogenic amino acids, generates structural diversity by extensive post-translational-modification. This strategy seems to be highly distributed in all kingdoms of life. One example for this is the lasso peptides, which are an emerging class of ribosomally assembled and post-translationally modified peptides (RiPPs) from bacteria that were first described in 1991. A wide range of interesting biological activities are known for these compounds, including antimicrobial, enzyme inhibitory, and receptor antagonistic activities. Since 2008, genome mining approaches allowed the targeted isolation and characterization of such molecules and helped to better understand this compound class and their biosynthesis. Their defining structural feature is a macrolactam ring that is threaded by the C-terminal tail and held in position by sterically demanding residues above and below the ring, resulting in a unique topology that is reminiscent of a lariat knot. The ring closure is achieved by an isopeptide bond formed between the N-terminal α-amino group of a glycine, alanine, serine, or cysteine and the carboxylic acid side chain of an aspartate or glutamate, which can be located at positions 7, 8, or 9 of the amino acid sequence. In this Account, we discuss the newest findings about these compounds, their biosynthesis, and their physicochemical properties. This includes the suggested mechanism through which the precursor peptide is enzymatically processed into a mature lasso peptide and crucial residues for enzymatic recognition. Furthermore, we highlight new insights considering the protease and thermal stability of lasso peptides and discuss why seven amino acid residue rings are likely to be the lower limit feasible for this compound class. To elucidate their fascinating three-dimensional structures, NMR spectroscopy is commonly employed. Therefore, the general methodology to elucidate these structures by NMR will be discussed and pitfalls for these approaches are highlighted. In addition, new tools provided by recent investigations to assess and prove the lasso topology without a complete structure elucidation will be summarized. These include techniques like ion mobility-mass spectrometry and a combined approach of thermal and carboxypeptidase treatment with subsequent LC-MS analysis. Nevertheless, even though much was learned about these compounds in recent years, their true native function and the exact enzymatic mechanism of their maturation remain elusive.

Molecular insights into frataxin-mediated iron supply for heme biosynthesis in Bacillus subtilis.

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen-deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.

Rational improvement of the affinity and selectivity of integrin binding of grafted lasso peptides.

Integrins moderate diverse important functions in the human body and are promising targets in cancer therapy. Hence, the selective inhibition of specific integrins is of great medicinal interest. Here, we report the optimization of a grafted lasso peptide, yielding MccJ25(RGDF), which is a highly potent and selective αvß3 integrin inhibitor. Furthermore, its NMR structure was elucidated and employed in a molecular dynamics approach, revealing information about the integrin binding mode and selectivity profile of MccJ25(RGDF).

Xanthomonins†I-III: a new class of lasso peptides with a seven-residue macrolactam ring.

Lasso peptides belong to the class of ribosomally synthesized and post-translationally modified peptides. Their common distinguishing feature is an N-terminal macrolactam ring that is threaded by the C-terminal tail. This lasso fold is maintained through steric interactions. The isolation and characterization of xanthomonins I-III, the first lasso peptides featuring macrolactam rings consisting of only seven amino acids, is now presented. The crystal structure of xanthomonin I and the NMR structure of xanthomonin II were also determined. A total of 25 variants of xanthomonin II were generated to probe different aspects of the biosynthesis, stability, and fold maintenance. These mutational studies reveal the limits such a small ring imposes on the threading and show that every plug amino acid larger than serine is able to maintain a heat-stable lasso fold in the xanthomonin II scaffold.

Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry.

Lasso peptides are natural products with a unique three dimensional structure resembling a lariat knot. They are from ribosomal origin and are post-translationally modified by two enzymes (B and C), one of which shares little similarity to enzymes outside of lasso peptide biosynthetic gene clusters and as such is a useful target for genome mining. In this study, we demonstrate a B protein-centric genome mining approach through which we were able to identify 102 putative lasso peptide biosynthetic gene clusters from a total of 87 different proteobacterial strains. Ten of these clusters were cloned into the pET41a expression vector, optimized through incorporation of a ribosomal binding site and heterologously expressed in Escherichia coli BL21(DE3). All 12 predicted lasso peptides (namely burhizin, caulonodin I, caulonodin II, caulonodin III, rhodanodin, rubrivinodin, sphingonodin I, sphingonodin II, syanodin I, sphingopyxin I, sphingopyxin II, and zucinodin) were detected by high-resolution Fourier transform mass spectrometry and their proposed primary structure was confirmed through tandem mass spectrometry. High yields (ranging from 0.4 to 5.2 mg/L) were observable for eight of these compounds, while thermostability assays revealed five new representatives of heat labile lasso peptides.

Radical S-adenosylmethionine enzyme catalyzed thioether bond formation in sactipeptide biosynthesis.

Sactipeptides represent a new emerging class of ribosomally assembled and posttranslationally modified peptides that show diverse bioactivities. Their common hallmark is an intramolecular thioether bond that crosslink the sulfur atom of a cysteine residue with the α-carbon of an acceptor amino acid. This review summarizes recent achievements concerning the biosynthesis of sactipeptides in general and with special focus on the common enzymatic radical SAM mechanism leading to the thioether linkage formation. In addition this mechanism is compared to the mechanism of thioether bond formation during lanthipeptide biosynthesis and to other radical based thioether bond forming reactions.

The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron.

Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of ferrous (Fe[II]) and ferric (Fe[III]) iron in the bacterium Bacillus subtilis. We show that the binding protein EfeO and the permease EfeU form a minimal complex for ferric iron uptake. The third component EfeB is a hemoprotein that oxidizes ferrous iron to ferric iron for uptake by EfeUO. Accordingly, EfeB promotes growth under microaerobic conditions where ferrous iron is more abundant. Notably, EfeB also fulfills a vital role in cell envelope stress protection by eliminating reactive oxygen species that accumulate in the presence of ferrous iron. In conclusion, the EfeUOB system contributes to the high-affinity uptake of iron that is available in two different oxidation states.

A tRNA-dependent two-enzyme pathway for the generation of singly and doubly methylated ditryptophan 2,5-diketopiperazines.

A large number of bioactive natural products containing a 2,5-diketopiperazine (DKP) moiety have been isolated from various microbial sources. Especially tryptophan-containing cyclic dipeptides (CDPs) show great structural and functional diversity, while little is known about their biosynthetic pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide synthase (CDPS)-containing gene cluster from Actinosynnema mirum spanning 2.9 kb that contains two putative DKP-modifying enzymes. We establish the biosynthetic pathway leading to two methylated ditryptophan CDPs through in vivo and in vitro analyses. Our studies identify the first CDPS (Amir_4627) that shows high substrate specificity synthesizing only one main product, cyclo(Trp-Trp) (cWW). It is the first member of the CDPS family that can form ditryptophan DKPs and the first prokaryotic CDPS whose main product constituents differ from the four amino acids (Phe, Leu, Tyr, and Met) usually found in CDPS-dependent CDPs. We show that after cWW formation a S-adenosyl-l-methionine-dependent N-methyltransferase (Amir_4628) conducts two successive methylations at the DKP-ring nitrogens and additionally show that it is able to methylate four other phenylalanine-containing CDPs. This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase. The large number of known modifying enzymes of bacterial and fungal origin known to act upon Trp-containing DKPs makes the identification of a potent catalyst for cWW formation, encoded by a small gene, valuable for combinatorial in vivo as well as chemoenzymatic approaches, with the aim of generating derivatives of known CDP natural products or entirely new chemical entities with potentially improved or new biological activities.

The astexin-1 lasso peptides: biosynthesis, stability, and structural studies.

Lasso peptides are a large family of natural products that owe their name to a unique structure formed by a side chain to backbone macrocyclization, resembling a knotted lasso. The unique structure has significant impact on their biological and physical properties, as lasso peptides are usually more stable than linear ones. Current work examines stability, structure, and biosynthesis of recently discovered lasso peptide astexin-1, a heat-sensitive lasso peptide. The obtained results revealed a new lasso structure with a tight loop and long tail as well as narrow specificity of the maturation machinery for some essential residues associated with the protease processing site, involved in macrolactam ring formation and entrapment of the tail. Using the astexin-1 structure, it was possible to rationally construct a thermostable variant of this lasso peptide.

Two [4Fe-4S] clusters containing radical SAM enzyme SkfB catalyze thioether bond formation during the maturation of the sporulation killing factor.

The sporulation killing factor (SKF) is a 26-residue ribosomally assembled and posttranslationally modified sactipeptide. It is produced by Bacillus subtilis 168 and plays a key role in its sporulation. Like all sactipeptides, SKF contains a thioether bond, which links the cysteine residue Cys4 with the α-carbon of the methionine residue Met12. In this study we demonstrate that this bond is generated by the two [4Fe-4S] clusters containing radical SAM enzyme SkfB, which is encoded in the skf operon. By mutational analysis of both cluster-binding sites, we were able to postulate a mechanism for thioether generation which is in agreement with that of AlbA. Furthermore, we were able to show that thioether bond formation is specific toward hydrophobic amino acids at the acceptor site. Additionally we demonstrate that generation of the thioether linkage is leader-peptide-dependent, suggesting that this reaction is the first step in SKF maturation.

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.