Isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of Burkholderia thailandensis E264

Lasso peptides are a structurally unique class of bioactive peptides characterized by a knotted arrangement, where the C-terminus threads through an N-terminal macrolactam ring. Although ribosomally synthesized, only the gene cluster for the best studied lasso peptide MccJ25 from Escherichia coli consisting of the precursor protein McjA and the processing and immunity proteins McjB, McjC, and McjD is known. Through genome mining studies, we have identified homologues of all four proteins in Burkholderia thailandensis E264 and predicted this strain to produce a lasso peptide. Here we report the successful isolation of the predicted peptide, named capistruin. Upon optimization of the fermentation conditions, mass spectrometric and NMR structural studies proved capistruin to adopt a novel lasso fold. Heterologous production of the lasso peptide in Escherichia coli showed that the identified genes are sufficient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related Burkholderia and Pseudomonas strains. In general, our rational approach should be widely applicable for the isolation of new lasso peptides to explore their high structural stability and diverse biological activity.     

Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino acid substitutions

Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to either the ring or tail portions of MccJ25 and discovered nearly 100 new MccJ25 variants that retain antimicrobial function. While multiple amino acid substitutions in the tail portion of the peptide are well-tolerated, mutagenesis of the ring portion of the peptide is detrimental to the antimicrobial function of MccJ25. We demonstrated that the decreased function of the ring variants is due to the inability of these variants to be transported to the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with improved efficacy toward the MccJ25-sensitive strains E. coli and Salmonella enterica serovar Newport with the best variants exhibiting a nearly 5-fold increase in potency. The results described here provide further evidence that diverse amino acid sequences can be tolerated by the rigid lasso peptide fold.     

Tying the knot: making of lasso peptides

In this issue of Chemistry & Biology, Duquesne and colleagues [1] describe the biosynthetic machinery of microcin J25, an antibacterial peptide that adopts an exceptional three-dimensional structure resembling a knot. The dedicated enzymes, McjB and McjC, were identified and employed to produce this modification in vitro.     

Topoisomer Differentiation of Molecular Knots by FTICR MS: Lessons from Class II Lasso Peptides

Lasso peptides constitute a class of bioactive peptides sharing a knotted structure where the C-terminal tail of the peptide is threaded through and trapped within an N-terminal macrolactam ring. The structural characterization of lasso structures and differentiation from their unthreaded topoisomers is not trivial and generally requires the use of complementary biochemical and spectroscopic methods. Here we investigated two antimicrobial peptides belonging to the class II lasso peptide family and their corresponding unthreaded topoisomers: microcin J25 (MccJ25), which is known to yield two-peptide product ions specific of the lasso structure under collision-induced dissociation (CID), and capistruin, for which CID does not permit to unambiguously assign the lasso structure. The two pairs of topoisomers were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) upon CID, infrared multiple photon dissociation (IRMPD), and electron capture dissociation (ECD). CID and ECD spectra clearly permitted to differentiate MccJ25 from its non-lasso topoisomer MccJ25-Icm, while for capistruin, only ECD was informative and showed different extent of hydrogen migration (formation of c•/z from c/z•) for the threaded and unthreaded topoisomers. The ECD spectra of the triply-charged MccJ25 and MccJ25-lcm showed a series of radical b-type product ions ( b￠_n ^· ) \left( {b{\prime}_n^{ \bullet }} \right) . We proposed that these ions are specific of cyclic-branched peptides and result from a dual c/z• and y/b dissociation, in the ring and in the tail, respectively. This work shows the potentiality of ECD for structural characterization of peptide topoisomers, as well as the effect of conformation on hydrogen migration subsequent to electron capture.     

Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail

Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.     

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.

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The role of a conserved threonine residue in the leader peptide of lasso peptide precursors

The conserved threonine (Thr) residue in the penultimate position of the leader peptide of lasso peptides microcin J25 and capistruin can be effectively replaced by several amino acids close in size and shape to Thr. These findings suggest a model for lasso peptide biosynthesis in which the Thr sidechain is a recognition element for the lasso peptide maturation machinery.     

Gene selection of rat hepatocyte proliferation using adaptive sparse group lasso with weighted gene co-expression network analysis

Grouped gene selection is the most important task for analyzing the microarray data of rat liver regeneration. Many existing gene selection methods cannot outstand the interactions among the selected genes. In the process of rat liver regeneration, one of the most important events involved in many biological processes is the proliferation of rat hepatocytes, so it can be used as a measure of the effectiveness of the method. Here we proposed an adaptive sparse group lasso to select genes in groups for rat hepatocyte proliferation. The weighted gene co-expression networks analysis was used to identify modules corresponding to gene pathways, based on which a strategy of dividing genes into groups was proposed. A strategy of adaptive gene selection was also presented by assessing the gene significance and introducing the adaptive lasso penalty. Moreover, an improved blockwise descent algorithm was proposed. Experimental results demonstrated that the proposed method can improve the classification accuracy, and select less number of significant genes which act jointly in groups and have direct or indirect effects on rat hepatocyte proliferation. The effectiveness of the method was verified by the method of rat hepatocyte proliferation.     

Rational generation of lasso peptides based on biosynthetic gene mutations and site-selective chemical modifications

Lasso peptides are a unique family of natural products whose structures feature a specific threaded fold, which confers these peptides the resistance to thermal and proteolytic degradation. This stability gives lasso peptides excellent pharmacokinetic properties, which together with their diverse reported bioactivities have garnered extensive attention because of their drug development potential. Notably, the threaded fold has proven quite inaccessible by chemical synthesis, which has hindered efficient generation of structurally diverse lasso peptides. We herein report the discovery of a new lasso peptide stlassin (1) by gene activation based on a Streptomyces heterologous expression system. Site-directed mutagenesis on the precursor peptide-encoding gene is carried out systematically, generating 17 stlassin derivatives (2–17 and 21) with residue-replacements at specific positions of 1. The solution NMR structures of 1, 3, 4, 14 and 16 are determined, supporting structural comparisons that ultimately enabled the rational production of disulfide bond-containing derivatives 18 and 19, whose structures do not belong to any of the four classes currently used to classify lasso peptides. Several site-selective chemical modifications are first applied on 16 and 21, efficiently generating new derivatives (20, 22–27) whose structures bear various decorations beyond the peptidyl monotonicity. The high production yields of these stlassin derivatives facilitate biological assays, which show that 1, 4, 16, 20, 21 and 24 possess antagonistic activities against the binding of lipopolysaccharides to toll-like receptor 4 (TLR4). These results demonstrate proof-of-concept for the combined mutational/chemical generation of lasso peptide libraries to support drug lead development.

A new class II lasso peptide stlassin (1) was discovered and stlassin derivatives (2–27) were rationally generated by biosynthetic gene mutations and site-selective chemical modifications, expanding the structural diversity of lasso peptides.     

Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide

Previous study showed that some Gram-negative bacteria possess human blood group activity. Among them, Escherichia coli O86 has high blood group B activity and weak blood group A activity. This is due to the cell surface O-antigen structure, which resembles that of human blood group B antigen. In this study, we sequenced the entire E. coli O86 antigen gene cluster and identified all the genes responsible for O-antigen biosynthesis by sequence comparative analysis. The blood group B-like antigen in E. coli O86 O-polysaccharide was synthesized by sequentially employing three glycosyltransferases identified in the gene cluster. More importantly, we identified a new bacterial glycosyltransferase (WbnI) equivalent to human blood group transferase B (GTB). The enzyme substrate specificity and stepwise enzymatic synthesis of blood group B-like antigen revealed that the biosynthetic pathway of B antigen is essentially the same in E. coli O86 as in humans. This new finding provides a model to study the specificity and structure relationship of blood group transferases and supports the hypothesis of anti-blood group antibody production by bacterial stimulation.     

A pH‐Sensitive Lasso‐Based Rotaxane Molecular Switch

The synthesis of a pH‐sensitive two‐station [1]rotaxane molecular switch by self‐entanglement of a non‐interlocked hermaphrodite molecule, containing an anilinium and triazole moieties, is reported. The anilinium was chosen as the best template for the macrocycle benzometaphenylene[25]crown‐8 (BMP25C8) and allowed the self‐entanglement of the molecule. The equilibrium between the hermaphrodite molecule and the pseudo[1]rotaxane was studied by ¹H NMR spectroscopy: the best conditions of self‐entanglement were found in the less polar solvent CD₂Cl₂ and at high dilution. The triazole moiety was then benzylated to afford a benzyltriazolium moiety, which then played a dual role. On one hand, it acts as a bulky gate to trap the BMP25C8, thus to avoid any self‐disentanglement of the molecular architecture. On another hand, it acts as a second molecular station for the macrocycle. At acidic pH, the BMP25C8 resides around the best anilinium molecular station, displaying the lasso [1]rotaxane in a loosened conformation. The deprotonation of the anilinium molecular station triggers the shuttling of the BMP25C8 around the triazolium moiety, therefore tightening the lasso.     

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.     

A molecular switch created by in vitro recombination of nonhomologous genes

We have created a molecular switch by the in vitro recombination of nonhomologous genes and subjecting the recombined genes to evolutionary pressure. The gene encoding TEM1 beta-lactamase was circularly permuted in a random fashion and subsequently randomly inserted into the gene encoding Escherichia coli maltose binding protein. From this library, a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose. Upon removal of maltose, RG13's catalytic activity returned to its premaltose level, illustrating that the switching is reversible. The modularity of RG13 was demonstrated by increasing maltose affinity while preserving switching activity. RG13 gave rise to a novel cellular phenotype, illustrating the potential of molecular switches to rewire the cellular circuitry.     

Lasso Proteins: Modular Design,Cellular Synthesis,and Topological Transformation

Entangled proteins have attracted significant research interest. Herein, we report the first rationally designed lasso proteins, or protein [1]rotaxanes, by using a p53dim-entwined dimer for intramolecular entanglement and a SpyTag-SpyCatcher reaction for side-chain ring closure. The lasso structures were confirmed by proteolytic digestion, mutation, NMR spectrometry, and controlled ligation. Their dynamic properties were probed by experiments such as end-capping, proteolytic digestion, and heating/cooling. As a versatile topological intermediate, a lasso protein could be converted to a rotaxane, a heterocatenane, and a “slide-ring” network. Being entirely genetically encoded, this robust and modular lasso-protein motif is a valuable addition to the topological protein repertoire and a promising candidate for protein-based biomaterials.     

Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system

Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.     

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.     

Lasso peptides: structure, function, biosynthesis, and engineering

Covering: up to May 2012Lasso peptides are a class of ribosomally-synthesized and posttranslationally-modified natural products with diverse bioactivities. This review describes the structure and function of all known lasso peptides (as of mid-2012) and covers our current knowledge about the biosynthesis of those molecules. The isolation and characterization of lasso peptides are also covered as are bioinformatics strategies for the discovery of new lasso peptides from genomic sequence data. Several studies on the engineering of new or improved function into lasso peptides are highlighted, and unanswered questions in the field are also described.     

Structure and Mechanism of the Sphingopyxin I Lasso Peptide Isopeptidase

Lasso peptides are natural products that assume a unique lariat knot topology. Lasso peptide isopeptidases (IsoPs) eliminate this topology through isopeptide bond cleavage. To probe how these enzymes distinguish between substrates and hydrolyze only isopeptide bonds, we examined the structure and mechanism of a previously uncharacterized IsoP from the proteobacterium Sphingopyxis alaskensis RB2256 (SpI‐IsoP). We demonstrate that SpI‐IsoP efficiently and specifically linearizes the lasso peptide sphingopyxin I (SpI) and variants thereof. We also present crystal structures of SpI and SpI‐IsoP, revealing a threaded topology for the former and a prolyl oligopeptidase (POP)‐like fold for the latter. Subsequent structure‐guided mutational analysis allowed us to propose roles for active‐site residues. Our study sheds light on lasso peptide catabolism and expands the engineering potential of these fascinating molecules.