Mimicry of a Non-ribosomally Produced Antimicrobial, Brevicidine, by Ribosomal Synthesis and Post-translational Modification.

The group of bacterial non-ribosomally produced peptides (NRPs) forms a rich source of antibiotics, such as daptomycin, vancomycin, and teixobactin. The difficulty of functionally expressing and engineering the corresponding large biosynthetic complexes is a bottleneck in developing variants of such peptides. Here, we apply a strategy to synthesize mimics of the recently discovered antimicrobial NRP brevicidine. We mimicked the molecular structure of brevicidine by ribosomally synthesized, post-translationally modified peptide (RiPP) synthesis, introducing several relevant modifications, such as dehydration and thioether ring formation. Following this strategy, in two rounds peptides were engineered in vivo, which showed antibacterial activity against Gram-negative pathogenic bacteria susceptible to wild-type brevicidine. This study demonstrates the feasibility of a strategy to structurally and functionally mimic NRPs by employing the synthesis and post-translational modifications typical for RiPPs. This enables the future generation of large genetically encoded peptide libraries of NRP-mimicking structures to screen for antimicrobial activity against relevant pathogens.

Antimicrobial peptides produced by Brevibacillus spp.: structure, classification and bioactivity: a mini review.

Species that are currently listed under the genus Brevibacillus (formerly, Bacillus brevis cluster) have been a rich source of antimicrobial peptides for many decades. The first known peptide antibiotic, gramicidin, is presumed to be produced by a Brevibacillus sp. Members of the genus are widely spread in nature. They can be found in a variety of environments including intestinal tracts of animals, seawater, and soil. Some Brevibacillus strains have been used commercially as probiotics. Bioactive peptides produced by Brevibacillus spp. include antibacterial, antifungal and anti-invertebrate agents. Brevibacillus antimicrobial peptides are synthesized through ribosomal or nonribosomal pathway; these two groups can be further categorized based on specific structural features such as cyclization and presence of lipid chain. Some of the antimicrobial compounds produced by this genus share structural similarities that were overlooked previously. For example, the structural similarity between BT peptide, brevibacillin, and bogorol was revealed only recently. Here we review and classify Brevibacillus antimicrobial peptides and summarize their bioactivities and potential applications.

ß-sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus.

The structures of two major tyrocidines, antibiotic peptides from Bacillus aneurinolyticus, in an aqueous environment were studied using nuclear magnetic resonance spectroscopy, restrained molecular dynamics (MD), circular dichroism, and mass spectrometry. TrcA and TrcC formed ß-structures in an aqueous environment. Hydrophobic and hydrophilic residues were not totally separated into nonpolar and polar faces of the peptides, indicating that tyrocidines have low amphipathicity. In all the ß-structures, residues Trp(4)/Phe(4) and Orn(9) were on the same face. The ability of the peptides to form dimers in aqueous environment was studied by replica exchange MD simulations. Both peptides readily dimerize, and predominant complex structures were characterized through cluster analysis. The peptides formed dimers by either associating sideways or stacking on top of each other. Dimers formed through sideways association were mainly stabilized by hydrogen bonding, while the other dimers were stabilized by hydrophobic interactions. The ability of tyrocidine peptides to form different types of dimers with different orientations suggests that they can form larger aggregates, as well.

An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis.

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.

Impact of epimerization domains on the intermodular transfer of enzyme-bound intermediates in nonribosomal peptide synthesis.

Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.

Fluorescence resonance energy transfer as a probe of peptide cyclization catalyzed by nonribosomal thioesterase domains.

Macrocyclization of synthetic peptides by thioesterase (TE) domains excised from nonribosomal peptide synthetases (NRPS) has been limited to peptides that contain TE-specific recognition elements. To alter substrate specificity of these enzymes by evolution efforts, macrocyclization has to be detected under high-throughput conditions. Here we describe a method to selectively detect cyclic peptides by fluorescence resonance energy transfer (FRET). Using this method, picomolar detection limits were easily realized, providing novel entry for kinetic studies of catalyzed macrocyclization. Application of this method also provides an ideal tool to track TE-mediated peptide cyclization in real time. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE and calcium-dependent antibiotic (CDA) TE. For the latter cyclase, this approach was combined with site-directed affinity labeling, opening the possibility for high-throughput enzymatic screening.

Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases.

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf). These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.

Evidence for a monomeric structure of nonribosomal Peptide synthetases.

Nonribosomal peptide synthetases (NRPS) are multimodular biocatalysts that bacteria and fungi use to assemble many complex peptides with broad biological activities. The same modular enzymatic assembly line principles are found in fatty acid synthases (FAS), polyketide synthases (PKS), and most recently in hybrid NRPS/PKS multienzymes. FAS as well as PKS are known to function as homodimeric enzyme complexes, raising the question of whether NRPS may also act as homodimers. To test this hypothesis, biophysical methods (size exclusion chromatography, analytical equilibrium ultracentrifugation, and chemical crosslinking) and biochemical methods (two-affinity-tag-system and complementation studies with enzymes being inactivated in different catalytic domains) were applied to NRPS subunits from the gramicidin S (GrsA-ATE), tyrocidine (TycB(1)-CAT and TycB(2-3)-AT.CATE), and enterobactin (EntF-CATTe) biosynthetic systems. These methods had revealed the dimeric structure of FAS and PKS previously, but all three NRPS systems investigated are functionally active as monomers.

Substrate spectrum of tyrocidine thioesterase probed with randomized peptide N-acetylcysteamine thioesters.

Apparent kinetic constants k(cat) and K(m) were determined for tyrocidine thioesterase (TycC TE) using randomized peptide N-acetylcysteamine thioesters as substrate analogues. The enzyme has been found to be adequately active for the synthesis of positional-scanning libraries for novel antibiotic screening with reduced k(cat)/K(m) in the range of 2 to 82 folds lower than that of the wild-type sequence

Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase.

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

Interrelationships between tyrocidine and gramicidin A' in their interaction with phospholipids in model membranes.

(1) The interaction of tyrocidine with different lipids is studied in model membranes and the results are compared to the gramicinid-lipid interaction. (2) The tyrocidine-dielaidoylphosphatidylethanolamine interaction gives rise to a population of phospholipids with a lower gel to liquid-crystalline transition temperature and to an abolition of the bilayer to HII phase transition, resulting in a macroscopic organization with dynamic and structural properties different from those of the pure lipid. (3) Tyrocidine has a strong fluidizing effect on the acyl chains of phosphatidylcholines, manifested by a decrease in enthalpy of the main thermotropic transition. (4) No evidence of a gramicidin A'-like lipid-structure modulating activity was found. However, tyrocidine inhibits the formation by gramicidin of an HII phase in dioleoylphosphatidylcholine model membranes. Instead, a cubic type of lipid organization is observed. (5) Tyrocidine greatly perturbs the barrier properties of dioleoylphosphatidylcholine model membrane. (6) Gramicidin A' reverses the effect of tyrocidine on membrane permeability by forming a complex in the model membrane with an apparent 1:1 stoichiometry. (7) The results suggest that both peptide antibiotics, which are produced by Bacillus brevis ATC 8185 prior to sporulation, show antagonism in their effect on membrane structure similar to their effect on superhelical DNA (Bogh, A. and Ristow, H. (1986) Eur. J. Biochem. 160, 587-591. The possible underlying basic mechanism is indicated.