Biochemical characterization of a cyanobactin arginine-N-prenylase from the autumnalamide biosynthetic pathway.

Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.

Flowering Poration-A Synergistic Multi-Mode Antibacterial Mechanism by a Bacteriocin Fold.

Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays, and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins.

Revealing Sources of Variation for Reproducible Imaging of Protein Assemblies by Electron Microscopy.

Electron microscopy plays an important role in the analysis of functional nano-to-microstructures. Substrates and staining procedures present common sources of variation for the analysis. However, systematic investigations on the impact of these sources on data interpretation are lacking. Here we pinpoint key determinants associated with reproducibility issues in the imaging of archetypal protein assemblies, protein shells, and filaments. The effect of staining on the morphological characteristics of the assemblies was assessed to reveal differential features for anisotropic (filaments) and isotropic (shells) forms. Commercial substrates and coatings under the same staining conditions gave comparable results for the same model assembly, while highlighting intrinsic sample variations including the density and heterogenous distribution of assemblies on the substrate surface. With no aberrant or disrupted structures observed, and putative artefacts limited to substrate-associated markings, the study emphasizes that reproducible imaging must correlate with an optimal combination of substrate stability, stain homogeneity, accelerating voltage, and magnification.

Synthesis of the natural product descurainolide and cyclic peptides from lignin-derived aromatics.

Alternative sources of potential feedstock chemicals are of increasing importance as the availability of oil decreases. The biopolymer lignin is viewed as a source of useful mono-aromatic compounds as exemplified by the industrial scale production of vanillin from this biomass. Alternative lignin-derived aromatics are available in pure form but to date examples of the use of these types of compounds are rare. Here we address this issue by reporting the conversion of an aromatic keto-alcohol to the anti- and syn-isomers of Descurainolide A. The key step involves a rhodium-catalyzed allylic substitution reaction. Enantio-enriched allylic alcohols were generated via an isothiourea-catalyzed kinetic resolution enabling access to both the (2R,3R) and (2S,3S) enantiomers of anti-Descurainolide A. In addition we show that the lignin-derived keto-alcohols can be converted into unnatural amino acid derivatives of tyrosine. Finally, these amino acids were incorporated into cyclic peptide scaffolds through the use of both chemical and an enzyme-mediated macrocylisation.

Synthesis of Hybrid Cyclopeptides through Enzymatic Macrocyclization.

Natural products comprise a diverse array of molecules, many of which are biologically active. Most natural products are derived from combinations of polyketides, peptides, sugars, and fatty-acid building blocks. Peptidic macrocycles have attracted attention as potential therapeutics possessing cell permeability, stability, and easy-to-control variability. Here, we show that enzymes from the patellamide biosynthetic pathway can be harnessed to make macrocycles that are hybrids of amino acids and a variety of manmade chemical building blocks, including aryl rings, polyethers, and alkyl chains. We have made macrocycles with only three amino acids, one of which can be converted to a thiazoline or a thiazole ring. We report the synthesis of 18 peptide hybrid macrocycles, nine of which have been isolated and fully characterized.

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP.

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C-terminal "follower" sequence, rather than the canonical N-terminal "leader" sequence. We report herein the structural and biochemical characterization of BotP, a leucyl-aminopeptidase-like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro- mycin.

Chemical Protein Synthesis Using a Second-Generation N-Acylurea Linker for the Preparation of Peptide-Thioester Precursors.

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C-terminal peptide-thioesters, key intermediates in NCL. We have developed an N-acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into N-acylurea peptides. Here, we introduce a new N-acylurea linker bearing an o-amino(methyl)aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired N-acyl-N'-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer.

Structural effects of proline substitution and metal binding on hexameric cyclic peptoids.

L-Proline and N-methoxyethyl glycine have been included in novel cyclic hexameric peptoids. Supramolecular coordination with Na(+) triggered the formation of the first 1D metal-organic framework based on peptoids.

Cyclopeptoids: a novel class of phase-transfer catalysts.

The synthesis, complexation properties and catalytic activities under phase-transfer (PT) conditions of differently substituted cyclohexapeptoids are reported. Association constants, for small cationic alkali, and catalytic performances, in a model nucleophilic substitution, are comparable to those of representative crown ethers. Noteworthy, the N-[2-(2-methoxyethoxy)ethyl] side chain derivative presents a catalytic efficiency comparable to that of crypt-222, and higher than some commonly used quaternary ammonium salts and crown ethers. Moreover its association constant for Na(+) complexation proved to be higher when compared with dicyclohexyl-18-crown-6. The synthesized cyclohexapeptoids represent the first example of these peptidomimetics in PT catalysis, anticipating interesting applications in biphasic PT methodology.