Bacteriocins represent a large family of ribosomally produced peptide antibiotics. Here we describe the discovery of a widely conserved biosynthetic gene cluster for the synthesis of thiazole and oxazole heterocycles on ribosomally produced peptides. These clusters encode a toxin precursor and all necessary proteins for toxin maturation and export. Using the toxin precursor peptide and heterocycle-forming synthetase proteins from the human pathogen Streptococcus pyogenes, we demonstrate the in vitro reconstitution of streptolysin S activity. We provide evidence that the synthetase enzymes, as predicted from our bioinformatics analysis, introduce heterocycles onto precursor peptides, thereby providing molecular insight into the chemical structure of streptolysin S. Furthermore, our studies reveal that the synthetase exhibits relaxed substrate specificity and modifies toxin precursors from both related and distant species. Given our findings, it is likely that the discovery of similar peptidic toxins will rapidly expand to existing and emerging genomes.antibiotics ͉ bacteriocin ͉ bioinformatics ͉ hemolytic ͉ streptolysin
Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide which exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [2H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as key to the mode of action. [2H]pb-A3 has a 2H-isotopically labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the drug mode of action. However, rotational-echo double resonance NMR characterization of [2H]pb-A3 bound to [1-13C]glycine labeled whole-cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane, but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [2H]pb-A3 during S. aureus growth resulted in an accumulation of Park’s nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.
Plusbacin A 3 is a lipodepsipeptide isolated from a fermentation broth of Pseudomonas sp. PB-6250 obtained from a soil sample collected in the Okinawa Prefecture, Japan (Figure 1). 1 The structure of plusbacin A 3 was established in 1992. It is a member of a family of lipodepsipeptide natural products that differ either in the structure of their respective fatty acid side chains or in substitution of L-proline for 3-hydroxy-L-proline residues. 2In a recent evaluation, plusbacin A 3 displayed strong antibiotic activity against methicillinresistant Staphylococcus aureus and VanA-type vancomycin-resistant enterococci with minimum inhibitory concentration (MIC) values from 0.78 to 3.13 μg/mL. 3 Plusbacin A 3 also inhibited incorporation of N-acetylglucosamine into staphylococcal cell wall peptidoglycan with a 50% inhibitory concentration (IC 50 ) that was close to its MIC value. Like vancomycin, plusbacin A 3 was found to inhibit nascent peptidoglycan formation; however, unlike vancomycin, plusbacin was also found to inhibit the formation of the lipid intermediates utilized in bacterial cell wall biosynthesis. Interestingly, the activity of plusbacin A 3 was not antagonized by the presence of N-acetyl-L-Lys-D-Ala-D-Ala, a tripeptide mimic of the binding domain for vancomycin, 3 suggesting that if plusbacin A 3 achieves its biological activity through binding to the lipid intermediates or to nascent peptidoglycan it does so at a site on these precursors that is not utilized by vancomycin itself. As a result, plusbacin A 3 possesses significant promise for use in the treatment of vancomycin-resistant infections.The amino acid sequences of the plusbacins were established through Edman degradation of their deacylated products and supported by mass spectrometric studies. Degradation experiments also suggested a lactone linkage between an L-threo-β -hydroxyaspartic acid residue and a 3-hydroxy fatty acid subunit. In the case of plusbacin A 3 , the fatty acid component is reported to be 3-hydroxyisopentadecanoic acid, although the stereochemical configuration at the hydroxyl stereocenter has yet to be assigned.Plusbacin A 3 also has several non-proteinogenic amino acids embedded in its peptide backbone. In addition to the L-threo-β -hydroxyaspartic acid residue mentioned above, other non-natural amino acids contained in plusbacin A 3 include D-threo-β -hydroxyaspartic acid, D-allo-threonine, and trans-3-hydroxy-L-proline. The presence of these non-natural amino acids, coupled with the base sensitivity of the lactone linkage, renders plusbacin A 3 a challenging target for total synthesis.Our retrosynthetic analysis for plusbacin A 3 with our selected disconnections is presented in Figure 1. We chose to divide the target molecule into four fragments of approximately equal complexity. This was done in order to provide a measure of flexibility over the amide bond to E-mail: msv@ucsd.edu. Supporting Information Available: Experimental details and spectroscopic data for all new compounds. This material is available free of char...
A simple strategy for linking biomolecules to porous Si surfaces and detecting peptide/drug binding is described. Porous Si is prepared using an electrochemical etch and then thermally oxidized by heating in ambient atmosphere. Bovine serum albumin (BSA) is then non-covalently adsorbed to the inner pore walls of the porous Si oxide (PSiO 2 ) matrix. The BSA layer is used as a linker for covalent attachment of the peptide Ac-L-Lysine-D-Alanine-D-Alanine (KAA) using published bioconjugation chemistry. BSA-coated surfaces functionalized with KAA display specificity for the glycopeptide vancomycin while resisting adsorption of non-specific reagents. While the biomolecule attachment strategy reported here is used to bind peptides, the scheme can be generalized to the linking of any primary amine-containing molecule to PSiO 2 surfaces.
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