Nathaniel I. Martin

Two-peptide bacteriocins produced by lactic acid bacteria

Bacteriocins from lactic acid bacteria are ribosomally produced peptides (usually 30-60 amino acids) that display potent antimicrobial activity against certain other Gram-positive organisms. They function by disruption of the membrane of their targets, mediated in at least some cases by interaction of the peptide with a chiral receptor molecule (e.g., lipid II or sugar PTS proteins). Some bacteriocins are unmodified (except for disulfide bridges), whereas others (i.e. lantibiotics) possess extensive post-translational modifications which include multiple monosulfide (lanthionine) bridges and dehydro amino acids as well as possible keto amide residues at the N-terminus. Most known bacteriocins are biologically active as single peptides. However, there is a growing class of two peptide systems, both unmodified and lantibiotic, which are fully active only when both partners are present (usually 1:1). In some cases, neither peptide has activity by itself, whereas in others, the activity of one is enhanced by the other. This review discusses the classification, structure, production, regulation, biological activity, and potential applications of such two-peptide bacteriocins.

An Escherichia coli expression–based method for heme substitution

Heme reconstitution with porphyrin analogs is a powerful approach toward understanding the molecular function of heme proteins; present methods, however, have not proven to be generally useful. Here we describe the development and application of an expression-based method for introducing modified porphyrins. The approach allows efficient incorporation of heme analogs using a widely available bacterial strain and offers an attractive alternative to present reconstitution methods that subject proteins to harsh, denaturing conditions.

Specificity of the transport of lipid II by FtsW in Escherichia coli

Background: The mechanism of FtsW-mediated Lipid II transport across the bacterial cytoplasmic membrane is unknown. Results: Transmembrane segment 4 and particularly two charged residues are required for the transport of Lipid II as well as a maximal size of the substrate. Conclusion: Lipid II is specifically transported possibly through a porelike structure. Significance: Elucidating how FtsW acts is crucial for understanding how lipid flippases function in general. Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg145 and Lys153) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.

Nη-Substituted Arginyl Peptide Inhibitors of Protein Arginine N-Methyltransferases

Protein arginine N-methyltransferases (PRMTs) catalyze the post-translational methylation of arginine residues within substrate proteins. Their roles in the epigenetic regulation of gene expression make them viable targets for drug discovery. Peptides containing a single arginine residue substituted at the guanidino nitrogen (N(η)) with an ethyl group bearing zero to three fluorine atoms (R1-1, -2, -3, and -4) have been synthesized and tested for methylation and inhibition activity with PRMT1, PRMT6, and CARM1. Only the nonfluorinated R1-1 peptide is methylated by PRMT1, demonstrating that the N(η)-substituted arginine is accommodated by its active site. The R1-1 ethyl-substituted guanidine N(η) was further identified as the methylation site via mass spectrometry. Although weak inhibitors of CARM1, R1-1, -2, -3, and -4 are potent inhibitors of PRMT1 and PRMT6. These peptides are more potent against PRMT1 than product inhibitor peptides, showing that N(η)-substituted arginyl peptides do not work by a purely product inhibitor mechanism. A trend of increasing potency with an increase in the number of fluorine atoms is observed for PRMT1, which may result from the corresponding change in the guanidino dipole moment. Modeling of the ethyl-arginine moiety of the R1-1 peptide demonstrates that the active site of PRMT1 accommodates such modifications. N(η)-Substituted arginyl peptides represent lead compounds for the further development of inhibitors that target the methyl-acceptor binding site of PRMTs.

Semisynthetic Lipopeptides Derived from Nisin Display Antibacterial Activity and Lipid II Binding on Par with That of the Parent Compound.

The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic constructs that display potent inhibition of bacterial growth, with activities approaching that of nisin itself. Most notable was the activity observed against clinically relevant bacterial strains including MRSA and VRE. Experiments with membrane models indicate that these constructs operate via a lipid II-mediated mode of action without causing pore formation. A lipid II-dependent mechanism of action is further supported by antagonization assays wherein the addition of lipid II was found to effectively block the antibacterial activity of the nisin-derived lipopeptides.

Preparation of NG-Substituted l-Arginine Analogues Suitable for Solid Phase Peptide Synthesis

A high-yielding and concise preparation of N(G)-substituted L-arginine analogues, suitably protected for use in solid phase peptide synthesis, is reported. The synthesis of each analogue employed an activated thiourea intermediate that was converted under mild conditions to the desired L-arginine analogue (10 examples, each in near quantitative yield). Subsequent allyl group removal provided each analogue in a form ideally suited for use in solid phase peptide synthesis.

A Novel in vivo Cell‐Wall Labeling Approach Sheds New Light on Peptidoglycan Synthesis in Escherichia coli

Peptidoglycan synthesis and turnover in relation to cell growth and division has been studied by using a new labeling method. This method involves the incorporation of fluorescently labeled peptidoglycan precursors into the cell wall by means of the cell‐wall recycling pathway. We show that Escherichia coli is able to import exogenous added murein tripeptide labeled with N‐7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl (AeK–NBD) into the cytoplasm where it enters the peptidoglycan biosynthesis route, resulting in fluorescent labels specifically located in the cell wall. When wild‐type cells were grown in the presence of the fluorescent peptide, peptidoglycan was uniformly labeled in cells undergoing elongation. Cells in the process of division displayed a lack of labeled peptidoglycan at mid‐cell. Analysis of labeling patterns in cell division mutants showed that the occurrence of unlabeled peptidoglycan is dependent on the presence of FtsZ, but independent of FtsQ and FtsI. Accumulation of fluorescence at the division sites of a triple amidase mutant (ΔamiABC) revealed that AeK–NBD is incorporated into septal peptidoglycan. AmiC was shown to be involved in the rapid removal of labeled peptidoglycan side chains at division sites in wild‐type cells. Because septal localization of AmiC is dependent on FtsQ and FtsI, this points to the presence of another peptidoglycan hydrolase activity directly dependent on FtsZ.

Oseltamivir analogues bearing N-substituted guanidines as potent neuraminidase inhibitors.

A series of oseltamivir analogues bearing an N-substituted guanidine unit were prepared and evaluated as inhibitors of neuraminidases from four strains of influenza. The two most potent analogues identified contain relatively small N-guanidine substituents (N-methyl and N-hydroxyl) and display enhanced inhibition with IC50 values in the low nanomolar range against neuraminidases from wild-type and oseltamivir-resistant strains. Potential advantages of including the N-hydroxyguanidine moiety in neuraminidase inhibitors are also discussed.

Hit 'em where it hurts: The growing and structurally diverse family of peptides that target lipid-II.

Understanding the mode of action of antibiotics is becoming more and more important in the time that microorganisms start to develop resistance. One very well validated target of several classes of antibiotics is the peptidoglycan precursor lipid II. In this review different classes of lipid II targeting antibiotics will be discussed in detail, including the lantibiotics, human invertebrate defensins and the recently discovered teixobactin. By hitting bacteria where it hurts, at the level of lipid II, we expect to be able to develop efficient antibacterial agents in the future. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Peptidic Partial Bisubstrates as Inhibitors of the Protein Arginine N-Methyltransferases

Protein arginine N-methyltransferases (PRMTs) catalyze the Nmethylation of guanidine moieties present in the arginine side chains of target proteins. This post-translational modification is implicated in epigenetic gene regulation and a number of disease states, which makes PRMTs attractive targets for the development of new therapeutics. Specifically, PRMTs make use of a bisubstrate mechanism whereby the methyl group donor, S-adenosyl-l-methionine (AdoMet), is employed in the selective methylation of substrate proteins/peptides. Given that the amino acid sequences constituting the AdoMet binding region in PRMTs are largely invariant, specificity in the binding of target proteins likely plays the major role in the substrate selective methylation performed by these enzymes. It is, therefore, conceivable that selective inhibition among PRMTs might be achieved with compounds containing a general AdoMet mimic coupled with a variable peptide scaffold that can be optimized for each different PRMT. In this regard, we have prepared new PRMT partial bisubstrate hybrids composed of a minimal AdoMet fragment (lacking adenosine) covalently linked to a model substrate peptide, and have characterized the inhibition profiles of these compounds against PRMT1, -4 (also known as coactivator-associated arginine methyltransferase 1, CARM1), and -6. The methylation of lysine and arginine residues in target proteins constitutes an important post-translational modification with numerous implications for both epigenetics and medicine. 2] Most notable are the roles played by the methyltransferases in chromatin remodeling by histone modification and the regulation of gene expression. 4] An increasing body of biochemical and biological evidence also points towards a direct role for these enzymes in inflammatory and neurodegenerative diseases as well as cancer and other pathogenic conditions. While more than fifty lysine N-methyltransferases have been described to date, a comparatively small number (ca. ten) of arginine N-methyltransferases are currently known. 2] PRMTs make use of the cofactor AdoMet for methylating target proteins while forming the byproduct Sadenosyl-l-homocysteine (AdoHcy; Scheme 1). The first methylation catalyzed by PRMTs yields monomethyl arginine (MMA),