Abstract: A series of HIV-Tat peptide analogues were synthesised and evaluated as substrates and inhibitors of three members of the PRMT family. This work provides new insights into the methylating activity and inhibition of PRMTs when interacting with highly positively charged, multiple-arginine-containing peptides.
Abstract: 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.
Abstract: 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.
Abstract: Lantibiotics are antimicrobial peptides containing the unique bis-amino acids lanthionine and beta-methyllanthionine. While previous syntheses of lanthionine have often involved the coupling of precursors derived from D-serine and L-cysteine, we here report an inverted strategy whereby D-cysteine and L-serine are employed as building blocks. This approach provides for a concise preparation of tetra-orthogonally protected (2R,6S)-lanthionines while allowing convenient introduction of orthogonal protecting groups not previously incorporated into lanthionines.
Abstract: 4,4-Difluoro-l-arginine and 4,4-difluoro-N(G)-hydroxy-l-arginine were synthesized and shown to be substrates for the inducible isoform of nitric oxide synthase (iNOS). Binding of both fluorinated analogues to the NOS active site was also investigated using a spectral binding assay employing a heme domain construct of the inducible NOS isoform (iNOS(heme)). 4,4-Difluoro-N(G)-hydroxy-arginine was found to bind at the NOS active site in a unique manner consistent with a model involving ligation of the Fe(III) heme center by the oxygen atom of the N(G)-hydroxy moiety.
Abstract: Nitric oxide synthase (NOS) is a P450 mono-oxygenase that catalyzes the oxidation of l-arginine to citrulline and NO through the stable intermediate N(G)-hydroxy-l-arginine (NHA). The oxidation of NHA by NOS is unique. There is little direct evidence in support of the nature of the heme bound oxidant [i.e., ferric-peroxo vs Fe(IV)O(por(*+))] responsible for this transformation. Previous work characterizing the H(2)O(2)-driven oxidation of NHA by NOS showed the formation of citrulline and the side product N(delta)-cyanoornithine (CN-orn). This led to the proposed involvement of a ferric-peroxo intermediate in the oxidation of NHA to citrulline. To test this hypothesis we used this model reaction to study the effects of pH, heme substitution, active site mutagenesis, and a fluorinated substrate analogue on the product distribution. Further, the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H(2)O(2) and iNOS(heme) was used to probe the protein-catalyzed breakdown of peroxide to the Fe(IV)O(por(*+)) intermediate. At pH 6.5, 7.5, and 8.5 the peroxide shunt reaction forms 26 +/- 2, 36 +/- 1, and 51 +/- 1% citrulline, respectively. The rate of peroxidase activity, however, was negatively correlated to pH, with a peroxide breakdown rate of 13.1 +/- 0.3, 8.3 +/- 0.2, and 4.2 +/- 0.1 M(-1) s(-1) at pH 6.5, 7.5, and 8.5, respectively. Mutation of active site valine 346 to an alanine shifted the product distribution to 5.2 +/- 0.5% citrulline while enhancing the peroxide cleavage rate to 14.3 +/- 0.7 M(-1) s(-1). Substitution of the heme cofactor with iron mesoporphyrin IX (Fe-MPIX) alters the product distribution from 36 +/- 1% citrulline to 22 +/- 3% citrulline. Metal substitution with Mn results in the formation of 64.7 +/- 0.8% citrulline. Conversely, the electrophilic 4,4-difluoro-N(G)-hydroxy-l-arginine substrate analogue shifted the product distribution to 68.6 +/- 0.6% 4,4-difluorocitrulline. The peroxidase data provide insight into the chemical features of NOS that control the processing of the ferric-peroxo species to the Fe(IV)O(por(*+)) intermediate and help interpret the product distributions observed for the peroxide shunt under various conditions. In all cases, the ability of the protein to break down peroxide is negatively correlated with the formation of citrulline by the peroxide shunt. These results support the high valent Fe(IV)O(por(*+)) intermediate as the species responsible for CN-orn formation and are consistent with the involvement of the ferric-peroxo intermediate in the oxidation of NHA to citrulline.
Abstract: A concise, general, and high-yielding method for the preparation of N(G)-aminoguanidines from primary amines is reported. Using available and readily prepared materials, primary amines are converted to protected N(G)-aminoguanidines in a one-pot procedure. The method has been successfully applied to a number of examples including the syntheses of four nitric oxide synthase (NOS) inhibitors. The inhibitors prepared were investigated as competitive inhibitors and as mechanistic inactivators of the inducible isoform of NOS (iNOS). In addition, one of the four inhibitors prepared, N(G)-amino-N(G)-2,2,2-trifluoroethyl-L-arginine 19, displays the unique ability to both inhibit NO formation and prevent NADPH consumption by iNOS without irreversible inactivation of the enzyme.
Abstract: 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.
Abstract: 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.
Abstract: Chemical synthesis of lantibiotic analogues wherein monosulfide bridges are replaced with other groups can shed light on structure-activity relationships and generate variants that are resistant to aerobic oxidation and have better metabolic stability. This work describes the first complete synthesis of a carbocyclic lantibiotic analogue 2, using sequential on-resin ring-closing olefin metathesis and solution-phase peptide synthesis. The methodology described should find wide application for the preparation of rigidified peptidomimetics containing multiple carbocyclic rings. [structure: see text].
Abstract: The role of nitric oxide (NO) as a biological signaling molecule is well established. NO is produced by the nitric oxide synthases (NOSs, EC 1.14.13.39), a class of heme proteins capable of converting l-arginine to NO and l-citrulline. Despite the large body of knowledge associated with the NOSs, mechanistic details relating to the unique oxidative chemistry performed by these enzymes remain to be fully elucidated. Furthermore, a number of disease states are associated with either the over- or underproduction of NO, making the NOS pathway an attractive target for the development of therapeutics. For these reasons, molecular tools capable of providing mechanistic insights into the production of NO and/or the inhibition of the NOSs remain of interest. We report here the stereospecific synthesis and testing of a number of new l-arginine analogues bearing a minimal substitution, methylation at position 5 of the amino acid side chain (such analogues have not been previously reported). The synthetic approach employed a modified photolysis procedure whereby irradiation of the appropriate diacylperoxide precursors at 254 nm gave access to the required unnatural amino acids in good yields. A heme domain construct of the inducible NOS isoform (iNOSheme) was used to assess the binding of each compound to the enzyme active site. The compounds were also investigated as either inhibitors of, or alternate substrates for, the inducible NOS isoform. The results obtained provide new insight into the steric and stereochemical tolerance of the enzyme active site. These findings also further support the role of a conserved active site water molecule previously proposed to be necessary for NOS catalysis.
Abstract: Lipid II is an essential cell-wall precursor required for the growth and replication of both Gram-positive and Gram-negative bacteria. Compounds that use lipid II to selectively target bacterial cells for destruction represent an important class of antibiotics. Clinically, vancomycin is the most important example of an antibiotic that operates in this manner. Despite being considered the 'antibiotic drug of last resort', significant bacterial resistance to vancomycin now manifests itself worldwide. In this paper we review recent progress made in understanding the lipid II-associated antibacterial characteristics of various naturally occurring compounds, with particular focus on the lantibiotic peptides.
Abstract: Soluble guanylate cyclase (sGC) is activated by the known benzylindazole derivative YC-1 [1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole]. YC-1 also acts synergistically with CO, activating sGC to a level comparable to that achieved upon binding of nitric oxide, the endogenous activator of sGC. We here describe the synthesis of a YC-1 phosphonate analogue with improved aqueous solubility as well as its effects on sGC.
Abstract: A concise and general method for the preparation of N(G)-hydroxyguanidines from primary amines is reported. Using available and readily prepared materials, primary amines are converted to protected N(G)-hydroxyguanidines in a one-pot procedure followed by deprotection under nonreducing conditions. The method has been successfully applied to a number of examples including a high-yielding preparation of N(G)-hydroxy-L-arginine, the intermediate in the enzymatic conversion of L-arginine to nitric oxide and L-citrulline by nitric oxide synthase.
Abstract: Carnobacterium maltaromaticum UAL26 produces the antimicrobial peptides (bacteriocins) piscicolin 126, first isolated from C. maltaromaticum JG126, and carnobacteriocin BM1, first isolated from C. maltaromaticum LV17. C. maltaromaticum UAL26 is especially inhibitory to strains of Listeria monocytogenes. Bacteriocin activity is not observable in the supernatant of cultures of UAL26 grown in liquid media at 25 degrees C, but at temperatures less than 19 degrees C bacteriocin activity can be detected. In contrast to JG126, the piscicolin 126 operon is downregulated in UAL26 at higher temperature, and piscicolin 126 mRNA is not detected when UAL26 is grown at 25 degrees C. Bacteriocin production in UAL26 grown at 15 degrees C can be induced by addition of 10(-10) M of chemically synthesized piscicolin 126 induction peptide (PisN). However, induction of bacteriocin production in UAL26 grown at 25 degrees C requires 10(-7) M of PisN. The sequence of the piscicolin 126 operon in UAL26 contains 34 single nucleotide differences compared with the piscicolin 126 operon in JG126, including single nucleotide differences in the immunity, histidine kinase, dedicated ABC-transporter and accessory genes, as well as a single nucleotide deletion in the transport accessory gene. This deletion causes a frameshift, resulting in truncation of the PisE transport accessory protein in UAL26.
Abstract: Lantibiotics are antibacterial peptides isolated from bacterial sources that exhibit activity toward Gram-positive organisms and are usually several orders of magnitude more potent than traditional antibiotics such as penicillin. They contain a number of unique structural features including dehydro amino acid and lanthionine (thioether) residues. Introduced following ribosomal translation of the parent peptide, these moieties render conventional methods of peptide analysis ineffective. We report herein a new method using nickel boride (Ni(2)B), in the presence of deuterium gas, to reduce dehydro side chains and reductively desulfurize lanthionine bridges found in lantibiotics. Using this approach, it is possible to identify and distinguish the original locations of dehydro side chains and lanthionine bridges by traditional peptide sequencing (Edman degradation) followed by mass spectrometry. The strategy was initially verified using nisin A, a structurally well characterized lantibiotic, and subsequently extended to the novel two-component lantibiotic, lacticin 3147, produced by Lactococcus lactis subspecies lactis DPC3147. The primary structures of both lacticin 3147 peptides were then fully assigned by use of multidimensional NMR spectroscopy, showing that lacticin 3147 A1 has a specific lanthionine bridging pattern which resembles the globular type-B lantibiotic mersacidin, whereas the A2 peptide is a member of the elongated type-A lantibiotic class. Also obtained by NMR were solution conformations of both lacticin 3147 peptides, indicating that A1 may adopt a conformation similar to that of mersacidin and that the A2 peptide adopts alpha-helical structure. These results are the first of their kind for a synergistic lantibiotic pair (only four such pairs have been reported to date).
Abstract: Mattacin is a nonribosomally synthesized, decapeptide antibiotic produced by Paenibacillus kobensis M. The producing strain was isolated from a soil/manure sample and identified using 16 S rRNA sequence homology along with chemical and morphological characterization. An efficient production and isolation procedure was developed to afford pure mattacin. Structure elucidation using a combination of chemical degradation, multidimensional NMR studies (COSY, HMBC, HMQC, ROESY), and mass spectrometric (MALDI MS/MS) analyses showed that mattacin is identical to polymyxin M, an uncommon antibiotic reported previously in certain Bacillus species by Russian investigators. Mattacin (polymyxin M) is cyclic and possesses an amide linkage between the C-terminal threonine and the side chain amino group of the diaminobutyric acid residue at position 4. It contains an (S)-6-methyloctanoic acid moiety attached as an amide at the N-terminal amino group, one D-leucine, six L-alpha,gamma-diaminobutyric acid, and three L-threonine residues. Transfer NOE experiments on the conformational preferences of mattacin when bound to lipid A and microcalorimetry studies on binding to lipopolysaccharide showed that its behavior was very similar to that observed in previous studies of polymyxin B (a commercial antibiotic), suggesting an identical mechanism of action. It was capable of inhibiting the growth of a wide variety of Gram-positive and Gram-negative bacteria, including several human and plant pathogens with activity comparable with purified polymyxin B. The biosynthesis of mattacin was also examined briefly using transpositional mutagenesis by which 10 production mutants were obtained, revealing a set of genes involved in production.
Abstract: 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.
