The Iterative Gramicidin S Thioesterase Catalyzes Peptide Ligation and Cyclization

Hoyer, Katharina M.; Mahlert, Christoph; Marahiel, Mohamed A.

doi:10.1016/j.chembiol.2006.10.011

Cited by 92 publications

(105 citation statements)

References 35 publications

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“…On the other hand, iterative phenomena have also been observed for specific domains. Nice examples are the repeated use of an unique adenylation domain involved in the biosynthesis of the antibiotic congocidine in Streptomyces ambofaciens [43] and the iterative bimodular PCP-thioesterase (TE) domain of gramicidin S synthetase B, which catalyzes the ligation of the pentapeptide intermediates prior to product release by cyclization [44]. In our work, fusaricidin synthetase exhibits an interesting iterative potential of the C-terminal alanine-specific module, which is able to perform both cyclization and modification of fusaricidins.…”

Section: Classification Of Fusaricidins Into Four Familiesmentioning

confidence: 99%

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

Vater

Niu

Dietel³

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. Paenibacillus polymyxa-M1 is a potent producer of bioactive compounds, such as lipopeptides, polyketides, and lantibiotics of biotechnological and medical interest. Genome sequencing revealed nine gene clusters for nonribosomal biosynthesis of such agents. Here we report on the investigation of the fusaricidins, a complex of cyclic lipopeptides containing 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid component by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). More than 20 variants of these compounds were detected and characterized in detail. Mass spectrometric sequence analysis was performed by MALDI-LIFT-TOF/TOF fragment analysis. The obtained product ion spectra show a specific processing in the fatty acid part. GHPD is cleaved between the α-and ß-position yielding two fragments a and b, one bearing the end-standing guanidine group and another one comprising the residual two C-atoms of GHPD with the attached peptide moiety. The complete sequence of all fusaricidins was derived from sets of b n -and y n -ions. The fusaricidin complex can be divided into four lipopeptide families, three of them showing variations of the amino acid in position 3, Val or Ile for the first and Tyr or Phe for families 2 and 3, respectively. A collection of novel fusaricidins was detected differing from those of families 1-3 by an additional residue of 71 Da (family 4). LIFT-TOF/TOF fragment spectra of these species imply that in their peptide moiety, an Ala-residue is attached by an ester bond to the free hydroxyl group of Thr 4 . More than 10 novel fusaricidins were characterized mass spectrometrically.

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Section: Classification Of Fusaricidins Into Four Familiesmentioning

confidence: 99%

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

Vater

Niu

Dietel³

et al. 2015

J. Am. Soc. Mass Spectrom.

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“…migulanus utilizes two nonribosomal peptide synthetases for GS biosynthesis, both having a modular architecture (17). GrsA synthetase functions as a phenylalanine-racemase and catalyzes the ATP-dependent transformation of L-phenylalanine into D-phenylalanine (30).…”

Section: Discussionmentioning

confidence: 99%

“…Polymerization begins with the transfer of D-phenylalanine on GrsA to the 4-phosphopanthetheine residue of GrsB peptidyl carrier protein (pan-PCP) and proceeds through a sequential thiolation and transpeptidation reaction via pan-PCP on GrsB (16). Penultimately, two pentapeptides, one bound as an ester to the active-site serine of the terminal thioesterase domain (TE) and the second bound as a thioester to the adjacent pan-PCP, are ligated to a decapeptidyl-pan-PCP, which is finally cyclized on TE (17). The effectiveness of this unusual way of biosynthesis depends on multiple factors, such as growth temperature, medium composition, and phenotype variability; therefore, GS production is not a trivial task (18).…”

Section: T He Cyclic Decapeptide Gramicidin S (Gs) Contains a Repeat mentioning

confidence: 99%

Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Berditsch

Afonin

Steineker

et al. 2015

Appl Environ Microbiol

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Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membranebound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13 C/ 15 N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13 C/ 15 N-labeled amino acids. The most cost-effective production of 13 (Fig. 1A). Since its discovery about 70 years ago, GS has been used as a topical antibiotic in Russia and neighboring countries, exploiting its pronounced antimicrobial activity against Gram-positive bacteria (1, 2). For example, GS is the bioactive agent in GrammidinNeo, the lozenge against sore throat and mouth ulcer produced by the Russian JSC Valenta Pharmaceuticals. Notably, despite a long medication history, no clinical cases of bacterial resistance against GS have been reported, which suggest that this peptide may be an exceptionally promising resistance-free antibiotic (3).The biological activity of GS was first described as membranolytic, which is believed to determine its profound antimicrobial as well as hemolytic activities in vitro. Less acknowledged are GS activities not directly related to interaction with membrane bilayers: the peptide has been shown to inhibit membrane enzymes, such as bacterial NADH dehydrogenase and cytochrome bd terminal oxidase (4), and to delocalize the peripheral cell division regulator MinD, the lipid II biosynthesis protein MurG, and cytochrome c (5).The main molecular target of GS is supposed to be the plasma membrane of susceptible bacterial species, where an accumulation of the amphiphilic peptide leads to a loss of the barrier function and an irreversible increase in permeability for ions and metabolites (6). This concentration-dependent effect leads to membrane depolarization (7) and an overall dysfunction of the cellular osmoregulation, which starts already at GS levels well below the respective MICs (8). However, the structure-function relationship of the peptide and its detailed molecular mechanism of action at the plasma membrane, i.e., its interaction with the lipid bilayer, are still lacking a consensus view.Over the last decades, the molecular structure of GS has been stud...

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“…Using several excised TEs from the gramicidin, 12 enterobactin, 13 surfactin 22 and tyrocidine 23 NRPS biosynthetic assembly module has helped to elucidate the activity involved during this process and corroborate their proposed function. These enzymes were analyzed with substrates that were esterified with N-acetylcysteamine (SNAC) to mimic their natural substrates and ultimately establish their substrate-tolerance.…”

Section: Chemoenzymatic Synthesis Of Echinomycin Derivatives Using a mentioning

confidence: 99%

“…Before synthesizing 12 for supplying the substrate during biosynthesis of 4, the absolute configuration of SW-163D (Figure 2, 4) was determined together with that of 12 as a constituent of its peptide backbone. 21 Then, the synthetic method of the unusual amino acid, ( )-(1S,2S)-norcoronamic acid (12) was developed for culture supplementation. 21 Subjecting culture extract isolated from the fermentation broth of the genetically engineered E. coli to a series of chromatographic steps, 11 was isolated at a final yield of 0.6 mg of compound per liter of culture.…”

Section: Synthesis Of Echinomycin Derivatives By Inactivation Of Specmentioning

confidence: 99%

Enzymatic Synthesis of Molecular Skeletons of Complex Antitumor Antibiotics with Non-ribosomal Peptide Synthetases

Watanabe

Oguri

Oikawa

2009

J. Synth. Org. Chem. Jpn.

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Nonribosomal peptide synthetase (NRPS) is a programmable modular machinery which produces a number of biologically active small-molecule peptides. Recent progress on understanding the catalytic mechanism of NRPS enabled us to engineer this complex catalytic system. We demonstrated both in vivo and in vitro enzymatic synthesis of complex natural antitumor peptides echinomycin and saframycin. An Escherichia coli-based expression system served as a flexible yet robust platform for producing complex natural products and their analogs by deletion, mutation and swapping of a specific subunit. The excised thioesterase domain of echinomycin exhibited remarkable substrate tolerance and created a cyclic peptide library. On the other hand, saframycin NRPS catalyzed highly unusual seven-step transformations to construct a pentacyclic tetrahydro-isoquinoline skeleton. According to the generally accepted mechanism (colinearity rule), 3 NRPS produces polypeptides different in size and amino acid-sequence with various modifications. An important strategy that this enzyme adopts is that condensation substrates are always bound to the enzyme (T) and that the chain-elongated intermediates are never released from NRPS until the final cleavage. Therefore, this system guarantees reliable and safe delivery of the substrates. This allows rather broad substrate specificity of the C-domain. Each module is a self-consistent portable component. This suggests that replacing A-domain or a specific module may create new peptides in a programmable manner. Hence, the rational design of NRPS makes it possible to synthesize novel peptides just in the same way as organic synthesis. In this account, we describe an enzymatic synthesis of two important families of antitumor peptide antibiotics, echinomycin and saframycin. Enzymatic Synthesis of Echinomycin and its DerivativesEchinomycin (1) belongs to quinomycin bis-intercalator antibiotics (Figure 2). 4 This class of antibiotics are dimeric cyclic peptides with a pair of chromophores and have potent DNA binding affinities at a level between nM to M with different sequence selectivities. Reflecting these distinct affinities, bis-intercalator antibiotics such as 1-4 show fairly different inhibitory activities against various enzymes such as DNA polymerase, RNA polymerase, reverse transcriptase and topoisomerase. Based on structure-activity relationships among this class of antibiotics, one can expect that minute structural difference may drastically change profiles between antitumor activity and toxicity. To examine this possibility, we became interested in synthesizing echinomycin derivatives.To identify and isolate the echinomycin biosynthetic gene cluster, a cosmid library was constructed using S. lasaliensis total DNA. Screening with the PCR product from degenerate primers for NRPS afforded plasmids encoding the 36-kb echinomycin biosynthetic gene clusters. 5 Homology of these genes enabled us to speculate their functions in the biosynthesis of quinoxaline-2-carboxylic acid (5, QXC) and ...

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The Iterative Gramicidin S Thioesterase Catalyzes Peptide Ligation and Cyclization

Cited by 92 publications

References 35 publications

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Enzymatic Synthesis of Molecular Skeletons of Complex Antitumor Antibiotics with Non-ribosomal Peptide Synthetases

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The Iterative Gramicidin S Thioesterase Catalyzes Peptide Ligation and Cyclization

Cited by 92 publications

References 35 publications

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

Fermentation and Cost-Effective 13 C/ 15 N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

Enzymatic Synthesis of Molecular Skeletons of Complex Antitumor Antibiotics with Non-ribosomal Peptide Synthetases

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Fermentation and Cost-Effective ¹³ C/ ¹⁵ N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis