Synthesis Of Peptide Antibiotics Research Articles

Induction of antimicrobial peptide synthesis by the fat body cells of maggots of Calliphora vicina R.-D. (Diptera, Calliphoridae)

Fat body is the major site of peptide antibiotic synthesis in insects. The goal of this work was to search in Calliphora vicina maggot for humoral factors that induce synthesis of antimicrobial peptides by the fat body cells in trauma. The preliminary analysis has shown that the activation factor revealed in hemolymph is a thermostable hydrophilic compound with a molecular mass lower than 3 kDa. The integument epitheliocytes were also found to release the humoral factors directly stimulating synthesis of antimicrobial peptides by the fat body.

Neurogenic and septic inductions of synthesis of peptide antibiotics in larvae of Calliphora vicina R.-D. (Diptera: Calliphoridae)

Synthesis of antimicrobial peptides in diapausing larvae Calliphora vicina can be induced by two different pathways. One pathway is well known in insects and includes recognition of microbial particles by the pattern-recognizing receptors. The other pathway includes perception and transduction of stress signal to immunocompetent cells by neuroendocrine system. This phenomenon consists in stimulation of synthesis of defensins, cecropins, and diptericins under effect of chromic stimulation of mechanoreceptors with ligature applied on the larva head end. Formation of immune response in brain is established to need less than 30 s, after which isolation of the neuroendocrine complex does not eliminate activation of immune response As judging from rate of the neurogenic induction, transduction of the stimulating signal from brain to the immune system cells can be connected with release into hemolymph of biogenic amines or other neurohormones stored preliminarily in the neurohemal organ. The nature of this inductor at present remains unknown, as analysis of role of octopamine, dopamine, and adipokynetic hormone did not reveal stimulating effect on synthesis of bactericidal peptides. Physiological mechanism of this phenomenon is not finally understood, its key links seem to be CNS, hormonal factor of cardial bodies, and system of antimicrobial peptides. Synthesis of antimicrobial peptides is directly regulated by the neuroendocrine system that can produce both stimulating and stress action by reminding in this aspect the known immunoneuroendocrine interrelations in vertebrates. The existence of similar integrating mechanisms in such polar animal kingdom groups which are insects and vertebrates indicate that they are more ancient than this was considered earlier.

Production of a New Thiopeptide Antibiotic, TP-1161, by a Marine Nocardiopsis Species

Twenty-seven marine sediment- and sponge-derived actinomycetes with a preference for or dependence on seawater for growth were classified at the genus level using molecular taxonomy. Their potential to produce bioactive secondary metabolites was analyzed by PCR screening for genes involved in polyketide and nonribosomal peptide antibiotic synthesis. Using microwell cultures, conditions for the production of antibacterial and antifungal compounds were identified for 15 of the 27 isolates subjected to this screening. Nine of the 15 active extracts were also active against multiresistant gram-positive bacterial and/or fungal indicator organisms, including vancomycin-resistant Enterococcus faecium and multidrug-resistant Candida albicans. Activity-guided fractionation of fermentation extracts of isolate TFS65-07, showing strong antibacterial activity and classified as a Nocardiopsis species, allowed the identification and purification of the active compound. Structure elucidation revealed this compound to be a new thiopeptide antibiotic with a rare aminoacetone moiety. The in vitro antibacterial activity of this thiopeptide, designated TP-1161, against a panel of bacterial strains was determined.

Functional characterization of 4'-phosphopantetheinyl transferase genes of bacterial and fungal origin by complementation of Saccharomyces cerevisiae lys5.

Lysine biosynthesis in yeast requires the posttranslational conversion of the alpha-aminoadipate semialdehyde reductase Lys2 by the 4'-phosphopantetheinyl transferase (PPTase) Lys5 from the inactive apo-form into the catalytically active holo-form. In this reaction, the peptidyl carrier domain of Lys2 is modified at a conserved serine residue side chain with the 4'-phosphopantetheine moiety derived from coenzyme A. We have deleted the lys5 gene in Saccharomyces cerevisiae to investigate the substrate specificity of various heterologous PPTase genes of bacterial and fungal origin by testing their ability to complement lys5 in trans. Genes encoding PPTases Sfp and Gsp from Bacillus spp., which are involved in non-ribosomal peptide antibiotic synthesis, complemented the lys5 deletion, whereas ydcB of Bacillus subtilis, which encodes the acyl carrier protein synthase involved in fatty acid synthesis, could not. Two yet uncharacterized fungal genes, q10474 of Schizosaccharomyces pombe, meanwhile annotated as the putative lys7 gene, and npgA of Aspergillus nidulans, also complemented the lys5 deletion and have thus been functionally characterized as PPTases. The complementation system described also provides the basis for a simple method of functional characterization of PPTase candidate genes and their cloning from chromosomal DNA or cDNA libraries of diverse origin.

Functional characterization of 4′-phosphopantetheinyl transferase genes of bacterial and fungal origin by complementation of Saccharomyces cerevisiae lys5

Lysine biosynthesis in yeast requires the posttranslational conversion of the α-aminoadipate semialdehyde reductase Lys2 by the 4′-phosphopantetheinyl transferase (PPTase) Lys5 from the inactive apo-form into the catalytically active holo-form. In this reaction, the peptidyl carrier domain of Lys2 is modified at a conserved serine residue side chain with the 4′-phosphopantetheine moiety derived from coenzyme A. We have deleted the lys5 gene in Saccharomyces cerevisiae to investigate the substrate specificity of various heterologous PPTase genes of bacterial and fungal origin by testing their ability to complement lys5 in trans. Genes encoding PPTases Sfp and Gsp from Bacillus spp., which are involved in non-ribosomal peptide antibiotic synthesis, complemented the lys5 deletion, whereas ydcB of Bacillus subtilis, which encodes the acyl carrier protein synthase involved in fatty acid synthesis, could not. Two yet uncharacterized fungal genes, q10474 of Schizosaccharomyces pombe, meanwhile annotated as the putative lys7 gene, and npgA of Aspergillus nidulans, also complemented the lys5 deletion and have thus been functionally characterized as PPTases. The complementation system described also provides the basis for a simple method of functional characterization of PPTase candidate genes and their cloning from chromosomal DNA or cDNA libraries of diverse origin.

Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis.

Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis.

Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping.

Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure.

Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA

Analysis of the primary structure of peptide synthetases involved in non-ribosomal synthesis of peptide antibiotics revealed a highly conserved and ordered domain structure. These functional units, which are about 1000 amino acids in length, are believed to be essential for amino acid activation and thioester formation. To delineate the minimal extension of such a domain, we have amplified and cloned truncated fragments of the grsA gene, encoding the 1098-amino acid multifunctional gramicidin S synthetase 1, GrsA. The overexpressed His6-tagged GrsA derivatives were affinity-purified, and the catalytic properties of the deletion mutants were examined by biochemical studies including ATP-dependent amino acid activation, carboxyl thioester formation, and the ability to racemize the covalently bound phenylalanine from L- to the D-isomer. These studies revealed a core fragment (PheAT-His) that comprises the first 656 amino acid residues of GrsA, which restored all activities of the native protein, except racemization of phenylalanine. A further deletion of about 100 amino acids at the C-terminal end of the GrsA core fragment (PheAT-His), including the putative thioester binding motif LGGHSL, produced a 556-amino acid fragment (PheA-His) that shows a phenylalanine-dependent aminoacyl adenylation, but almost no thioester formation. A 291-amino acid deletion at the C terminus of the native GrsA, that contains a putative racemization site resulted in complete loss of racemization ability (PheATS-His). However, it retained the functions of specific amino acid activation and thioester formation. The results presented defined biochemically the minimum size of a peptide synthetase domain and revealed the locations of the functional modules involved in substrate recognition and ATP-dependent activation as well as in thioester formation and racemization.

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin.

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.

Use of microbial enzyme and cell preparations to synthesise oligopeptide antibiotics

AbstractMicrobial enzymes and cells (in free or immobilised form) have so far been applied mainly in hydrolysis and bioconversion reactions, but recently there has been an increasing interest in the use of such ‘controlled catalytic biomass’ to produce fermentation products and to apply the principle to organic synthesis. Particular attention is given here to the use of immobilised enzymes and cells for producing known or new peptide antibiotics. An evaluation is presented of the applicability of this concept in the fascinating field of peptide antibiotic bioconversions and fermentations. As an important example of the use of enzymes or cells to convert peptide antibiotics into useful derivatives, free and immobilised penicillin acylases, producing the penicillin nucleus 6‐amino‐penicillanic acid, are reviewed as well as their potential to synthesise semi‐synthetic penicillins and cephalosporins. The action of acetylesterase and α‐amino acid ester hydrolase on cephalosporin compounds is also discussed. Use of enzymes and cells to synthesise antibiotics as an alternative method to conventional fermentation is subsequently reviewed. The concept of total enzymic synthesis in vitro of antibiotics is illustrated in the exploitation of the ‘multi‐enzyme thiotemplate mechanism’ of Bacillus brevis, to produce gramicidin S. Total synthesis of peptide antibiotics with immobilised living cells or mycelium as catalysts has been demonstrated recently in the cases of penicillin and bacitracin. These examples of complex multi‐step enzyme reactions might indicate the vast potential of immobilised enzyme and cell technology in fermentation, in biotechnological processes and in organic synthesis.

Induction of sporulation in Bacillus brevis by peptide antibiotics.

THE regulatory mechanisms of the process of bacterial sporulation are unknown, although some authors have speculated that sporulation and the synthesis of peptide antibiotics are somehow connected1–4. Paulus and coworkers proposed that tyrocidine and gramicidin D, both synthesised by Bacillus brevis, may interact with the transcriptional process during sporogenesis5. They have isolated a mutant of B. brevis which is unable to synthesise gramicidin D and which produces defective spores. The defects could be cured by exogenous gramicidin D, suggesting that this peptide is involved in sporogenesis6. In this report we demonstrate that sporulation of early vegetative growing B. brevis cells can be induced by exogenous tyrothricin, a mixture of tyrocidine and gramicidin D, provided that the cells are exposed to a culture medium lacking the nitrogen source. The induction of sporulation occurs concomitantly with a significant increase in RNA synthesis. The results suggest that sporulation and the action of these two peptides are causally connected. Both peptides may play a positive regulatory role in cell differentiation.

Kinetics of the enzymatic synthesis of peptide antibiotics.

Biotechnology and BioengineeringVolume 19, Issue 8 p. 1225-1232 Communications to the EditorFree Access Kinetics of the enzymatic synthesis of peptide antibiotics D. I. C. Wang, D. I. C. Wang Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139Search for more papers by this authorB. K. Hamilton, B. K. Hamilton Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139Search for more papers by this author D. I. C. Wang, D. I. C. Wang Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139Search for more papers by this authorB. K. Hamilton, B. K. Hamilton Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139Search for more papers by this author First published: August 1977 https://doi.org/10.1002/bit.260190813Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 C. H. Tzeng, K. D. Thrasher, J. P. Montgomery, B. K. Hamilton, and D. I. C. Wang, Biotechnol. Bioeng., 17, 143 (1975). 2 H. Koischwitz and H. Kleinkauf, Biochim. Biophys. Acta, 429, 1041 (1976). 3 M. Dixon and E. C. Webb, Enzymes, Academic, New York, 1964. 4 J. R. Whitaker, Principles of Enzymology for the Food Sciences, Marcel Dekker, New York, 1972. 5 E. Katz, Pure Appl. Chem., 28, 551 (1971). 6 F. Lipmann, Accounts Chem. Res., 6, 361 (1973). 7 S. G. Laland and T. L. Zimmer, Essays Biochem., 9, 31 (1973). 8 K. Kurahashi, Ann. Rev. Biocherm., 43, 445 (1974). 9 F. G. Heineken, H. M. Tsuchiya, and H. Aris, Math. Biosci., 1, 95 (1967). Citing Literature Volume19, Issue8August 1977Pages 1225-1232 ReferencesRelatedInformation

Aminoacyl thiol esters and the origins of genetic specificity

Reasons for believing that primitive mechanisms of translation may have employed thiol esters of the amino acids rather than oxygen esters are summarized. It is suggested that coenzyme A (HSCoA), which fulfills the role of aminoacyl transfer in the synthesis of peptide antibiotics, is a primitive analogue of tRNA which performs a similar role in protein synthesis. HSCoA—an adenylic acid moiety containing phosphates esterified at the 3′ and 5′ positions and linked to a peptide-like structure terminating in a reactive thiol—possesses chemical features suggestive of both peptides and polynucleotides. Examination of the chemistry of HSCoA-like molecules shows that a rather similar compound can carry out a repeating intramolecular peptide synthesis in the absence of enzymes. Condensation of further nucleotides onto the adenylic acid moiety gives rise to parallel modes of peptide and oligonucleotide synthesis. A “self-improving” ability to select available amino acids is inherent in the proposed mechanism of peptide synthesis. The hypothesis plausibly explains the universal occurrence of a sulphur-containing amino acid at the N terminus of nascent proteins.

Synthesis of Peptide Antibiotics

Synthesis of Peptide Antibiotics

Biosynthesis of polypeptide antibiotics.

Abstract

Metabolism of Bacillus thuringiensis in Relation to Spore and Crystal Formation

A general pattern of metabolism was determined for Bacillus thuringiensis grown in a glucose-yeast extract-salts medium. The pattern did not differ significantly from that of B. cereus grown in a similar medium. Acetic acid produced from glucose during exponential growth was further catabolized in the early sporulation phase of growth, at which time the specific activity of aconitate hydratase increased markedly. Fluoroacetate and alpha-picolinate prevented the removal of accumulated acid, and the resulting low pH inhibited spore and crystal synthesis. Neither crystal-related antigens nor insect toxicity was shown by cells whose crystal synthesis was inhibited in this way. alpha-Picolinate prevented the normal increase in specific activity of aconitate hydratase without inhibiting exponential growth. It also inhibited aconitate hydratase in vitro, but only if preincubated with the enzyme. alpha-Picolinate did not inhibit the increase in specific activity of aconitate hydratase or spore and crystal synthesis in a medium buffered near neutrality. Chloramphenicol and actinomycin D inhibited crystal enlargement and sporulation when added to cells in which small crystals had already begun to form. Typical messenger ribonucleic acid-dependent protein synthesis, rather than the type associated with peptide antibiotic synthesis, is thus indicated for the synthesis of crystal peptide subunits.