Biosynthesis Of Gramicidin Research Articles

First Draft Genome Sequence of Thermophilic Laceyella tengchongensis BKK01, Isolated from Municipal Solid Waste in Thailand.

Laceyella tengchongensis BKK01 is a thermophilic bacterium isolated from municipal solid waste. The genome of L. tengchongensis BKK01 includes a gene putatively encoding gramicidin S synthase. Gramicidin S has antibiotic activity against some bacteria and fungi. The newly sequenced 3.44-Mb draft genome of L. tengchongensis BKK01 will shed some light on the biosynthesis of gramicidin S.

Antimicrobial peptide gramicidin S is accumulated in granules of producer cells for storage of bacterial phosphagens

Many antimicrobial peptides are synthesized non-ribosomally in bacteria, but little is known about their subcellular route of biosynthesis, their mode of intracellular accumulation, or their role in the physiology of the producer cells. Here, we present a comprehensive view on the biosynthesis of gramicidin S (GS) in Aneurinibacillus migulanus, having observed a peripheral membrane localization of its synthetases. The peptide gets accumulated in nano-globules, which mature by fusion into larger granules and end up within vacuolar structures. These granules serve as energy storage devices, as they contain GS molecules that are non-covalently attached to alkyl phosphates and protect them from dephosphorylation and premature release of energy. This finding of a fundamentally new type of high-energy phosphate storage mechanism can explain the curious role of GS biosynthesis in the physiology of the bacterial producer cells. The unknown role of the GrsT protein, which is part of the non-ribosomal GS synthetase operon, can thus be assumed to be responsible for the biosynthesis of alkyl phosphates. GS binding to alkyl phosphates may suggest its general affinity to phosphagens such as ATP and GTP, which can represent the important intracellular targets in pathogenic bacteria.

Parallel interrogation of covalent intermediates in the biosynthesis of gramicidin S using high‐resolution mass spectrometry

For determination of multiple covalent intermediates bound to the ultra-large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry (MS) is a promising method to provide new mechanistic insight. Application of a quadrupole-Fourier-transform instrument (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestions of recombinant proteins afforded detection of two active site peptides (both ~13 kDa) that provided direct evidence for modules copurifying with their preferred amino acid substrates. Given the ability to detect multiple covalent intermediates in tandem, a competition experiment among several nonnatural substrates in parallel was performed using the first module. This defined mixture of acyl-enzyme intermediates was used to probe the selectivity of the condensation step producing a diversity of noncognate dipeptides on the second module.

Characterization of the Binding Site of the Tripeptide Intermediated-Phenylalanyll-Prolyl-l-Valine in Gramicidin S Biosynthesis

The tripeptide intermediate D-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of either L-[14C]phenylalanine or L-[14C]valine in an in vitro biosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that D-Phe-Pro-Val is attached to the 4'-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426-15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component.

The effect of positive end-expiratory pressure on the cardiac output and organ blood volumes accompanied with increase in intraabdominal pressure

Current studies on biosynthesis of gramicidin S have revealed that the peptide is synthesized by cell-free system from Bacillus brevis and the mechanism of its formation is different from that in usual protein synthesis, i.e. there is no participation of any RNA or influence of any inhibitor on protein biosynthesis (Yukioka et al., 1965; Bhagavan et al., 1966; Spaeren et al., 1967; Tomino et al., 1967). The activations of the five constituent amino acids were each shown to occur by amino acid dependent PPi-ATP exchange reactions, but amino acyl-tRNA formation was not observed (Gevers et al., 1968). Among these reactions, it is most interesting to study the mechanism of activation of ornithine, since ornithine is not involved in usual protein biosynthesis.In this paper, a procedure for partial purification of the ornithine activating enzyme from extracts of Bacillus brevis Nagano, some properties of the enzyme and also the relation of this enzyme to the formation of gramicidin S are presented.

Biosynthesis of gramicidin S with the aid of dipeptides by gramicidin S synthetase.

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.

The presence of 4′-phosphopantetheine in the bacitracin synthetase

A soluble enzyme complex participates in the biosynthesis of bacitracin which is an antibiotic dodecapeptide produced by a strain of Bacillus licheniformis and the peptide is synthesized from amino acids bdund to enzyme protein through thioester linkages [ 1,2] . These features of bacitracin synthetase resemble those in the biosynthesis of gramicidin S and tyrocidine [3]. Recently, it was reported that gramicidin S and tyrocidine synthetases contained one and two moles of 4’-phosphopantetheine pr mole of synthetase, respectively [4,5], and that phosphopantetheine arm participated in peptide chain elongation [6]. Bacitracin synthetase has beeri separated into two complementary fractions (Peaks I and II) by column chromatography on hydroxyapatite [2]. The present work demonstrates that Peak II fraction was further separated into two components and that each of the three components responsible for bacitracin biosynthesis contains approximately one equivalent of 4’-phosphopantetheine.

43] Gramicidin S synthetase

Publisher Summary This chapter discusses the assay and purification procedure of gramicidin S synthetases. The following enzymic activities may be used for assaying gramicidin S synthetase: (1) synthesis of gramicidin S; (2) amino acid-dependent ATP- 32 PPi exchange; (3) amino acid-dependent ATP-[ 14 C] AMP exchange; (4) thioesterbonding of the individual amino acids, (5) ATP-dependent racemization of phenylalanine. Purification procedure involves cultivation of the microorganism, preparation of crude extract, streptomycin sulfate precipitation, ammonium sulfate precipitation, and chromatography on DEAE sephadex A-50, chromatography on sephadex G-200 alternative method or the separation of light and heavy enzyme: affinity chromatography. The biosynthesis of gramicidin S is the result of the concomitant functioning of a large number of catalytic activities. For instance, in the case of the heavy enzyme it has been estimated that about 18–20 different catalytic activities are involved. During the purification of gramicidin S synthetases that gives an almost homogeneous preparation of heavy enzyme, some 97% of its ability to synthesize the antibiotic is lost. The loss is particularly great during fractionation on the DEAE Sephadex G-50 and Sephadex G-200. Of the many catalytic activities involved in the biosynthesis, the catalytic activities responsible for amino acid activation are remarkably stable.

On the use of affinity chromatography in demonstrating the transfer of thioester-bound D-phenylalanine from the light enzyme of gramicidin S synthetase to an acceptor site for this amino acid on the heavy enzyme.

When a mixture of the light enzyme of gramicidin S synthetase containing thioester‐bound d‐[14C]phenylalanine and the heavy enzyme is passed through a Sepharose column containing carboxyl‐bound proline (substrate amino acid) as ligand, the two enzymes are retained as the result of the affinity between the heavy enzyme and the ligand and the affinity between the two enzymes. Upon elution and separation of the two enzymes it was demonstrated that the d‐[14C]phenylalanyl groups had been transferred to the heavy enzyme. This transfer did not take place when similar experiments were carried out using as ligands valine, ornithine or leucine which are also substrates for the heavy enzyme. Hence binding of proline to the heavy enzyme is required for transfer to take place. Upon incubation of the heavy enzyme containing the transferred d‐[14C]phenylalanyl group with the light enzyme, ATP and the five unlabelled substrate amino acids, radioactivity is incorporated into gramicidin S. This suggests that in the biosynthesis of gramicidin S, the d‐phenylalanyl group is transferred to an acceptor site on the heavy enzyme and subsequently to the imino group of thioester‐bound proline. In view of what is known about gramicidin S synthesis it seems likely that the acceptor site is a thiol site.

Biosynthesis of valinomycin

Received 29 March 1974 1. Introduction Valinomycin is a cyclododecadepsipeptide consis- ting of three residues each of L-valine, ~-D-hydroxy- isovaleric acid, D-valine and L-lactic acid joined to- gether by alternating amide and ester linkages [1 ]. The results on the biosynthesis of gramicidin S [2], tyrocidine [3] and alamethicin [4] suggest that valinomycin is formed by a similar non-ribosomal pep- tide synthesizing mechanism. In this report we des- cribe the isolation of an enzyme complex that is ca- pable of synthesizing the complete molecule of valino- mycin from the precursors L-valine and L-alanine or L-threonine.

Gramicidin S synthetase, an enzyme with an unusually large number of catalytic functions.

THE biosynthesis of gramicidin S consists in joining five different amino-acids, each occurring twice, into the cyclic structure: illustration Open image in new window

Starting point and direction of biosynthesis of gramicidin S

Starting point and direction of biosynthesis of gramicidin S

Ornithine activating enzyme from [formula omitted

Current studies on biosynthesis of gramicidin S have revealed that the peptide is synthesized by cell-free system from Bacillus brevis and the mechanism of its formation is different from that in usual protein synthesis, i.e. there is no participation of any RNA or influence of any inhibitor on protein biosynthesis ( Yukioka et al. , 1965 ; Bhagavan et al. , 1966 ; Spaeren et al. , 1967 ; Tomino et al. , 1967 ). The activations of the five constituent amino acids were each shown to occur by amino acid dependent PPi-ATP exchange reactions, but amino acyl-tRNA formation was not observed ( Gevers et al. , 1968 ). Among these reactions, it is most interesting to study the mechanism of activation of ornithine, since ornithine is not involved in usual protein biosynthesis. In this paper, a procedure for partial purification of the ornithine activating enzyme from extracts of Bacillus brevis Nagano, some properties of the enzyme and also the relation of this enzyme to the formation of gramicidin S are presented.

The presence of protein bound intermediates in the biosynthesis of gramicidin S

The presence of protein bound intermediates in the biosynthesis of gramicidin S

A peptide conjugate with the sequence Phe-Pro-Val-Orn and its relationship to the biosynthesis of gramicidin S

A peptide conjugate with the sequence Phe-Pro-Val-Orn and its relationship to the biosynthesis of gramicidin S

Studies on the biosynthesis of gramicidin S in a cell-free system from Bacillus brevis. Further attempts to elucidate its mechanism of synthesis.

1. The pH optima for the incorporation of (14)C-labelled amino acids into gramicidin S by an 11000g cell-free extract from Bacillus brevis have been determined. The pH optima for leucine, proline, phenylalanine, ornithine and valine were 7.5-7.7, 7.5-7.7, 7.7-7.9, 7.7-7.9 and 8.0-8.2 respectively. Hence the greatest difference in pH optima existed between leucine and valine, where it was 0.5pH unit. 2. The 11000g cell-free extract incorporated into gramicidin S only the l-isomers of valine, proline and ornithine. However, both isomers of leucine are utilized and the experiments indicate that a leucine racemase exists in the 11000g cell-free extract. With phenylalanine the l-isomer is utilized much more effectively than the d-isomer. This is noteworthy since it is the d-isomer that occurs in gramicidin S. The experiments indicate that conversion of the l-isomer into the d-form takes place at a stage beyond that of the free amino acid.

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis.

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.

Biosynthesis of gramicidin S by a cell-free system of bacillus brevis

Several Studies on the biosynthesis of gramicidin S by Bacillus brevis have been reported in recent years from different laboratories, such as those by Barry and Ichihara (1958), Winnick et al . (1961) and Eikhom et al . (1963, 1964) . However, most of these were concerned with experiments on intact cells of this organism. Kurahashi (1961) reported the formation of D -phenylalanyl- L -prolyl diketopiperazine as the starting peptide of gramicidin S synthesis by a cell-free system of B . Brevis Nagano, and Tomino and Kurahashi (1964) obtained D -phenylalanyl- L -prolyl- L -valine with the same system. In our laboratory, gramicidin S has been synthesized by a cell-free system of B . Brevis Nagano and part of this work has already been presented by Yukioka et al . (1963, 1964) . This communication is on some features of the enzymatic formation of gramicidin S.

Studies on the biosynthesis of gramicidin S in whole cells of Bacillus brevis

1. 1. When dl-[1- 14C]phenylalanine and dl-[5- 14C]ornithine respectively, were used, it was found that the synthesis of gramicidin S that occurred in whole cells of Bacillus brevis in the presence of chloramphenicol, and that was sufficient to prevent protein synthesis, represented synthesis de novo. 2. 2. The specific activity of gramicidin S in different cell fractions after brief labelling with dl-[1- 14C]phenylalanine was determined. Gramicidin S in the 105 000 × g fraction had by far the highest specific activity. 3. 3. Synthesis of gramicidin S de novo in the presence of chloramphenicol suggests a route of synthesis which differs from that of bacterial proteins. However, the high specific activity of gramicidin S in the 105 000 × g fractions suggests some relationship between the ribosomes and the synthesis of gramicidin S. 4. 4. Addition of mitomycin C (1 μg/ml) to a growing culture of B. brevis inhibited protein synthesis whereas gramicidin S synthesis continued with unchanged rate for another 2 h and therefore represents another instance in which the synthesis of gramicidin S continues in the absence of protein synthesis.

Biosynthesis of gramicidin and tryocidine in the Dubos strain of Bacillus brevis. I. Experiments with growing cultures.

Okuda, Kiyoshi (Department of Biochemistry and Biophysics, University of Hawaii, Honolulu), Gordon C. Edwards, and Theodore Winnick. Biosynthesis of gramicidin and tyrocidine in the Dubos strain of Bacillus brevis. I. Experiments with growing cultures. J. Bacteriol. 85:329-338. 1963.-A simple chromatographic method was developed for the isolation of gramicidin and tyrocidine from tyrothricin of Bacillus brevis. A Tryptone-yeast extract-glucose medium containing mineral salts gave the best yields of peptides in a stationary culture of the organism. The incorporation of suitable C(14)-labeled amino acids into gramicidin and tyrocidine was studied Several analogues of tyrocidine amino acids (beta-hydroxyglutamic acid, pipecolic acid, beta-2-thienylalanine, p-fluorophenylalanine, and phenyl glycine) selectively reduced tyrocidine synthesis, when added to the nutrient medium. At the same time, the production of gramicidin was augmented. Growth and protein synthesis were not affected. Two analogues in isotopic form, beta-2-thienylalanine and isoleucine, were shown to give rise to high degrees of labeling in tyrocidine.