Dependence of in vivo inactivation kinetics of gramicidin S synthetase on cell physiology

Gramicidin S synthetase, the enzyme complex catalyzing the biosynthesis of the antibiotic gramicidin S in Bacillus brevis, is subject to O(2)-dependent in vivo inactivation during exponential aerobic growth after reaching a peak in specific activity. The five amino acid substrates of the synthetase are capable of stabilizing its activity to varying degrees in whole cells shaken aerobically. Depending on the time of cell harvesting before, during, or after the peak in intracellular gramicidin S synthetase specific activity, the enzyme has a long, medium, or short half-life, respectively. The kinetic profiles of gramicidin S synthetase in B. brevis cells indicate that both the kinetics of synthetase loss and the degree of its amino-acid-mediated stabilization are a strong function of the cells' physiological development.     

A comparative study of sulfhydryl groups required for the catalytic activity of gramicidin S synthetase and isoleucyl tRNA synthetase

The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.     

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.     

Proteinchemical and kinetic features of gramicidin S synthetase

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S.     

On the domain construction of the multienzyme gramicidin S synthetase 2. Isolation of domains activating valine and leucine

The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multienzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined.     

Oxygen-dependent inactivation of gramicidin S synthetase in Bacillus brevis.

Incorporation of L-[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 37 degrees C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The L-ornithine- and D-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts.     

The proline-activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115-kDa tryptic fragment

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.     

Acidity and solubility of gramicidin S in water

The acid-base transformations of the gramicidin S molecule in water were studied. The protonization constants of the antibiotic amino group were calculated by the data of the potentiometric titration and the antibiotic distribution in the system of chloroform-water: K1 1.55 X 10(10), K2 1.38 X 10(6), the logarithm of the distribution coefficient of gramicidin S in the system of chloroform-water (1:1) lg alpha G 4.10. By the same data the constants of water solubility of gramicidin S base (1.02 X 10(7) mol/l), gramicidin S monohydrate (1.06 X 10(-4) mol/l) and gramicidin S dihydrochloride (2.08 X 10(-4) mol/l) were calculated.     

The nucleotide sequence for a proline-activating domain of gramicidin S synthetase 2 gene from Bacillus brevis

A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PPi exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

High productivity tank fermentation for gramicidin S synthetases

A high productivity tank fermentation for gramicidin S synthetases has been developed to supply biocatalyst for a preparative-scale ATP-driven cell-free enzymatic synthesis employing the polypeptide antibiotic, gramicidin S, as a model product. A rich, complex medium supports rapid and dense growth of the enzyme-producing microorganism, Bacillus brevis ATCC 9999, accompanied by the appearance of excellentenzyme activities. Under conditions used, the two enzyme fractions of the gramicidin S synthesizing system, as well as the total enzymatic activity for synthesis of gramicidin S, all reach their maxima simultaneously at the point where growth enters the stationary phase. Successful batch enzyme fermentations have been performed at the bench (14 liter) and pilot (180 liter)scales.     

Kinetic analysis of three activated phenylalanyl intermediates generated by the initiation module PheATE of gramicidin S synthetase

The three-domain initiation module PheATE (GrsA) of Bacillus brevis gramicidin S synthetase catalyzes the activation, thiolation and epimerization of L-phenylalanine (L-Phe), the first amino acid incorporated into the decapeptide antibiotic gramicidin S. There are three activated intermediates in the PheATE catalyzed chemical pathway: L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), L-Phe-S-4'-phosphopantetheine(Ppant)- and D-Phe-S-4'-Ppant-acyl enzyme. In this study, we examined PheATE in single-turnover catalysis using rapid chemical quench techniques. Kinetic modeling of the process of disappearance of the substrate L-Phe, transient appearance and disappearance of L-Phe-AMP and the ad seriatim formation and equilibration of the L- and D-Phe-S-Ppant-acyl enzyme adducts allowed evaluation of the microscopic rate constants for the three chemical reactions in the initiation module PheATE. This study provides the first transient-state kinetic analysis of a nonribosomal peptide synthetase (NRPS) module.     

Studies on gramicidin S synthetase. Purification of the heavy enzyme obtained from some mutants of Bacillus brevis.

The heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20,W value of 12.2 at a protein concentration of 2.5 mg per ml. These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.     

Dissolved oxygen levels and the in vivo stability of gramicidin S synthetase

Summary In the course of our studies on the oxidative inactivation of gramicidin S (GS) synthetase in vivo, we examined the involvement of dissolved oxygen (D.O.) levels in agitated and aerated cell suspensions. The previously discovered retardation of synthetase inactivation in the presence of glycerol, which is normally accompanied by lower D.O. than in glycerol-free controls, was also observed when D.O. was forcibly maintained at control levels. Stabilization of synthetase in cell suspensions supplemented with amino acid substrates was accompanied by similar levels of D.O. as in unsupplemented controls. These experiments show that carbon source-mediated retardation of synthetase inactivation is not brought about by decreased D.O. and that low D.O. is not necessary for stabilization of the enzyme. However, D.O. concentrations constantly maintained at or above 100% air saturation do accelerate in vivo inactivation of GS synthetase.     

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.     

Structure-activity relationship of gramicidin S analogues on membrane permeability

The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608-619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(-X-D-Leu-D-Lys-D-Leu-L-Pro-)2, where X = Gly, D-Leu and D-cyclohexylalanine (D-cHxAla); (2) N,N'-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a beta-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = D-cHxAla induced the efflux of K+ through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K+ from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide.     

Cell wall components of gramicidin S resistant Staphylococcus aureus]

Comparative study of two staphylococcus aureus 209P strains--resistant and susceptible to gramicidin S demonstrated that peptidoglycanes of two strains differ by ratio glycine/serine at peptide bridges. Besides peptidoglycanes significantly differ by amidation of alfa-carboxyles of glutamic acid in muropeptide. This peptidoglycane modification of resistant cells along with enhanced content of etherized D-alanine in teichoic acid provides lower negative charge of cell wall components. It may influence the cell wall ability to react with positively charged gramicidin molecules. It was shown that isolated cell walls and peptidoglycane of resistant cells binds significantly less gramicidin than cell walls and peptodoglyce of susceptable cells. Simultaneous determination of gramicidin binding by intact S. aureus cells and their killing revealed that lower ability of resistant cells to bind gramicidin is significant but not critical factor of gramicidin resistance.