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PubMed1980

Nuclear Overhauser effect and cross-relaxation rate determinations of dihedral and transannular interproton distances in the decapeptide tyrocidine A.

The following interproton distances are reported for the decapeptide tyrocidine A in solution: (a) r(phi) distances between NH(i) and H alpha (i), (b) r(psi) distances between NH (i + 1) and H alpha (i), (c) r(phi psi) distances between NH(i + 1) and NH(i), (d) NH in equilibrium NH transannular distances, (e) H alpha in equilibrium H alpha transannular distances, (f) r x 1 distances between H alpha and H beta protons, (g) NH(i) in equilibrium H beta (i) distances, (h) NH (i + 1) in equilibrium H beta (i) distances, (i) carboxamide-backbone protons and carboxamide-side chain proton distances, (j) side chain proton-side chain proton distances. The procedures for distance calculations were: NOE ratios and calibration distances, sigma ratios and calibration distances, and correlation times and sigma parameters. The cross-relaxation parameters were obtained from the product, say, of NOE 1 leads to 2 and the monoselective relaxation rate of proton 2; the NOEs were measured by NOE difference spectroscopy. The data are consistent with a type I beta-turn/ type II' beta-turn/ approximately antiparallel beta-pleated sheet conformation of tyrocidine A in solution and the NOEs, cross-relaxation parameters, and interproton distances serve as distinguishing criteria for beta-turn and beta-pleated sheet conformations. It should be borne in mind that measurement of only r phi and r psi distances for a decapeptide only defines the ( phi, psi)-space in terms of 4(10) possible conformations; the distances b-j served to reduce the degeneracy in possible (phi, psi)-space to one tyrocidine A conformation. The latter conformation is consistent with that derived from scalar coupling constants, hydrogen bonding studies, and proton-chromophore distance measurement, and closely resembles the conformation of gramicidin S.

Amino Acids↗
PubMed1979

Determination of individual side-chain conformations, tertiary conformations, and molecular topography of tyrocidine A from scalar coupling constants and chemical shifts.

We report for the decapeptide tyrocidine A: (a) H alpha and H beta chemical shifts and scalar coupling constants for most residues of tyrocidine A in methanol-d4 and dimethyl-d6 sulfoxide (Me2so-d6) and the H alpha and H beta chemical shifts for other residues; (b) scalar coupling constants 3J alpha beta for nine side chains in methanol-d4 but only seven side chains in Me2SO-d6, due to chemical shift degeneracy; the Gln9 and Tyr10 side chains in methanol-d4 were only approximately analyzed; (c) a total spin-spin analysis of Pro5 in Me2SO-d6 and, partly by comparison, also in methanol-d4; (d) conversion of 3J alpha beta values to side-chain conformations for all residues in methanol-d4; comparisons, where possible, led to the conclusion that side-chain conformations are similar in methanol-d4 and Me2SO-d6; (e) an absolute conformational analysis of Pro5 from 3J values and a method of assigning all pro-R,S protons; Pro5 has a Ramachandran B, C2-Cexo-Cendo conformation; (f) chi 1, chi 2 conformations of several aromatic residues based upon proton-chromophore distance measurement from anomalous chemical shifts and Johnson-Bovey diagrams; (g) pro-R and pro-S assignments of H beta's from anomalous chemical shifts, high-temperature dependence of anomalous chemical shifts, and backbone side-chain nuclear Overhauser effects; (h) most tertiary conformations of the whole tyrocidine A molecule possessing residues 4--8 and 10 in highly preferred (ca. 90%) chi 1 conformations, but residues 1--3 and 9 having at least two chi 1 rotamers; (2) description of three topographical regions of the molecule--a hydrophobic region, a flat hydrophilic surface on the other side of the molecule, and a hydrophilic region consisting of two peptide backbone units and the side chains of Asn8, Gln9, and Tyr10; (j) proposed side chain, beta-turn, and beta-pleated sheet conformations that readily account for all "normal" and anomalous chemical shifts.

Amino Acids↗
PubMed2000

Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase.

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

Bacillus↗
PubMed1986

DNA-supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin.

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces 'packaging' of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5',8-trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.-E. (1980) Cell 21, 773-783]. Relaxed DNA is not affected by tyrocidine whereas linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine-induced relaxation as well as the packaging, an observation which might be of biological relevance.

DNA, Superhelical↗
PubMed1974

Isolation of a peptidyl-pantetheine-protein from tyrocidine-synthesizing polyenzymes.

The polyenzyme complex responsible for the synthesis of tyrocidine in Bacillus brevis (ATCC 8185) was found to contain 4'-phosphopantetheine, which appeared to be connected with the production of growing peptide chains. Confirmation of this assumption has now been obtained by purifying from bacterial lysates a polyenzyme-dissociation product; this was labeled with [(14)C]pantothenic acid and peptide chains containing tritiated amino acids, and had a molecular weight of 17,000. To obtain these results, organisms were grown udner conditions favorable for incorporation of radioactive pantothenic acid into tyrocidine-synthesizing enzymes. A crude lysate of the [(14)C]pantothenic acid-labeled organisms was preincubated with the tritiated amino acids to form enzyme-bound growing peptide chains. The doubly labeled fragments were purified from the polyenzyme-dissociation products produced by prolonged lysis. In a second set of experiments, the three enzymes responsible for tyrocidine synthesis, including the two polyenzymes containing pantetheine, were purified and incubated with radioactive amino acids and ATP to form polyenzyme-bound peptide chains. Thereupon, a Triton X-100 extract of the 20,000 x g fraction of crude homogenate was added to dissociate the purified polyenzymes. The dissociation products were purified and yielded, on dodecyl sulfate gel electrophoresis, peptidyl-marked products ranging in molecular weight from 90,000 to 17,000, the latter being most abundant. Electrophoresis of analogous preparations after preincubation with higher concentrations of dodecyl sulfate and dithiothreitol at 100 degrees yielded a single product of 17,000 molecular weight, indicating that the larger molecular weight fractions were aggregates thereof.

Amino Acids↗
PubMed1989

Structure of tyrocidine micelles in isotropic aqueous solution.

Aqueous solutions of tyrocidine B, iodoacetyltyrocidine B, and diiodotyrocidine B in 40% H2O:60% ethanol (w/w) were investigated by analytical ultracentrifugation, light scattering, and small-angle X-ray scattering techniques. A reasonable hydrodynamic description of the aggregates of molecular weight 28,600 is a rod with a length of 170 A and a diameter of 30 A; this description is consistent with the X-ray scattering data. Over a broad range of concentrations, inelastic light scattering measurements and small-angle X-ray scattering experiments provide the same hydrodynamic values (e.g., Stokes radii and frictional ratios). The positions of the iodines were resolved and found at a radius (R) of 16 A. So, the iodine-iodine distance across the cross section is 34 A, indicating that labeled tyrocidine B is not affected during the aggregational process.

Chemical Phenomena↗
PubMed2003

Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst.

Nonribosomal peptides (NRP) such as the antibiotic tyrocidine have D-amino acids, introduced by epimerase (E) domains embedded within modules of the enzymatic assembly lines. We predict that the peptide bond-forming condensation (C) domains immediately downstream of E domains are D-specific for the peptidyl donor and L-specific for the aminoacyl acceptor ((D)C(L)). To validate this prediction and establish that the C(5) domain of tyrocidine synthetase is indeed (D)C(L), the apoT (thiolation) forms of module 4 (TycB(3) AT(4)E) and module 5 (TycC(1) C(5)AT(5)) were expressed. T(5) was posttranslationally primed with CoASH to introduce the HS-pantetheinyl group and autoaminoacylated with radiolabeled L-Asn* or L-Asp*. Alternate donor substrates were introduced by priming apo AT(4)E with synthetically prepared tetrapeptidyl-CoA's differing in the chirality of Phe-4, D-Phe-L-Pro-L-Phe-L-Phe-CoA, and D-Phe-L-Pro-L-Phe-D-Phe-CoA. The tetrapeptidyl-S-T(4) and L-Asp-S-T(5) were studied for peptide bond formation and chain translocation by C(5) to yield pentapeptidyl-S-T(5), whose chirality (D-L-L-D-L- vs D-L-L-L-L-) was assayed by thioester cleavage and chiral chromatography of the released pentapeptides. Only the D-Phe-4 pentapeptidyl-S-T(5) was generated, implying that only D-L-L-D-S-T(4) was utilized, proving C(5) is indeed a (D)C(L) catalyst. Furthermore, a mutant with an inactive E domain transferred tetrapeptide only when loaded with D-Phe-4 tetrapeptidyl donor, not L-Phe-4, confirming that in the wild-type assembly line C(5) only transfers D-L-L-L-tetrapeptidyl-S-T(4) after in situ epimerization by the E domain. These results contrast the observation that C(5) can make both L-Phe-L-Asn and D-Phe-L-Asn when assayed with Phe as the donor substrate. Hence, utilizing an aminoacyl-S-T(4) versus the natural peptidyl-S-T(4) donor produced misleading information regarding the specificity of the condensation domain.

Amino Acid Substitution↗
PubMed1979

Total assignments, including four aromatic residues, and sequence confirmation of the decapeptide tyrocidine A using difference double resonance. Qualitative nuclear overhauser effect criteria for beta turn and antiparallel beta-pleated sheet conformations.

The complete assignments of all the proton magnetic resonance signals from each NH-CalphaH-CbetaH2 moiety in a complex peptide containing several residues of the same type has not yet been achieved without specific or stereospecific isotopic enrichment. We report the sequencing and proton magnetic resonance spectral assignments, including those of 4 aromatic residues, of tyrocidine A, an analog of the decapeptide gramicidin S. Two complementary methods, proton-proton nuclear Overhauser enhancements and scalar decoupling, evaluated by two distinct forms of difference double resonance, were used. All chemical shifts, scalar coupling constants, and [1H:1H] nuclear Overhauser enhancements for the backbone protons are reported. The [1H:1H] nuclear Overhauser enhancements are consistent with tyrocidine A possessing a beta-I turn/beta-II' turn/antiparallel beta-pleated sheet conformation. In addition to the previously proposed nuclear Overhauser enhancement criteria for beta turns and antiparallel beta sheets, another criterion for identifying the antiparallel beta sheet is demonstrated; namely, the nuclear Overhauser enhancement between 2 CalphaH protons of the central resisdues, in this case the Phe7CalphaH and Orn2CalphaH.

Amino Acid Sequence↗
PubMed2005

Relationship between activating and editing functions of the adenylation domain of apo-tyrocidin synthetase 1 (apo-TY1).

Tyrocidine synthetase 1 (TY1), the initial monomodular constituent of the tyrocidine biosynthetic system, exhibits relaxed substrate specificity, however an efficient editing of the mis-activated amino acid provides for fidelity of product formation. We chose to analyse the consequence of single amino acid substitutions, in the amino acid activation site of apo-TY1, on the editing functions of the enzyme. Discrimination between L-Phe and D-Phe by apo-TY1 depends primarily on the editing reaction. Distraction of unnatural amino acid substrates, such as L-PheSer, implies that editing is not designated to select a specific mis-activated amino acid, but instead to discriminate all mis-activated amino acid analogues. It was shown that active site residues which interact with the adenylate are essential for both activation and editing. Substitution of Lys186 with arginine substantially reduces the editing capacity of the protein. Loss of amino acid discrimination ability by the apo-K186T and apo-R416T mutant proteins suggests a role of active site residues in maintaining the structural determinants for substrate selection. Inadequate conformational changes, induced by non-cognate amino acid substrates, promote ATP breakdown yielding P(i) and ADP. Replacement of residue Lys186 or Arg416 enhances ATP hydrolysis implying a role in binding or adjusting of the triphosphate chain for adenylate formation and pyrophosphate cleavage.

Adenosine Monophosphate↗
PubMed2004

Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent.

Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.

Amino Acid Substitution↗
PubMed1975

Studies of individual amino acid residues of the decapeptide tyrocidine A by proton double-resonance difference spectroscopy in the correlation mode.

The cyclic decapeptide antibiotic tyrocidine A was studied by two relatively new methods, viz., correlation proton magnetic resonance (pmr) spectroscopy and double-resonance difference pmr spectroscopy. The correlation method of spectral accumulation provided pmr spectra of good resolution, and in addition the signal-to-noise ratio achieved per unit time of accumulation was much higher than that achieved by use of the conventional continuous wave (cw) method. Furthermore, when protonated solvents are used, the correlation mode of accumulation has a distinct advantage over pulse and fast Fourier transform (fft) methods currently in use. Double-resonance difference (drd) spectra of individual amino acid residues in tyrocidine A were obtained by the correlation method when the decoupling frequency was maintained at the center frequency of the appropriate C-alpha proton multiplet and at a level of power that totally decoupled vicinal C-alpha and C-beta protons; the resolution of these spectra was good, and the signal-to-noise ratio was high. The distinct patterns and spectral positions of the drd spectra were characteristic of the particular type of amino acid residue and, therefore, could be used as the basis for making assignments. Furthermore, the drd spectra revealed the spectral positions of individual C-alpha and C-beta proton transitions and therefore, upon spectral analysis, could provide the chemical shifts and coupling constants of these protons. Positions of transitions were revealed even though they were hidden by overlap in the corresponding conventional single- or double-resonance spectra.

Amino Acids↗
PubMed1969

On the association of tyrocidine with acetylcholinesterase.

In studies of several polypeptide antibiotics with a high affinity for a variety of biological membranes, tyrocidine was found to bind specifically to acetylcholinesterase, an enzyme localized in excitable membranes. Several other polypeptides tested were not bound. Tyrocidine reversibly inhibits acetylcholinesterase formed by homogeneous protein, but seems to have no effect on the activity of the enzyme bound to the eel electroplax membrane. This inhibition is accompanied by a reversible association of the soluble enzyme in to ordered and rapidly sedimenting aggregates of large molecular weight. Electron micrographs of acetylcholinesterase are shown which are consistent with the chemical evidence of the existence of four subunits.

Animals↗
PubMed1971

Pantetheine-linked peptide intermediates in gramicidin S and tyrocidine biosynthesis.

To study the function of pantetheine in gramicidin S and tyrocidine biosynthesis, pepsin digests of the polymerizing enzymes, of which only the heavy ones contain pantetheine, were analyzed. The digests of gramicidin S enzymes charged with either [(14)C]proline or with D-phenylalanyl-[(14)C]proline, were analyzed by thin-layer chromatography; only the dipeptide showed a derivative associated with pantetheine. Similar results were obtained from the heavy tyrocidine enzyme charged with either [(14)C]asparagine alone or with the pentapeptide D-Phe-Pro-Phe-D-Phe-[(14)C]Asn. Several radioactive products appeared on the thin-layer chromatograms of both these digests; association with pantetheine was found only in the case of the pentapeptide. Exposure of the chromatogram from the pentapeptide-labeled digest to performic acid and development in a second direction separated the peptide from pantetheine, indicating that a nascent peptide was originally linked to the cofactor by a thioester bond. The connection of pantetheine only with peptide residues appears to confirm its role in transpeptidation during peptide chain growth.

Asparagine↗
PubMed1989

Gene cluster containing the genes for tyrocidine synthetases 1 and 2 from Bacillus brevis: evidence for an operon.

From a genomic library of the tyrocidine producer Bacillus brevis ATCC 8185 constructed in the bacteriophage vector EMBL3, a recombinant phage which contains the structural genes coding for tyrocidine synthetases 1 and 2, TycA and TycB, was identified. The location of the tycA gene within the 16-kilobase insert of this clone, EMBL25-1, was mapped by hybridization studies by using the previously isolated tycA DNA as a probe. Restriction analyses, the construction of subclones, and the analysis of proteins encoded by the subclones located the tycB gene at the 3' end of the tycA gene and revealed that the two genes are transcribed in the same direction. Nuclease S1 protection studies and DNA sequencing studies of the intergenic region indicated that tycA and tycB are separated by a 94-base-pair noncoding region and suggested that these genes are organized as an operon.

Amino Acid Sequence↗
PubMed1965

Effect of polymyxin B, tyrocidine, gramicidin D, and other antibiotics on the enzymatic hydrolysis of poly-beta-hydroxybutyrate.

Merrick, J. M. (State University of New York, Buffalo). Effect of polymyxin B, tyrocydine, gramicidin D, and other antibiotics on the enzymatic hydrolysis of poly-beta-hydroxybutyrate. J. Bacteriol. 90:965-969. 1965.-Previous studies have demonstrated that native poly-beta-hydroxybutyrate (PHB) granules isolated from Bacillus megaterium are surrounded by a discrete membranelike structure. Morphological alterations of PHB granules are mainly characterized by membrane fragmentation, and can be correlated with decreased susceptibility of the polymer to enzymatic hydrolysis by soluble factors. In the present investigation, the inhibitory effect of a variety of surface-active and other antibiotics on the enzymatic depolymerization of PHB was examined. The most potent inhibitors were polymyxin B, tyrocidine, and gramicidin D. These polypeptide antibiotics are known to attack other types of membranous structures. The results, therefore, support previous evidence that the membrane or similar constituents of PHB granules are intimately involved in its metabolism. Chlortetracycline was also found to be a potent inhibitor of the depolymerization, but its mechanism of action may be different from the other antibiotics. The polymer-synthesizing enzyme(s), also localized on the granules, is inhibited by tyrocidine and gramicidin D but not by polymyxin B or chlortetracycline.

Antimycin A↗
PubMed2004

Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase.

The 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(beta-D-Gal) and Tyr(beta-D-Glc) as well as with Ser-containing beta-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization.

Bacillus subtilis↗
PubMed2005

Chimeric streptogramin-tyrocidine antibiotics that overcome streptogramin resistance.

Streptogramin antibiotics are comprised of two distinct chemical components: the type A polyketides and the type B cyclic depsipeptides. Clinical resistance to the type B streptogramins can occur via enzymatic degradation catalyzed by the lyase Vgb or by target modification through the action of Erm ribosomal RNA methyltransferases. We have prepared through chemical and chemo-enzymatic approaches a series of chimeric antibiotics composed of elements of type B streptogramins and the membrane-active antibiotic tyrocidine that evade these resistance mechanisms. These new compounds show broad antibiotic activity against gram-positive bacteria including a number of important pathogens, and chimeras appear to function by a mechanism that is distinct from their parent antibiotics. These results allow for the development of a brand new class of antibiotics with the ability to evade type B streptogramin-resistance mechanisms.

Anti-Bacterial Agents↗
PubMed2002

Substrate spectrum of tyrocidine thioesterase probed with randomized peptide N-acetylcysteamine thioesters.

Apparent kinetic constants k(cat) and K(m) were determined for tyrocidine thioesterase (TycC TE) using randomized peptide N-acetylcysteamine thioesters as substrate analogues. The enzyme has been found to be adequately active for the synthesis of positional-scanning libraries for novel antibiotic screening with reduced k(cat)/K(m) in the range of 2 to 82 folds lower than that of the wild-type sequence

Acetylcysteine↗