Structure of bacillomycin F, a new peptidolipid antibiotic of the iturin group

The structure of a new antibiotic of the iturin group, bacillomycin F, has been demonstrated. It is a mixture of homologous peptidolipids, essentially C51H80N12O14 and C52N82N12O14. The lipid moiety consists of minor isoC15, anteisoC15 beta-amino acids and major isoC16, isoC17 and anteisoC17 beta-amino acids. The peptide sequence was determined by studying the peptides obtained after mild HCl hydrolysis and by Edman degradation of bacillomycin F treated with N-bromosuccinimide. The sequence was confirmed by two-dimensional NMR spectrometry and fast-atom-bombardment mass spectrometry gave the molecular masses of the homologous compounds. Bacillomycin F is a cyclic peptidolipid; its complete structure is given in the paper.     

Structure of iturine A, a peptidolipid antibiotic from Bacillus subtilis.

A mixture of iturines extracted from Bacillus subtilis gave, on column chromatography, iturine A, iturine B, and iturine C. Iturine A has the entire antifungal activity. It is a mixture of two homologous peptidolipids C48,H74N12O14 and C49H76N12O14 (mp 177 degrees C, [alpha]D-1.7 degrees in methanol (c 0.05 g/mL); mol wt 1042 and 1056). The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid. The peptide moiety contains 7 mol of amino acids: D-Asn2, L-Asn, L-Gln, L-Pro, L-Ser, and D-Tyr. A cyclic structure for iturine A with the serine residue linked to the fatty amino acids through a peptide bond has been domonstrated. By mild HCl hydrolysis, lipid-soluble and water-soluble peptides were obtained. They were analyzed by chemical methods and by mass spectrometry. Permethylated and perdeuteriomethylated derivatives of iturine A were also subjected to mass spectrometric analysis. Both chemical analysis and mass spectrometry led to the cyclic structure I for iturine A.     

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Aims:  Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages.
Methods and Results:  A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16.
Conclusions:  The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F.
Significance and Impact of the Study:  The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100°C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.     

Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D

Bacillus subtilis AU195 produces bacillomycin D, a cyclic lipopeptide that is an inhibitor of the aflatoxin producing fungus Aspergillus flavus. Sequence analysis of the bacillomycin D operon revealed four ORFs with the structural organization of the peptide synthetases. Disruption of ORF 2, which links the amino acid moiety to the b-amino fatty acid, resulted in the loss of antifungal activity. By comparing the sequence of bacillomycin D, iturin A and mycosubtilin operons, our results showed that intergenic module replacement have occurred between B. subtilis lipopeptide synthetases including the iturin family and the plipastatin and fengycin family.     

Interactions of antibiotics of the iturin group with human erythrocytes

The peptidolipid antibiotics, iturin A and bacillomycin L have a disrupting effect on erythrocyte membrane leading to a simultaneous release of K+ ions and hemoglobin. The formation of ghosts is accompanied by a partial solubilisation of lipid components. Binding experiments with radioactive antibiotics show that about 7 X 10(7) molecules of iturin A and 4 X 10(7) molecules of bacillomycin L are bound to one erythrocyte at the concentration giving 100% hemolysis. This concentration is reduced by about 20% after treatment of erythrocytes by phospholipase A2. Scatchard plots show that the affinity for erythrocyte membrane is higher with bacillomycin L than with iturin A. This difference is probably correlated to the differences in their peptide moieties. The substitution of tyrosyl residue leads to a ten-fold increase of the concentrations giving 100% hemolysis, probably due to a low distribution coefficient of derivatives between the membrane and the solvent. This result and the humped Scatchard curves obtained with both antibiotics may be related to the self-association of the compounds in aqueous solutions.     

Synthesis of a coumarin based fluorescent amino acid

L-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid (L-Adp), as a non-proteinogenic fluorescent amino acid has been synthesized by a highly stereoselective routine (>99.5%). This fluorescent amino acid, as fluorophore-quencher pair, may be used to study peptide assays. For enantiomeric excess determination, the racemic D-Adp (D-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid) has also been synthesized.     

Biosynthesis of peptide inhibitor MR-387 by Streptomyces neyagawaensis

Biosynthesis of MR-387 by Streptomyces neyagawaensis SL-387 was studied by testing the incorporation of radio-active precursors and the detection of biosynthetic enzyme. L-Phenylalanine, L-valine, L-proline, and acetic acid were efficiently incorporated into MR-387, but phenylethylamine and oxalic acid were not. These results suggest that the (2 S,3 R)-3-amino-2-hydroxy-4-phenylbutanoic acid moiety of MR-387 is synthesized from phenylalanine and acetic acid but not directly via the phenylethylamine. Synthesis of MR-387 is mediated by a peptide synthetase with a novel activity.     

Effect of phenylalanine and tyrosine analogues on Bactrocera oleae Gmelin (Dipt., Tephritidae) reproduction

The effect of nine phenylalanine and tyrosine analogues at various concentrations upon the reproduction of adult olive fruit fly Bactrocera oleae Gmelin (Diptera, Tephritidae), was tested. Fecundity (eggs/female/day) and percentage egg hatchability was significantly reduced by the following anti-amino acids (in parentheses are indicated the antagonized amino acid): p-fluoro- DL -phenylalanine (phe), p-amino- DL - and - L -phenylalanine (tyr), 3-amino- L -tyrosine (tyr) and L -mimosine (tyr), at concentrations of 0.1, 0.25, 0.05 and 0.5% in the diet, respectively. Hatchability was also affected by two other analogues of phenylalanine and tyrosine, p-bromo- DL -phenylalanine at a concentration of 10% and m-fluoro- DL -tyrosine at a concentration of 1.5%. Insect survival was affected only by p-fluorophenylalanine and 3-amino- L -tyrosine at concentrations 0.25 and 6%, respectively. No significant differences were observed between the survival of the two sexes. Finally, β-2-thienyl- DL -alanine (phe) and α-methyl- DL -p-tyrosine (tyr) did not affect any of the parameters tested. Electron microscopy examination of the follicles and the egg-shell structure of eggs laid by females fed with diets containing p-amino- L -phenylalanine and 3-amino- L -tyrosine, revealed abnormalities related to the egg-shell fine structure.     

Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus

AIMS: In a search for an antifungal peptide with a high activity against Aspergillus flavus, Bacillus subtilis AU195 was selected from a collection of isolates with antagonistic activity against A. flavus. METHODS AND RESULTS: To identify the antifungal peptides, a protein purification scheme was developed based on the detection of the antifungal activity in purified fractions against A. flavus. Two lipopeptides were purified with anion exchange and gel filtration chromatography. Their masses were determined to be 1045 and 1059 m/z with mass spectrometry, and their peptide moiety was identical to bacillomycin D. CONCLUSION: AU195 synthesized a mixture of two antifungal bacillomycin D analogues with masses of 1045 and 1059, the 14 mass unit difference representing the difference between a C15 and a C16 lipid chain. SIGNIFICANCE AND IMPACT OF THE STUDY: Both bacillomycin D analogues were active at the same concentration against A. flavus, but the different lipid chain length apparently affected the activity of the lipopeptide against other fungi.     

Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor.

The gene coding for sensory rhodopsin I (SR-I) has been identified in a restriction fragment of genomic DNA from the Halobacterium halobium strain L33. Of the 1014 nucleotides whose sequence was determined, 720 belong to the structural gene of SR-I. In the 5' non-coding region two putative promoter elements and a ribosomal binding site have been identified. The 3' flanking region bears a potential terminator structure. The SR-I protein moiety carries no signal peptide and is not processed at its N terminus. The C terminus, however, lacks the last aspartic acid residue encoded by the gene. Analysis of the primary structure of SR-I reveals no consistent homology with the eukaryotic photoreceptor rhodopsin, but 14% homology with the halobacterial ion pumps, bacteriorhodopsin (BR) and halorhodopsin (HR). Residues conserved in all three proteins are discussed with respect to their contribution to secondary structure, retinal binding and ion translocation. The aspartic acid residue which mediates in BR the reprotonation of the Schiff base (D96) is replaced in SR-I by a tyrosine (Y87). This amino acid replacement is proposed to be of crucial importance in the evolution of the slow-cycling photosensing pigment SR-I.     

NMR structure of active and inactive forms of the sterol-dependent antifungal antibiotic bacillomycin L.

The antifungal antibiotic lipopeptide bacillomycin L [cyclo-(L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6++ +-L-Thr7-beta-amino fatty acid)] from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol-phospholipid dependence on biomembranes. This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (L-Asp1, D-Tyr2) di-O-methylated form. The structures were calculated under NMR-derived restraints using molecular-dynamic simulated-annealing protocols starting from a random array of atoms. The structure of the native antibiotic is spread over different conformers in which two families are recognized. It was found that most structures have dihedral phi and psi angles defining a type-II' beta-turn including amino acids 5-8, in certain cases stabilized by a 8HN-5CO hydrogen bond, whereas a minority of structures adopt an inverse gamma-turn including amino acids 6-8, stabilized in all cases by an 8HN-6CO hydrogen bond. The di-O-methylation of L-Asp1 and D-Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures. The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved D-Tyr2 position.     

Synthesis of a coumarin based fluorescent amino acid

Summary l-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid (l-Adp), as a non-proteinogenic fluorescent amino acid has been synthesized by a highly stereoselective routine (>99.5%). This fluorescent amino acid, as fluorophorequencher pair, may be used to study peptide assays. For enantiomeric excess determination, the racemicdl-Adp (dl-2-amino-3-(6,7-dimethoxy-4-coumaryl)-propionic acid) has also been synthesized.     

Immunochemical characteristics of a lipopolysaccharide from Pseudomonas aurantiaca

A lipopolysaccharide was isolated from Pseudomonas aurantiaca IMB 31 by extraction with aqueous phenol and purified by ultracentrifugation. The lipopolysaccharide was confined to the phenol phase. Fucosamine (2-amino-2,6-dideoxygalactose) (36%) and bacillosamine (2,4-diamino-3,4,6-trideoxyglucose) (23%) were identified as hypothetic components of the O-chain in the carbohydrate moiety of the macromolecule using the techniques of paper chromatography, gas-liquid chromatography and ion-exchange chromatography on an amino acid analyser. Rhamnose, glucose, galactose, glucosamine and galactosamine were detected as hypothetical components of the core in the lipopolysaccharide composition, as well as 2-keto-3-deoxyoctonic acid, heptose, alpha-alanine and phosphorus, usual components of the core in Pseudomonas. The following predominant fatty acids were identified in the composition of lipid A using the techniques of gas-liquid chromatography with standard compounds and gas-liquid mass spectrometry: 3-OH C10:0 (14.4%), C12:0 (30.5%), 2-OH C12:0 (14.9%), 3-OH C12:0 (17.4%), C16:0 (9.9%). The serological relationship between P. aurantiaca strains was studied, and their phylogenetic relationship with P. fluorescens is discussed.     

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.     

A new lipophilic fluorescent probe for interaction studies of bioactive lipopeptides with membrane models

The new fluorescent lipophilic moiety 11-[(7-amino-4-methyl-2-oxo-2H-1-benzopyran-3-acetyl)amino]undecanoic acid (AMCA-omegaAud-OH) was introduced by SPPS at the N-terminus of the immunodominant epitope GpMBP(74-85). FRET experiments using the new fluorescent lipopeptide demonstrate that the peptide interacts with much more affinity with the membrane compared to the lipid free analogue.     

Design and synthesis of BACE1 inhibitors containing a novel norstatine derivative (2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid

A novel norstatine derivative, phenylthionorstatine [(2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid; Ptns], containing a hydroxymethylcarbonyl (HMC) isostere was designed, synthesized, and stereochemically determined. Then, Ptns was introduced into the structure of BACE1 inhibitors at the P(1) position. Finally, Ptns was found as a suitable P(1) moiety for potent BACE1 inhibitor design.     

Conformational restriction of the Tyr53 side-chain in the decapeptide HE.

A series of conformationally restricted analogs of the hen egg lysozyme (HEL) decapeptide 52-61 in which the conformationally flexible Tyr53 residue was replaced by several more constrained tyrosine and phenylalanine analogs was prepared. Among these tyrosine and phenylalanine analogs were 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (Htc), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 4-amino- 1,2,4,5-tetrahydro-8-hydroxy-2-benzazepine-3-one (Hba), 4-amino-1,2,4,5-tetrahydro-2-benzazepine-3-one (Aba), 2-amino-6-hydroxytetralin-2-carboxylic acid (Hat) and 2-amino-5-hydroxyindan-2-carboxylic acid (Hai) in which the rotations around Calpha-Cbeta and Cbeta-Cgamma were restricted because of cyclization of the side-chain to the backbone. Synthesis of Pht-Hba-Gly-OH using a modification of the Flynn and de Laszlo procedure is described. Analogs of beta-methyltyrosine (beta-MeTyr) in which the side-chains were biased to particular side-chain torsional angles because of substitution at the beta-hydrogens were also prepared. These analogs of HEL[52-61] peptide were tested for their ability to bind to the major histocompatibility complex class II I-Ak molecule and to be recognized in this context by two T-cell hybridomas, specific for the parent peptide HEL[52-61]. The data showed that the conformation and also the configuration of the Tyr53 residue influenced both the binding of the peptide to I-Ak and the recognition of the peptide/I-Ak complex by a T-cell receptor.     

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives

(S-2-amino-5-(aminooxy)pentanoic acid (L -homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L -homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.     

A crystallographic determination of a chemical structure: 6-amino-10-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine, an example of an unusual D-ribose conformation.

The crystal and molecular structure of 6-amino-10-(beta-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine has been determined by single crystal X-ray diffraction methods. The crystals are triclinic, of noncentric space group Pl, with cell dimensions a equals 5.434 (5), b equals 12.269 (19), c equals 4.574 (4) A, alpha equals 92.3 (1), beta equals 94.0 (1), gamma equals 95.3 degrees (1) and Z equals 1. The structure has been refined to an R value of 0.049 (Rw equals 0.063), by use of counter measured intensity data for 1063 observed reflections. The pyrimidopyrimidine ring is planar. The sugar moiety is in the envelope conformation with O-1'-endo (0E), and there is an intramolecular hydrogen bond (2.58 A) (O-3'-H-O3'...O-2'). All oxygen atoms except O-1' ring oxygen-atom are involved in hydrogen bonding. The pyrimidopyrimidine rings lie in planes 3.4 A apart.