1H NMR study on the conformation of bacitracin A in aqueous solution.

The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other.     

Further evidence for the presence of a thiazoline ring in the isoleucylcysteine dipeptide intermediate in bacitracin biosynthesis

Isoleucylcysteine dipeptide, a first intermediate peptide in bacitracin biosynthesis, was liberated from the enzyme protein and oxidized with manganese dioxide in dimethylsulfoxide. The resulting oxidation product was identified by thin-layer chromatography as 2-(2-methyl-l-oxobutyl)-thiazole-4-carboxylic acid which has been isolated from the hydrolysate of bacitracin F. This result shows that the intermediate dipeptide contains a thiazoline ring, and that the thiazoline ring is synthesized at the dipeptide stage in the process of peptide chain elongation in bacitracin biosynthesis. Improbability of non-enzymatic dehydrative cyclization of the dipeptide is discussed.     

Proton NMR studies of Co(II) complexes of the peptide antibiotic bacitracin and analogues: insight into structure-activity relationship

Bacitracin is a widely used metal-dependent peptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(II) for its biological activity, and has been reported to bind several other transition metal ions, including Co(II), Ni(II), and Cu(II). Despite the wide use of bacitracin, a structure-activity relationship for this drug has not been established, and the structure of its metal complexes has not been fully determined. We report here one- and two-dimensional nuclear magnetic resonance (NMR) studies of the structure of the metal complexes of several bacitracin analogues by the use of paramagnetic Co(II) as a probe. The Co(II) complex of this antibiotic exhibits many well-resolved isotropically shifted (1)H NMR signals in a large spectral window ( approximately 200 ppm) due to protons near the metal, resulting from both contact and dipolar shift mechanisms. The assignment of the isotropically shifted (1)H NMR features concludes that bacitracin A(1), the most potent component of the bacitracin mixture, binds to Co(II) via the His-10 imidazole ring N(epsilon), the thiazoline nitrogen, and the monodentate Glu-4 carboxylate to form a labile complex in aqueous solutions. The free amine of Ile-1 does not bind Co(II). Several different analogues of bacitracin have also been isolated or prepared, and the studies of their Co(II) binding properties further indicate that the antimicrobial activity of these derivatives correlates directly to their metal binding mode. For example, the isotropically shifted (1)H NMR spectral features of the high-potent bacitracin analogues, including bacitracins A(1), B(1), and B(2), are virtually identical. However, Glu-4 and/or the thiazoline ring does not bind Co(II) in the bacitracin analogues with low antibiotic activities, including bacitracins A(2) and F.     

A nuclear magnetic resonance study of the metal binding sites in bacitracin.

Carbon-13 nuclear magnetic resonance spectroscopy has been used to identify sites in bacitracin which bind Cu2+ and Mn2+. Results are presented which implicate the free carboxyl groups of the aspartic and glutamic acid residues and the imidazole ring of the histidine residue as metal complexation sites between pH 6 and 8. Evidence is presented which also indicates that the thiazoline ring of bacitracin binds Mn2+. Bacitracin does not bind Cu2+ or Mn2+ at pH values of 2.5 or less.     

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.     

In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.

The action of a sporulation-specific seryl protease on antibiotic-free extracts of Bacillus licheniformis cells yields a peptide that is identified as bacitracin by its biological activity, its spectral properties, and its comigration with genuine bacitracin in both paper and thin-layer chromatography. During proteolysis, a chemical structure is generated with the spectral properties of a delta-2 thiazoline ring. The yield in vitro, 4 microgram of bacitracin per mg of protein, is less than the maximal yield from sporulating cells, 75 microgram of bacitracin per mg of cell protein, but is a linear function of the amount of protein in the reaction system. Approximately 30% of the protein yielding the antibiotic is ribosomal associated, and only 25% of that amount can be removed by washing with 1 M NH4Cl. The substrate protein is a constant fraction of the cell protein throughout exponential growth and very early sporulation stages of culture development.     

Sulphur-aromatic interactions in proteins

The geometry of sulphur-aromatic interactions in globular proteins has been analysed using crystallographic data derived from 36 proteins, solved to resolutions of 2 Å or better. About half of all sulphur atoms from cyst(e)ine and methionine residues are in contact ( 6 Å from ring centroid) with an aromatic ring (phenylalanine, tyrosine or tryptophan). Compared to carbon and nitrogen atoms the interacting sulphur atoms express an affinity towards the edge of the aromatic rings, and avoid the region above the ring in the vicinity of the π-electrons. This preference is similar to that previously found for oxygen atoms around phenylalanine rings, and may be electrostatic in origin.

Sulfw-aromatic interaction Protein Side-chain contact Cyst(e)ine Methionine Packing     

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.     

CCK8 peptide derivatized with diphenylphosphine for rhenium labelling: synthesis and molecular mechanics calculations.

A novel CCK8 derivative bearing a chelating agent at its N- end and its oxo-rhenium(V) complex have been synthesized and characterized. The chelating agent N-[N-13-(diphenylphosphino)propionyl]glycyl]cysteine (PN2S) ligand, the coordination set of which is made by the phosphorus atom of phosphine, the nitrogen atoms of the two amido groups and the sulphur atom of cysteine, has been used due to its high affinity towards the oxo-rhenium(V) moiety. Molecular modelling studies indicate that the CCK8 peptide adopts the right conformation for cholecystokinin receptor binding, and that modifications on the N-terminal side of CCK8 obtained by introducing chelating agents and its metal complexes should not affect the interaction with CCK(A) receptor.     

In vivo anticancer, anti-inflammatory, and toxicity studies of mixed-ligand Cu(II) complexes of dien and its Schiff dibases with heterocyclic aldehydes and 2-amino-2-thiazoline. Crystal structure of [Cu(dien)(Br)(2a-2tzn)](Br)(H(2)O)

A new series of complexes of the type [Cu(dien)(2a-2tzn)Y(2)] and [Cu(dienXX)(2a-2tzn)Y(2)], where dien=diethylenetriamine and dienXX=Schiff dibase of diethylenetriamine formed with 2-furaldehyde (dienOO), 2-thiophenecarboxaldehyde (dienSS), or pyrrol-2-carboxaldehyde (dienNN); Y=Cl, Br or NO(3); and 2a-2tzn=2-amino-2-thiazoline, were synthesized and their structure established by C, H, N and Cu analysis; IR and electronic spectra; magnetic susceptibility; and molar conductivity. The isolated complexes are monomers, paramagnetic, and electrolytes of types 1:1 or 1:2. In both types of solid state complexes, [Cu(dien)(2a-2tzn)Y(2)] and [Cu(dienXX)(2a-2tzn)Y(2)], dien and its Schiff dibases are bonded to Cu(II) in a tridentate fashion through 3N atoms. The coordination sphere is completed by the endocyclic nitrogen of the thiazoline moiety and by two Cl, Br, or NO(3) groups with distorted octahedral geometry. The proposed structure of these compounds was supported by X-ray analysis of [Cu(dien)(Br)(2a-2tzn)](Br)(H(2)O). The coordination polyhedron around the copper atom can be described as a distorted square pyramid [Cu(dien)(Br)(2a-2tzn)](+). Its basal plane is occupied by the four nitrogen atoms of the dien and thiazoline ligands with Cu-N distances ranging between 1.996(6) and 2.032(3)A, and the axial position is occupied by one of the two bromine atoms (Br1) with a Cu1-Br1 bond distance of 2.782(1)A. The second bromine atom (Br2) is 4.694(2)A from the copper atom, which exists as a discrete anion and is responsible for the cationic nature of the complex. Results regarding toxicity, antitumor, and anti-inflammatory activities of the investigated compounds are promising and allow the selection of a lead compound for further biological studies.     

Interaction centres of purine nucleotides: adenosine-5'-diphosphate and adenosine-5'-triphosphate in their reactions with tetramines and Cu(II) ions

The interactions between the nucleotides: adenosine-5'-diphosphate (ADP) and adenosine-5'-triphosphate (ATP) with spermine (Spm) and 1,11-diamine-4,8-diazaundecane (3,3,3-tet), as well as Cu(II) ions are studied. In the metal-free systems nucleotide-polyamine molecular complexes have been found to form, in which the interaction centres are the nitrogen atoms of the purine ring N(1) and N(7), oxygen atoms of the phosphate group of the nucleotide (for 3,3,3-tet) and protonated nitrogen atoms of the polyamine. Significant differences in the mode of metallation between the systems with Spm and 3,3,3-tet have been established. In the systems with Spm, the main products are protonated species with [N(7),O] chromophore and the nitrogen N(1) is involved in the intramolecular interaction additionally stabilising the complex. In the systems with 3,3,3-tet the formation of metal-ligand-ligand (MLL) species has been observed, in which the oxygen atoms from the phosphate group and the nitrogen atoms from the polyamine are involved in the metallation, while the N(1) and N(7) atoms from the purine ring of the nucleotide remain outside the inner coordination sphere of the copper ion. The main centre of metallation in the nucleotide, both with Spm and 3,3,3-tet, is the phosphate group of the nucleotide.     

Interaction centres of pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) in their reactions with tetramines and Cu(II) ions

The interactions between pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) and Cu(II) ions, spermine (Spm) and 1,11-diamino-4,8-diazaundecane (3,3,3-tet) have been studied. The composition and stability constants of the complexes formed have been determined by means of the potentiometric method, while the centres of interactions in the ligands have been identified by the spectral methods (UV-Vis, Ultraviolet and Visible spectroscopy; EPR, electron spin resonance; NMR). In the systems without metal, formation of the molecular complexes nucleotide-polyamine with the interaction centres at the endocyclic nitrogen atom of purine ring N3, the oxygen atoms of the phosphate group from the nucleotide and protonated nitrogen atoms of the polyamine have been detected. Significant differences have been found in the metallation between the systems with Spm and with 3,3,3-tet. In the systems with spermine, mainly protonated species are formed with the phosphate group of the nucleotide and deprotonated nitrogen atoms of the polyamine making the coordination centres, while the donor nitrogen atom of the nucleotide N3 is involved in the intramolecular interligand interactions, additionally stabilising the complex. In the systems with 3,3,3-tet, the MLL' type species are formed in which the oxygen atoms of the phosphate group and nitrogen atoms of the polyamine are involved in metallation, whereas the N3 atom from the pyrimidine ring of the nucleotide is located outside the inner coordination sphere of copper ion. The main centre of Cu(II) interaction in the nucleotide, both in the system with Spm and 3,3,3-tet is the phosphate group of the nucleotide.     

Complexation properties of aminophosphites bearing phosphorus and nitrogen atoms included in six-membered cycles

A series of amino phosphites having phosphorus or/and nitrogen atoms, which are included in a six-membered cycle, has been synthesized. The presence of such cycles in these P,N-bidentate ligands has been shown to strongly influence the ways of their coordination with Rh(I) and Pd(II) atoms. The resulting chelate complexes have been found to be less stable when the six-membered chelate metalacycle formed and the initial cycle of the ligand molecule comprise a bicyclic system with the phosphorus or nitrogen atom being as a spiro atom.     

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.     

Potentiometric and spectroscopic studies of the Cu(II) complexes of Ala-Arg8-vasopressin and oxytocin: two vasopressin-like peptides.

The results are reported of a potentiometric and spectroscopic study of the H+ and Cu2+ complexes of Ala-Arg8-vasopressin (Ala-AVP) and oxytocin at 25 degrees C and an ionic strength of 0.10 mol dm-3 (KNO3). The coordination chemistry of oxytocin and Cu(II) has been shown to be virtually identical to that of Arg8-vasotocin, forming unusually stable complexes with four nitrogen coordination (4N complexes) below pH 7. Spectroscopic evidence suggests weak interaction between Cu(II) and the sulphur atom of the -Cys6- residue in the 2N species (pH congruent to 6) but this is absent in the 4N complex. Evidence is also presented for perturbation of electronic transitions within the aromatic ring of the Tyr residue by Cu(II). While the physiological potency of Ala-AVP is very high, its coordination chemistry differs significantly from that of Arg8-vasopressin. With Cu(II) it forms complexes of similar stability to those with tetraglycine, demonstrating that the addition of an alanyl residue to the amino-terminal of the peptide destroys the conformation which is particularly favorable for rapid 4N coordination.     

Platinum(II) complexes with fluoroquinolones: Synthesis and characterization of unusual metal-piperazine chelates

The fluoroquinolones constitute an important class of synthetic antimicrobial agents. The interaction between these compounds and metallic cations has been investigated primarily with the goal of identifying the functional groups directly linked to the metal. Furthermore, there has been some additional work investigating the effect of metal ions upon antibacterial activity. The vast majority of the metallic complexes reported in the literature show coordination of the fluoroquinolone through the carboxylate and the carbonyl oxygen atoms. In this work, we report the synthesis and characterization of platinum(II) complexes with ciprofloxacin, levofloxacin, ofloxacin, sparfloxacin, and gatifloxacin. The resulting complexes present the fluoroquinolone coordinated through the nitrogen atoms of the piperazine ring, which is unusual. The proposed structures were supported by spectroscopic evidence (IR, NMR, and mass spectra) as well as by theoretical studies.     

Coordination mode of adenosine 5'-diphosphate in ternary systems containing Cu(II), Cd(II) or Hg(II) ions and polyamines

The interactions of adenosine 5'-diphosphate (ADP) with some polyamines (PA) (1,3-diaminopropane (tn), 1,4-diaminobutane (Put), 1,7-diamino-4-azaheptane (3,3-tri) and 1,8-diamino-4-azaoctane (Spd)) both in presence and in the absence of metal ions (Cu(II), Cd(II) and Hg(II)) have been studied. In the metal-free systems the formation of adducts (ADP)Hx(PA) has been observed, in which the main reaction centres are the endocyclic nitrogen atoms of the purine ring, the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine. The effectiveness of the phosphate group in formation of adducts has been found to decrease in the series Put > Spd > Spm and to be lower than in the reactions with shorter homologues of biogenic amines. In the ternary systems with metal ions the formation of molecular complexes (ML L' type) has been evidenced in which the protonated polyamine interacts with the nitrogen atoms N(1) or N(7) of the purine ring of the nucleotide. In the ternary systems Cu(II)/ADP/polyamine the coordination dichotomy observed in the binary system Cu(II)/ADP disappears. In the systems with Hg(II) ions the pH range of the dichotomy is extended, while for the systems Cd(II)/ADP/polyamine no changes of the range relative to the binary system Cd(II)/ADP have been noted.     

Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin

Bacitracin is a widely used metallopeptide antibiotic produced by Bacillus subtilis and Bacillus licheniformis with a potent bactericidal activity directed primarily against Gram-positive organisms. This antibiotic requires a divalent metal ion such as Zn(2+) for its biological activity, and has been reported to bind several other transition metal ions, including Mn(2+), Co(2+), Ni(2+), and Cu(2+). Despite the widespread use of bacitracin since its discovery in the early 1940s, the structure-activity relationship of this drug has not been established and the coordination chemistry of its metal complexes was not fully determined until recently. This antibiotic has been suggested to influence cell functioning through more than one route. Since bacterial resistance against bacitracin is still rare despite several decades of widespread use, this antibiotic can serve as an ideal lead for the design of potent peptidyl antibiotics lacking bacterial resistance. In this review, the results of physical (including NMR, EPR, and EXAFS) and molecular biological studies regarding the synthesis and structure of bacitracin, the coordination chemistry of its metal derivatives, the mechanism of its antibiotic actions, its influence on membrane function, and its structure and function relationship are discussed.     

Interactions of 1,12-diamino-4,9-dioxadodecane (OSpm) and Cu(II) ions with pyrimidine and purine nucleotides: adenosine-5'-monophosphate (AMP) and cytidine-5'-monophosphate (CMP)

The interactions of Cu(II) ions with adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP) and 1,12-diamino-4,9-dioxadodecane (OSpm) were studied. A potentiometric method was applied to determine the composition and stability constants of complexes formed, while the mode of interactions was analysed by spectral methods (ultraviolet and visible spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), (13)C NMR, (31)P NMR). In metal-free systems, molecular complexes nucleotide-polyamine (NMP)H(x)(OSpm) were formed. The endocyclic nitrogen atoms of the purine ring N(1), N(7), the nitrogen atom of the pyrimidine ring N(3), the oxygen atoms of the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine were the reaction centres. The mode of interaction of the metal ion with OSpm and the nucleotides (AMP or CMP) in the coordination compounds was established. In the system Cu(II)/OSpm the dinuclear complex Cu(2)(OSpm) forms, while in the ternary systems Cu(II)/nucleotide/OSpm the species type MH(x)LL' and MLL' appear. In the MH(x)LL' type species, the main centres of copper (II) ion binding in the nucleotide are the phosphate groups. The protonated amino groups of OSpm are involved in non-covalent interaction with the nitrogen atoms N(1), N(7) or N(3) of the purine or pyrimidine ring, whereas at higher pH, deprotonated nitrogen atoms of polyamine are engaged in metallation in MLL' species.