Enniatins and Beauvericin

The structure of the enniatin B complex with potassium iodide has been studied by X-ray crystallography Unfortunately, from this investigation it could not be concluded with certainty whether the metal ion is entrapped in the central cavity or, instead, occupies a site between two adjacent ligand molecules. An arrangement of the latter type has been observed in the crystal structure of the 1 1 complex between RbNCS and the synthetic LDLLDL isomer of enniatin B In this case, Rb ions are coordinated by five carbonyl oxygens (three of the upper and two of the lower depsipeptide molecules) and the nitrogen atom of the isocyanate anion, thus forming infinite sandwiches. 

Evidence is added to the assumption of a binding of the metal cation outside the macrocyclic cavity also in the 1 1 complexes by the fact that these, though being more stable than sandwich-type aggregates, are apparently ineffective in membrane transport This suggests that the metal ion is not sufficiently shielded from solvent or counter ion interactions which would be in agreement with a metal ion position outside the macrocyclic cavity. 

In comparison to the 1 1 complexes, the enniatin sandwiches display a higher ion selectivity, their actual stability constants decreasing in the order Cs Na. Besides adducts of 2 1 stoichiometry, a 3 2 club sandwich has been proposed for the Cs complex 

Among the enniatin antibiotics, beauvericin is the one characterized in greater detail. This carrier is most interesting with respect to an anion-dependence of its transport properties Moreover, in contrast to valinomycin, it is capable of com-plexing alkaline earth as well as alkali metal ions . A study of the effects of beauvericin on the conductivity of artificial lipid membranes in the presence of both mono- and divalent cations revealed a second-order relationship between conductance and antibiotic concentration Finally, Prince, Crofts, and Steinrauf detected an apparent charge of plus one for calcium in the beauvericin-mediated transport across bacterial chromatophore membranes 

These most unusual findings could be well explained after elucidation of the crystal structure of the beauvericin complex with barium picrate This adduct turned out to be a 2 2 dimer structure of the form (Bv Ba Picj Ba Bv) Pic (Fig. 8) which is very unique inasmuch as three of the four picrate anions are incorporated into the space between the antibiotic molecules. Both the Ba ions 

---

Fig. 8. Stmcture of (a) valinomycin and (J3) and enniatins and beauvericin. Hov = a-hydroxy-isovaleric acid and Lac = lactic acid. The /V-methylamino acid for enniatin A is isoleucine enniatin B, valine enniatin C, leucine and beauvericin, phenylalanine.

Figure 1 Structures of enniatins and beauvericin. Enniatin A, R, = R2 = R3 = sec-butyl enniatin Al, Ri = isopropyl, R2 = R3 =. sec-butyl enniatin B, R3 = R2 = R3 = isopropyl enniatin Bl, Ri = R2 = isopropyl, R3 =. sec-butyl beauvericin, Ri = R2 = R3 = benzyl.

Valinomycin and the Enniatins. Neutral ionophores such as the cycHc dodecadepsipeptide valinomycin [2001-95-8] C H QN O g, (Fig. 8) from StreptomjcesJulvissimus (179), and the cycHc hexadepsipeptides enniatin [11113-62-5] and beauvericin [26048-05-5] from fungi (180—182),... 

The enniatins enniatin A [2503-13-17, C gH N Og, enniatin B [917-13-5] C23H yN202, enniatin C [19893-23-3], C Hg N Og, and beauvericin (Fig. 8) are 700—800 molecular weight cycHc hexadepsipeptides. They form valinomycinlike hydrophilic cavities surrounded by outer lipophilic regions, but they have more flexible stmctures than those seen with valinomycin and therefore have less specificity for potassium over sodium ion than valinomycin (186,187). 

Effects of the enniatins at lipid bilayers have been studied by several groups133,197,268>28s), Considerably higher concentrations of these antibiotics are required to obtain membrane conductance and potential values comparable with those induced by valinomycin and the macrotetrolides. Ivanov et al. observed first, second, and third power dependence of membrane conductance on enniatin B concentration, but only, first power dependence on the concentration of bis-enniatin B (cf. Section 5.1). They concluded that complexes with 1 1,2 1, and 3 2 carrier-cation stiochiometry are involved in the ion transport mediated by enniatin B and beauvericin. In the limiting case, stacking of many enniatin complex molecules can be postulated, which could lead to a channel mechanism of cation transfer by enniatin133. ... 

A comparison of beauvericin, enniatin and valinomycin as calcium transporting agents in liposomes and chromatophores... 

Benz. R. Alkali ion transport through lipid bilayer membranes mediated by enniatin A and B and beauvericin. J. Membr. Biol. 1978, 43. 367- 394. 

Barley-associated fungi are also responsible for the production of bioactive compounds called emerging or minor mycotoxins. This group includes toxins such as enniatins (ENNs) and beauvericin (BEA). Recently, the occurrence and fate of these toxins in the barley-to-beer chain have gained attention (Hu, Gastl, Linkmeyer, Hess, Rychlik, 2014 Vaclavikova et al., 2013). 

Enniatins belong to the class of A-mcthylatcd cyclopeptides which are produced by various strains of the genus Fusarium [12], As shown in Figure 1, enniatins consist of alternating residues of D-2-hydroxyisovaleric acid (D-Hiv) and a branched chain A-methyl-L-amino acid, linked by peptide and ester bonds. In the case of beauvericin the branched chain L-amino acid is substituted by L-phenylalanine (Fig. 1). 

Beauvericin is a structural homolog of enniatins in which the branched-chain L-amino acid is substituted by the aromatic amino acid L-phenylalanine. Beauvericin synthetase, which has been isolated from the fungus Beauveria bassiana [54] and various strains of Fusaria [55], strongly resembles Esyn with respect to its molecular size and the reaction mechanism. In contrast to Esyn, which is only able to incorporate aliphatic amino acids, beauvericin synthetase exhibits high substrate specificity for aromatic amino acids such as phenylalanine. This capability is obviously caused by mutational alterations in the adenylation domain of this enzyme. 

Cyclosporins, produced by the filamentous fungus Tolypocladium niveum and by numerous strains of Fusaria and Neocosmospora, are a class of cyclic undecapep-tides which are composed of hydrophobic aliphatic amino acids [73-75], They exhibit antiinflammatory, immunosuppressive, antifungal, and antiparasitic properties [74], The main metabolite, cyclosporin A, is in clinical use worldwide under the trade name SANDIMMUN to prevent allograft rejection [77,78], Besides cyclosporin A, there are 24 naturally occuring cyclosporins which have substitutions of amino acids in positions 1, 2, 4, 5, 7, and 11 and/or contain unmethylated peptide bonds in positions 1,4,6,9,10, or 11 [79-82], Cyclosporin A contains three nonproteinogenic amino acids D-alanine in position 8, L-a-aminobutyric acid in position 2, and, in position 1, the unusual amino acid 4(R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (Bmt) (Fig. 8). All three amino acids have to be synthesized by a pathway independent of the primary metabolism. In addition, several peptide bonds of the cyclosporin molecule are A -mcthylated similar to the depsipeptides enniatin, beauvericin and PF1022A-related peptides. 

JF Grove, M Pople. The insecticidal activity of beauvericin and the enniatin complex. Mycopathologia 70 103-105, 1980. 

Enniatins A and B were isolated in 1947 by Plattner and coworkers from the culture fluid of several Fusarium species238. Beauvericin is a metabolite of the fungus Beau-veria bassiana117 Syntheses of the enniatins were first reported in 1963239,246). Beauvericin was synthesized in 1971212. The structures of the three closely related antibiotics are shown in Fig. 27. 

The enniatins are closely related to beauvericin, having the general formula (LMeX-DHyv)3, and also form complexes with a number of cations. A preliminary paper on the structure of enniatin B (where X = Val) shows that the backbone of the molecule has a conformation very similar to that of beauvericin (Tishchenko et al., 1976). The cavity has a diameter of -- 2.7 A. All six isopropyl groups are pseudoequatorial and form a lipophilic perimeter on the disk-shaped molecule. 

Finally, the dlllll stereoisomer of enniatin B retains the characteristic disk shape of beauvericin and the ldldld enniatin, with the isopropyl groups directed to the periphery of the disk and a sizable cavity within the ring. The principal difference in the dlllll stereoisomer is the occurrence of one cis N-methyl peptide group (Shishova and Simonov, 1977). This group of molecules does not possess any NH groups available for hydrogen bond formation. 

The strength of the ion-dipole interaction depends not only on the M+-C =O distance but also on the angle between these groups. In the enniatin, beauvericin, complexes the angles are < 180 (unfavorable), and the shifts are much smaller than in the valinomycin complex, despite the comparable M -C =0 distance. Replacement of K+ by the smaller ion Na+ in the beauvericin cavity allows an angle closer to 180 to be obtained and the shifts are larger on complexation (1.7 and 2.3 ppm downfield). 

Enniatins A (also called lateritiin I) (Fig. 2), B, C, D, E, and are antibiotics synthesized by various strains of the genus FusariumP Like valinomycin, they are cyclic compounds, but they differ in several respects. First, the enniatins are cyclohexadepsipeptides, so they are half the size of valinomycin. Second, their ring nitrogen atoms are methylated. In contrast to valinomycin, they show poor ion selectivity. The prototype of this family is enniatin B, a cyclohexadepsipeptide assembled by threefold repetition of the L-N-methylvaline D-hydroxyisova-leric acid subunit. When t-A-methylvaline is replaced by L-N-methylphenylalanine in this repeating sequence, beauvericin results. 

Steinrauf. L.M. Beauvericin and the other enniatins. Met. Ions Biol. Syst. 1985. 19, 139-171. 

In the cases of valinomycin and enniatin depsipeptides described in Section 2. the explanation for the structural origins of the more or less pronounced ion selectivities exhibited by these antibiotics was, though being plausible, somewhat tentative, as completed X-ray analyses were available only for complexes with a single ion species, i.e. with for valinomycin and Ba " for beauvericin. However, a detailed discussion of the structural features that lead to metal ion selectivities should be based on a whole set of comparable data on complex structures with various metal ions of... 

The Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) (http //www.efsa.europa.eu/en/panels/contam.htm) deals with contaminants in the food chain. Scientific opinions on health risks are prepared by this panel. It is anticipated that the number of mycotoxins with regulatory status will increase in the future. Other Fusarium mycotoxins with possible regulatory interest in the future are fusarenone-x (an acetylated form of nivalenol), fusarin C, enniatins, beauvericin, diacetoxyscirpenol and moniliformin. More scientific opinions and risk assessments on mycotoxins can be found in the EFSA webpages (http //www.efsa.europa.eu/en/topics/topic/mycotoxins.htm). 

Logrieco, A., Rizzo, A., Ferracane, R., Ritieni, A. (2002). Occurrence of beauvericin and enniatins in wheat affected by Fusarium avenaceum head bhght. Applieed and Environmental Microbiology, 68, 82-85. 

The ion binding strength as evaluated from the Kg values in TABLE IV is comparable to that of beauvericin [12], an eighteen-membered depsipeptide belonging to enniatins, but it is much weaker than that of nonactin [13]. For both of the macrocyclic oligoesters, the Kg values decreased in the order, Cs" > Rb > K+ > Na+ > Li , and Ba " > Ca. ... 

---