Phleomycin F

Phleomycin is one of the anticancer glycopeptide antibiotics which cause DNA cleavage. It is commonly used as a copper(II) complex. Therefore, it is important to study the metal ion binding process and to define the coordination mode. In this paper, we describe the acid-base properties of phleomycin and the coordination sphere of the Cu(II) cation. In the metal binding process up to five nitrogen donor atoms can be involved. Four of them in the same plane deriving from: the pyrimidine ring, secondary amine of β-aminoalanine, imidazole and amide of the nearest peptide bond (from β-hydroxyhistidine) and in the apical position from the α-amino functional group of β-aminoalanine, resulting complex has a square-pyramidal geometry. Phleomycin complexes are able to induce single- and double-stranded DNA damage when they are accompanied by one-electron reductants, such as dithiothreitol, glutathione, 2-mercaptoethanol or ascorbic acid. In such conditions they produce reactive oxygen species which are responsible for DNA cleavage. The metal ion binding site is relatively close to the nucleic acid interacting moiety. This supports the hypothesis that copper ion is important in the anticancer activity which involves DNA degradation.

In order to genetically map and eventually isolate avirulence genes, parasexual crosses between different races of Fusarium oxysporum f. sp. lycopersici were performed by means of protoplast fusion. Two wild-type strains, race 1 Fol004 (A1a2a3) and race 3 Fol029 (a1a2A3), were transformed with phleomycin and hygromycin resistance genes, respectively. In total 32 fusion products were selected by screening for the presence of both marker genes. The presence of either avirulence gene A1 or A3 in the fusion products was determined by plant bioassays. Segregation of avirulence revealed a bias for the presence of A1. Two recombinants for the avirulence phenotype were observed, each with a new association of avirulence genes never observed to exist in the wild. Electrophoretic karyotype analysis revealed that chromosome patterns were different for all fusion products. Hybridization patterns using various probes indicated that chromosome rearrangements and recombination had occurred. Karyotype analysis of the two avirulence recombinants revealed hybrid karyotypes resulting from a massive exchange of parental DNA. This indicates that the present population of recombinants can be used for gene mapping in the asexual fungus F. oxysporum f. sp. lycopersici.

Bleomycin (Bm), a 1.4 kDa glycopeptide excreted by Streptomyces verticillus, is a natural antibacterial compound used in therapy as antineoplastic drug. To counteract its biological activity, cells have developed several resistance mechanisms, one of these based on proteins able to tightly bind Bm. In this paper, the interaction of Zn(2+)-Bm with the Streptoalloteichus hindustanus Bm resistance protein (ShBle) has been investigated by solution state NMR. Sequential nOe and chemical shift index have shown that the fold of the protein (in absence or presence of Bm) is identical to the previously published X-ray structure. The dimeric nature of ShBle is confirmed by the diffusion tensor as determined by NMR relaxation data. Using isotope filtered nOe experiment, intermolecular nOes between Bm and ShBle have been observed as used for modeling. While the interaction of the Bm metal binding site with ShBle appears to be uniquely defined, several conformations of the bithiazole moieties are compatible with the NMR data. Binding of Bm also induces changes of the local dynamics (stretch N85-G91), as shown by (15)N relaxation data. These results are discussed in the context of several Bm analogues able to interact with ShBle and of the recently published X-rays structures.